Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative me... more Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecancoated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-b1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-b1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.
Injuries to articular cartilage and menisci can lead to cartilage degeneration that ultimately re... more Injuries to articular cartilage and menisci can lead to cartilage degeneration that ultimately results in arthritis. Different forms of arthritis affect ~50 million people in the USA alone, and it is therefore crucial to identify methods that will halt or slow the progression to arthritis, starting with the initiating events of cartilage and meniscus defects. The surgical approaches in current use have a limited capacity for tissue regeneration and yield only short-term relief of symptoms. Tissue engineering approaches are emerging as alternatives to current surgical methods for cartilage and meniscus repair. Several cell-based and tissue-engineered products are currently in clinical trials for cartilage lesions and meniscal tears, opening new avenues for cartilage and meniscus regeneration. This Review provides a summary of surgical techniques, including tissue-engineered products, that are currently in clinical use, as well as a discussion of state-of-the-art tissue engineering strategies and technologies that are being developed for use in articular cartilage and meniscus repair and regeneration. The obstacles to clinical translation of these strategies are also included to inform the development of innovative tissue engineering approaches.
This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musc... more This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages-for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc-are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.
Journal of Tissue Engineering and Regenerative Medicine, 2014
Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not... more Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into coculture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.
Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been ... more Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been well described, its cellular and mechanical effects on native and engineered articular cartilage remain unclear. For the repair of articular cartilage defects, fresh autologous and allogenic cartilage grafts are commonly used, and engineered cell-based therapies are emerging. The outcome of grafting therapies aimed at repairing damaged cartilage relies largely on maintaining proper viability and mechanical suitability of the donor tissues. Purpose: To investigate the in vitro effects of single bupivacaine exposure on the viability and mechanics of 2 cartilage graft types: native articular cartilage and engineered neocartilage. Study Design: Controlled laboratory study. Methods: Articular cartilage explants were harvested from the bovine stifle femoral condyles, and neocartilage constructs were engineered from bovine stifle chondrocytes using the self-assembling process, a scaffold-free a...
Engineering extracellular matrices that utilize the body's natural healing capacity enable the pr... more Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob-protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants (K D) obtained using surface plasmon resonance indicate that these fusions have μM binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob-pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.
Background: Every human being carries with them a collection of microbes, a collection that is li... more Background: Every human being carries with them a collection of microbes, a collection that is likely both unique to that person, but also dynamic as a result of significant flux with the surrounding environment. The interaction of the human microbiome (i.e., the microbes that are found directly in contact with a person in places such as the gut, mouth, and skin) and the microbiome of accessory objects (e.g., shoes, clothing, phones, jewelry) is of potential interest to both epidemiology and the developing field of microbial forensics. Therefore, the microbiome of personal accessories are of interest because they serve as both a microbial source and sink for an individual, they may provide information about the microbial exposure experienced by an individual, and they can be sampled non-invasively. Findings: We report here a large-scale study of the microbiota found on cell phones and shoes. Cell phones serve as a potential source and sink for skin and oral microbiota, while shoes c...
Nasal cartilage pathologies are common; for example, deviated nasal septum conditions afflict up ... more Nasal cartilage pathologies are common; for example, deviated nasal septum conditions afflict up to 80% of people. Because cartilage provides the supportive framework of the nose, afflicted patients suffer low quality of life. To correct pathologies, graft cartilage is often required. Grafts are currently sourced from the patient's septum, ear, or rib. However, their use yields donor site morbidity and is limited by tissue quantity and quality. Additionally, rhinoplasty revision rates exceed 15%, exacerbating the shortage of graft cartilage. Alternative grafts, such as irradiated allogeneic rib cartilage, are associated with complications. Tissue-engineered neocartilage holds promise to address the limitations of current grafts. The engineering design process may be used to create suitable graft tissues. This process begins by identifying the surgeon's needs. Second, nasal cartilages properties must be understood to define engineering design criteria. Limited investigations have examined nasal cartilage properties; numerous additional studies need to be performed to examine topographical variations, for example. Third, tissue-engineering processes must be applied to achieve the engineering design criteria. Within the recent past, strategies have frequently utilized human septal chondrocytes. As autologous and allogeneic rib graft cartilage is used, its suitability as a cell source should also be examined. Fourth, quantitative verification of engineered neocartilage is critical to check for successful achievement of the engineering design criteria. Finally, following the FDA paradigm, engineered neocartilage must be orthotopically validated in animals. Together, these steps delineate a path to engineer functional nasal neocartilages that may, ultimately, be used to treat human patients.
