Tissue Eng Regen Med Online ISSN 2212-5469

https://doi.org/10.1007/s13770-018-0135-9 Print ISSN 1738-2696

REVIEW ARTICLE

Collagen Scaffolds in Cartilage Tissue Engineering and Relevant

Approaches for Future Development
Vincent Irawan1 • Tzu-Cheng Sung2 • Akon Higuchi2 • Toshiyuki Ikoma1

Received: 17 April 2018 / Revised: 30 May 2018 / Accepted: 15 June 2018

Ó The Korean Tissue Engineering and Regenerative Medicine Society and Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract
BACKGROUND: Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through
the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of ‘matured’ constructs
is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as
scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval.
However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity
and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in
the CTE applications.
METHODS: Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and
mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds
crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by
blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are
combined in non-blending manner is detailed.
RESULTS: Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited
chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II
collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are
shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs.
Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites.
However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition.
Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural
polymer.
CONCLUSION: Considering the previous improvements of physical and compositional properties of collagen scaffolds
and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach
to advance further collagen-based scaffolds in CTE.

Keywords Cartilage tissue engineering
Type I collagen
Articular chondrocytes
Mesenchymal stem cells
Hybrid
scaffolds

& Toshiyuki Ikoma 2

Department of Chemical and Materials Engineering, National
[email protected] Central University, No. 300 Jung Da Rd., Chung-Li,
Taoyuan 320, Taiwan
1
Department of Materials Science and Engineering, Tokyo
Institute of Technology, 2 Chome-12-1, Meguro-ku,
Tokyo 152-8550, Japan

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1 Introduction to articular cartilage tissue relatively good initial clinical results, several complica-
engineering tions are associated with these treatments, such as donor
site morbidity, graft hypertrophy, and inconsistent repair
Articular cartilage is an avascular tissue mainly function to tissue [5, 6].
allow frictionless movement between articulated bones. To overcome these limitations, a concept of cartilage
Due to the mechanical loads, extracellular matrices tissue engineering (CTE) is introduced (Fig. 1C) [6]. CTE
(ECMs) of articular cartilage are highly organized and was defined as the attempt to reconstitute damaged carti-
maintained in homeostatic state by the residing articular lages by combining relevant cells, three-dimensional (3-D)
chondrocytes (ACs) (Fig. 1A, B). However, the avascular scaffolds and extraneous signals in a harmonious manner
nature of articular cartilage leads to the poor self-healing [3, 6]. Before implantation, CTE constructs can be matured
capability [1, 2]. Healing process is also impaired as pro- partially or fully in vitro to achieve similar level of bio-
genitor cells gain no access to the injury site [3]. Host ACs physical and biochemical properties with native cartilage
near lesions are burdened with simultaneous tasks to tissue. The obtained CTE products are expected to elimi-
replace the dead cells and rebuild the secreted matrices at nate the necessity of using whole-tissue transplant [3, 7].
the same time [4]. Spontaneous healing is generally There are two relevant cell types in regards to CTE: ACs
absence for chondral lesions beyond critical size (C 3 cm2) and mesenchymal stem cells (MSCs), each associated with
and thus surgical interventions are necessary [5]. potencies and challenges. ACs are the preferable option for
Several medical interventions are available for treating treating chondral defects because their molecular profile is
large chondral lesions: microfracture (MF), osteochondral similar with surrounding host cells [8]. As the adult cells,
transplant (OCT), and autologous chondrocytes implanta- ACs are also actively secreting cartilage-related ECMs.
tion (ACI) [3, 5, 6]. MF relies on the innate healing Prevalent usage of ACs suffers from their scarce avail-
capability of patients to repair the injury. In this procedure, ability. The cell number of isolated AC per biopsy of
hole is drilled near lesions area to penetrate subchondral articular cartilage (5 mm 9 10 mm of full-thickness area)
bone and to allow influx of stem cells and/or growth factors is around 200,000–300,000 cells [8]. The numbers are
to the defect site. However, MF was inconsistent in clinical relatively small compared with the required cells per cm2
results and inappropriate for treating elder patients [5]. In of lesion (0.5–5 million cells/cm2) [6, 9]. Thus, it is com-
contrast, OCT and ACI essentially exploit healthy cartilage mon to expand cells for several passages before seeding
tissue or ACs for treating chondral lesions. Despite into 3-D scaffolds. Nonetheless, ACs gradually lose their

