Cell delivery therapeutics for musculoskeletal regeneration☆

Advanced Drug Delivery Reviews 62 (2010) 765–783 Contents lists available at ScienceDirect Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r Cell delivery therapeutics for musculoskeletal regeneration☆ Ulrich Nöth a, Lars Rackwitz a, Andre F. Steinert a, Rocky S. Tuan b,⁎ a b Orthopaedic Center for Musculoskeletal Research, Department of Orthopaedic Surgery, König-Ludwig-Haus, Julius-Maximilians-University, Würzburg, Germany Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA a r t i c l e i n f o Article history: Received 30 January 2010 Accepted 8 April 2010 Available online 14 April 2010 Keywords: Cell-based therapy Cell delivery Mesenchymal stem cells Embryonic stem cells Natural and synthetic biomaterials Gene delivery Musculoskeletal regeneration a b s t r a c t The last decade has witnessed the development of cell-based therapy as a major biomedical research area, including the treatment of musculoskeletal diseases. Both differentiated and undifferentiated stem cells have been used as starting cell sources. In particular, the use of multipotent adult mesenchymal stem cells holds great promise for future therapeutic strategies. In addition to the cell type used, the cell delivery system is also of critical importance in cell-based therapy. Cell delivery may be achieved by direct cell injection or by grafting engineered constructs derived by cell seeding into natural or synthetic biomaterial scaffolds. While direct injection is the most direct and convenient means of cell delivery, the latter approach is capable of producing three-dimensional engineered tissues with mechanical properties compatible with those of various musculoskeletal tissues. This review will focus on the functional approach of using biomaterial scaffold materials as cell carriers for musculoskeletal applications, as well as the use of cell-based gene therapy for tissue engineering and regeneration. © 2010 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. 5. Introduction . . . . . . . . . . . . . . . . . . . . . Potential of stem cells for musculoskeletal regeneration 2.1. Mesenchymal stem cells . . . . . . . . . . . . 2.2. Embryonic stem cells . . . . . . . . . . . . . Scaffold-based cell delivery . . . . . . . . . . . . . . 3.1. Natural biomaterials . . . . . . . . . . . . . . 3.1.1. Collagen . . . . . . . . . . . . . . . 3.1.2. Gelatin . . . . . . . . . . . . . . . . 3.1.3. Fibrin . . . . . . . . . . . . . . . . 3.1.4. Hyaluronan . . . . . . . . . . . . . . 3.1.5. Chondroitin sulfate . . . . . . . . . . 3.1.6. Chitosan . . . . . . . . . . . . . . . 3.1.7. Alginate . . . . . . . . . . . . . . . 3.2. Synthetic biomaterials . . . . . . . . . . . . . 3.2.1. Poly(α-hydroxy esters) . . . . . . . . 3.2.2. Ceramics . . . . . . . . . . . . . . . Ex vivo gene transfer strategies . . . . . . . . . . . . Tissue-specific cell delivery . . . . . . . . . . . . . . 5.1. Articular cartilage . . . . . . . . . . . . . . . 5.1.1. Focal articular cartilage defects . . . . 5.1.2. Osteoarthritis and rheumatoid arthritis. 5.2. Bone . . . . . . . . . . . . . . . . . . . . . 5.2.1. Critical size segmental bone defects and 5.2.2. Osteonecrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . non-unions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766 767 767 767 767 767 768 768 769 769 769 769 769 769 769 771 771 772 772 772 772 773 773 774 ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Therapeutic Cell Delivery for in situ Regenerative Medicine”. ⁎ Corresponding author. Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 221, Pittsburgh, PA 15219, USA. Tel.: + 1 412 648 2603; fax: + 1 412 624 5544. E-mail address: [email protected] (R.S. Tuan). 0169-409X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2010.04.004 766 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 5.3. Ligament and tendon. . . . . . . 5.3.1. Anterior cruciate ligament 5.3.2. Tendon . . . . . . . . . 5.4. Meniscus . . . . . . . . . . . . 5.5. Spine . . . . . . . . . . . . . . 5.5.1. Intervertebral disc . . . . 5.5.2. Spinal fusion . . . . . . 6. Conclusion . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction For cell-based therapy, for example in musculoskeletal medicine, the most convenient and direct method of cell delivery is direct injection of a suspension of cells. The suspension can be injected directly into the diseased tissue, where the cells might function via secretion of bioactive factors and eventually integrate into the tissue with or without further differentiation (Fig. 1). Such a direct approach may be used for diseases such as osteoarthritis (OA), tendinitis of the Achilles tendon or rotator cuff injuries, where the cells can be easily introduced by intraarticular, intratendinous or intramuscular injection to the diseased target tissues [1–3]. In other cases involving substantial structural defects that require bridging or filling, such as segmental bone or focal articular cartilage defects, cells need to be delivered in a matrix-guided manner into the target tissue [4,5]. For such cell delivery therapeutics, a large number of natural or synthetic materials, as well as composites consisting of two or more different biomaterials (Table 1), have been investigated for their potential as cell delivery vehicles for almost every tissue of the musculoskeletal system [6–11]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775 775 775 776 776 776 777 778 778 778 In particular, novel processing technologies (e.g., electrospinning) have been adapted to fabricate scaffolds with optimized ultrastructure, surface chemistry and biomechanical properties to best suit the targeted tissue and utilized cell type [12–14]. The essential function of a cell delivery scaffold is to provide a temporary three-dimensional template for the seeded cells to adhere and subsequently to synthesize new target-tissue-specific extracellular matrix (ECM) in a shape and form guided, at least initially, by the scaffold. The main criteria for scaffold design include controlled biodegradability, suitable mechanical strength and appropriate surface chemistry, as well as the ability to regulate cellular activities, such as proliferation, cell–cell and cell–matrix interactions, and directed differentiation [12,13]. Another key requirement is sufficient scaffold porosity to facilitate nutrition, proliferation, migration of cells, and integration into the native tissue, and to ensure cell colonization of the entire carrier. Naturally, in addition to the specific characteristics of the carrier system, the nature and biological activities of the delivered cell type are of critical importance to the functionality of the cell-based construct. Fig. 1. Delivery of cells to diseased musculoskeletal tissue. (A) Cell-based therapy for musculoskeletal tissue engineering. Cells in suspension can be injected directly into the diseased tissue, where they may function via secretion of trophic mediators to elicit regenerative effects. Also, cells can be delivered in matrix-guided approach into the target tissue after ex vivo conditioning with biochemical and/or mechanobiological stimulation. (B) Gene therapy approach for diseased musculoskeletal tissues. Expression constructs of genes can be directly injected for in vivo gene delivery or primary cells are used as vehicles for ex vivo gene delivery. In the latter, successfully transduced cells can be applied either by injection as a suspension, or seeded within a matrix that can be delivered into the target tissue. Depending on the delivery approach chosen, ubiquitous or local transgene expression is induced by the ex vivo genetically modified cells or resident cells are transduced via vector application into the target tissue. 767 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 Table 1 Commonly used biomaterials for cell delivery in musculoskeletal tissue engineering.a Natural Synthetic a Biomaterial Scaffold Target tissues Ref. Collagen type I type II gelatin Fibrin Hyaluronan Chondroitin sulfate Chitosan Alginate Hydrogel, sponge, membrane, fleece, nanofibers Cartilage, bone, skin, intervertebral disc, adipose, skin, drug delivery [37,41–61] Gel, nanofibers, tubes Gel, fibers, membrane, nanofibers Hydrogel, sponge Hydrogel, sponge, nanofibers Hydrogel (beads) Cardiovascular, cartilage, spinal cord, bone Cartilage, vascular, skin, adipose, bone Cartilage, vascular, bone, heart valve, kidney Cartilage, bone, skin, nerve, vascular, periodontal bone Cartilage, intervertebral disc, bone, vascular, liver, pancreas, nerve [62–65] [66–69] [70–75] [76–78] [79–83] Biomaterial Scaffold Target tissues Ref. Poly (α-hydroxyesters) polylactic acid (PLA) polyglycolic acid (PGA) polycaprolactone (PCL) Ceramics Calcium phosphate Calcium sulfate Bioactive glass Nanofibers, sponge, membrane, fibers, tubes Cartilage, bone, tendon, adipose, muscle [8,11,12,14,84–89] Macro-/micro-porous scaffolds in desired shapes from blocks to granules, tubes Bone, cartilage [9,11,90–92] For extensive biomaterial review see Malafaya et al. [78] and Dawson et al. [8]. 2. Potential of stem cells for musculoskeletal regeneration 2.1. Mesenchymal stem cells Both differentiated and undifferentiated stem cells have been used as the starting cell type in cell-based therapies. While primary differentiated cells, such as chondrocytes, are often limited in quantity, adult mesenchymal stem cells (MSCs) can be easily obtained from a bone marrow aspirate or other mesenchymal tissues [15]. MSCs have a high expansion capacity, the potential to differentiate along all mesenchymal lineages, and have emerged as a candidate cell type with high potential for cell-based musculoskeletal regeneration [16]. MSCs are commonly isolated by adherence to cell culture plastic or density-gradient fractionation, and MSC cultures thus generally represent a heterogeneous population of cells. Although no definitive MSC marker(s) has been identified so far, an immunophenotype positive for STRO-1, CD73, CD146 and CD106, and negative for CD11b, CD45, CD34, CD31 and CD117, has been shown to most reliably characterize the MSC population [13,17]. While the exact mechanisms that guide tissue homing of delivered MSCs are not known, it is clear that MSCs themselves secrete a broad spectrum of bioactive molecules that have immunoregulatory [18,19] and/or regenerative activities [20]. The secreted bioactive factors have been shown to inhibit tissue scarring, suppress apoptosis, stimulate angiogenesis, and enhance mitosis of tissue-intrinsic stem or progenitor cells. This complex, multifaceted, “pro-regenerative” activity of the secretory function of MSCs has been referred to as “trophic activity”, distinct from the capacity of MSCs to differentiate [21]. MSCs are potent modulators of immune response, exhibiting antiproliferative capacities. They inhibit the proliferation of T lymphocytes induced by allergens, mitogens or anti-CD3 and -CD28 antibodies. They also modulate the function of the major immune cell populations including CD8+ cytotoxic T lymphocyte, B lymphocytes and NK cells [22]. The immunosuppressive activity of MSCs is induced by a combination of inflammatory cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)1α and -1β [23]. Also, MSCs can inhibit the proliferation and activation of B cells, similar to their effect on T cells [24]. These features might play a key role especially for the treatment of systemic musculoskeletal disease, such as OA or rheumatoid arthritis (RA). Targeted gene therapy might further enhance the activities of MSCs (Fig. 1). Direct injection of vectors can be used for in vivo gene delivery. Also, stem or differentiated cells can be used as vehicles for ex vivo gene delivery. The transduced cells can be applied either by injection as a cell suspension or seeded within a scaffold material. Both ubiquitous and local transgene expression can be induced by the ex vivo genetically modified cells or resident cells can be transduced via vector application into the musculoskeletal tissue. 2.2. Embryonic stem cells Derived from the inner cell mass of blastocysts, human embryonic stem cells (ESCs) represent another potential cell source for the repair of diseased human musculoskeletal tissues [25,26]. ESCs exhibit unlimited in vitro self-renewal capacity and pluripotency, as they can differentiate into any cell type of the three germ layers [27,28]. In vitro and in vivo experiments have shown the osteogenic and chondrogenic differentiation capacity of ESCs [25,29,30]. The predisposition of ESCs to form teratomas, even after predifferentiation ex vivo, because of residual undifferentiated cell populations complicates their possible clinical applications [31,32]. Also, in contrast to MSCs, ESCs are by necessity not autologous, so that an allogenic cell delivery is likely to lead to immunological rejection, even with HLA (human leukocyte antigen)-match of donor and recipient cells. In addition, present political and ethical complicates concerning ESC-harvest and therapeutic tenability continue to raise unresolved issues. To circumvent the use of allogenic cells, a major advancement has recently been achieved whereby adult human dermal fibroblasts have been successfully reprogrammed into an ESC-like, pluripotent state. These cells have been termed induced pluripotent stem cells (iPS cells) are created via retroviral transduction with different transcription factor genes (c-MYC+ Klf4, Nanog+ Lin28, Oct 3/4 + SOX-2 or Oct 4 + SOX-2) that regulate the maintenance of cell pluripotency and proliferation [33–36]. At present, the tumorigenicity and transformed nature of iPS cells represent a major limitation of their benchside to bedside applicability. Further work demonstrating a controlled chondrogenic and/or osteogenic differentiation and improved protocols and techniques without the use of retroviral transfection is needed to advance the use of iPS cells for musculoskeletal tissue regeneration. 3. Scaffold-based cell delivery 3.1. Natural biomaterials The advantage of employing natural biomaterials, such as collagen [37], fibrin [38], hyaluronan (HA) [39], or chondroitin sulfate (CS) 768 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 [40], for cell delivery scaffolding, is their ability to mimic certain aspects of native ECM, thus facilitating cell adherence, migration, differentiation and ECM deposition, while exhibiting optimal biocompatibility and biodegradability. On the other hand, the limitations of natural polymers, in terms of range of physicochemical properties, requirement of extensive purification protocols, and potential pathogen contamination when harvested from animal or human source, mark the disadvantages in using natural materials. 3.1.1. Collagen Collagen is the major structural component of the ECM of many different connective tissues, including tendon/ligament, cartilage, bone, skin, and regulates essential cellular events, such as proliferation, migration and differentiation via cell–matrix interactions [37]. Collagen contains specific adhesion domain sequences (i.e. RGD) that may function in retaining the cell phenotype and regulating important cellular events via integrin binding [41]. Specifically, collagen type I has been extensively investigated as a scaffold material and can be processed as nanofibrous non-woven meshes [42], sponges [43], membranes [44], fleeces [45] and hydrogels [46]. Hydrogels, in particular, permit homogeneous cell distribution throughout the scaffold, thus supporting an even deposition of newly synthesized ECM compared to meshes or fleeces, in which cell seeding is often limited to superficial regions of the scaffold. Our studies have utilized collagen type I extensively for the fabrication of articular cartilage, bone, tendon, ligament, meniscus and vertebral disc constructs (Fig. 2) [47–51]. Collagen type II is the main structural ECM molecule of articular cartilage and therefore of special interest for matrix-based cartilage repair strategies. Type II collagen has been demonstrated to exhibit induction of chondrogenic differentiation in MSCs [52] and superior cartilage specific ECM deposition by chondrocytes when compared to a type I collagen scaffold [53]. 