It is crucial that the properties of repair cartilages, including engineered neocartilage, match ... more It is crucial that the properties of repair cartilages, including engineered neocartilage, match surrounding healthy cartilage to promote the functional restoration of a cartilage injury. To accurately assess the quality of neocartilage, it's properties must be evaluated against healthy native cartilage. Fetal ovine cartilage has emerged as a promising and translationally relevant cell source with which to engineer neocartilage, yet, it is largely non-characterized. The influence of biomechanics during articular cartilage development, as well as their potential impact on structure-function relationships in utero in motivates additional study of fetal cartilage. Toward providing cartilage tissue engineering design criteria and elucidating fetal cartilage structure-function relationships, 11 locations across four regions of the fetal ovine stifle were characterized. Locational and regional differences were found to exist. Although differences in GAG content were observed, compressive stiffness did not vary or correlate with any biochemical component. Tensile stiffness and strength of the patella were significantly greater than those of the medial condyle. Tensile modulus and UTS significantly correlated with pyridinoline content. More advanced zonal organization, intense collagen II staining, and greater collagen and pyridinoline contents in the trochlear groove and patella suggest that these regions exhibit a more advanced maturational state than others. Regional differences in functional properties and their correlations suggest that structure-function relationships emerge in utero. These data address the dearth of information of the fetal ovine stifle, may serve as a repository of information for cartilage engineering strategies, and may help elucidate functional adaptation in fetal articular cartilage. Dear Editor, We are excited to present the research article entitled "Structure-function Relationships of Fetal Ovine Articular Cartilage." Fetal ovine chondrocytes have emerged as a promising and translationally relevant cell source with which to engineer neocartilage. However, fetal ovine articular cartilage is largely noncharacterized. The role of biomechanical stimuli in articular cartilage development and its influence on structure-function relationships further motivates study of fetal cartilage. In light of this, we comprehensively characterized 11 locations across four regions of the fetal ovine stifle with the goal of providing a benchmark for cartilage engineering efforts and to elucidate structure-function relationships in fetal articular cartilage. Importantly, locational and regional differences in fetal ovine cartilage were found to exist. The data presented in this manuscript suggest that endochondral ossification drives functional adaptation in articular cartilage and that functional adaptation begins in utero, much earlier than previously thought. These data may clarify the order of development of cartilage functional properties. We anticipate that this study will be of broad interest to the Acta Biomaterialia's readership.
Modern advances in sequencing technology have enabled the census of microbial members of many nat... more Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the "buildings" in which we travel through space will take on increasing importance, as plans for human exploration continue, with the possibility of colonization of other planets and moons. Sterile swabs were used to sample 15 sur...
Bone-to-bone integration can be obtained by osteoconductive ceramics such as hydroxyapatite (HAp)... more Bone-to-bone integration can be obtained by osteoconductive ceramics such as hydroxyapatite (HAp) and beta-tricalcium phosphate (β-TCP), but cartilage-to-cartilage integration is notoriously difficult. Many cartilage repair therapies, including microfracture and mosaicplasty, capitalize on the reparative aspects of subchondral bone due to its resident population of stem cells and vascularity. A strategy of incorporating tissue engineered neocartilage into a ceramic to form an osteochondral construct may serve as a suitable alternative to achieve cartilage graft fixation. The use of a tissue engineered osteochondral construct to repair cartilage defects may also benefit from the ceramic's proximity to underlying bone and abundant supply of progenitor cells and nutrients. The objective of the first study was to compare HAp and β-TCP ceramics, two widely used ceramics in bone regeneration, in terms of their ability to influence neocartilage interdigitation at an engineered osteocho...