A
Femur

Articular
Cartilage
Lesion C
Biopsies ACs Expansion

Fibula
Implantation
Cell seeding
In vitro
maturation 3D Scaffolds

Fig. 1 Illustration of articular cartilage tissues and concept of indicating presence of proteoglycan. Inset shown AC (dotted circle)
cartilage tissue engineering. A Schematic representation of articular embedded in cartilage matrix. Scale bar: 20 lm. C Flow of cartilage
cartilage with a lesion in knee. B Articular cartilage consists of tissue engineering started with biopsy of cartilage tissue and ended
multiple zones and is separated with subchondral bone by calcified with implantation of mature scaffolds into lesions. (Parts of
zones. Some areas are stained with red Safranin O/Fast staining, figure B adapted with permission from Armiento et al. [6])

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chondrogenic characteristics with passage numbers (de-dif- advancing further type I collagen-based scaffolds, espe-
ferentiation), effectively limiting the cell number of feasible cially in the context of CTE. Firstly, the interaction of type
passages [10]. In contrast, MSCs serve as the more abundant I/II collagen with ACs and MSCs is discussed. Secondly,
alternative of ACs, owing to their numerous extraction sites physical features of scaffolds necessary to optimize chon-
(e.g., bone marrow [11], adipose tissues [12]). drogenic activities of ACs and MSCs are highlighted.
To be useful in CTE, it is corollary to differentiate Thirdly, studies aspiring to improve collagen matrices
MSCs in the chondrogenic lineage through culture in through blending with other materials are discussed.
inducing medium. Yet, MSCs apparently possessed variety Lastly, concept of hybrid scaffolds as the next promising
of chondrogenic capacity dependent on its extraction strategy for CTE is detailed.
sources [13]. MSCs might also undergo hypertrophic dif-
ferentiation that will be deemed detrimental for cartilage
regeneration. Important markers of ACs and MSCs corre- 2 Interaction of extracellular matrix and cells
sponding to CTE are listed in Table 1.
Type I collagen is the crucial material commonly used Unlike synthetic materials, ECMs-based scaffolds allow
as scaffolds for CTE products in clinical market [7]. direct attachment of cells, owing to the presence of unique
Preference of type I collagen in CTE is largely attributed to ligand (e.g. amino acid sequences) capable of binding the
its general biocompatibility and safety approvals granted specific cell receptor (Fig. 2). The activated cell receptor
by various agencies (e.g. Food and Drug Agency, Phar- triggers intracellular signaling pathway responsible for a
maceuticals and Medical Devices Agency). Short- and distinct cellular response (e.g., active proliferation, phenotype
mid-term outcomes of the collagen-based CTE generally maintenance) [47–49]. As tissue engineering mainly depends
showed improvement in clinical and histological outcomes on the particular cell activities (e.g. matrix deposition or
compared with the control group (MF) (Table 2). remodeling), prudent choice of ECMs substrates is essential to
Nonetheless, long-term performance of pure type I colla- draw out desirable cellular responses. The following sections
gen may be compromised by significant shrinkage [19], mainly focus on studies investigating interaction of type I/II
weak mechanical property [40], and limited chondrogenic collagen with AC/MSC and the relevant cellular responses in
capacity [23]. Furthermore, recent investigation revealed regards of cartilage tissue engineering.
the continuing de-differentiation of chondrocytes in colla-
gen-based CTE constructs even after implantation [41], 2.1 Interaction of articular chondrocytes (ACs)
suggesting the necessity for future development. with type I/II collagen
Considering the aforementioned works, type I collagen
serves as an essential platform for next generation of CTE Type I collagen is a fibril forming protein mainly dis-
scaffolds. This paper aims to give insights necessary for tributed as the major component in mammalian flesh and

Table 1 Important markers of ACs and MSCs in regards to the chondrogenic performance
Cells Markers