3.1.2. Gelatin Gelatin is derived from collagen via a denaturing process by either alkaline or acid treatment, resulting in a charged polyelectrolyte with different isoelectric point values of between 5.0 and 9.0. These positively or negatively charged polyelectrolytes can interact with oppositely charged biomolecules to form polyionic complexes [54,55], offering the possibility to bind and release growth factors (e.g., TGFβ1 and IGF-1) or incorporate proteins and peptides (e.g., vitronectin, fibronectin and RGD peptides) that can influence cell adhesion and growth in a controlled manner [56–58]. Although both types of gelatin are soluble in water, more stable hydrogels can be formed via Fig. 2. Fabrication of tissue engineered constructs using collagen type I hydrogels for musculoskeletal repair. (A) Articular cartilage. (Left) Autologous Chondrocyte Implantation (ACI) using collagen type I hydrogels as a delivery vehicle (matrix-guided ACI). The size and shape of the collagen hydrogel can be adjusted intraoperatively to the articular cartilage defect using a special punch. (Right) Fabrication of a multiphasic composite scaffold with an upper collagen type I fibrous layer for articular cartilage repair, separated by a hydrophobic interface from a lower polylactic acid (PLA) part for bone repair. The upper layer was seeded with human MSCs suspended in a collagen type I hydrogel for homogeneous cell distribution. (B) Bone. The collagen type I hydrogel was used to engineer a cell laden medical grade ε-polycaprolactone (PCL)-hydrogel construct for segmental bone repair. (C) Ligament (Top). MSC-laden anterior cruciate ligament-(ACL) like construct from a collagen type I hydrogel and non-demineralized bone cylinders at each end. The construct mimics a patellar tendon (bone/patellar tendon/bone) graft. Tendon (Bottom). Fabrication of a long tendon like construct from human MSCs and a collagen type I hydrogel. Cells were suspended in a collagen type I hydrogel, polymerized in a defined glass cylinder and cultured under cyclic stretching conditions. (D) Meniscus. An MSC-laden collagen type I hydrogel was injected into an artificially created bucket handle defect of a human lateral meniscus, which was harvested during total knee arthroplasty. (E) Intervertebral disc. Fabrication of an intervertebral disc from human MSCs and a collagen type I hydrogel. The cell laden hydrogel was polymerized in a custom-designed mould according to the shape of a human intervertebral disc of the lower spine. U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 chemically cross-linking with various bifunctional agents, including glutaraldehyde or water-soluble carbodiimide [55]. In addition, gelatin exhibits lower antigenicity compared to natural collagen due to the denaturing process. Because of these desirable physicochemical characteristics, gelatin has been widely used in drug delivery systems and tissue engineering approaches targeting bone, cartilage, skin and fat tissue [55–61]. 3.1.3. Fibrin Fibrin is a protein matrix that is derived from fibrinogen under the enzymatic action of thrombin and forms a nano-/micro-fibrillar meshwork in the formation of blood clots [62]. Because of its biomimetic and physical properties, fibrin glue (fibrinogen plus thrombin) represents an injectable biomaterial that can be mixed with cells and is rapidly invaded, remodeled and replaced by the transplanted or host cells [63,64]. In addition, electrospinning of fibrin has been shown to be a suitable method to fabricate nanofibrous, nonwoven meshes that can be utilized as a predefined scaffold for ex vivo tissue regeneration [65]. 3.1.4. Hyaluronan HA, a non-sulfated glycosaminoglycan, is a major ECM macromolecule of many connective tissues. The physiological role of HA is associated with ECM fluid regulation, and structural integrity of the tissue and, in cartilage, accounts for its viscoelastic properties [66]. HA can be applied as an injectable, gel-like, cell carrier [67] or as a preshaped (nano)fibrous scaffold [68,69]. Application of HA as a cell delivery vehicle has been investigated primarily for cartilage, bone and osteochondral regeneration. 3.1.5. Chondroitin sulfate CS is another glycosaminoglycan that is present in the ECM of many connective tissues, but in contrast to HA is covalently linked via a link protein to a core protein to form proteoglycans [66,70]. As other glycosaminoglycans, CS modulates the binding of growth factors and cytokines, protease inhibition, and thus regulates cell adhesion, migration, proliferation and differentiation [71–73]. The applicability of CS as a solid, pre-shaped scaffold is impaired by its water-soluble nature, and thus cross-linking techniques are needed to tailor the physical properties of CS-based scaffolds [74]. Alternatively, CS is combined with other natural or synthetic biomaterials [74,75] in order to obtain a more stable design and retain the favorable characteristics of CS. 3.1.6. Chitosan Chitosan, a cationic polymer, is the partially or fully deacylated form of chitin, a natural polysaccharide making up the shell of 769 crustacean and insects [76]. Due to its renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional qualities, chitosan reflects an attractive biomaterial for use in a variety of tissue engineering and drug delivery approaches [76,77]. Chitosan reveals certain structural similarities to glycosaminoglycans, which represent the major non-collagenous component of the ECM of articular cartilage, contributing to the particular interest of the use of chitosan in cartilage repair strategies [77]. Additionally, chitosan can be processed as fibers, granules, sponge or hydrogel, due to its biochemical properties [76–78]. 3.1.7. Alginate Alginate is a polysaccharide polymer harvested principally from marine brown algae. In aqueous solution, upon interaction with divalent cations, such as Ca2+, alginates undergo reversible gelation to form a hydrogel [79], thus offering the possibility of homogeneous encapsulation of cells and/or retention of growth factors within the hydrogel. Alginate hydrogel beads have been shown to promote cell growth and deposition of newly synthesized extracellular matrix for primary chondrocytes and MSCs in vitro and in vivo [80–83]. 3.2. Synthetic biomaterials Synthetic biomaterials, such as poly(α-hydroxy esters) and ceramics, commonly offer a higher primary stability and are more amenable to macro-/microstructure formation than natural biomaterials. 3.2.1. Poly(α-hydroxy esters) Poly(α-hydroxy esters), such as polyglycolic acid (PGA), polylactic acid (PLA), their copolymer poly(lactic-co-glycolic acid) (PLGA), and poly-ε-caprolactone (PCL), are the most commonly used synthetic polymer and have gained FDA approval for human use in a variety of applications [8,11,12]. These polymers are degraded through bulk erosion by hydrolysis of ester bonds, and the non-toxic degradation products, lactic and glycolic acid, are physiologically eliminated from the body via metabolic pathways to form carbon dioxide and water [11]. However, rapid degradation in vivo might lead to local accumulation of lactic and glycolic acid, thus, impairing cell growth and differentiation, as well as causing inflammatory reactions [84,85] in the adjacent tissue. Degradation rate can be adjusted from weeks to several years by altering the initial molecular weight, crystallinity, and the co-polymer ratio [8,11]. Due to the thermoplastic nature of poly(α-hydroxy esters), threedimensional scaffolds with desired micro-architecture, porosity, biomechanical properties and gross shape can be manufactured using various techniques, including crystal leaching, porogen melting, gas-foaming, sintering and nanofiber electrospinning [11,14]. Electrospinning, in Fig. 3. Scanning electron microscopy view of electrospun collagen type I nanofibers. (A) Electrospinning produced randomly oriented nanofibers when utilizing a stationary target and (B) aligned nanofibers when electrospinning was performed using a rotating mandrel (7 m/s). Bar = 10 µm. 770 Table 2 Non-viral and viral vectors for orthopaedic gene therapy applications.* Non-viral and viral vectors for orthopaedic gene therapy applications Description Advantages Disadvantages Naked or uncomplexed DNA Plasmid DNA delivered in a phospholipid vesicle that merges with host cell DNA-injection, Biolistics (gene gun), Electroporation, Ca/P precipitation dsDNA virus 35 kb genome Episomal Delivers DNA Divided in 100 map units (E1–E4) 7.5 kb capacity Multiple serotypes ssDNA virus 8 serotypes, with AAV-2 with highest chondrocyte and MSC tropism Wild-type AAV integrates recombinant AAV appears to be non-integrating 4 kb capacity Easy to manufacture Non-infectious (safety) Low transfection efficiency Transient transgene expression (less than 1 week) Inflammatory Infects dividing and non-dividing cells High efficiency High levels of transgene expression Straightforward production High titer Approved for use in clinical trials Transient transgene expression Immunogenicity of transduced cells Cytotoxic at high doses Infects dividing and non-dividing cells No viral protein expression in infected cells Not known to cause disease in humans Biologically relevant transgene expression after direct i.a. delivery Transient transgene expression Moderate transduction efficiency Moderate levels of transgene expression Difficult to manufacture Small capacity Transient transgene expression Viral protein expression in infected cells Cytotoxic Immunogenic Possible insertional mutagenesis Low titre with FV Adenovirus Adeno-associated virus (AAV) Herpes simplex virus (HSV) dsDNA virus Delivers episomal DNA 40 kb capacity Spumavirus/foamyvirus (FV) RNA virus Integrates in genome 10–13 kb capacity Moloney murine leukemia virus RNA virus (momlv) Integrating 4–6 kb capacity Lentivirus RNA virus Integrates in genome 4–6 kb capacity *modified from [96]. Infects dividing and non-dividing cells Very high transduction efficiency Very high levels of transgene expression Large capacity Large capacity Persistent transgene expression No viral protein expression in infected cells Favorable integration pattern Foamy/adeno hybrid vectors possible Persistent transgene expression No viral protein expression in infected cells Infects dividing and non-dividing cells High transduction efficiency and persistent transgene expression No viral protein expression in infected cells Only infects dividing cells Possible insertional mutagenesis Possible insertional mutagenesis U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 Vector Naked DNA Liposomes Others 771 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 particular, has been recognized as a suitable method to produce scaffolds, from synthetic and natural biomaterials (i.e. collagen, fibrin), consisting of nano- to micro-scaled fibers. While the use of a stationary target will allow the fabrication of randomly oriented nanofibrous scaffolds, the introduction of a rotating mandrel as a collecting target will yield, depending on the rotational speed, highly aligned nanofibers mimicking the anisotropic architectural aspects of native extracellular matrix, such as in ligaments (Fig. 3) [14,86–89]. Thus, adjustable fiber diameter and alignment offers the possibility to custom-fabricate threedimensional cell carriers for target-tissue-specific regulation of cell proliferation, differentiation and ECM synthesis. 3.2.2. Ceramics Ceramics, such as calcium phosphates, calcium sulfates, and bioactive glass, are made from an inorganic, nonmetallic material that can possess crystalline structure [9,11]. These substances exhibit high compressive strength, variable degradation time from weeks (calcium sulfate) to non-degradable (crystalline hydroxyapatite) and have been used mainly in bone tissue engineering, especially calcium phosphate, due to the analogy with the inorganic bone component — calcium hydroxyapatite [90,91]. Calcium phosphates reveal a high protein binding affinity and can stimulate the formation, precipitation, and deposition of calcium phosphate from solution, resulting in enhanced bone–matrix interface strength [92]. 4. Ex vivo gene transfer strategies To facilitate tissue regeneration, cells may also be employed as vehicles for gene delivery. As exogenous DNA is not spontaneously taken up and sufficiently expressed by cells, genes are transferred to cells with the aid of vectors, which fall into two broad categories: those that have been developed from viruses and those that are not. Gene transfer with the aid of a viral vector is known as transduction, while the term transfection is restricted to the delivery of plasmid DNA. The most common virus vector systems being used in human clinical trials comprise retrovirus, adenovirus, adeno-associated virus (AAV) and herpes simplex virus, but also other types of vectors or hybrid vectors have also been explored experimentally. The fundamental characteristics of any viral vector type from an ex vivo gene delivery perspective include its host range, ability to infect nondividing cells, immunogenicity, integration capacity into the host genomic DNA, attainable vector titer and overall safety [93]. Unfortunately, there is no ideal universal vector system; instead, the choice of vector depends on the ex vivo gene delivery application. The specific properties of each available viral vector system have been reviewed extensively elsewhere [93–95], and are summarized in Table 2 [96]. Non-viral gene transfer is known as transfection. Nonviral vector systems, comprehensively reviewed in [97–99], may be as simple as naked or polymer-linked plasmid DNA, or delivered by liposomes or via physical methods, such as electroporation. Generally speaking, non-viral vectors are simpler, safer and cheaper than viral vectors, but are significantly less efficient and inflammatory (Table 2). Although direct application of free vector is simple and cheap, it has not been the preferred mode of gene delivery in clinical trials to date, because of the uncontrolled release of the vector in the body and associated safety concerns. Thus, ex vivo strategies of gene transfer have been preferentially explored in clinical trials, as they permit control and expansion of the transduced cells, as well as exhaustive safety testing prior to reimplantation. However, in general, they are more invasive, expensive and technically tedious. In trying to combine the advantages of both modes of gene delivery, namely the simplicity of in vivo gene delivery with the safety of ex vivo delivery, we and others have begun to focus on the development of an expedited ex vivo gene transfer approach for applications in tissue regeneration of the musculoskeletal system [100,101]. This approach entails the use of an endogenous matrix and cells capable of tissue regeneration that can be recovered intraoperatively, transduced and returned to the patient in one operative setting [101]. In this context we have begun to use vector-laden coagulated bone marrow aspirates for gene delivery to osteochondral defects [102], a technology that can potentially be transferred to the repair of all other orthopaedic tissues [100,101]. These strategies all have their respective merits and their effectiveness will depend on a number of variables, including the anatomy and physiology of the target organ, the pathophysiology of the underlying disease, the transgene, and the vector, among others. Recent investigations have identified several bioactive factors that might be functional in augmenting different aspects of cell-based musculoskeletal regeneration. Of particular interest are morphogens and transcription factors that promote differentiation along specific mesenchymal lineages, inhibitors of dedifferentiation, growth factors that promote tissue matrix synthesis, and antagonists that inhibit apoptosis, senescence or responses to catabolic cytokines. Several of these substances have shown promise in animal models of tissue repair and regeneration. However, their clinical application is hindered by delivery problems [103,104]. In particular, because of the limited half-lives of many proteins in vivo, they are difficult to administer to sites of tissue damage at therapeutic concentrations in a sustained fashion. Furthermore, the localized delivery of these agents without involvement of non-target organs has also proven to be problematic [105]. Therefore, it has been suggested that appropriate gene delivery strategies might be adopted to overcome these limitations. By using ex vivo approaches, the cells and proteins of interest are delivered locally and are presented to the microenvironment in a natural fashion. Moreover, proteins that are produced in bacteria may have altered activity, since they may not be posttranslationally modified in an appropriate manner compared to molecules produced by a mammalian cell [106]. Examples of potentially useful classes of cDNAs for cell-based musculoskeletal tissue repair comprise factors that induce tissuespecific differentiation, ECM synthesis, and phenotype maintenance, Table 3 Clinical evolution of three generations of modalities of cell-based approaches for the treatment of focal cartilage defect. Generation Technique I “Classical” autologous chondrocyte implantation (ACI) Chondrocyte suspension + periosteal flap Chondrocyte suspension + collagen type I membrane Matrix-associated autologous chondrocyte implantation (MACITM) Chondrocyte seeded biomaterial collagen membrane collagen hydrogel hyaluronan polymer fleece Autologous matrix-induced chondrogenesis (AMIC) Cell free biomaterial + microfracturing Cell free biomaterial A B II III A B Clinical applications Ref. In clinical use since late 1980s, long-term data [116,301] [302] In clinical use since late 1990s, mid term data [303,304] [305–307] [308,309] [310] First clinical study, mid term data Preclinical data, (CartiPlug™) [311] 772 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 including secreted proteins that act as tissue morphogens, signal transduction molecules, transcription factors, or regulating small interfering (si) RNAs. As many of these molecules function completely intracellularly, they cannot be delivered in soluble form, and gene transfer might be the only way to harness these factors for repair. Alternatively, delivery and expression of cDNAs encoding specific ECM components such as collagen types I and II, tenascin, or cartilage oligomeric matrix protein (COMP), may also be used to support production and maintenance of the proper tissue matrix [107]. Maintenance of neo-issue formation and prevention of tissue loss may also require the inhibition of the actions of certain proinflammatory cytokines, such as IL-1β and TNF-α, as these are important mediators of matrix degradation and apoptosis after trauma and disease [108]. In order to maintain cell populations at the injury site, inhibitors of apoptosis or senescence, such as