Although numerous cartilage engineering methods have been described, few report generation of con... more Although numerous cartilage engineering methods have been described, few report generation of constructs greater than 4 cm2, which is the typical lesion size considered for cell-based therapies. Furthermore, current cell-based therapies only target focal lesions, while treatment of large non-isolated lesions remains an area of great demand. The objective of this study was to scale-up fabrication of self-assembled neocartilage from standard sizes of 0.2 cm2 to greater than 8 cm2. Passaged sheep articular chondrocytes were self-assembled into 5 mm or 25 mm dia. scaffoldless neocartilage constructs. The 25 mm dia. constructs grew up to 9.3 cm2 (areal scale-up of 23) and possessed properties similar to those of the 5 mm dia. constructs; unfortunately, these large constructs were deformed and are unusable as a potential implant. A novel neocartilage fabrication strategy - employing mechanical confinement, a minute deadweight, and chemical stimulation (cytochalasin D, TGF-β1, chondroitina...
Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue e... more Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were obs...
Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell ty... more Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell types for tissue engineering applications. However, their use in tissue engineering may be hindered via contamination by undesirable cell types. These include blood-associated cells, as well as unwanted resident cell types, found both in healthy and pathologic donor tissues. Ammonium-chloride-potassium lysing buffer (ACK buffer) is used to lyse red blood cells during the isolation of stem cell populations, but has not been explored for the purification of fully differentiated cells. This study sought to investigate the effect of ACK buffer treatment of freshly isolated, fully differentiated cells to increase cell purity and enhance the formation of biofunctional, engineered neotissues; this was tested in the well-established cartilage tissue engineering model of the self-assembling process using fetal ovine articular chondrocytes (foACs) and juvenile bovine articular chondrocytes (jbACs). ...
Background.While significant attention has been paid to the potential risk of pathogenic microbes... more Background.While significant attention has been paid to the potential risk of pathogenic microbes aboard crewed spacecraft, the non-pathogenic microbes in these habitats have received less consideration. Preliminary work has demonstrated that the interior of the International Space Station (ISS) has a microbial community resembling those of built environments on Earth. Here we report the results of sending 48 bacterial strains, collected from built environments on Earth, for a growth experiment on the ISS. This project was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS).Results.Of the 48 strains sent to the ISS, 45 of them showed similar growth in space and on Earth using a relative growth measurement adapted for microgravity. The vast majority of species tested in this experiment have also been found in culture-independent surveys of the ISS. Only one bacterial strain showed significantly different growth in space.Bac...
As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Li... more As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. This article provides a review of important steps toward regeneration of articular cartilage with suitable biomechanical properties. As a first step, biomechanical and biochemical characterization studies at the tissue level were used to provide design criteria for engineering neotissues. Extending this work to the single cell and subcellular levels has helped to develop biochemical and mechanical stimuli for tissue engineering studies. This strong mechanobiological foundation guided studies on regenerating hyaline arti...
Journal of tissue engineering and regenerative medicine, Jan 29, 2017
Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articu... more Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articular cartilage tissue engineering. Limited knowledge exists, however, on costal cartilage characteristics. This study describes the characterization of costal cartilage and articular cartilage properties and compares neocartilage engineered with costal chondrocytes to native articular cartilage, all within a sheep model. Specifically, we 1) quantitatively characterized the properties of costal cartilage in comparison to patellofemoral articular cartilage, and 2) evaluated the quality of neocartilage derived from costal chondrocytes for potential use in articular cartilage regeneration. Ovine costal and articular cartilages from various topographical locations were characterized mechanically, biochemically, and histologically. Costal cartilage was stiffer in compression but softer and weaker in tension than articular cartilage. These differences were attributed to high amounts of glycosami...
Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic ... more Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O 2) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O 2) or normoxic (20% O 2) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p,0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p,0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.
Engineering extracellular matrices that utilize the body's natural healing capacity enable the pr... more Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob-protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants (K D ) obtained using surface plasmon resonance indicate that these fusions have μM binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob-pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.
This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musc... more This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages-for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc-are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.
Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative me... more Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecancoated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-b1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-b1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.