ACs Differentiated markers

1. Cellular rounded morphology [14, 15], high proteoglycan synthesize rate [14, 16]
2. Gene upregulation of Sox9 [17], Col2A1 [18], aggrecan (ACAN) [19], and cartilage oligomeric protein (COMP) [20]
3. Protein high secretion of type II collagen (Type II/I collagen ratio) [21, 22] and sulfated glycosaminoglycan (sGAG) [14, 23], high
expression of integrin a10b1 [24, 25], diffused actin organization [26, 27]
De-differentiated markers
1. Cellular flattened morphology [14, 15], high proliferation rate [18, 28]
2. Gene upregulation of Col1A2, Col2A1 [29]
3. Protein organized actin filament [26, 27]
MSCs Chondrogenic markers
1. Gene expression of Sox9, Col2A1, ACAN, and COMP [30–32]
2. Protein secretion of type II collagen (Type II/I collagen ratio) [33, 34] and sGAG [35, 36]
Hypertrophic markers
Transcription and synthesize of type X collagen [37, 38], confirmed with analysis of osteogenic marker (e.g., type I collagen, alkaline
phosphatase (ALP), mineral deposition) [34, 39]

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Table 2 Clinical trials of CTE products employing type I collagen scaffolds

Product name Markers Main results References

NOVOCARTÒ3D Type I collagen sponges (n = 28) Significant improvement of clinical results after 24 [42]
with bilayer structure months follow up compared to baseline, based on subjective
scoring system: IKDCa (36.0 ± 15.0 ) and Noyes sport ratinga
(38.9 ± 31.0) MOCARTb showed low score up to 6 months
(60.3 ± 17.4), but it exhibited development of cartilage repair
after 24 months (73.2 ± 12.4)
MACIÒ Membrane of type I/III (n = 144) MACIÒ showed more effective cartilage healing than [43]
collagenà microfracture (MF) based on KOOSa scoring system of pain
(MACIÒ: 82.5 ± 16.2 vs MF 70.9 ± 24.2) and function
(MACIÒ: 60.9 ± 27.8 vs MF: 48.7 ± 30.3)
CaReSÒ Type I collagen gelà (n = 17) All three scoring systems of clinical outcome showed [44]
significant improvement of CaReSÒ treated patient from
baseline IKDCa (31.7 ± 12.7 to 61.3 ± 18.2), Lysholma
(43.6 ± 11.4 to 64.7 ± 17.1), Cincinnatia (31.5 ± 13.1 to
56.2 ± 13.9)). No significant differences (IKDCa) were
observed between CaReSÒ (61.3 ± 18.2) and MF groups
(50.1 ± 24.9)
NeoCartÒ Type I collagen gel loaded (n = 49) Pain scoring system of KOOSa showed the significant [45]
into sponges of same improvement for NeoCartÒ-treated group compared to
materialà baseline at six, twelve, and twenty-four months. The
improvement from baseline is greater for NeoCartÒ-treated
group than MF-treated group
Transferred to Japan Tissue Type I atelocollagen gel (n = 28) Lysholm scoresa showed significant improvement from [46]
Engineering Co., Ltd. with brand the baseline over 25 months. Arthroscopic evaluation revealed
name JACCÒ 26 of 28 knees (93%) were graded as good or excellent.
Biomechanical test revealed that transplants had similar
stiffness with nearby cartilage
à
Controlled with MF
a
Clinical evaluation: KOOS, Knee injury and Osteoarthritis Outcome Score; IKDC, International Knee Documentation Committee; Lysholm;
Cincinnati; Noyes sport rating
b
Cartilage evaluation: MOCARTÒ, Modified magnetic resonance observation of cartilage repair tissue