Bcl-2, Bcl-XL, hTERT, i(NOS) and others, may also be beneficially employed [109–113]. Different candidate cDNAs might also be administered in combination, especially when favoring complementary therapeutic responses. For example, the combined administration of an anabolic growth factor (e.g., IGF-1) together with an inhibitor of the catabolic action of inflammatory cytokines (e.g., IL-1Ra) has the potential to control ECM degradation as well as to allow partial restoration of the damaged tissue matrix [114,115]. The moderate success of some of these studies suggests that this technology may have application in treating a number of musculoskeletal disorders for which current treatment modalities are unsatisfactory. In contrast to the treatment of a genetic or chronic disease, where most likely a lifelong expression of a corrective transgene is required, the use of gene transfer techniques to facilitate musculoskeletal tissue repair offers perhaps an immediate opportunity for a clinical application of gene therapy, as it may only require transient, localized expression of a specific transgene product. Some specific approaches to cell and gene-based musculoskeletal tissue regeneration are illustrated below as well as comments on the pros and cons of the respective approaches to repair, as well as future challenges. flap or a collagen membrane is sutured over the defect to create a cavity in which the culture-expanded chondrocytes can be injected. The long-term results of this technique have shown predominantly good to very good clinical results in more than 70% of the patients [117,118]. However, first generation ACI reveals several disadvantages, such as transplant hypertrophy, calcification, delamination and cell leakage [119]. Given these shortcomings, recent experimental and clinical research has been directed towards the development of second generation ACI procedures, using biocompatible scaffolds as vehicles for secure cell delivery. Promising clinical outcome data using collagen type I membranes or hydrogels, copolymers of PGA/PLA, polyglactin/vicryl and polydioxanone, as well as hyaluronic acid sponges have been reported [120]. Specifically, the use of collagen hydrogels has several advantages. They are biodegradable, can be metabolized by the cells via endogenous collagenases, cause a very limited if any inflammatory reaction, and offer a three-dimensional surrounding, that is similar to that of hyaline cartilage [46]. Also, collagen gels may be readily rendered into specific shapes and therefore adapt to any defect size (Fig. 4). In contrast to meshes or fleeces, where cell seeding is normally limited to the surface or periphery of the scaffold material, chondrocytes embedded in hydrogels show an even distribution, which allows homogeneous synthesis of ECM components immediately after transplantation [46]. First clinical results for the transplantation of MSCs seeded with collagen type I hydrogels for the repair of focal full-thickness cartilage defects were reported by Wakitani et al. [121]. Two patients with a patella defect were treated with collagen gel MSC-constructs, which were covered with a periosteal flap, and fibrocartilaginous defect filling was found after one year, as well as significantly improved patient outcome. In another case report from the same group, a fullthickness cartilage defect in the medial femoral condyle was treated [122]. Histologically, the defect was filled with a hyaline-like type of cartilage tissue, which stained positively with Safranin O and one year after surgery, the clinical symptoms had improved significantly. The clinical evolution of the three generations of ACIis outlined in Table 3. 5. Tissue-specific cell delivery 5.1.2. Osteoarthritis and rheumatoid arthritis In contrast to focal articular cartilage lesions, which can result from acute injury or osteochondrosis dissecans (OD), regenerative approaches for the treatment of OA and RA must take into consideration that cartilage damage arises from an underlying disease process. In OA and RA, treatment requires in most cases more than one compartment or even the entire articulating surface. Also, inflammatory conditions in the joint will lead to degradation of any engineered cartilage [123]. Consequently, unless the underlying disease is treated effectively, any cell-based treatment in OA and RA is unlikely to be of long-term benefit. 5.1. Articular cartilage 5.1.1. Focal articular cartilage defects An established procedure for the treatment of articular cartilage defects of the knee and ankle joint is the first generation of Autologous Chondrocyte Implantation (ACI) [116]. First, a cartilage biopsy is taken arthroscopically from a non-load bearing area. Second, after enzymatic isolation and chondrocyte expansion, the joint is opened via an arthrotomy. After debridement of the diseased cartilage, a periosteal Fig. 4. Autologous chondrocyte implantation (ACI) using a collagen type I hydrogel as a cell delivery system. (A) Typical collagen type I hydrogel (Arthro Kinetics, Krems, Austria) of 8 mm in height was used clinically for matrix-based ACI. (B) Microscopic appearance of the chondrocyte laden collagen type I hydrogel after three days in cell culture. The chondrocytes maintained their round chondrogenic phenotype and are normally cultured for 12 days before implantation. (C) Reconstructed full-thickness articular cartilage defect of the medial condyle in a 28-year-old male. The hydrogel showed excellent bonding to the host cartilage, mostly because its size could be reduced by reduced by more than 50% with gentle compression, e.g., by compressing with the back of forceps after implantation. U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 Intra-articular injection of MSCs into a joint is the simplest approach for their application in rheumatic diseases. Following delivery, MSCs should be distributed throughout the joint space to interact with all available, receptive cells and surfaces. Since the synovium lines all the internal surfaces of the joint, except for cartilage and meniscus, and is highly cellular, it is likely to be a primary tissue for the interaction with MSCs. Only few studies on the direct intra-articular injection of MSCs using animal models of OA have been performed so far. One study describes the delivery of autologous MSCs via a dilute solution of sodium HA in knee joints using a goat OA model, induced by a total medial meniscectomy and resection of the anterior cruciate ligament [124]. In cell-treated joints, there was evidence of marked regeneration of the medial meniscus, and implanted cells were detected in the newly formed tissue. Articular cartilage degeneration, osteophytic remodeling, and subchondral sclerosis also were reduced. Whether the changes observed in the MSC treated joints result from repair tissue formation by the transplanted cells or from the interaction of MSCs with host synovial fibroblasts at the site of injury remains unclear. In another study, a freshly created partial thickness cartilage defect in the knee joint of the mini pig was treated with a direct intraarticular injection of MSC suspended in HA [125]. The results demonstrated that the cell-treated group showed improved cartilage healing compared to the control group without cells. The authors postulated that HA might facilitate the migration and adherence of MSCs, probably derived from the synovium, to the defect. This may possibly explain the fact that groups treated with HA alone demonstrated some form of partial healing at 6 weeks. However, this repair tissue was of inferior quality, possibly due to insufficient MSCs localized to the site of injury, and this was shown to further deteriorate by 12 weeks. The application of genetically modified cell delivery to joints was pioneered by Evans et al., as a means to treat arthritic disorders [126]. For the treatment of RA, biologic therapies that suppress the activities of pro-inflammatory cytokines, such as tumor TNF-α or IL-1β have shown efficacy. However, such therapies are costly, require repeated administration, with only less than half of patients achieve a robust therapeutic response, and the issue of side-effects remains of concern. Experimental approaches of this technology have focused on evaluation of methods for gene delivery and identification of appropriate anti-arthritic genes. Although, systemic gene delivery was initially considered as an option, most attention has since been focused on local, intra-articular administration using ex vivo and in vivo modes of gene delivery. Genes encoding certain anti-inflammatory cytokines, such as IL-4, -10 and -13, antagonists of IL-1 and TNF, or anti-angiogenic proteins have shown efficacy in several animal models of RA [105,127–130]. Collectively, these studies have established a convincing proof of principle that has led to the development of human gene therapy protocols for RA, seven of which have entered the clinic [131]. The first clinical protocol selected an IL-1 blocker, the IL-1 receptor antagonist (IL-1Ra), as the transgene [126], which was delivered in an ex vivo fashion, using a retrovirus, to the metacarpophalangeal joints of nine individuals with severe RA. This phase I trial was successfully completed without incident [126]. Although, this phase I study was not designed to determine efficacy, intra-articular gene delivery and transgene expression was detected in all treated joints [126]. An almost similar phase I protocol including one patient has been completed in Germany, with results very similar to those from the trial in Pittsburgh, USA [132]. A phase I protocol involving the direct, intra-articular injection of a recombinant AAV2 vector into 15 individuals, carrying a cDNA encoding a fusion protein comprising two TNF soluble receptors (sTNF-R) combined with an immunoglobulin molecule is now closed, and was converted to a first phase II clinical trial [131]. This was temporarily halted by FDA due to the death of one individual enrolled in the study, which was caused by a 773 severe histoplasmosis infection [133]. However, the FDA determined that the vector was not to blame and allowed the trial to continue [131]. The only clinical trial of gene therapy in RA using non-viral gene delivery employs the genetic synovectomy approach using DNA encoding herpes simplex thymidine kinase, with one individual treated [131]. This trial is now closed. Two other phase I trials in Korea and USA facilitated the use of ex vivo delivery of transforming growth factor (TGF) β1 via retrovirus vectors, and 16 individuals have been involved without adverse events thus far [133]. As IL-1 is also an important mediator for cartilage breakdown in OA, its inhibitors were considered likewise useful targets for gene interventions. Several animal studies have confirmed the promise of IL-1Ra gene delivery in treating OA [134]. Ex vivo delivery of IL-1Ra cDNA via retrovirus vectors, and direct delivery of IL-1Ra via plasmid DNA to OA knee joints of dogs [135] and rabbits [136], respectively, were shown to slow cartilage loss. Remarkably, in a similar study in horses exploring the effects of adenoviral-mediated gene delivery of IL-1Ra in experimental OA, reduced lameness of the horses receiving gene therapy was observed [137]. Of note is that the localized pathology in OA makes it better suited for local, intra-articular delivery of gene transfer vectors compared to RA, where a systemic condition is typically present. However, in late stages of human OA, arresting the progress of the disease with an anti-inflammatory and chondroprotective gene, such as IL-1Ra, may be insufficient. In addition, it may be necessary to repair damaged cartilage, possibly using the gene therapy approaches to restore full joint function, which has been extensively reviewed elsewhere [96,120]. 5.2. Bone 5.2.1. Critical size segmental bone defects and non-unions Although bone can spontaneously heal and restore function without significant scarring, there are several conditions where this ability is compromised, including critical sized defects through traumatic injury, osteomyelitis or bone tumor resections. Healing of bone may also be impaired in much smaller defects, and non-union following fracture occurs in 5–10% of cases. Reconstruction and healing of such problematic bone defects is one of the central goals of current research in the field of orthopaedics. Additional research into the biology of bone formation has identified several potent osteogenic proteins, of which human recombinant BMP-2 and BMP-7 (OP-1) have recently been approved by the FDA, for restricted clinical use. Such factors along with administration of a combination of appropriate carrier materials and MSCs provide a promising approach to reconstructive therapy [138]. For restoration of large critical sized bone defects, MSCs have been successfully tested in several small and large animal models of disease [139–142]. Although different animal models, types of defects and biomaterials were used, improved healing of critical sized bone defects following MSC administration compared to no MSC controls was evident. These preclinical data set the stage for an initial phase I clinical trial, in which 4–7 cm large segmental bone defects of three patients were successfully treated with MSCs and a hydroxyapatite carrier material [143]. However, instead of the resorbable carrier material, a non-resorbable more rigid biomaterial might also be effectively employed, as it provides enough primary stability at the defect site to engender healing. Individualized biodegradable carrier materials can be fabricated by “Fused Deposition Modeling“ (FDM), a material production technique derived from rapid prototyping technologies, that facilitates scaffold fabrication through layers of melting polymers [144]. Using such manufacturing methods, large bone defects can be reconstructed with exact fit via computed tomography (CT) and/or magnetic resonance imaging (MRI) scans. This technology is regarded as one the most promising scaffold fabrication techniques for tissue engineering applications on the market [145,146]. For the reconstruction of large 774 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 craniofacial parietal bone defects, rapid prototyping constructs made of biodegradable PCL carrier materials and MSCs were successfully tested in animal experiments [147]. However, to date there is unfortunately neither clinical, nor in vivo experimental data on the use of stem cell-based PCL-constructs for the healing of critical sized segmental bone defects available. In cooperation with Professor D. Hutmacher (University of Queensland, Bisbane, Australia), we have begun to develop and fabricate an MSC-based biodegradable PCLconstruct for the reconstruction of segmental bone defects in an animal model. Using CT-scan data of 6-month-old New Zealand White rabbits, PCL-scaffolds have been fabricated by FDM for the reconstruction of segmental bone defects of critical size and 1.5 cm length. 