Injuries to articular cartilage and menisci can lead to cartilage degeneration that ultimately re... more Injuries to articular cartilage and menisci can lead to cartilage degeneration that ultimately results in arthritis. Different forms of arthritis affect ~50 million people in the USA alone, and it is therefore crucial to identify methods that will halt or slow the progression to arthritis, starting with the initiating events of cartilage and meniscus defects. The surgical approaches in current use have a limited capacity for tissue regeneration and yield only short-term relief of symptoms. Tissue engineering approaches are emerging as alternatives to current surgical methods for cartilage and meniscus repair. Several cell-based and tissue-engineered products are currently in clinical trials for cartilage lesions and meniscal tears, opening new avenues for cartilage and meniscus regeneration. This Review provides a summary of surgical techniques, including tissue-engineered products, that are currently in clinical use, as well as a discussion of state-of-the-art tissue engineering strategies and technologies that are being developed for use in articular cartilage and meniscus repair and regeneration. The obstacles to clinical translation of these strategies are also included to inform the development of innovative tissue engineering approaches.
This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musc... more This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages-for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc-are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.
Journal of Tissue Engineering and Regenerative Medicine, 2014
Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not... more Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into coculture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.
Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been ... more Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been well described, its cellular and mechanical effects on native and engineered articular cartilage remain unclear. For the repair of articular cartilage defects, fresh autologous and allogenic cartilage grafts are commonly used, and engineered cell-based therapies are emerging. The outcome of grafting therapies aimed at repairing damaged cartilage relies largely on maintaining proper viability and mechanical suitability of the donor tissues. Purpose: To investigate the in vitro effects of single bupivacaine exposure on the viability and mechanics of 2 cartilage graft types: native articular cartilage and engineered neocartilage. Study Design: Controlled laboratory study. Methods: Articular cartilage explants were harvested from the bovine stifle femoral condyles, and neocartilage constructs were engineered from bovine stifle chondrocytes using the self-assembling process, a scaffold-free a...
Engineering extracellular matrices that utilize the body's natural healing capacity enable the pr... more Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob-protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants (K D) obtained using surface plasmon resonance indicate that these fusions have μM binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob-pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.
Background: Every human being carries with them a collection of microbes, a collection that is li... more Background: Every human being carries with them a collection of microbes, a collection that is likely both unique to that person, but also dynamic as a result of significant flux with the surrounding environment. The interaction of the human microbiome (i.e., the microbes that are found directly in contact with a person in places such as the gut, mouth, and skin) and the microbiome of accessory objects (e.g., shoes, clothing, phones, jewelry) is of potential interest to both epidemiology and the developing field of microbial forensics. Therefore, the microbiome of personal accessories are of interest because they serve as both a microbial source and sink for an individual, they may provide information about the microbial exposure experienced by an individual, and they can be sampled non-invasively. Findings: We report here a large-scale study of the microbiota found on cell phones and shoes. Cell phones serve as a potential source and sink for skin and oral microbiota, while shoes c...
Nasal cartilage pathologies are common; for example, deviated nasal septum conditions afflict up ... more Nasal cartilage pathologies are common; for example, deviated nasal septum conditions afflict up to 80% of people. Because cartilage provides the supportive framework of the nose, afflicted patients suffer low quality of life. To correct pathologies, graft cartilage is often required. Grafts are currently sourced from the patient's septum, ear, or rib. However, their use yields donor site morbidity and is limited by tissue quantity and quality. Additionally, rhinoplasty revision rates exceed 15%, exacerbating the shortage of graft cartilage. Alternative grafts, such as irradiated allogeneic rib cartilage, are associated with complications. Tissue-engineered neocartilage holds promise to address the limitations of current grafts. The engineering design process may be used to create suitable graft tissues. This process begins by identifying the surgeon's needs. Second, nasal cartilages properties must be understood to define engineering design criteria. Limited investigations have examined nasal cartilage properties; numerous additional studies need to be performed to examine topographical variations, for example. Third, tissue-engineering processes must be applied to achieve the engineering design criteria. Within the recent past, strategies have frequently utilized human septal chondrocytes. As autologous and allogeneic rib graft cartilage is used, its suitability as a cell source should also be examined. Fourth, quantitative verification of engineered neocartilage is critical to check for successful achievement of the engineering design criteria. Finally, following the FDA paradigm, engineered neocartilage must be orthotopically validated in animals. Together, these steps delineate a path to engineer functional nasal neocartilages that may, ultimately, be used to treat human patients.