ECMs-based Scaffolds
Seeded Cells
encoded by COL1A1 gene, while the latter chain is enco-
Ligand ded by COL1A2 gene [51]. Adhesion of type I collagen and
ECMs
ECMs ACs is mediated by integrin: strongly by a2b1 integrin and
weakly by a10b1 integrin [52, 53]. Type I collagen possess
Ligand
Receptor multiple binding sites, such as GFOGER and GROGER
Pores Cells Receptor
Cells ECMs (amino acid sequences with single letter amino acid
nomenclature), capable to be recognized by a2 I and a10 I
Cellular Responses Receptor Ligand domains of integrin [52, 53], respectively.
• Proliferation, differentiation
Type I collagen has benefit of promoting the cell pro-
• Phenotype maintenance Ligand-Receptor Complex
• ECM-deposition / -remodeling liferation of ACs without significantly compromising their
chondrogenic traits [17, 54]. Kino-Oka et al. [54] investi-
Fig. 2 Schematic illustration of the attachment of cells to ECMs- gated ACs on the different substrates [uncoated monolayer
based scaffolds. Cells attach to the ECMs molecules by formation of
ligand-receptor complex. Cells express various receptors capable of
polystyrene (PS) and type I collagen-coated PS substrates
recognizing various type of ligand presented by ECMs molecules. (CL)]. ACs cultured both on PS and CL groups showed
Activated cell receptor subsequently modify various responses of similar level of population doubling for a period of
cells (proliferation, ECMs deposition) 18 days. Yet, chondrocytes in CL group retained higher
fraction of rounded cells compared with majority of flat-
connective tissues (e.g., bone, skin, or scar tissue) [45, 50]. tened chondrocytes in the PS group. To assess the preser-
The collagen is a heterotrimeric molecule consisting of two vation of chondrogenic phenotype, passaged cells were
a1 chains and one a2 chain [51]. The former chain is further transferred into collagen gel (3-D environment).

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ACs of CL group significantly secreted glycosaminogly- regulating endochondral ossification [61]. Interaction of
cans (GAG) and type II collagen in contrast with ACs of PS DDR-2 and monomeric type II collagen apparently induce
group, demonstrating the capacity of type I collagen in ACs to upregulate the expression of cartilage-degrading
suppressing de-differentiation of chondrocytes. Nonethe- enzymes (e.g. matrix metallopeptidase (MMP-13, MMP-14)
less, type I collagen could not fully stabilize ACs for a long- and pro-inflammatory cytokines (e.g. interleukin (IL)-1b,
term culture. The loss of chondrogenic phenotype (altering IL-6), suggesting signaling role of type II collagen in phys-
of COL2A1 and COL1A2 expression) was found after iological cartilage turnover process [62, 63].
1000-fold expansion (* 35 to 50 days) in type I collagen Shakibaei et al. [15] showed that ACs quickly adopt
gels [18]. ACs only regained its capability on producing type fibroblastic morphology over 5 days of monolayer culture on
II collagen after application of re-differentiating factors the plastic substrate, whereas it retained round morphology
(insulin and bone morphogenic protein-2) [18]. for more than 2 weeks culture on type II collagen coated
The unnatural existence of type I collagen in articular substrate. Furthermore, type II collagen-based scaffolds
cartilage raises concern whether it could destabilize the apparently induced favorable chondrocyte phenotype in 3-D
differentiated phenotype of chondrocytes. It was described culture, on the basis of higher deposition of type II collagen,
that chondrocytes greatly destabilized when it was seeded GAG, larger fraction of spherical chondrocytes, and higher
in type I collagen gels of high concentration (1 g/ml) and expression of GAG/DNA ratio, compared with its type I
slightly destabilized in the gels at lower concentration collagen counterpart [13, 15, 27, 64]. Nevertheless, con-
(10 mg/ml) [55]. The negative impact of type I collagen flicting results were reported in which chondrocytes were
can be explained by extensive contraction of collagen gel indifferent when cultured on monolayer or 3-D scaffold of
at low concentration, which in turn would encourage the type I/II collagen [23, 65, 66]. Such inconsistencies could
condensation of chondrocytes [56]. It requires experi- originate from the experimental variabilities (interspecies
ments capable of decoupling the concentration and difference, culture procedure, etc.) [67] or it might indicate
shrinkage effects to elucidate whether type I collagen that complete interactions between pure type II collagen and
would be detrimental for differentiated phenotype of ACs requires additional factors [68]. It is interesting to note
chondrocytes. that previous studies demonstrating the superior chondro-
Type II collagen is a major component of articular genic performance of type II collagen were conducted by
cartilage. Helical part of the collagen is homotrimer con- adding chondroitin sulfate (CS) into the final matrices,
sisting of three a1(II) chains encoded by COL2A1 gene resulting in scaffolds with composition approaching native
[51]. Due to its native nature, type II collagen has been cartilage tissue [13, 22].
expected to provide signaling cues necessary for ACs to
retain its differentiated morphology and related secretion 2.2 Interaction of mesenchymal stem cells (MSCs)
activities. with type I/II collagen
ACs possess several receptors capable of recognizing
type II collagen: (1) integrin receptor (a10b1 and a2b1) and Either type I or II collagen can support the attachment,
(2) non-integrin receptor (human discoidin domain recep- proliferation and chondrogenic differentiation of MSCs
tor-2 (DDR-2) or annexin V) [22, 54, 55]. b1-integrins [33, 69], whereas head-to-head studies revealed that type II
mainly bind to the D-4 segment of type II collagen [57]. collagen matrices grant more extensive chondrogenic dif-
Removal of D-4 section significantly reduced attachment ferentiation of MSCs on the basis of higher secretion of
of ACs onto type II collagen and diminished their ability to type II collagen, proteoglycan and larger production ratio
migrate to the inner part of 3-D scaffolds [57]. Annexin-V of GAG/DNA compared with its type I counterpart
and DDR-2 largely adhered to N-propeptide [58] and D-2 [29, 33, 70]. Nevertheless, the chondrogenic stimulation of
segment of type II collagen [59], respectively. type II collagen for MSCs was absence at the gene level,
ACs may elicit different responses dependent on the type meaning no significant differences of chondrogenic RNA
of activated cellular receptors. The a10b1 is the newly dis- expression (Sox9, COMP, COL1, COL2, COL10) for a
covered integrin indicated to play important roles in proper period of 21 days in type I and II collagen gels [30]. Other
cartilage development [60]. In the knock-out study, mice studies confirmed the similar trend in which upregulation
lacking a10b1 integrin showed abnormal chondrocytes (in- of chondrogenic markers were prominent at protein level,
creased apoptosis, reduced proliferation, change of shapes) yet insignificant at gene level [33, 70]. The discrepancy
and imperfect cartilage tissue [60]. However, it is currently between gene and protein production is frequently occurred
unknown whether cell–matrix interaction involving a10b1 in in vitro [71] or some of the early upregulated genes, such
integrin is beneficial to stabilize phenotype of ACs. Binding as Sox9 and Runx2, are returned to the basal level at later
of annexin-V and type II collagen was shown to increase time, thus giving no difference at late stage analysis [34].
uptake of Ca2? by chondrocyte, suggesting its roles in Chondro-inductive properties of type II collagen would