5.2.2. Osteonecrosis Cell-based procedures not only have great therapeutic potential for the treatment of large bone defects, but are also promising modes of therapy in osteonecrosis, bone death that results from poor blood supply to an area of bone [138,148]. An operative procedure for the treatment of hip osteonecrosis that can be readily adapted for stem cell applications is core decompression, described 30 years ago by Ficat and Arlet [149]. This mode of therapy aims to lower the elevated levels of intraosseous pressure and to enhance repair capacities at the site of necrosis via stimulation of neovascularization [150,151]. The application of depressurization causes pain relief for the patient, although recent MRI and histomorphometric studies have shown no positive effect of core decompression alone on the repair capacity of the necrosis area [152]. A combination of core decompression procedures together with application of potentially osteogenic or angiogenic cell populations might provide a reasonable mode of therapy in the future. As carrier material for the cells, allograft bone (e.g. demineralized bone matrix, DBM) or biodegradable synthetic materials (e.g. β-tricalcium phosphate, β-TCP) might be adequately employed, while avoiding any donor site morbidity [153]. In initial clinical trials different cell preparations from bone marrow have been applied percutaneously via smaller drill holes and without carrier material to necrotic areas of femoral heads (Fig. 5) [154,155]. Furthermore, a recent publication reported a cell-based study using β-TCP biomaterial in combination with core decompression via one single drill canal in three patients [156]. A relatively similar procedure using autologous bone marrow cells (Tissue Repair Cells = TRCs, Aastrom, Ann Arbor, MI, USA) together with β-TCP matrices (Vitoss, Orthovita) have been successfully developed and used by our group for treatment of hip osteonecrosis stage ARCO II in three patients [157]. Six weeks after transplantation of the autologous TRC/β-TCP grafts, radiography showed intact femoral heads and completely filled bone canals, while MRI scans revealed obvious filling of the former necrotic area. At 3 and 12 months follow-up, there was adverse outcome, due to either the grafts or fractures. It remains to be proven, in large scale randomized controlled clinical trials compared to no-cell controls, whether such procedures can improve long-term clinical outcome after core decompression. It is conceivable, that other osteogenic or angiogenic agents, such as growth factors (e.g. bone morphogenetic proteins — BMP-2 and BMP-7), might also be successfully administered via this approach to elicit a greater healing response at the osseous defect site. The first set of animal data using BMP-2 in combination with core depression support this view and show promising results using this technology [158]. A key challenge in the application of growth factors is their inefficient delivery, and several gene-based approaches to bone regeneration have therefore been evaluated experimentally [159]. Cell-based gene delivery approaches to induce bone formation have been pioneered by the Lieberman laboratory [160,161], using bone marrow stromal cells genetically modified to deliver bone morphogenetic protein-2 (BMP-2) cDNA to heal critical sized femoral defects in the femora of rats [162]. In this study, healing achieved by BMP-2 gene transfer was superior to that achieved with recombinant BMP-2 protein by histological criteria [162]. Other investigators have confirmed this approach, using osteoprogenitor cells derived from periosteum, muscle, fat and skin [163]. Other transgenes, such as BMP-4, are also effective in these models [164–167], and it is to be expected that additional genes encoding osteogenic proteins such as BMPs -6, -7 and -9 [168–172], or VEGF [173] will also be successful. Furthermore, retrovirus [174], lentivirus [175–177] and AAV [175– 178] were successfully employed as alternative vector systems using Fig. 5. Cell delivery for the treatment of avascular necrosis of the femoral head. (A) β-TCP granula (Vitoss, Stryker, Belgium). (B) An autologous MSC suspension is injected onto the granula and becomes completely absorbed. (C) Autologous serum is used to clot the granula for better handling. (D) After approximately 30 min, the clotted MSC/β-TCP construct can be handled with a forceps or placed in a syringe and inserted into the bone defect (E). (F) Postoperative X-ray showing a 10 mm drill hole after core decompression completely filled with the MSC/β-TCP construct in a 32-year-old patient suffering from primary avascular necrosis of the femoral head. U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 this approach. Interestingly, a comparison between short term adenovirus-mediated BMP transgene expression with long-term lentiviral-mediated transgene expression for bone regeneration revealed equivalent robust bone formation for both types of vectors used [179,180]. Apart from bone marrow stromal cells, other cell types such as muscle-derived cells have proven beneficial for bone tissue regeneration approaches in small animal models [181]. In contrast to the use of ex vivo delivery methods, several groups have focussed on the healing of osseous defects by in vivo delivery of genes to the lesion, which have been extensively reviewed elsewhere [163,182–184]. There are also data that indicate beneficial effects for gene therapy to enhance bone healing at sites of osteoporotic [182] and infected fractures [185], non-unions, as well as to augment allografted bone [186] or spinal fusion [186–189]. Collectively, the data from all these studies are remarkable and suggest the feasibility of future clinical application of this technology, once existing safety concerns are sufficiently met. However, an issue that has not been addressed to date is whether the osteogenic response of these treatments in humans, especially of those who are older, diabetic, traumatized or smokers, will be as vigorous as that in the otherwise healthy young experimental animals investigated preclinically. 5.3. Ligament and tendon The ECM of ligaments and tendons mainly exhibit parallel aligned collagen type I fibers and, to a lesser extent, collagen type III, elastin, fibronectin, decorin, and tenascin [190]. The beneficial effect of MSCdelivery to the defect side in tendon-/ligament healing has previously been demonstrated [191–193]. When MSCs are directly delivered to the defect site via injection, cell integration within the targeted ligament and an accelerated tissue healing response has been demonstrated [192,194]. Ligament differentiation is strongly influenced by mechanical stimulation of MSCs [195], as this reflects the natural mechanical environment of tenocytes, where cyclic stretching [196,197] and the additional influence of growth factors [198–200] can function synergistically. Additionally, MSCs can contribute in tendon healing when delivered in a matrix-based approach to the injury site [201,202]. Finally, the recent discovery of tendon stem cells [203,204] presents another alternative cell source for tissue repair. Although the basic principles of cell-based strategies for tendon and ligament healing are essentially similar, different approaches designed for different anatomical aspects and biomechanical needs must be considered. 5.3.1. Anterior cruciate ligament The anterior cruciate ligament (ACL), as an intra-articular ligament of the knee joint, regulates the gliding/sliding movement of tibiofemoral articulation and provides biomechanical stability. Hence, ACL rupture causes significant instability of the knee joint exceeding the biomechanical properties of primary ligament sutures counteracting the healing response. Autologous tendon grafts (i.e., muscularis semitendinosus or ligamentum patellae) have been employed as standard therapy in ACL replacement and can render joint stability to a certain extent. However, these autografts fail to reconstitute the complex architecture and biomechanical properties of the natural ACL in the long-term, and may not preserve the early onset of OA [205]. MSCs have been the primary cell type used in an effort to ameliorate ACL-reconstruction using different experimental approaches. Ex vivo engineered MSC-laden scaffolds fabricated from collagen type I hydrogel or PLA confirmed the tenogenic or ligamentogenic differentiation of the seeded cells. Unfortunately, a possible in vivo application of these cell constructs was impeded by the inferior biomechanical properties [198]. The additional utilization of MSC on knitted poly(α-hydroxy ester) that have been augmented 775 with autologous fascia lata for the support of a primary ACL suture in rabbits has shown superior histological results compared to unseeded scaffold. However, the use of MSCs did not result in a significant improvement in biomechanical properties of the reconstructed ACL after 20 weeks [206]. Using a rabbit ACL in vivo model, Li et al. delivered MSCs seeded onto de-cellularized Achilles tendon allografts. Histological analysis at 12 weeks after surgery exhibited accelerated cellular infiltration into the ACL and enhanced collagen deposition compared to the use of allografts alone [207]. Using another approach for ACL repair in rabbits, Lim et al. have shown beneficial effects in tendon graft osseointegration when hamstring autograft were coated with fibrin glue-entrapped MSCs. Eight weeks after ACL replacement, the MSC-augmented autografts showed a significantly higher failure load of the tendon–bone interface compared to the control group without MSC application [208]. Fibroblasts derived from ligament and tendon have been transduced by a variety of viral and non-viral vectors, and marker genes have been delivered by direct and indirect gene transfer strategies to ligaments and tendons in vivo [209,210]. Delivery of cDNAs encoding growth factors, such as TGFβ1 [211] or insulin-like growth factor (IGF) 1 [212], platelet-derived growth factor (PDGF) [213] or vasculoendothelial growth factor (VEGF) [214], have been found to promote cell division and the deposition of ECM in vitro and in situ [101,184,215– 217]. Although experimental proof of principle for the potential utility of cell-based gene delivery to heal and regenerate ligaments and tendons has been shown using different cell types including MSCs, fibroblasts and myoblasts [218], relatively few studies have shown efficient transfer of therapeutic factors to ligaments [218,219] and tendons [220–223] in vivo. Of particular interest are growth and differentiation factors 5–7 (BMP-12–14) or Smad8 are, as they promote the differentiation of MSCs into tissue with the phenotypic appearance of ligament and tendon [200,224–226]. In a chick tendon laceration model, BMP-12 gene transfer doubled the tensile strength and stiffness of the repaired tendons [221], and also showed strongly enhanced healing of Achilles tendon in a rat model [227]. Another strategy that has been shown effective for improving the healing of ligaments and tendons is to reduce the synthesis of small proteoglycans such as decorin, as these are known to limit the diameter of collagen fibrils and act as TGFβ antagonists. By inhibiting decorin production using antisense oligodenucleotides (ODN) in healing medial collateral ligaments in a rabbit model, it was found that the mechanical properties of the treated ligaments improved substantially [219]. 5.3.2. Tendon In a similar manner, MSCs have been shown to effectively enhance tendon healing in vitro and in vivo. MSC seeded onto collagen sponges showed mechanically induced upregulation in gene expression in vitro after implantation into explants of sheep patella tendon [228]. When comparing acellular and MSC-seeded collagen hydrogels, that were applied to an experimental defect at the patellar tendon of rabbits, Awad et al. observed significantly improved biomechanical properties in the cell-based groups as early as 4 weeks after surgery. By 26 weeks, repairs augmented with MSC–collagen composites revealed one-fourth of the maximum mechanical strength of native patellar tendon. Interestingly, the density of cell seeding into the collagen gels (1–8 × 106 cells/ml) did not significantly influence the development of biomechanical properties. [229,230]. When the MSCladen collagen hydrogel was augmented with a collagen sponge the resulting repair stiffness and maximum force could be improved, and mechanical stimulation of these constructs ex vivo further accelerated repair biomechanics [231]. An improved healing response of suture supported Achilles tendon defects in rabbits in terms of superior histological and biomechanical parameters has also been described for the delivery of MSCs in both collagen and fibrin scaffolds [232,233]. Initial in vitro studies for the ex vivo reconstruction of 776 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 long finger tendons have been published by Nöth et. al. [51] using a pre-shaped collagen hydrogel after MSC encapsulation. Successful cell delivery to the rotator cuff (-muscles) in rats via injection has also been demonstrated previously. The injected muscle-derived stem cells were integrated into the native collagen bundles, and showed a spindle shaped morphology and postive vimentin expression [2]. was observed [251]. Once more data on this technology become available, in particular on the most relevant growth factor cDNAs, cell systems and modes of delivery, a clearer picture will emerge on the most expeditious method of harnessing such factors for clinical use in the repair of meniscus tissue. 5.5. Spine 5.4. Meniscus Due to the greater understanding of the importance of the menisci in recent years, with numerous studies reporting early onset of degenerative changes within the knee joint following meniscectomy, orthopaedic surgeons aim to preserve as much functional meniscus tissue as possible during surgery [234]. Unfortunately, the majority of meniscal tears occur in the inner, avascular two-thirds which do not heal adequately even with surgical repair, and thus approaches for meniscus regeneration and replacement are increasingly appreciated [235–238]. Several cell-based approaches facilitated with various growth factors and matrices have been proposed as ways to stimulate a healing response in this region of the meniscus [235,236,238]. For example, collagen meniscus implants seeded with autologous fibrochondrocytes have been used successfully for meniscus replacements in a sheep model [239]. Furthermore, MSCs have been suggested as an attractive alternative cell source to mature meniscus cells, which must be isolated from a limited supply of healthy meniscus tissue with associated donor site morbidity [124,240–242]. Several biomaterials have been found to be appropriate carriers from meniscus cells or MSCs for the purpose of meniscus regeneration, including collagen sponges [241,243] and porous polymer scaffolds [244,245], and aligned biodegradable nanofibers [87], among others [246]. To trigger MSCs toward a fibrocartilaginous phenotype and to maintain fibrocartilage matrix production in transplanted meniscus cells, certain mechanical stimuli have been found to be crucial [247]; numerous cytokines have also been evaluated for their ability to stimulate various aspects of repair, including cell outgrowth, cell division and matrix synthesis [235,236,238,242]. In this respect, TGFβ1, IGF-1, fibroblast growth factor-2 (FGF2), PDGF, hepatocyte growth factor (HGF), and certain BMPs emerge as among the most promising candidates [235,236,238,242]. However, the clinical application of growth factors such as these is hampered by delivery problems. Notably, the delivery of a recombinant protein into meniscus lesions has not yet resulted in sustained regeneration of meniscus tissue to date, indicating that stimulation provided by a single bolus of soluble growth factor is not sufficient to promote differentiation of the implanted cells in vivo [101,217]. Therefore, gene delivery approaches have been suggested to be the most expeditious method of harnessing such factors for clinical use in the repair of meniscus tissues [101,217], although to date only few studies have been conducted on gene transfer approaches to meniscal repair. Direct delivery of both marker genes and the TGFβ1 cDNA to meniscal tissue [248,249] and to meniscus allografts [250] was shown to be feasible; however, direct delivery of vector may not be practical for the repair of meniscal tears, where there is extensive cell loss, since it does not substantially increase the number of meniscal cells within the lesion and the target cells are embedded in a dense collagenous matrix. Such obstacles may be overcome using ex vivo delivery, in which autologous cells are harvested, expanded in culture and genetically modified under controlled conditions prior to their implantation to the defect site. We have previously shown that transfer of TGFβ1 cDNA to bovine meniscus cells and MSCs and cultured in collagen-glycosaminoglycan matrices was useful for tissue regeneration in the avascular zone of the meniscus in an explant culture system in vitro [241]. In vivo, genetically modified meniscal cells to express HGF have been seeded onto scaffolds and implanted into nude mice, where development of vascularized meniscal tissue 5.5.1. Intervertebral disc Intervertebral disc (IVD) degeneration increases with age so that around 10% of 50-year-old discs and 60% of 70-year-old discs are severely degenerated [252]. Their major role is mechanical, and they consist of a thick outer ring of fibrous cartilage termed the annulus fibrosus, which surrounds a more gelatinous core known as the nucleus pulposus. The collagen network formed mostly of collagen type I and type II fibrils and making up approximately 70% and 20% of the dry weight of the annulus and nucleus, respectively, provides tensile strength to the disc and anchors the tissue to the bone [253]. Aggrecan, the major proteoglycan of the disc, is responsible for maintaining tissue hydration through the osmotic pressure provided by its constituent chondroitin and keratan sulfate chains [254]. The molecules of the ECM are continually being broken down by proteinases, such as the matrix metalloproteinase (MMPs) and aggrecanases, which are also synthesized by disc cells [255]. The balance between synthesis, breakdown, and accumulation of matrix macromolecules determines the quality and integrity of the matrix, and thus the mechanical behavior of the disc itself. The IVD is often likened to articular cartilage and there are certain similarities between the current biological approaches to disc and cartilage repair [256]. Like articular cartilage, the nucleus pulposus of the IVD consists of proteoglycan and type II collagen, while in contrast, the annulus fibrosus is rich in type I collagen [257]. However, there are differences between the two tissues, one of these being the composition and structure of aggrecan. IVD aggrecan is more highly substituted with keratan sulfate than that found in the deep zone of articular cartilage. In addition, the aggrecan molecules are less aggregated (30%) and more heterogeneous, with smaller, more degraded fragments in the disc than in articular cartilage (80% aggregated) from the same individual [258]. Investigators have studied the effects of cell injection, seeded matrices, growth factors and certain genes for regeneration of the IVD. Disc cell injection has been performed in 112 patients in the Euro Disc Randomized Trial. In the interim analyses, patients who received autologous disc cell transplantation had greater pain reduction at 2 years compared with patients who did not receive cells following discectomy. Also, discs in patients that received cells demonstrated a significant difference in the fluid content of their treated disc when compared to a control group. The study shows, that autologous discderived cell transplantation is technically feasible and biologically relevant to repair disc damage and retarding disc degeneration [259]. Of high interest in this context is the injection of