It is crucial that the properties of repair cartilages, including engineered neocartilage, match ... more It is crucial that the properties of repair cartilages, including engineered neocartilage, match surrounding healthy cartilage to promote the functional restoration of a cartilage injury. To accurately assess the quality of neocartilage, it's properties must be evaluated against healthy native cartilage. Fetal ovine cartilage has emerged as a promising and translationally relevant cell source with which to engineer neocartilage, yet, it is largely non-characterized. The influence of biomechanics during articular cartilage development, as well as their potential impact on structure-function relationships in utero in motivates additional study of fetal cartilage. Toward providing cartilage tissue engineering design criteria and elucidating fetal cartilage structure-function relationships, 11 locations across four regions of the fetal ovine stifle were characterized. Locational and regional differences were found to exist. Although differences in GAG content were observed, compressive stiffness did not vary or correlate with any biochemical component. Tensile stiffness and strength of the patella were significantly greater than those of the medial condyle. Tensile modulus and UTS significantly correlated with pyridinoline content. More advanced zonal organization, intense collagen II staining, and greater collagen and pyridinoline contents in the trochlear groove and patella suggest that these regions exhibit a more advanced maturational state than others. Regional differences in functional properties and their correlations suggest that structure-function relationships emerge in utero. These data address the dearth of information of the fetal ovine stifle, may serve as a repository of information for cartilage engineering strategies, and may help elucidate functional adaptation in fetal articular cartilage. Dear Editor, We are excited to present the research article entitled "Structure-function Relationships of Fetal Ovine Articular Cartilage." Fetal ovine chondrocytes have emerged as a promising and translationally relevant cell source with which to engineer neocartilage. However, fetal ovine articular cartilage is largely noncharacterized. The role of biomechanical stimuli in articular cartilage development and its influence on structure-function relationships further motivates study of fetal cartilage. In light of this, we comprehensively characterized 11 locations across four regions of the fetal ovine stifle with the goal of providing a benchmark for cartilage engineering efforts and to elucidate structure-function relationships in fetal articular cartilage. Importantly, locational and regional differences in fetal ovine cartilage were found to exist. The data presented in this manuscript suggest that endochondral ossification drives functional adaptation in articular cartilage and that functional adaptation begins in utero, much earlier than previously thought. These data may clarify the order of development of cartilage functional properties. We anticipate that this study will be of broad interest to the Acta Biomaterialia's readership.
Modern advances in sequencing technology have enabled the census of microbial members of many nat... more Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the "buildings" in which we travel through space will take on increasing importance, as plans for human exploration continue, with the possibility of colonization of other planets and moons. Sterile swabs were used to sample 15 sur...
Bone-to-bone integration can be obtained by osteoconductive ceramics such as hydroxyapatite (HAp)... more Bone-to-bone integration can be obtained by osteoconductive ceramics such as hydroxyapatite (HAp) and beta-tricalcium phosphate (β-TCP), but cartilage-to-cartilage integration is notoriously difficult. Many cartilage repair therapies, including microfracture and mosaicplasty, capitalize on the reparative aspects of subchondral bone due to its resident population of stem cells and vascularity. A strategy of incorporating tissue engineered neocartilage into a ceramic to form an osteochondral construct may serve as a suitable alternative to achieve cartilage graft fixation. The use of a tissue engineered osteochondral construct to repair cartilage defects may also benefit from the ceramic's proximity to underlying bone and abundant supply of progenitor cells and nutrients. The objective of the first study was to compare HAp and β-TCP ceramics, two widely used ceramics in bone regeneration, in terms of their ability to influence neocartilage interdigitation at an engineered osteocho...