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originate through b1 integrin-mediated Rho A/Rock sig- 3 Influence of physical features of collagen
naling, which in turn promote the morphological change of scaffolds to chondrogenic activities of ACs
MSCs to round shapes [16, 35, 45]. Type II collagen could and MSCs
promote the chondrogenesis by aggregation of MSCs;
however, denaturated type II collagen never showed such 3-D collagen scaffolds are fabricated in three forms, each
aggregation, suggesting the importance of maintaining possessing unique internal structure: (1) hydrogels, (2) porous
native helical structure of type II collagen [36]. sponges, and (3) nanofibers (Fig. 3A, C, E) [32, 50, 73–78].
In the same manner with ACs, type II collagen apparently Hydrogels include loose bundle of collagen fibers forming the
requires additional factors to unlock the chondrogenic potency. branched three-dimensional network [50, 74, 75]. Porous
Lu et al. compared MSCs cultured in type I and II collagen gels sponges consist of membrane-like wall and microscopic pores
in the absence and presence of nucleus pulposus cells (NPCs). [32, 73], and nanofibers have the nonwoven network of col-
No difference of chondrogenic markers were observed for lagen fibers with tenth to hundredth nanometer in diameter,
MSCs cultured in these gels when NPCs were absence [72]. mimicking structural protein fibers in the human body
Strikingly, co-culturing with NPCs caused MSCs grown in type [76–78].
II collagen to significantly upregulate the chondrogenic Hydrogels and porous sponges are fabricated by a
markers over MSCs of type I collagen group. The selective common initial route (fibrillogenesis), in which pH of
improvements may originate from soluble factors released by collagen solution is neutralized and followed with incu-
NPCs. These factors were assumed to work specifically with bation below the denaturation temperature [32, 45, 73, 79].
type II collagen to promote chondrogenic differentiation [72]. Collagen molecules are self-assembled as nano-sized fibrils
In summary, type II collagen is a promising CTE scaf- in a staggered arrangement [51]. Since collagen possesses
fold alternative of type I collagen (Table 3). However, type abundant water-binding amino acids [50, 80], collagen
II collagen is a potential arthritogenic agent [60, 61] and attracts a lot of water molecules and subsequently formed
has not been widely approved by various health agencies, hydrogels [80, 81]. Porous sponges is obtained by freeze-
thus limiting its usage in clinical setting [40]. drying the hydrogels or directly freeze-drying collagen
solution [73, 79]. Microstructural observations of both
hydrogel and porous sponges generally require freeze-
drying as sample preparation step, therefore it is common
to dehydrate the hydrogels with ethanol series and