MSCs to treat IVD regeneration. Autologous adipose tissue derived stem cells have been used in a dog model for IVD repair. The results were effective in promoting disc regeneration, as evidenced by disc matrix production and overall disc morphology [260]. Also, it has been shown that MSCs can be effectively stimulated toward IVD cell phenotypes in vitro [261] and in vivo [262], especially when certain growth factors such as BMP2 or TGF-β1 are used [263]. MSCs, as well as an alternative cell source, muscle-derived stem cells, have also been used effectively to arrest IVD regeneration, especially when co-cultures with nucleus pulposus cells were used [264]. Specifically, we have recently found that conditioned medium derived from notochordal cells isolated from the nucleus pulposus stimulated MSCs to differentiate toward an IVD tissue phenotype [Korecki et al., manuscript submitted for publication]. Cell transplantation via fibrin matrices to the nucleus pulposus have proven useful U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 to immobilize the transplanted cells compared to suspension delivery that resulted in rapid loss of the majority of cells [265]. Finally, a recent application of a unique biphasic HA-nanofiber amalgam scaffold, seeded with bone marrow derived MSCs induced to undergo chondrogenesis, suggests the feasibility of ex vivo IVD tissue engineering [266]. Nucleus pulposus cells have been efficiently transduced using several non-viral [267] and viral vector systems [268–273]. Investigators have studied the effects of overexpressing genes for transcription factors such as SOX-9 [274,275], and anabolic growth factors such as BMPs [276,277], growth differentiation factor-5 (GDF5) [278], or TGFβ1 [279] that promote extracellular matrix synthesis. Also, overexpression of catabolic inhibitors, such as tissue inhibitor of metalloproteinase-1 (TIMP-1) [280] in IVD cells has been found to reverse disc degeneration. In addition, a combination of TGFβ1, BMP-2 and IGF-1 has shown beneficial effects to stimulate nucleus pulposus matrix synthesis [281]. One interesting and unexpected finding in these investigations was the remarkable stability of transgene expression after intradiscal injection of recombinant adenoviral vectors [272], which may be attributed to the immunologically protected environment of the IVD and the non-dividing state of its cells. However safety testing experiments using genetic approaches for IVD regeneration indicate caution, as intradural injection of very high doses of adenoviral vectors expressing TGFβ1 and BMP-2 resulted in paralysis of lower extremities in a rabbit model [282,283]. Taken together, these promising features of cell-based therapy approaches to IVD regeneration support the feasibility of the development of these treatments to clinical use. 5.5.2. Spinal fusion Iliac crest bone grafting reflects today's gold standard technique in spinal fusion approaches [284] Notwithstanding clinical effectiveness, numerous reports have indicated pronounced donor site morbidity, including postoperative pain, hematoma, and meralgia paresthetica, occurring in 19–41% of the patients [285,286]. In order to circumvent harvest associated morbidity, recent research has targeted on identifying alternative strategies, by introducing osteoconductive scaffolds in combination with osteoinductive growth factors, such as BMP, and MSCs [287–291]. Clinical application of collagen and other bioabsorbable ceramics, such as demineralized bone matrix, hydroxyapatite, and calcium phosphate ceramic, in combination with osteoinductive proteins, have shown sufficient spinal fusion rates, comparable to those achieved by autologous bone grafting [287,288,292,293]. However, in order to obtain sufficient bone formation, high doses of BMP are clinically applied, thus accounting for local inflammatory reactions, such as soft tissue edema, postoperative radiculitis and ectopic bone formation with the risk of neurological deficits [294,295]. The additional delivery of MSCs is thought to accelerate bony fusion via osteogenic differentiation and consecutive bone matrix deposition. However, fresh bone marrow aspirate, due to its low (1 of 100,000–1,000,000 cells) concentration of MSCs, may not have the potential to promote sufficient osteogenesis [289,290]. Therefore, ex vivo culture-expanded MSCs have been used in various preclinical studies to evaluate their potential to augment spinal fusion. Performing a posterolateral lumbar spine fusion in rabbits, Nakajima et al. demonstrated a superior spinal fusion rate (80%) of osteogenic pre-differentiated MSCs in a collagen–hydroxyapatite scaffold when compared to undifferentiated MSCs (33%) or iliac crest bone grafting (67%) [290]. Using the same animal model, Minamide et al. have demonstrated the importance of the number of cells delivered to the fusion site [291]. Whereas no fusion was observed when 1 × 106 differentiated MSCs in a collagen–hydroxyapatite scaffold were applied, 71% of the animals treated with 100 × 106 MSCs showed solid fusion and mature bone formation after 6 weeks [291]. In general, the augmentation of scaffold-based spinal fusion approaches 777 with differentiated MSC results in similar fusion rates as that achieved by iliac crest bone grafting without the risk of donor site morbidity [288]. Over the last decade novel gene therapy approaches aiming for a prolonged, local expression of osteoinductive proteins to enhance bone formation have been investigated in preclinical spinal fusion models. The field was pioneered by Boden et al. [187,188] focusing on the adenoviral-mediated delivery of LIM mineralization protein-1 (LMP-1), an intracellular osteogenic protein. Performing an abbreviated ex vivo approach to gene delivery, bone marrow derived MSCs or intraoperatively harvested buffy-coat cells from autologous blood were briefly incubated with the adenoviral transgene vector and subsequently delivered on a collagen-ceramic sponge to the fusion site. In rabbits with a single-level posterolateral arthrodesis of the lumbar spine, this procedure resulted in full spinal fusion within 4 to 5 weeks, while none of the control rabbits underwent spinal fusion [296]. Genes encoding other osteogenic proteins, such as BMP-2, -6, -7, and -9, have also been proven to accelerate spinal fusion or paravertebral bone formation that led to solid spinal arthrodesis in preliminary animal models [186,288,297–300]. Zhu et al. showed a synergistic effect via co-administration of adenoviral vectors encoding BMP-2 and -7 loaded on allografts and implanted at the posterolateral Table 4 Present status of cell delivery therapeutics in musculoskeletal regeneration. Application Status Rheumatoid arthritis Cell delivery +/− proteins Efficacy in various animal models Cell + gene delivery Efficacy in various animal models using synoviocytes Phase I human clinical trials using ex vivo gene delivery in synoviocytes Phase II gene therapy protocols using ex vivo gene transfer in synoviocytes Cell delivery +/− proteins Efficacy in various animal models using MSCs Cell + gene delivery Efficacy in various animal models using ex vivo gene delivery in MSCs Cell delivery +/− proteins Efficacy in various animal models using MSCs and osteoblasts Cell + gene delivery Efficacy in various animal models using MSCs and osteoblasts Efficacy in various animal models using ex vivo gene delivery in MSCs Cell delivery +/− proteins Broad clinical application of ACI Phase I human trials using MSCs Efficacy in various animal models using MSCs and chondrocytes Cell + gene delivery Promising preclinical data in animal models using matrix-guided delivery of genetically modified MSCs and chondrocytes Cell delivery +/− proteins Efficacy in various animal models using MSCs and meniscus cells Cell + gene delivery Preliminary preclinical data in animal models using MSCs Cell delivery +/− proteins Efficacy in various animal models using MSCs Cell + gene delivery Preliminary preclinical data in animal models using MSCs Cell delivery +/− proteins Efficacy in various animal models using MSCs Cell + gene delivery Preliminary preclinical data in animal models in vivo using MSCs and fibroblasts Osteoarthritis Bone healing and spine fusion Articular cartilage repair Meniscus repair Intervertebral disc repair Ligament and Tendon 778 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 lumbar spine of rats. After 8 weeks. 73% of the animals treated with a vector combination of BMP-2/-7 revealed a mechanically stable spinal fusion, while only 8% and 16% spinal fusions were found in BMP-2 or -7 vector treated animals, respectively [298]. Despite these encouraging data, clinical application of gene therapy approaches for spinal fusion is still considered as premature and extremely ambiguous due to unsolved safety issues and the nonlife-threatening nature of spine degeneration. 6. Conclusion Although ex vivo cell culture is thought to be elaborate and costly, especially under GMP conditions, there is tremendous scope for using cell delivery therapeutics for regeneration of the musculoskeletal system, the current status of which is listed in Table 4. In the majority of cases, cells are used to deliver a space filling entity via a biomaterial to repair an injured tissue, although in some cases it has been found to be beneficial as a therapy alone to treat a chronic condition such as OA and RA. In some cases, such as ACI, cell-based therapies have shown efficacy in large scale human studies and have therefore been advanced to broad clinical application. In next-generation applications, including matrixbased delivery of MSCs for the repair of bone and cartilage, cells have shown safety and efficacy in phase I clinical trials. In contrast cells have only been used experimentally in other arenas (e.g. meniscus and disc repair). Cell therapeutics for musculoskeletal regeneration is thought to be improved by the use of factors that aid certain aspects of repair. Despite early successful phase I and II clinical trials report on the efficacy of cell-mediated protein and gene delivery for the treatment of arthritis, such therapies have not become standard modes of treatment to date, largely due to the safety concerns associated with such procedures in non-life-threatening conditions. However, the most rapid progress is likely to come from arenas, where the application of cells via biomaterials may be successful, without the use of additional factors. Bone healing, in particular, is very responsive to cell therapy. As most musculoskeletal indications are not life-threatening, safety will be a key issue for any clinical application of cell therapy. Once these concerns are addressed satisfactorily, cell therapy is likely to be added to the orthopaedic armamentarium at a significant scale in the near future. Acknowledgement This work is supported by the Deutsche Forschungsgemeinschaft (DFG RA1820/2-1, LR and UN; DFG STE1051/2-1, AFS and UN), the Interdisziplinäres Zentrum für Klinische Forschung (IZKF Z-3/8, LR; IZKF D-137, UN and LR; IZKF D-23/1, D-101AFS), Bayerische Forschungsstiftung (FORZEBRA TP2WP5, AFS and UN), and the Commonwealth of Pennsylvania Department of Health (RST). References [1] U. Noth, A.F. Steinert, R.S. Tuan, Technology insight: adult mesenchymal stem cells for osteoarthritis therapy, Nat. Clin. Pract. Rheumatol. 4 (2008) 371–380. [2] D. Pelinkovic, J.Y. Lee, M. Engelhardt, M. Rodosky, J. Cummins, F.H. Fu, J. Huard, Muscle cell-mediated gene delivery to the rotator cuff, Tissue Eng. 9 (2003) 143–151. [3] R.K. Smith, Mesenchymal stem cell therapy for equine tendinopathy, Disabil. Rehabil. 30 (2008) 1752–1758. [4] M. Marcacci, E. Kon, V. Moukhachev, A. Lavroukov, S. Kutepov, R. Quarto, M. Mastrogiacomo, R. Cancedda, Stem cells associated with macroporous bioceramics for long bone repair: 6- to 7-year outcome of a pilot clinical study, Tissue Eng. 13 (2007) 947–955. [5] M. Brittberg, Autologous chondrocyte implantation—technique and long-term follow-up, Injury 39 (Suppl 1) (2008) S40–S49. [6] P.X. Ma, Biomimetic materials for tissue engineering, Adv. Drug Deliv. Rev. 60 (2008) 184–198. [7] J.I. Dawson, R.O. Oreffo, Bridging the regeneration gap: stem cells, biomaterials and clinical translation in bone tissue engineering, Arch. Biochem. Biophys. 473 (2008) 124–131. [8] E. Dawson, G. Mapili, K. Erickson, S. Taqvi, K. Roy, Biomaterials for stem cell differentiation, Adv. Drug Deliv. Rev. 60 (2008) 215–228. [9] Y. Khan, M.J. Yaszemski, A.G. Mikos, C.T. Laurencin, Tissue engineering of bone: material and matrix considerations, J. Bone Joint Surg. Am. 90 (Suppl 1) (2008) 36–42. [10] J.F. Mano, G.A. Silva, H.S. Azevedo, P.B. Malafaya, R.A. Sousa, S.S. Silva, L.F. Boesel, J.M. Oliveira, T.C. Santos, A.P. Marques, et al., Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends, J. R. Soc. Interface 4 (2007) 999–1030. [11] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering, Biomaterials 27 (2006) 3413–3431. [12] C.K. Kuo, W.J. Li, R.L. Mauck, R.S. Tuan, Cartilage tissue engineering: its potential and uses, Curr. Opin. Rheumatol. 18 (2006) 64–73. [13] F.H. Chen, K.T. Rousche, R.S. Tuan, Technology Insight: adult stem cells in cartilage regeneration and tissue engineering, Nat. Clin. Pract. Rheumatol. 2 (2006) 373–382. [14] W.J. Li, R.S. Tuan, Fabrication and application of nanofibrous scaffolds in tissue engineering, Curr. Protoc. Cell Biol. Chapter 25 (2009) Unit 25 2. [15] D. Baksh, L. Song, R.S. Tuan, Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy, J. Cell. Mol. Med. 8 (2004) 301–316. [16] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [17] C.M. Kolf, E. Cho, R.S. Tuan, Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation, Arthritis Res. Ther. 9 (2007) 204. [18] X. Chen, M.A. Armstrong, G. Li, Mesenchymal stem cells in immunoregulation, Immunol. Cell Biol. 84 (2006) 413–421. [19] A. Uccelli, V. Pistoia, L. Moretta, Mesenchymal stem cells: a new strategy for immunosuppression? Trends Immunol. 28 (2007) 219–226. [20] I. Kan, E. Melamed, D. Offen, Autotransplantation of bone marrow-derived stem cells as a therapy for neurodegenerative diseases, Handb. Exp. Pharmacol. (2007) 219–242. [21] A.I. Caplan, J.E. Dennis, Mesenchymal stem cells as trophic mediators, J. Cell. Biochem. 98 (2006) 1076–1084. [22] D. Noel, F. Djouad, C. Bouffi, D. Mrugala, C. Jorgensen, Multipotent mesenchymal stromal cells and immune tolerance, Leuk. Lymphoma 48 (2007) 1283–1289. [23] C. Ren, S. Kumar, D. Chanda, J. Chen, J.D. Mountz, S. Ponnazhagan, Therapeutic potential of mesenchymal stem cells producing interferon-alpha in a mouse melanoma lung metastasis model, Stem Cells 26 (2008) 2332–2338. [24] A. Corcione, F. Benvenuto, E. Ferretti, D. Giunti, V. Cappiello, F. Cazzanti, M. Risso, F. Gualandi, G.L. Mancardi, V. Pistoia, et al., Human mesenchymal stem cells modulate B-cell functions, Blood 107 (2006) 367–372. [25] J.A. Thomson, J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, J.M. Jones, Embryonic stem cell lines derived from human blastocysts, Science 282 (1998) 1145–1147. [26] J.M. Jukes, C.A. van Blitterswijk, J. de Boer, Skeletal tissue engineering using embryonic stem cells, J. Tissue Eng. Regen. Med. 4 (2010) 165–188. [27] M.R. Alison, R. Poulsom, S. Forbes, N.A. Wright, An introduction to stem cells, J. Pathol. 197 (2002) 419–423. [28] B.E. Reubinoff, M.F. Pera, C.Y. Fong, A. Trounson, A. Bongso, Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro, Nat. Biotechnol. 18 (2000) 399–404. [29] R.C. Bielby, A.R. Boccaccini, J.M. Polak, L.D. Buttery, In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells, Tissue Eng. 10 (2004) 1518–1525. [30] C. Fecek, D. Yao, A. Kacorri, A. Vasquez, S. Iqbal, H. Sheikh, D.M. Svinarich, M. Perez-Cruet, G.R. Chaudhry, Chondrogenic derivatives of embryonic stem cells seeded into 3D polycaprolactone scaffolds generated cartilage tissue in vivo, Tissue Eng. Part A 14 (2008) 1403–1413. [31] S. Wakitani, K. Takaoka, T. Hattori, N. Miyazawa, T. Iwanaga, S. Takeda, T.K. Watanabe, A. Tanigami, Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint, Rheumatology (Oxford) 42 (2003) 162–165. [32] J.M. Jukes, L. Moroni, C.A. van Blitterswijk, J. de Boer, Critical steps toward a tissue-engineered cartilage implant using embryonic stem cells, Tissue Eng. Part A 14 (2008) 135–147. [33] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, S. Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell 131 (2007) 861–872. [34] M. Nakagawa, M. Koyanagi, K. Tanabe, K. Takahashi, T. Ichisaka, T. Aoi, K. Okita, Y. Mochiduki, N. Takizawa, S. Yamanaka, Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts, Nat. Biotechnol. 26 (2008) 101–106. [35] A. Meissner, M. Wernig, R. Jaenisch, Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells, Nat. Biotechnol. 25 (2007) 1177–1181. [36] J. Yu, M.A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J.L. Frane, S. Tian, J. Nie, G.A. Jonsdottir, V. Ruotti, R. Stewart, et al., Induced pluripotent stem cell lines derived from human somatic cells, Science 318 (2007) 1917–1920. [37] W. Friess, Collagen—biomaterial for drug delivery, Eur. J. Pharm. Biopharm. 45 (1998) 113–136. [38] C. Scotti, A. Pozzi, L. Mangiavini, F. Vitari, F. Boschetti, C. Domeneghini, G. Fraschini, G.M. Peretti, Healing of meniscal tissue by cellular fibrin glue: an in vivo study, Knee Surg. Sports Traumatol. Arthrosc. 