Although numerous cartilage engineering methods have been described, few report generation of con... more Although numerous cartilage engineering methods have been described, few report generation of constructs greater than 4 cm2, which is the typical lesion size considered for cell-based therapies. Furthermore, current cell-based therapies only target focal lesions, while treatment of large non-isolated lesions remains an area of great demand. The objective of this study was to scale-up fabrication of self-assembled neocartilage from standard sizes of 0.2 cm2 to greater than 8 cm2. Passaged sheep articular chondrocytes were self-assembled into 5 mm or 25 mm dia. scaffoldless neocartilage constructs. The 25 mm dia. constructs grew up to 9.3 cm2 (areal scale-up of 23) and possessed properties similar to those of the 5 mm dia. constructs; unfortunately, these large constructs were deformed and are unusable as a potential implant. A novel neocartilage fabrication strategy - employing mechanical confinement, a minute deadweight, and chemical stimulation (cytochalasin D, TGF-β1, chondroitina...
Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue e... more Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were obs...
Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell ty... more Juvenile and fetal, primary, fully differentiated cells are widely considered to be ideal cell types for tissue engineering applications. However, their use in tissue engineering may be hindered via contamination by undesirable cell types. These include blood-associated cells, as well as unwanted resident cell types, found both in healthy and pathologic donor tissues. Ammonium-chloride-potassium lysing buffer (ACK buffer) is used to lyse red blood cells during the isolation of stem cell populations, but has not been explored for the purification of fully differentiated cells. This study sought to investigate the effect of ACK buffer treatment of freshly isolated, fully differentiated cells to increase cell purity and enhance the formation of biofunctional, engineered neotissues; this was tested in the well-established cartilage tissue engineering model of the self-assembling process using fetal ovine articular chondrocytes (foACs) and juvenile bovine articular chondrocytes (jbACs). ...
Background.While significant attention has been paid to the potential risk of pathogenic microbes... more Background.While significant attention has been paid to the potential risk of pathogenic microbes aboard crewed spacecraft, the non-pathogenic microbes in these habitats have received less consideration. Preliminary work has demonstrated that the interior of the International Space Station (ISS) has a microbial community resembling those of built environments on Earth. Here we report the results of sending 48 bacterial strains, collected from built environments on Earth, for a growth experiment on the ISS. This project was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS).Results.Of the 48 strains sent to the ISS, 45 of them showed similar growth in space and on Earth using a relative growth measurement adapted for microgravity. The vast majority of species tested in this experiment have also been found in culture-independent surveys of the ISS. Only one bacterial strain showed significantly different growth in space.Bac...
As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Li... more As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. This article provides a review of important steps toward regeneration of articular cartilage with suitable biomechanical properties. As a first step, biomechanical and biochemical characterization studies at the tissue level were used to provide design criteria for engineering neotissues. Extending this work to the single cell and subcellular levels has helped to develop biochemical and mechanical stimuli for tissue engineering studies. This strong mechanobiological foundation guided studies on regenerating hyaline arti...
Journal of tissue engineering and regenerative medicine, Jan 29, 2017
Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articu... more Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articular cartilage tissue engineering. Limited knowledge exists, however, on costal cartilage characteristics. This study describes the characterization of costal cartilage and articular cartilage properties and compares neocartilage engineered with costal chondrocytes to native articular cartilage, all within a sheep model. Specifically, we 1) quantitatively characterized the properties of costal cartilage in comparison to patellofemoral articular cartilage, and 2) evaluated the quality of neocartilage derived from costal chondrocytes for potential use in articular cartilage regeneration. Ovine costal and articular cartilages from various topographical locations were characterized mechanically, biochemically, and histologically. Costal cartilage was stiffer in compression but softer and weaker in tension than articular cartilage. These differences were attributed to high amounts of glycosami...
Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic ... more Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O 2) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O 2) or normoxic (20% O 2) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p,0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p,0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.
Engineering extracellular matrices that utilize the body's natural healing capacity enable the pr... more Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob-protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants (K D ) obtained using surface plasmon resonance indicate that these fusions have μM binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob-pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.
This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musc... more This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages-for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc-are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.
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Papers by Wendy Brown