17 (2009) 645–651. [39] S. Nehrer, R. Dorotka, S. Domayer, D. Stelzeneder, R. Kotz, Treatment of fullthickness chondral defects with hyalograft C in the knee: a prospective clinical U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] case series with 2 to 7 years' follow-up, Am. J. Sports Med. 37 (Suppl 1) (2009) 81S–87S. Z. Ferdous, K.J. Grande-Allen, Utility and control of proteoglycans in tissue engineering, Tissue Eng. 13 (2007) 1893–1904. E. Ruoslahti, RGD and other recognition sequences for integrins, Annu. Rev. Cell Dev. Biol. 12 (1996) 697–715. L.S. Sefcik, R.A. Neal, S.N. Kaszuba, A.M. Parker, A.J. Katz, R.C. Ogle, E.A. Botchwey, Collagen nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human adipose stem cells, J. Tissue Eng. Regen. Med. 2 (2008) 210–220. T. Fujisato, T. Sajiki, Q. Liu, Y. Ikada, Effect of basic fibroblast growth factor on cartilage regeneration in chondrocyte-seeded collagen sponge scaffold, Biomaterials 17 (1996) 155–162. M. Steinwachs, New technique for cell-seeded collagen-matrix-supported autologous chondrocyte transplantation, Arthroscopy 25 (2009) 208–211. C. Danielsson, S. Ruault, A. Basset-Dardare, P. Frey, Modified collagen fleece, a scaffold for transplantation of human bladder smooth muscle cells, Biomaterials 27 (2006) 1054–1060. U. Noth, L. Rackwitz, A. Heymer, M. Weber, B. Baumann, A. Steinert, N. Schutze, F. Jakob, J. Eulert, Chondrogenic differentiation of human mesenchymal stem cells in collagen type I hydrogels, J. Biomed. Mater. Res. A (2007). U. Nöth, S. Siebenlist, L. Rackwitz, B. Schreiber, A. Steinert, T. Barthel, J. Eulert, Matrix-based autologous chondrocyte transplantation for the treatment of large osteochondral defects., Eurpean Musculoskeltal Review 2006. Touch Briefings. (2006) 62–64. J.C. Reichert, A. Heymer, A. Berner, J. Eulert, U. Noth, Fabrication of polycaprolactone collagen hydrogel constructs seeded with mesenchymal stem cells for bone regeneration, Biomed. Mater. 4 (2009) 65001. A. Heymer, G. Bradica, J. Eulert, U. Noth, Multiphasic collagen fibre-PLA composites seeded with human mesenchymal stem cells for osteochondral defect repair: an in vitro study, J. Tissue Eng. Regen. Med. 3 (2009) 389–397. U. Noth, K. Schupp, A. Heymer, S. Kall, F. Jakob, N. Schutze, B. Baumann, T. Barthel, J. Eulert, C. Hendrich, Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel, Cytotherapy 7 (2005) 447–455. S. Kall, U. Noth, K. Reimers, C.Y. Choi, T. Muehlberger, C. Allmeling, S. Jahn, A. Heymer, P.M. Vogt, In vitro fabrication of tendon substitutes using human mesenchymal stem cells and a collagen type I gel, Handchir. Mikrochir. Plast. Chir. 36 (2004) 205–211. C.W. Chen, Y.H. Tsai, W.P. Deng, S.N. Shih, C.L. Fang, J.G. Burch, W.H. Chen, W.F. Lai, Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells, J. Orthop. Res. 23 (2005) 446–453. S. Nehrer, H.A. Breinan, A. Ramappa, S. Shortkroff, G. Young, T. Minas, C.B. Sledge, I.V. Yannas, M. Spector, Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro [published erratum appears in J Biomed Mater Res 1997 Winter;38(4):288], J. Biomed. Mater. Res. 38 (1997) 95–104. Y. Ikada, Y. Tabata, Protein release from gelatin matrices, Adv. Drug Deliv. Rev. 31 (1998) 287–301. S. Young, M. Wong, Y. Tabata, A.G. Mikos, Gelatin as a delivery vehicle for the controlled release of bioactive molecules, J. Control. Release 109 (2005) 256–274. T.A. Holland, Y. Tabata, A.G. Mikos, Dual growth factor delivery from degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds for cartilage tissue engineering, J. Control. Release 101 (2005) 111–125. T.A. Holland, Y. Tabata, A.G. Mikos, In vitro release of transforming growth factor-beta 1 from gelatin microparticles encapsulated in biodegradable, injectable oligo(poly(ethylene glycol) fumarate) hydrogels, J. Control. Release 91 (2003) 299–313. A. Ito, A. Mase, Y. Takizawa, M. Shinkai, H. Honda, K. Hata, M. Ueda, T. Kobayashi, Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering, J. Biosci. Bioeng. 95 (2003) 196–199. H.A. Awad, M.Q. Wickham, H.A. Leddy, J.M. Gimble, F. Guilak, Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds, Biomaterials 25 (2004) 3211–3222. Y. Kimura, M. Ozeki, T. Inamoto, Y. Tabata, Adipose tissue engineering based on human preadipocytes combined with gelatin microspheres containing basic fibroblast growth factor, Biomaterials 24 (2003) 2513–2521. R.G. Payne, J.S. McGonigle, M.J. Yaszemski, A.W. Yasko, A.G. Mikos, Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 3. Proliferation and differentiation of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate), Biomaterials 23 (2002) 4381–4387. A.S. Wolberg, Thrombin generation and fibrin clot structure, Blood Rev. 21 (2007) 131–142. M.R. Neidert, E.S. Lee, T.R. Oegema, R.T. Tranquillo, Enhanced fibrin remodeling in vitro with TGF-beta1, insulin and plasmin for improved tissue-equivalents, Biomaterials 23 (2002) 3717–3731. H. Schmoekel, J.C. Schense, F.E. Weber, K.W. Gratz, D. Gnagi, R. Muller, J.A. Hubbell, Bone healing in the rat and dog with nonglycosylated BMP-2 demonstrating low solubility in fibrin matrices, J. Orthop. Res. 22 (2004) 376–381. M.C. McManus, E.D. Boland, H.P. Koo, C.P. Barnes, K.J. Pawlowski, G.E. Wnek, D.G. Simpson, G.L. Bowlin, Mechanical properties of electrospun fibrinogen structures, Acta Biomater. 2 (2006) 19–28. Y.H. Liao, S.A. Jones, B. Forbes, G.P. Martin, M.B. Brown, Hyaluronan: pharmaceutical characterization and drug delivery, Drug Deliv. 12 (2005) 327–342. 779 [67] H. Tan, C.M. Ramirez, N. Miljkovic, H. Li, J.P. Rubin, K.G. Marra, Thermosensitive injectable hyaluronic acid hydrogel for adipose tissue engineering, Biomaterials 30 (2009) 6844–6853. [68] T.G. Kim, H.J. Chung, T.G. Park, Macroporous and nanofibrous hyaluronic acid/ collagen hybrid scaffold fabricated by concurrent electrospinning and deposition/leaching of salt particles, Acta Biomater. 4 (2008) 1611–1619. [69] A. Facchini, G. Lisignoli, S. Cristino, L. Roseti, L. De Franceschi, E. Marconi, B. Grigolo, Human chondrocytes and mesenchymal stem cells grown onto engineered scaffold, Biorheology 43 (2006) 471–480. [70] R.L. Jackson, S.J. Busch, A.D. Cardin, Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes, Physiol. Rev. 71 (1991) 481–539. [71] P.A. McCourt, B. Ek, N. Forsberg, S. Gustafson, Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan, J. Biol. Chem. 269 (1994) 30081–30084. [72] T.C. Laurent, U.B. Laurent, J.R. Fraser, Functions of hyaluronan, Ann. Rheum. Dis. 54 (1995) 429–432. [73] L. Lippiello, Glucosamine and chondroitin sulfate: biological response modifiers of chondrocytes under simulated conditions of joint stress, Osteoarthritis Cartilage 11 (2003) 335–342. [74] S. Tang, S.M. Vickers, H.P. Hsu, M. Spector, Fabrication and characterization of porous hyaluronic acid–collagen composite scaffolds, J. Biomed. Mater. Res. A 82 (2007) 323–335. [75] J.S. Pieper, A. Oosterhof, P.J. Dijkstra, J.H. Veerkamp, T.H. van Kuppevelt, Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate, Biomaterials 20 (1999) 847–858. [76] E. Khor, L.Y. Lim, Implantable applications of chitin and chitosan, Biomaterials 24 (2003) 2339–2349. [77] A. Di Martino, M. Sittinger, M.V. Risbud, Chitosan: a versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (2005) 5983–5990. [78] P.B. Malafaya, G.A. Silva, R.L. Reis, Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications, Adv. Drug Deliv. Rev. 59 (2007) 207–233. [79] M. George, T.E. Abraham, Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review, J. Control. Release 114 (2006) 1–14. [80] C. Gaissmaier, J. Fritz, T. Krackhardt, I. Flesch, W.K. Aicher, N. Ashammakhi, Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures, Biomaterials 26 (2005) 1953–1960. [81] K. Akeda, H.S. An, M. Okuma, M. Attawia, K. Miyamoto, E.J. Thonar, M.E. Lenz, R.L. Sah, K. Masuda, Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis, Osteoarthritis Cartilage 14 (2006) 1272–1280. [82] A. Steinert, M. Weber, A. Dimmler, C. Julius, N. Schutze, U. Noth, H. Cramer, J. Eulert, U. Zimmermann, C. Hendrich, Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate, J. Orthop. Res. 21 (2003) 1090–1097. [83] C.A. Simmons, E. Alsberg, S. Hsiong, W.J. Kim, D.J. Mooney, Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells, Bone 35 (2004) 562–569. [84] E.J. Bergsma, F.R. Rozema, R.R. Bos, W.C. de Bruijn, Foreign body reactions to resorbable poly(L-lactide) bone plates and screws used for the fixation of unstable zygomatic fractures, J. Oral Maxillofac. Surg. 51 (1993) 666–670. [85] C. Martin, H. Winet, J.Y. Bao, Acidity near eroding polylactide-polyglycolide in vitro and in vivo in rabbit tibial bone chambers, Biomaterials 17 (1996) 2373–2380. [86] W.J. Li, J.A. Cooper Jr., R.L. Mauck, R.S. Tuan, Fabrication and characterization of six electrospun poly(alpha-hydroxy ester)-based fibrous scaffolds for tissue engineering applications, Acta Biomater. 2 (2006) 377–385. [87] W.J. Li, R.L. Mauck, J.A. Cooper, X. Yuan, R.S. Tuan, Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering, J. Biomech. 40 (2007) 1686–1693. [88] W.J. Li, Y.J. Jiang, R.S. Tuan, Chondrocyte phenotype in engineered fibrous matrix is regulated by fiber size, Tissue Eng. 12 (2006) 1775–1785. [89] W.J. Li, Y.J. Jiang, R.S. Tuan, Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies, Tissue Eng. Part A 14 (2008) 639–648. [90] D.J. Hak, The use of osteoconductive bone graft substitutes in orthopaedic trauma, J. Am. Acad. Orthop. Surg. 15 (2007) 525–536. [91] M.R. Sarkar, N. Wachter, P. Patka, L. Kinzl, First histological observations on the incorporation of a novel calcium phosphate bone substitute material in human cancellous bone, J. Biomed. Mater. Res. 58 (2001) 329–334. [92] E. Goyenvalle, N.J. Guyen, E. Aguado, N. Passuti, G. Daculsi, Bilayered calcium phosphate coating to promote osseointegration of a femoral stem prosthesis, J. Mater. Sci. Mater. Med. 14 (2003) 219–227. [93] C.E. Thomas, A. Ehrhardt, M.A. Kay, Progress and problems with the use of viral vectors for gene therapy, Nat. Rev. Genet. 4 (2003) 346–358. [94] P.D. Robbins, S.C. Ghivizzani, Viral vectors for gene therapy, Pharmacol. Ther. 80 (1998) 35–47. [95] A. Rethwilm, Foamy virus vectors: an awaited alternative to gammaretro- and lentiviral vectors, Curr. Gene Ther. 7 (2007) 261–271. [96] A.F. Steinert, U. Noth, R.S. Tuan, Concepts in gene therapy for cartilage repair, Injury 39 (Suppl 1) (2008) S97–S113. [97] K. Anwer, Formulations for DNA delivery via electroporation in vivo, Methods Mol. Biol. 423 (2008) 77–89. [98] G. Romano, Current development of nonviral-mediated gene transfer, Drug News Perspect. 20 (2007) 227–231. 780 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 [99] X. Gao, K.S. Kim, D. Liu, Nonviral gene delivery: what we know and what is next, AAPS J. 9 (2007) E92–E104. [100] C.H. Evans, G. Palmer, A. Pascher, A. Steinert, O. Betz, S.C. Ghivizzani, Genetically enhanced tissue engineering without cell culture or artificial scaffolds, in: A. Grodzinski, L. Sandell, Eds, Orthopaedic Tissue Engineering, American Academy of Orthopaedic Surgeons, Rosemong 2004, pp. 389–394. [101] C.H. Evans, G.D. Palmer, A. Pascher, R. Porter, F.N. Kwong, E. Gouze, J.N. Gouze, F. Liu, A. Steinert, O. Betz, et al., Facilitated endogenous repair: making tissue engineering simple, practical, and economical, Tissue Eng. 13 (2007) 1987–1993. [102] A. Pascher, G.D. Palmer, A. Steinert, T. Oligino, E. Gouze, J.N. Gouze, O. Betz, M. Spector, P.D. Robbins, C.H. Evans, et al., Gene delivery to cartilage defects using coagulated bone marrow aspirate, Gene Ther. 11 (2004) 133–141. [103] M. Lotz, Cytokines in cartilage injury and repair, Clin. Orthop. Relat. Res. (2001) S108–S115. [104] C.H. Evans, P.D. Robbins, Genetically augmented tissue engineering of the musculoskeletal system, Clin. Orthop. (1999) S410–S418. [105] C.H. Evans, S.C. Ghivizzani, P.D. Robbins, Progress and prospects: genetic treatments for disorders of bones and joints, Gene Ther. 16 (2009) 944–952. [106] S.S. Makarov, J.C. Olsen, W.N. Johnston, S.K. Anderle, R.R. Brown, A.S. Baldwin Jr., J.S. Haskill, J.H. Schwab, Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 402–406. [107] R.M. Dharmavaram, G. Liu, R.S. Tuan, D.G. Stokes, S.A. Jimenez, Stable transfection of human fetal chondrocytes with a type II procollagen minigene: expression of the mutant protein and alterations in the structure of the extracellular matrix in vitro, Arthritis Rheum. 42 (1999) 1433–1442. [108] P.D. Robbins, C.H. Evans, Y. Chernajovsky, Gene therapy for arthritis, Gene Ther. 10 (2003) 902–911. [109] D.D. D'Lima, S. Hashimoto, P.C. Chen, C.W. Colwell Jr., M.K. Lotz, Impact of mechanical trauma on matrix and cells, Clin. Orthop. Relat. Res. (2001) S90–S99. [110] C.W. Colwell Jr., D.D. D'Lima, H.R. Hoenecke, J. Fronek, P. Pulido, B.A. Morris, C. Chung, D. Resnick, M. Lotz, In vivo changes after mechanical injury, Clin. Orthop. Relat. Res. (2001) S116–S123. [111] D.D. D'Lima, S. Hashimoto, P.C. Chen, C.W. Colwell Jr., M.K. Lotz, Human chondrocyte apoptosis in response to mechanical injury, Osteoarthritis Cartilage 9 (2001) 712–719. [112] D.D. D'Lima, S. Hashimoto, P.C. Chen, M.K. Lotz, C.W. Colwell Jr., In vitro and in vivo models of cartilage injury, J. Bone Joint Surg. Am. 83-A (Suppl 2) (2001) 22–24. [113] D.D. D'Lima, S. Hashimoto, P.C. Chen, M.K. Lotz, C.W. Colwell Jr., Cartilage injury induces chondrocyte apoptosis, J. Bone Joint Surg. Am. 83-A (Suppl 2) (2001) 19–21. [114] J.L. Haupt, D.D. Frisbie, C.W. McIlwraith, P.D. Robbins, S. Ghivizzani, C.H. Evans, A.J. Nixon, Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model, J. Orthop. Res. 23 (2005) 118–126. [115] A.J. Nixon, J.L. Haupt, D.D. Frisbie, S.S. Morisset, C.W. McIlwraith, P.D. Robbins, C.H. Evans, S. Ghivizzani, Gene-mediated restoration of cartilage matrix by combination insulin-like growth factor-I/interleukin-1 receptor antagonist therapy, Gene Ther. 12 (2005) 177–186. [116] M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, L. Peterson, Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, N. Engl. J. Med. 331 (1994) 889–895. [117] L. Peterson, T. Minas, M. Brittberg, A. Lindahl, Treatment of osteochondritis dissecans of the knee with autologous chondrocyte transplantation: results at two to ten years, J. Bone Joint Surg. Am. 85-A (Suppl 2) (2003) 17–24. [118] U. Horas, D. Pelinkovic, G. Herr, T. Aigner, R. Schnettler, Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial, J. Bone Joint Surg. Am. 85-A (2003) 185–192. [119] P. Niemeyer, M. Steinwachs, C. Erggelet, P.C. Kreuz, N. Kraft, W. Kostler, A. Mehlhorn, N.P. Sudkamp, Autologous chondrocyte implantation for the treatment of retropatellar cartilage defects: clinical results referred to defect localisation, Arch. Orthop. Trauma Surg. 128 (2008) 1223–1231. [120] J. Iwasa, L. Engebretsen, Y. Shima, M. Ochi, Clinical application of scaffolds for cartilage tissue engineering, Knee Surg. Sports Traumatol. Arthrosc. 17 (2009) 561–577. [121] S. Wakitani, T. Mitsuoka, N. Nakamura, Y. Toritsuka, Y. Nakamura, S. Horibe, Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports, Cell Transplant. 13 (2004) 595–600. [122] R. Kuroda, K. Ishida, T. Matsumoto, T. Akisue, H. Fujioka, K. Mizuno, H. Ohgushi, S. Wakitani, M. Kurosaka, Treatment of a full-thickness articular cartilage defect in the femoral condyle of an athlete with autologous bone-marrow stromal cells, Osteoarthritis Cartilage 15 (2007) 226–231. [123] F. Djouad, L. Rackwitz, Y. Song, S. Janjanin, R.S. Tuan, ERK1/2 activation induced by inflammatory cytokines compromises effective host tissue integration of engineered cartilage, Tissue Eng. Part A 15 (2009) 2825–2835. [124] J.M. Murphy, D.J. Fink, E.B. Hunziker, F.P. Barry, Stem cell therapy in a caprine model of osteoarthritis, Arthritis Rheum. 48 (2003) 3464–3474. [125] K.B. Lee, J.H. Hui, I.C. Song, L. Ardany, E.H. Lee, Injectable mesenchymal stem cell therapy for large cartilage defects—a porcine model, Stem Cells 25 (2007) 2964–2971. [126] C.H. Evans, P.D. Robbins, S.C. Ghivizzani, M.C. Wasko, M.M. Tomaino, R. Kang, T.A. Muzzonigro, M. Vogt, E.M. Elder, T.L. Whiteside, et al., Gene transfer to human joints: progress toward a gene therapy of arthritis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 8698–8703. [127] S.C. Ghivizzani, E. Gouze, J.N. Gouze, J.D. Kay, M.L. Bush, R.S. Watson, P.P. Levings, D.M. Nickerson, P.T. Colahan, P.D. Robbins, et al., Perspectives on the use of gene therapy for chronic joint diseases, Curr. Gene Ther. 8 (2008) 273–286. [128] S.C. Ghivizzani, R. Kang, H.I. Georgescu, E.R. Lechman, D. Jaffurs, J.M. Engle, S.C. Watkins, M.H. Tindal, M.K. Suchanek, L.R. McKenzie, et al., Constitutive intraarticular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis, J. Immunol. 159 (1997) 3604–3612. [129] S.C. Ghivizzani, E.R. Lechman, R. Kang, C. Tio, J. Kolls, C.H. Evans, P.D. Robbins, Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 4613–4618. [130] S.C. Ghivizzani, T.J. Oligino, J.C. Glorioso, P.D. Robbins, C.H. Evans, Direct gene delivery strategies for the treatment rheumatoid arthritis, Drug Discov. Today 6 (2001) 236–244. [131] C.H. Evans, S.C. Ghivizzani, P.D. Robbins, Gene therapy of the rheumatic diseases: 1998 to 2008, Arthritis Res. Ther. 11 (2009) 209. [132] P. Wehling, J. Reinecke, A.A. Baltzer, M. Granrath, K.P. Schulitz, C. Schultz, R. Krauspe, T. Whiteside, E. Elder, S.C. Ghivizzani, et al., Clinical responses to gene therapy in joints of two subjects with rheumatoid arthritis, Hum. Gene Ther. 20 (2008) 97–101. [133] C.H. Evans, S.C. Ghivizzani, P.D. Robbins, Arthritis gene therapy's first death, Arthritis Res. Ther. 10 (2008) 110. [134] C.H. Evans, S.C. Ghivizzani, P.D. Robbins, Orthopaedic gene therapy, Clin. Orthop. Relat. Res. (2004) 316–329. [135] J.P. Pelletier, J.P. Caron, C. Evans, P.D. Robbins, H.I. Georgescu, D. Jovanovic, J.C. Fernandes, J. Martel-Pelletier, In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy, Arthritis Rheum. 40 (1997) 1012–1019. [136] J. Fernandes, G. Tardif, J. Martel-Pelletier, V. Lascau-Coman, M. Dupuis, F. Moldovan, M. Sheppard, B.R. Krishnan, J.P. Pelletier, In vivo transfer of interleukin-1 receptor gene in osteoarthritic rabbit knee joints: prevention of osteoarthritis progression, Am. J. Physiol. 154 (1999) 1159–1169. [137] D.D. Frisbie, S.C. Ghivizzani, P.D. Robbins, C.H. Evans, C.W. McIlwraith, Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene, Gene Ther. 9 (2002) 12–20. [138] R. Cancedda, G. Bianchi, A. Derubeis, R. Quarto, Cell therapy for bone disease: a review of current status, Stem Cells 21 (2003) 610–619. [139] S.P. Bruder, K.H. Kraus, V.M. Goldberg, S. Kadiyala, The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects, J. Bone Joint Surg. Am. 80 (1998) 985–996. [140] E. Kon, A. Muraglia, A. Corsi, P. Bianco, M. Marcacci, I. Martin, A. Boyde, I. Ruspantini, P. Chistolini, M. Rocca, et al., Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in criticalsize defects of sheep long bones, J. Biomed. Mater. Res. 49 (2000) 328–337. [141] S. Kadiyala, R.G. Young, M.A. Thiede, S.P. Bruder, Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro, Cell Transplant. 6 (1997) 125–134. [142] H. Petite, V. Viateau, W. Bensaid, A. Meunier, C. de Pollak, M. Bourguignon, K. Oudina, L. Sedel, G. Guillemin, Tissue-engineered bone regeneration, Nat. Biotechnol. 18 (2000) 959–963. [143] R. Quarto, M. Mastrogiacomo, R. Cancedda, S.M. Kutepov, V. Mukhachev, A. Lavroukov, E. Kon, M. Marcacci, Repair of large bone defects with the use of autologous bone marrow stromal cells, N. Engl. J. Med. 344 (2001) 385–386. [144] D.W. Hutmacher, M. Sittinger, M.V. Risbud, Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems, Trends Biotechnol. 22 (2004) 354–362. [145] D.W. Hutmacher, T. Schantz, I. Zein, K.W. Ng, S.H. Teoh, K.C. Tan, Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling, J. Biomed. Mater. Res. 55 (2001) 203–216. [146] S. Yang, K.F. Leong, Z. Du, C.K. Chua, The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques, Tissue Eng. 8 (2002) 1–11. [147] J.T. Schantz, D.W. Hutmacher, H. Chim, K.W. Ng, T.C. Lim, S.H. Teoh, Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology, Cell Transplant. 11 (2002) 125–138. [148] P. Hernigou, F. Beaujean, Treatment of osteonecrosis with autologous bone marrow grafting, Clin. Orthop. Relat. Res. (2002) 14–23. [149] F. Ficat, J. Arlet, Biopsy-forage in the treatment of primary femoro-capital osteonecrosis, Presse Med. 79 (1971) 581. [150] D.S. Hungerford, Pathogenetic considerations in ischemic necrosis of bone, Can J Surg 24 (1981) 583–7, 590. [151] T. Kiaer, N.W. Pedersen, K.D. Kristensen, H. Starklint, Intra-osseous pressure and oxygen tension in avascular necrosis and osteoarthritis of the hip, J. Bone Joint Surg. Br. 72 (1990) 1023–1030. [152] H. Plenk Jr., S. Hofmann, M. Breitenseher, M. Urban, Pathomorphological aspects and repair mechanisms of femur head necrosis, Orthopade 29 (2000) 389–402. [153] F.A. Petrigliano, J.R. Lieberman, Osteonecrosis of the hip: novel approaches to evaluation and treatment, Clin. Orthop. Relat. Res. 465 (2007) 53–62. [154] V. Gangji, J.P. Hauzeur, Treatment of osteonecrosis of the femoral head with implantation of autologous bone-marrow cells. Surgical technique, J. Bone Joint Surg. Am. 87 (Suppl 1) (2005) 106–112. [155] P. Hernigou, A. Poignard, O. Manicom, G. Mathieu, H. Rouard, The use of percutaneous autologous bone marrow transplantation in nonunion and avascular necrosis of bone, J. Bone Joint Surg. Br. 87 (2005) 896–902. U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 [156] K. Kawate, H. Yajima, H. Ohgushi, N. Kotobuki, K. Sugimoto, T. Ohmura, Y. Kobata, K. Shigematsu, K. Kawamura, K. Tamai, et al., Tissue-engineered approach for the treatment of steroid-induced osteonecrosis of the femoral head: transplantation of autologous mesenchymal stem cells cultured with beta-tricalcium phosphate ceramics and free vascularized fibula, Artif. Organs 30 (2006) 960–962. [157] U. Noth, J. Reichert, S. Reppenhagen, A. Steinert, L. Rackwitz, J. Eulert, J. Beckmann, M. Tingart, Cell based therapy for the treatment of femoral head necrosis, Orthopade 36 (2007) 466–471. [158] T.T. Tang, B. Lu, B. Yue, X.H. Xie, Y.Z. Xie, K.R. Dai, J.X. Lu, J.R. Lou, Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats, J. Bone Joint Surg. Br. 89 (2007) 127–129. [159] R.T. Franceschi, Biological approaches to bone regeneration by gene therapy, J. Dent. Res. 84 (2005) 1093–1103. [160] D.A. Oakes, J.R. Lieberman, Osteoinductive applications of regional gene therapy: ex vivo gene transfer, Clin. Orthop. Relat. Res. (2000) S101–S112. [161] J.R. Lieberman, S.C. Ghivizzani, C.H. Evans, Gene transfer approaches to the healing of bone and cartilage, Mol. Ther. 6 (2002) 141–147. [162] J.R. Lieberman, A. Daluiski, S. Stevenson, L. Wu, P. McAllister, Y.P. Lee, J.M. Kabo, G.A. Finerman, A.J. Berk, O.N. Witte, The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats, J. Bone Joint Surg. Am. 81 (1999) 905–917. [163] A.W. Baltzer, J.R. Lieberman, Regional gene therapy to enhance bone repair, Gene Ther. 11 (2004) 344–350. [164] S.L. Hall, S.T. Chen, R. Gysin, D.S. Gridley, S. Mohan, K.H. Lau, Stem cell antigen-1+ cell-based bone morphogenetic protein-4 gene transfer strategy in mice failed to promote endosteal bone formation, J. Gene Med. 11 (2009) 877–888. [165] X. Jiang, S.A. Gittens, Q. Chang, X. Zhang, C. Chen, Z. Zhang, The use of tissueengineered bone with human bone morphogenetic protein-4-modified bonemarrow stromal cells in repairing mandibular defects in rabbits, Int. J. Oral Maxillofac. Surg. 35 (2006) 1133–1139. [166] X.Q. Jiang, J.G. Chen, S. Gittens, C.J. Chen, X.L. Zhang, Z.Y. Zhang, The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits, Chin. Med. J. (Engl) 118 (2005) 281–288. [167] T. Rose, H. Peng, A. Usas, C. Josten, F.H. Fu, J. Huard, Ex-vivo gene therapy with BMP-4 for critically sized defects and enhancement of fracture healing in an osteoporotic animal model, Unfallchirurg 108 (2005) 25–34. [168] J.Z. Li, H. Li, T. Sasaki, D. Holman, B. Beres, R.J. Dumont, D.D. Pittman, G.R. Hankins, G.A. Helm, Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat, Gene Ther. 10 (2003) 1735–1743. [169] F.Z. Lu, M. Fujino, Y. Kitazawa, T. Uyama, Y. Hara, N. Funeshima, J.Y. Jiang, A. Umezawa, X.K. Li, Characterization and gene transfer in mesenchymal stem cells derived from human umbilical-cord blood, J. Lab. Clin. Med. 146 (2005) 271–278. [170] J.A. Jane Jr., B.A. Dunford, A. Kron, D.D. Pittman, T. Sasaki, J.Z. Li, H. Li, T.D. Alden, H. Dayoub, G.R. Hankins, et al., Ectopic osteogenesis using adenoviral bone morphogenetic protein (BMP)-4 and BMP-6 gene transfer, Mol. Ther. 6 (2002) 464–470. [171] A.L. Bertone, D.D. Pittman, M.L. Bouxsein, J. Li, B. Clancy, H.J. Seeherman, Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model, J. Orthop. Res. 22 (2004) 1261–1270. [172] P. Varady, J.Z. Li, M. Cunningham, E.J. Beres, S. Das, J. Engh, T.D. Alden, D.D. Pittman, K.M. Kerns, D.F. Kallmes, et al., Morphologic analysis of BMP-9 gene therapy-induced osteogenesis, Hum. Gene Ther. 12 (2001) 697–710. [173] H. Iwaguro, J. Yamaguchi, C. Kalka, S. Murasawa, H. Masuda, S. Hayashi, M. Silver, T. Li, J.M. Isner, T. Asahara, Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration, Circulation 105 (2002) 732–738. [174] T. Rose, H. Peng, A. Usas, R. Kuroda, H. Lill, F.H. Fu, J. Huard, Gene therapy to improve osteogenesis in bone lesions with severe soft tissue damage, Langenbecks Arch. Surg. 388 (2003) 356–365. [175] Y. Chen, K.D. Luk, K.M. Cheung, W.W. Lu, X.M. An, S.S. Ng, M.C. Lin, H.F. Kung, Combination of adeno-associated virus and adenovirus vectors expressing bone morphogenetic protein-2 produces enhanced osteogenic activity in immunocompetent rats, Biochem. Biophys. Res. Commun. 317 (2004) 675–681. [176] K.D. Luk, Y. Chen, K.M. Cheung, H.F. Kung, W.W. Lu, J.C. Leong, Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation, Biochem. Biophys. Res. Commun. 308 (2003) 636–645. [177] Y. Chen, K.D. Luk, K.M. Cheung, R. Xu, M.C. Lin, W.W. Lu, J.C. Leong, H.F. Kung, Gene therapy for new bone formation using adeno-associated viral bone morphogenetic protein-2 vectors, Gene Ther. 10 (2003) 1345–1353. [178] Y. Gafni, G. Pelled, Y. Zilberman, G. Turgeman, F. Apparailly, H. Yotvat, E. Galun, Z. Gazit, C. Jorgensen, D. Gazit, Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system, Mol. Ther. 9 (2004) 587–595. [179] M.S. Virk, A. Conduah, S.H. Park, N. Liu, O. Sugiyama, A. Cuomo, C. Kang, J.R. Lieberman, Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model, Bone 42 (2008) 921–931. [180] B.T. Feeley, A.H. Conduah, O. Sugiyama, L. Krenek, I.S. Chen, J.R. Lieberman, In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation models, J. Orthop. Res. 24 (2006) 1709–1721. [181] T. Rose, H. Peng, H.C. Shen, A. Usas, R. Kuroda, H. Lill, F.H. Fu, J. Huard, The role of cell type in bone healing mediated by ex vivo gene therapy, Langenbecks Arch. Surg. 388 (2003) 347–355. 781 [182] M. Egermann, E. Schneider, C.H. Evans, A.W. Baltzer, The potential of gene therapy for fracture healing in osteoporosis, Osteoporos. Int. 16 (Suppl 2) (2005) S120–S128. [183] V.M. Betz, O.B. Betz, M.B. Harris, M.S. Vrahas, C.H. Evans, Bone tissue engineering and repair by gene therapy, Front Biosci. 13 (2008) 833–841. [184] C.H. Evans, S.C. Ghivizzani, P.D. Robbins, Orthopedic gene therapy in 2008, Mol. Ther. 17 (2009) 231–244. [185] L.L. Southwood, D.D. Frisbie, C.E. Kawcak, S.C. Ghivizzani, C.H. Evans, C.W. McIlwraith, Evaluation of Ad-BMP-2 for enhancing fracture healing in an infected defect fracture rabbit model, J. Orthop. Res. 22 (2004) 66–72. [186] C. Hidaka, K. Goshi, B. Rawlins, O. Boachie-Adjei, R.G. Crystal, Enhancement of spine fusion using combined gene therapy and tissue engineering BMP-7expressing bone marrow cells and allograft bone, Spine (Phila Pa 1976) 28 (2003) 2049–2057. [187] S.T. Yoon, S.D. Boden, Spine fusion by gene therapy, Gene Ther. 11 (2004) 360–367. [188] C.W. Cha, S.D. Boden, Gene therapy applications for spine fusion, Spine (Phila Pa 1976) 28 (2003) S74–S84. [189] M. Miyazaki, O. Sugiyama, J. Zou, S.H. Yoon, F. Wei, Y. Morishita, C. Sintuu, M.S. Virk, J.R. Lieberman, J.C. Wang, Comparison of lentiviral and adenoviral gene therapy for spinal fusion in rats, Spine (Phila Pa 1976) 33 (2008) 1410–1417. [190] D.M. Doroski, K.S. Brink, J.S. Temenoff, Techniques for biological characterization of tissue-engineered tendon and ligament, Biomaterials 28 (2007) 187–202. [191] Z. Ge, J.C. Goh, E.H. Lee, Selection of cell source for ligament tissue engineering, Cell Transplant. 14 (2005) 573–583. [192] K.A. Hildebrand, F. Jia, S.L. Woo, Response of donor and recipient cells after transplantation of cells to the ligament and tendon, Microsc. Res. Tech. 58 (2002) 34–38. [193] F. Van Eijk, D.B. Saris, J. Riesle, W.J. Willems, C.A. Van Blitterswijk, A.J. Verbout, W.J. Dhert, Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source, Tissue Eng. 10 (2004) 893–903. [194] N. Watanabe, S.L. Woo, C. Papageorgiou, C. Celechovsky, S. Takai, Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation, Microsc. Res. Tech. 58 (2002) 39–44. [195] C.K. Kuo, R.S. Tuan, Mechanoactive tenogenic differentiation of human mesenchymal stem cells, Tissue Eng. Part A 14 (2008) 1615–1627. [196] G.H. Altman, R.L. Horan, I. Martin, J. Farhadi, P.R. Stark, V. Volloch, J.C. Richmond, G. Vunjak-Novakovic, D.L. Kaplan, Cell differentiation by mechanical stress, FASEB J. 16 (2002) 270–272. [197] I.C. Lee, J.H. Wang, Y.T. Lee, T.H. Young, The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system, Biochem. Biophys. Res. Commun. 352 (2007) 147–152. [198] L. Heckmann, J. Fiedler, T. Mattes, M. Dauner, R.E. Brenner, Interactive effects of growth factors and three-dimensional scaffolds on multipotent mesenchymal stromal cells, Biotechnol. Appl. Biochem. 49 (2008) 185–194. [199] A. Hoffmann, G. Gross, Tendon and ligament engineering: from cell biology to in vivo application, Regen. Med. 1 (2006) 563–574. [200] J. Lou, Y. Tu, F.J. Ludwig, J. Zhang, P.R. Manske, Effect of bone morphogenetic protein-12 gene transfer on mesenchymal progenitor cells, Clin. Orthop. (1999) 333–339. [201] M. Hairfield-Stein, C. England, H.J. Paek, K.B. Gilbraith, R. Dennis, E. Boland, P. Kosnik, Development of self-assembled, tissue-engineered ligament from bone marrow stromal cells, Tissue Eng. 13 (2007) 703–710. [202] L. Heckmann, H.J. Schlenker, J. Fiedler, R. Brenner, M. Dauner, G. Bergenthal, T. Mattes, L. Claes, A. Ignatius, Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering, J. Biomed. Mater. Res. B Appl. Biomater. 81 (2007) 82–90. [203] Y. Bi, D. Ehirchiou, T.M. Kilts, C.A. Inkson, M.C. Embree, W. Sonoyama, L. Li, A.I. Leet, B.M. Seo, L. Zhang, et al., Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche, Nat. Med. 131 (2007) 1219–1227. [204] J. Zhang, J.H. Wang, Mechanobiological response of tendon stem cells: implications of tendon homeostasis and pathogenesis of tendinopathy, J. Orthop. Res. 28 (2010) 639–643. [205] T. Zantop, W. Petersen, J.K. Sekiya, V. Musahl, F.H. Fu, Anterior cruciate ligament anatomy and function relating to anatomical reconstruction, Knee Surg. Sports Traumatol. Arthrosc. 14 (2006) 982–992. [206] Z. Ge, J.C. Goh, E.H. Lee, The effects of bone marrow-derived mesenchymal stem cells and fascia wrap application to anterior cruciate ligament tissue engineering, Cell Transplant. 14 (2005) 763–773. [207] F. Li, H. Jia, C. Yu, ACL reconstruction in a rabbit model using irradiated Achilles allograft seeded with mesenchymal stem cells or PDGF-B gene-transfected mesenchymal stem cells, Knee Surg. Sports Traumatol. Arthrosc. 15 (2007) 1219–1227. [208] J.K. Lim, J. Hui, L. Li, A. Thambyah, J. Goh, E.H. Lee, Enhancement of tendon graft osteointegration using mesenchymal stem cells in a rabbit model of anterior cruciate ligament reconstruction, Arthroscopy 20 (2004) 899–910. [209] A. Hoffmann, G. Gross, Tendon and ligament engineering in the adult organism: mesenchymal stem cells and gene-therapeutic approaches, Int. Orthop. 31 (2007) 791–797. [210] K.A. Hildebrand, C.B. Frank, D.A. Hart, Gene intervention in ligament and tendon: current status, challenges, future directions, Gene Ther. 11 (2004) 368–378. [211] A. Pascher, A.F. Steinert, G.D. Palmer, O. Betz, J.N. Gouze, E. Gouze, C. Pilapil, S.C. Ghivizzani, C.H. Evans, M.M. Murray, Enhanced repair of the anterior cruciate ligament by in situ gene transfer: evaluation in an in vitro model, Mol. Ther. 10 (2004) 327–336. 782 U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 [212] A.F. Steinert, M. Weber, M. Kunz, G.D. Palmer, U. Noth, C.H. Evans, M.M. Murray, In situ IGF-1 gene delivery to cells emerging from the injured anterior cruciate ligament, Biomaterials 29 (2008) 904–916. [213] X.T. Wang, P.Y. Liu, J.B. Tang, Tendon healing in vitro: genetic modification of tenocytes with exogenous PDGF gene and promotion of collagen gene expression, J. Hand. Surg. Am. 29 (2004) 884–890. [214] X.T. Wang, P.Y. Liu, J.B. Tang, Tendon healing in vitro: modification of tenocytes with exogenous vascular endothelial growth factor gene increases expression of transforming growth factor beta but minimally affects expression of collagen genes, J. Hand. Surg. Am. 30 (2005) 222–229. [215] S.C. Scherping Jr., C.C. Schmidt, H.I. Georgescu, C.K. Kwoh, C.H. Evans, S.L. Woo, Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits, Connect. Tissue Res. 36 (1997) 1–8. [216] C.C. Schmidt, H.I. Georgescu, C.K. Kwoh, G.L. Blomstrom, C.P. Engle, L.A. Larkin, C.H. Evans, S.L. Woo, Effect of growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments, J. Orthop. Res. 13 (1995) 184–190. [217] C.H. Evans, S.C. Ghivizzani, J.H. Herndon, P.D. Robbins, Gene therapy for the treatment of musculoskeletal diseases, J. Am. Acad. Orthop. Surg. 13 (2005) 230–242. [218] J. Menetrey, C. Kasemkijwattana, C.S. Day, P. Bosch, F.H. Fu, M.S. Moreland, J. Huard, Direct-, fibroblast- and myoblast-mediated gene transfer to the anterior cruciate ligament, Tissue Eng. 5 (1999) 435–442. [219] N. Nakamura, D.A. Hart, R.S. Boorman, Y. Kaneda, N.G. Shrive, L.L. Marchuk, K. Shino, T. Ochi, C.B. Frank, Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo, J. Orthop. Res. 18 (2000) 517–523. [220] N. Nakamura, K. Shino, T. Natsuume, S. Horibe, N. Matsumoto, Y. Kaneda, T. Ochi, Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament, Gene Ther. 5 (1998) 1165–1170. [221] J. Lou, Y. Tu, M. Burns, M.J. Silva, P. Manske, BMP-12 gene transfer augmentation of lacerated tendon repair, J. Orthop. Res. 19 (2001) 1199–1202. [222] T.G. Gerich, R. Kang, F.H. Fu, P.D. Robbins, C.H. Evans, Gene transfer to the patellar tendon, Knee Surg. Sports Traumatol. Arthrosc. 5 (1997) 118–123. [223] T.G. Gerich, H.P. Lobenhoffer, F.H. Fu, P.D. Robbins, C.H. Evans, Virally mediated gene transfer in the patellar tendon. An experimental study in rabbits, Unfallchirurg 100 (1997) 354–362. [224] N.M. Wolfman, G. Hattersley, K. Cox, A.J. Celeste, R. Nelson, N. Yamaji, J.L. Dube, E. DiBlasio-Smith, J. Nove, J.J. Song, et al., Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family, J. Clin. Invest. 100 (1997) 321–330. [225] Y.P. Wong, S.C. Fu, Y.C. Cheuk, K.M. Lee, M.W. Wong, K.M. Chan, Bone morphogenetic protein 13 stimulates cell proliferation and production of collagen in human patellar tendon fibroblasts, Acta Orthop 76 (2005) 421–427. [226] Q.W. Wang, Z.L. Chen, Y.J. Piao, Mesenchymal stem cells differentiate into tenocytes by bone morphogenetic protein (BMP) 12 gene transfer, J. Biosci. Bioeng. 100 (2005) 418–422. [227] M. Majewski, O. Betz, P.E. Ochsner, F. Liu, R.M. Porter, C.H. Evans, Ex vivo adenoviral transfer of bone morphogenetic protein 12 (BMP-12) cDNA improves Achilles tendon healing in a rat model, Gene Ther. 15 (2008) 1139–1146. [228] K.W. Li, D.P. Lindsey, D.R. Wagner, N.J. Giori, D.J. Schurman, S.B. Goodman, R.L. Smith, D.R. Carter, G.S. Beaupre, Gene regulation ex vivo within a wrap-around tendon, Tissue Eng. 12 (2006) 2611–2618. [229] H.A. Awad, D.L. Butler, G.P. Boivin, F.N. Smith, P. Malaviya, B. Huibregtse, A.I. Caplan, Autologous mesenchymal stem cell-mediated repair of tendon, Tissue Eng. 5 (1999) 267–277. [230] H.A. Awad, G.P. Boivin, M.R. Dressler, F.N. Smith, R.G. Young, D.L. Butler, Repair of patellar tendon injuries using a cell-collagen composite, J. Orthop. Res. 21 (2003) 420–431. [231] D.L. Butler, N. Juncosa-Melvin, G.P. Boivin, M.T. Galloway, J.T. Shearn, C. Gooch, H. Awad, Functional tissue engineering for tendon repair: a multidisciplinary strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation, J. Orthop. Res. 26 (2008) 1–9. [232] R.G. Young, D.L. Butler, W. Weber, A.I. Caplan, S.L. Gordon, D.J. Fink, Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair, J. Orthop. Res. 16 (1998) 406–413. [233] A.K. Chong, A.D. Ang, J.C. Goh, J.H. Hui, A.Y. Lim, E.H. Lee, B.H. Lim, Bone marrowderived mesenchymal stem cells influence early tendon-healing in a rabbit achilles tendon model, J. Bone Joint Surg. Am. 89 (2007) 74–81. [234] C. Starke, S. Kopf, W. Petersen, R. Becker, Meniscal repair, Arthroscopy 25 (2009) 1033–1044. [235] S.B. Adams Jr., M.A. Randolph, T.J. Gill, Tissue engineering for meniscus repair, J. Knee Surg. 18 (2005) 25–30. [236] P. Buma, N.N. Ramrattan, T.G. van Tienen, R.P. Veth, Tissue engineering of the meniscus, Biomaterials 25 (2004) 1523–1532. [237] P. Buma, T. van Tienen, R. Veth, The collagen meniscus implant, Expert Rev. Med. Devices 4 (2007) 507–516. [238] M.A. Sweigart, K.A. Athanasiou, Toward tissue engineering of the knee meniscus, Tissue Eng. 7 (2001) 111–129. [239] V. Martinek, P. Ueblacker, K. Braun, S. Nitschke, R. Mannhardt, K. Specht, B. Gansbacher, A.B. Imhoff, Second generation of meniscus transplantation: in-vivo study with tissue engineered meniscus replacement, Arch. Orthop. Trauma Surg. 126 (2006) 228–234. [240] T. Yamasaki, M. Deie, R. Shinomiya, Y. Izuta, Y. Yasunaga, S. Yanada, P. Sharman, M. Ochi, Meniscal regeneration using tissue engineering with a scaffold derived [241] [242] [243] [244] [245] [246] [247] [248] [249] [250] [251] [252] [253] [254] [255] [256] [257] [258] [259] [260] [261] [262] [263] [264] [265] [266] [267] [268] [269] [270] from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow, J. Biomed. Mater. Res. A 75 (2005) 23–30. A.F. Steinert, G.D. Palmer, R. Capito, J.G. Hofstaetter, C. Pilapil, S.C. Ghivizzani, M. Spector, C.H. Evans, Genetically enhanced engineering of meniscus tissue using ex vivo delivery of transforming growth factor-beta 1 complementary deoxyribonucleic acid, Tissue Eng. 13 (2007) 2227–2237. A.J. Schoenfeld, W.J. Landis, D.B. Kay, Tissue-engineered meniscal constructs, Am. J. Orthop. 36 (2007) 614–620. S.M. Mueller, T.O. Schneider, S. Shortkroff, H.A. Breinan, M. Spector, alphasmooth muscle actin and contractile behavior of bovine meniscus cells seeded in type I and type II collagen-GAG matrices, J. Biomed. Mater. Res. 45 (1999) 157–166. T.G. Tienen, R.G. Heijkants, P. Buma, J.H. De Groot, A.J. Pennings, R.P. Veth, A porous polymer scaffold for meniscal lesion repair—a study in dogs, Biomaterials 24 (2003) 2541–2548. T.G. Tienen, R.G. Heijkants, J.H. de Groot, A.J. Pennings, A.J. Schouten, R.P. Veth, P. Buma, Replacement of the knee meniscus by a porous polymer implant: a study in dogs, Am. J. Sports Med. 34 (2006) 64–71. T.G. van Tienen, G. Hannink, P. Buma, Meniscus replacement using synthetic materials, Clin. Sports Med. 28 (2009) 143–156. A.C. AufderHeide, K.A. Athanasiou, Mechanical stimulation toward tissue engineering of the knee meniscus, Ann. Biomed. Eng. 32 (2004) 1161–1174. H. Goto, F.D. Shuler, C. Niyibizi, F.H. Fu, P.D. Robbins, C.H. Evans, Gene therapy for meniscal injury: enhanced synthesis of proteoglycan and collagen by meniscal cells transduced with a TGFbeta(1)gene, Osteoarthritis Cartilage 8 (2000) 266–271. H. Goto, F.D. Shuler, C. Lamsam, H.D. Moller, C. Niyibizi, F.H. Fu, P.D. Robbins, C.H. Evans, Transfer of lacZ marker gene to the meniscus, J. Bone Joint Surg. Am. 81 (1999) 918–925. V. Martinek, A. Usas, D. Pelinkovic, P. Robbins, F.H. Fu, J. Huard, Genetic engineering of meniscal allografts, Tissue Eng. 8 (2002) 107–117. C. Hidaka, C. Ibarra, J.A. Hannafin, P.A. Torzilli, M. Quitoriano, S.S. Jen, R.F. Warren, R.G. Crystal, Formation of vascularized meniscal tissue by combining gene therapy with tissue engineering, Tissue Eng. 8 (2002) 93–105. J.A. Miller, C. Schmatz, A.B. Schultz, Lumbar disc degeneration: correlation with age, sex, and spine level in 600 autopsy specimens, Spine (Phila Pa 1976) 13 (1988) 173–178. P. Adams, D.R. Eyre, H. Muir, Biochemical aspects of development and ageing of human lumbar intervertebral discs, Rheumatol. Rehabil. 16 (1977) 22–29. B. Johnstone, M.T. Bayliss, The large proteoglycans of the human intervertebral disc. Changes in their biosynthesis and structure with age, topography, and pathology, Spine (Phila Pa 1976) 20 (1995) 674–684. S. Roberts, B. Caterson, J. Menage, E.H. Evans, D.C. Jaffray, S.M. Eisenstein, Eisenstein, Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc, Spine (Phila Pa 1976) 25 (2000) 3005–3013. C. Evans, Potential biologic therapies for the intervertebral disc, J. Bone Joint Surg. Am. 88 (Suppl 2) (2006) 95–98. F.M. Phillips, H. An, J.D. Kang, S.D. Boden, J. Weinstein, Biologic treatment for intervertebral disc degeneration: summary statement, Spine (Phila Pa 1976) 28 (2003) S99. P.J. Donohue, M.R. Jahnke, J.D. Blaha, B. Caterson, Characterization of link protein (s) from human intervertebral-disc tissues, Biochem. J. 251 (1988) 739–747. C. Hohaus, T.M. Ganey, Y. Minkus, H.J. Meisel, Cell transplantation in lumbar spine disc degeneration disease, Eur. Spine J. 17 (Suppl 4) (2008) 492–503. T. Ganey, W.C. Hutton, T. Moseley, M. Hedrick, H.J. Meisel, Intervertebral disc repair using adipose tissue-derived stem and regenerative cells: experiments in a canine model, Spine (Phila Pa 1976) 34 (2009) 2297–2304. E. Steck, H. Bertram, R. Abel, B. Chen, A. Winter, W. Richter, Induction of intervertebral disc-like cells from adult mesenchymal stem cells, Stem Cells 23 (2005) 403–411. S. Sobajima, G. Vadala, A. Shimer, J.S. Kim, L.G. Gilbertson, J.D. Kang, Feasibility of a stem cell therapy for intervertebral disc degeneration, Spine J. 8 (2008) 888–896. S.U. Kuh, Y. Zhu, J. Li, K.J. Tsai, Q. Fei, W.C. Hutton, S.T. Yoon, Can TGF-beta1 and rhBMP-2 act in synergy to transform bone marrow stem cells to discogenic-type cells? Acta Neurochir. (Wien) 150 (2008) 1073–1079 discussion 1079. G. Vadala, S. Sobajima, J.Y. Lee, J. Huard, V. Denaro, J.D. Kang, L.G. Gilbertson, In vitro interaction between muscle-derived stem cells and nucleus pulposus cells, Spine J. 8 (2008) 804–809. H. Bertram, M. Kroeber, H. Wang, F. Unglaub, T. Guehring, C. Carstens, W. Richter, Matrix-assisted cell transfer for intervertebral disc cell therapy, Biochem. Biophys. Res. Commun. 331 (2005) 1185–1192. L.J. Nesti, W.J. Li, R.M. Shanti, Y.J. Jiang, W. Jackson, B.A. Freedman, T.R. Kuklo, J.R. Giuliani, R.S. Tuan, Intervertebral disc tissue engineering using a novel hyaluronic acid-nanofibrous scaffold (HANFS) amalgam, Tissue Eng. Part A 14 (2008) 1527–1537. M.E. Morrey, P.A. Anderson, G. Chambers, R. Paul, Optimizing nonviral-mediated transfection of human intervertebral disc chondrocytes, Spine J. 8 (2008) 796–803. C.J. Wallach, L.G. Gilbertson, J.D. Kang, Gene therapy applications for intervertebral disc degeneration, Spine (Phila Pa 1976) 28 (2003) S93–S98. K. Nishida, L.G. Gilbertson, P.D. Robbins, C.H. Evans, J.D. Kang, Potential applications of gene therapy to the treatment of intervertebral disc disorders, Clin. Orthop. Relat. Res. (2000) S234–S241. S.H. Moon, L.G. Gilbertson, K. Nishida, M. Knaub, T. Muzzonigro, P.D. Robbins, C.H. Evans, J.D. Kang, Human intervertebral disc cells are genetically modifiable by U. Nöth et al. / Advanced Drug Delivery Reviews 62 (2010) 765–783 [271] [272] [273] [274] [275] [276] [277] [278] [279] [280] [281] [282] [283] [284] [285] [286] [287] [288] [289] [290] [291] adenovirus-mediated gene transfer: implications for the clinical management of intervertebral disc disorders, Spine (Phila Pa 1976) 25 (2000) 2573–2579. K. Nishida, J.D. Kang, J.K. Suh, P.D. Robbins, C.H. Evans, L.G. Gilbertson, Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration, Spine (Phila Pa 1976) 23 (1998) 2437–2442 discussion 2443. S. Sobajima, J.S. Kim, L.G. Gilbertson, J.D. Kang, Gene therapy for degenerative disc disease, Gene Ther. 11 (2004) 390–401. S.T. Yoon, The potential of gene therapy for the treatment of disc degeneration, Orthop. Clin. North Am. 35 (2004) 95–100. Y. Zhang, D.G. Anderson, F.M. Phillips, E.J. Thonar, T.C. He, D. Pietryla, H.S. An, Comparative effects of bone morphogenetic proteins and Sox9 overexpression on matrix accumulation by bovine anulus fibrosus cells: implications for anular repair, Spine (Phila Pa 1976) 32 (2007) 2515–2520. R. Paul, R.C. Haydon, H. Cheng, A. Ishikawa, N. Nenadovich, W. Jiang, L. Zhou, B. Breyer, T. Feng, P. Gupta, et al., Potential use of Sox9 gene therapy for intervertebral degenerative disc disease, Spine (Phila Pa 1976) 28 (2003) 755–763. H. Gu, G. Lv, L. Liu, Adenovirus-mediated human bone morphogenetic protein 2 gene transferred to rabbit intervertebral disc cells in vitro, Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 20 (2006) 820–824. Y. Zhang, Z. Li, E.J. Thonar, H.S. An, T.C. He, D. Pietryla, F.M. Phillips, Transduced bovine articular chondrocytes affect the metabolism of cocultured nucleus pulposus cells in vitro: implications for chondrocyte transplantation into the intervertebral disc, Spine (Phila Pa 1976) 30 (2005) 2601–2607. H. Wang, M. Kroeber, M. Hanke, R. Ries, C. Schmid, W. Poller, W. Richter, Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells, J. Mol. Med. 82 (2004) 126–134. K. Nishida, J.D. Kang, L.G. Gilbertson, S.H. Moon, J.K. Suh, M.T. Vogt, P.D. Robbins, C.H. Evans, Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene, Spine (Phila Pa 1976) 24 (1999) 2419–2425. C.J. Wallach, S. Sobajima, Y. Watanabe, J.S. Kim, H.I. Georgescu, P. Robbins, L.G. Gilbertson, J.D. Kang, Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs, Spine (Phila Pa 1976) 28 (2003) 2331–2337. S.H. Moon, K. Nishida, L.G. Gilbertson, H.M. Lee, H. Kim, R.A. Hall, P.D. Robbins, J.D. Kang, Biologic response of human intervertebral disc cells to gene therapy cocktail, Spine (Phila Pa 1976) 33 (2008) 1850–1855. C.J. Wallach, J.S. Kim, S. Sobajima, C. Lattermann, W.M. Oxner, K. McFadden, P.D. Robbins, L.G. Gilbertson, J.D. Kang, Safety assessment of intradiscal gene transfer: a pilot study, Spine J. 6 (2006) 107–112. E.A. Levicoff, J.S. Kim, S. Sobajima, C.J. Wallach, J.W. Larson III, P.D. Robbins, X. Xiao, L. Juan, G. Vadala, L.G. Gilbertson, et al., Safety assessment of intradiscal gene therapy II: effect of dosing and vector choice, Spine (Phila Pa 1976) 33 (2008) 1509–1511 discussion 1517. S.D. Boden, J.H. Schimandle, Biologic enhancement of spinal fusion, Spine (Phila Pa 1976) 20 (1995) 113S–123S. R.C. Sasso, J.C. LeHuec, C. Shaffrey, Iliac crest bone graft donor site pain after anterior lumbar interbody fusion: a prospective patient satisfaction outcome assessment, J. Spinal Disord. Tech. 18 (2005) S77–S81 Suppl. P.D. Sawin, V.C. Traynelis, A.H. Menezes, A comparative analysis of fusion rates and donor-site morbidity for autogeneic rib and iliac crest bone grafts in posterior cervical fusions, J. Neurosurg. 88 (1998) 255–265. B. Kwon, L.G. Jenis, Carrier materials for spinal fusion, Spine J. 5 (2005) 224S–230S. O.N. Gottfried, A.T. Dailey, Mesenchymal stem cell and gene therapies for spinal fusion, Neurosurgery 63 (2008) 380–391 discussion 391-2. M.C. Gupta, T. Theerajunyaporn, S. Maitra, M.B. Schmidt, C.E. Holy, S. Kadiyala, S.P. Bruder, Efficacy of mesenchymal stem cell enriched grafts in an ovine posterolateral lumbar spine model, Spine (Phila Pa 1976) 32 (2007) 720–726 discussion 727. T. Nakajima, H. Iizuka, S. Tsutsumi, M. Kayakabe, K. Takagishi, Evaluation of posterolateral spinal fusion using mesenchymal stem cells: differences with or without osteogenic differentiation, Spine (Phila Pa 1976) 32 (2007) 2432–2436. A. Minamide, M. Yoshida, M. Kawakami, S. Yamasaki, H. Kojima, H. Hashizume, S.D. Boden, The use of cultured bone marrow cells in type I collagen gel and porous hydroxyapatite for posterolateral lumbar spine fusion, Spine (Phila Pa 1976) 30 (2005) 1134–1138. 783 [292] E. Carlisle, J.S. Fischgrund, Bone morphogenetic proteins for spinal fusion, Spine J. 5 (2005) 240S–249S. [293] J.R. Dimar, S.D. Glassman, K.J. Burkus, L.Y. Carreon, Clinical outcomes and fusion success at 2 years of single-level instrumented posterolateral fusions with recombinant human bone morphogenetic protein-2/compression resistant matrix versus iliac crest bone graft, Spine (Phila Pa 1976) 31 (2006) 2534–2539 discussion 2540. [294] D.A. Wong, A. Kumar, S. Jatana, G. Ghiselli, K. Wong, Neurologic impairment from ectopic bone in the lumbar canal: a potential complication of off-label PLIF/TLIF use of bone morphogenetic protein-2 (BMP-2), Spine J. 8 (2008) 1011–1018. [295] L.B. Shields, G.H. Raque, S.D. Glassman, M. Campbell, T. Vitaz, J. Harpring, C.B. Shields, Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion, Spine (Phila Pa 1976) 31 (2006) 542–547. [296] M. Viggeswarapu, S.D. Boden, Y. Liu, G.A. Hair, J. Louis-Ugbo, H. Murakami, H.S. Kim, M.T. Mayr, W.C. Hutton, L. Titus, Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo, J. Bone Joint Surg. Am. 83-A (2001) 364–376. [297] J.C. Wang, L.E. Kanim, S. Yoo, P.A. Campbell, A.J. Berk, J.R. Lieberman, Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats, J. Bone Joint Surg. Am. 85-A (2003) 905–911. [298] W. Zhu, B.A. Rawlins, O. Boachie-Adjei, E.R. Myers, J. Arimizu, E. Choi, J.R. Lieberman, R.G. Crystal, C. Hidaka, Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model, J. Bone Miner. Res. 19 (2004) 2021–2032. [299] K.D. Riew, J. Lou, N.M. Wright, S.L. Cheng, K.T. Bae, L.V. Avioli, Thoracoscopic intradiscal spine fusion using a minimally invasive gene-therapy technique, J. Bone Joint Surg. Am. 85-A (2003) 866–871. [300] R.J. Dumont, H. Dayoub, J.Z. Li, A.S. Dumont, D.F. Kallmes, G.R. Hankins, G.A. Helm, Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents, Neurosurgery 51 (2002) 1239–1244 discussion 1244–5. [301] L. Peterson, M. Brittberg, I. Kiviranta, E.L. Akerlund, A. Lindahl, Autologous chondrocyte transplantation. Biomechanics and long-term durability, Am. J. Sports Med. 30 (2002) 2–12. [302] O. Haddo, S. Mahroof, D. Higgs, L. David, J. Pringle, M. Bayliss, S.R. Cannon, T.W. Briggs, The use of chondrogide membrane in autologous chondrocyte implantation, Knee 11 (2004) 51–55. [303] W. Bartlett, J.A. Skinner, C.R. Gooding, R.W. Carrington, A.M. Flanagan, T.W. Briggs, G. Bentley, Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study, J. Bone Joint Surg. Br. 87 (2005) 640–645. [304] M. Steinwachs, P.C. Kreuz, Autologous chondrocyte implantation in chondral defects of the knee with a type I/III collagen membrane: a prospective study with a 3-year follow-up, Arthroscopy 23 (2007) 381–387. [305] S. Andereya, U. Maus, K. Gavenis, R. Muller-Rath, O. Miltner, T. Mumme, U. Schneider, First clinical experiences with a novel 3D-collagen gel (CaReS) for the treatment of focal cartilage defects in the knee, Z. Orthop. Ihre Grenzgeb. 144 (2006) 272–280. [306] U. Maus, U. Schneider, S. Gravius, R. Muller-Rath, T. Mumme, O. Miltner, D. Bauer, C. Niedhart, S. Andereya, Clinical results after three years use of matrixassociated ACT for the treatment of osteochondral defects of the knee, Z. Orthop. Unfall. 146 (2008) 31–37. [307] M. Ochi, Y. Uchio, K. Kawasaki, S. Wakitani, J. Iwasa, Transplantation of cartilagelike tissue made by tissue engineering in the treatment of cartilage defects of the knee, J. Bone Joint Surg. Br. 84 (2002) 571–578. [308] A. Pavesio, G. Abatangelo, A. Borrione, D. Brocchetta, A.P. Hollander, E. Kon, F. Torasso, S. Zanasi, M. Marcacci, Hyaluronan-based scaffolds (Hyalograft C) in the treatment of knee cartilage defects: preliminary clinical findings, Novartis Found. Symp. 249 (2003) 203–217 discussion 229–33, 234–8, 239–41. [309] M. Marcacci, M. Berruto, D. Brocchetta, A. Delcogliano, D. Ghinelli, A. Gobbi, E. Kon, L. Pederzini, D. Rosa, G.L. Sacchetti, et al., Articular cartilage engineering with Hyalograft C: 3-year clinical results, Clin. Orthop. Relat. Res. (2005) 96–105. [310] C. Erggelet, M. Sittinger, A. Lahm, The arthroscopic implantation of autologous chondrocytes for the treatment of full-thickness cartilage defects of the knee joint, Arthroscopy 19 (2003) 108–110. [311] J. Gille, E. Schuseil, J. Wimmer, J. Gellissen, A.P. Schulz, P. Behrens, Mid-term results of Autologous Matrix-Induced Chondrogenesis for treatment of focal cartilage defects in the knee, Knee Surg. Sports Traumatol. Arthrosc. (2010) (Electronic publication ahead of print).