Table 3 Advantages and disadvantages of type I and II collagen in CTE

Type I collagen Type II collagen

Advantages Promotion of proliferation of chondrocytes [18] Stabilization of the morphology of AC [23]

Wide safety approvals [3, 40] Support of chondrogenic differentiation of MSCs
[72]
Disadvantages Unable to support the chondrogenic performance of ACs and MSCs Potentially arthritogenic [62, 63]
[55, 72]
Limited safety approvals [40]

Fig. 3 Schematic illustration of

macroscopic and internal
A Hydrogels B Porous Sponges C Nanofibers
structures of collagen A, B
hydrogels, C, D sponges, and E,
F nanofibers. (F was reprinted
with permission from Yeo et al.
[123]. Copyright (2008)
American Chemical Society)

D E F

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t-butanol to preserve the fibrillar structure (Fig. 3B, D). On intended for CTE. However, as each form of scaffolds
the other hand, nanofibers of collagen are obtained through shows different internal structures, it is not feasible to
the electrospinning process [45, 77, 78]; collagen solution directly compare one scaffold and the others.
was ejected through a spinning nozzle towards a metal In the next sections, we addressed the effects of stiffness
collector by applying high voltage between them and the on three forms of the scaffolds to the differentiated phe-
stable jets of collagen gradually accumulated on the col- notype and secretion activity of ACs and MSCs. In the
lector to form the nonwoven nanofibers (Fig. 3F) [40]. same manner, we also focused on pore size effect of
Several physical features of the scaffolds are controlled scaffolds and discussed its importance to ACs and MSCs.
by modifying fabrication parameters [32, 77, 82], such as
collagen concentration to change diameters of fibers [77], 3.1 Influence of matrices stiffness to differentiated
or freezing temperature to change pore sizes in the phenotype of ACs
hydrogels of the porous sponges [32, 82]. These scaffolds
might be subjected to various types of crosslinking in order Stiffness—intrinsic resistance of materials to deforma-
to impart sufficient stiffness and robustness for the future tion—is an important environmental cue to control cellular
handling [83, 84]. activities [89]. It is common practice to express stiffness
The internal structures of the scaffolds are of great (elasticity) of elastic or viscoelastic scaffolds with Young’s
importance in designing ideal scaffolds because they modulus and shear modulus [91]. Generally, anchorage-
influence behavior of cell by presenting cells with unique dependent cells exert contraction forces onto substrates and
microenvironment and distinct bulk properties (e.g., sur- respond to the stiffness of the substrates by adjusting its
face area, pore interconnectivity, gas or fluid permeability) adhesion strength and cytoskeletal formation [89].
[74–76, 85, 86]. ACs, environmentally sensitive cells, Adjustment of binding state subsequently affects the cel-
would be easily affected by the internal structures because lular activities, such as proliferation, differentiation, and
it would lose or regain their differentiated phenotype secretion activities [89].
depends on two dimensional, two-dimensional (2-D), Since the freshly isolated ACs had stiffness of
(flattened morphology) or 3-D, (rounded morphology) 0.7–4 kPa, scaffolds with the stiffness at these values were
culturing system [10]. expected to provide correct cues for ACs [25, 92, 93].
Hydrogels are more superior to porous sponges in sup- Schuh et al. [26] cultured monolayer ACs on polyacry-
porting differentiated traits of ACs. The wall in porous lamide with different stiffness (* 4, * 10, * 40,
sponges would be considered as flat area for ACs, while the * 100 kPa) for 7 days; only ACs on the gel at * 4 kPa
network structure forced the embedded ACs to assume retained the differentiated phenotype as evidenced by the
round shape [74, 87]. In contrast, porous sponges were diffused organization of actin, round morphology, and
superior to hydrogels in promoting the viability and significantly higher expression of type II collagen and
chondrogenic differentiation of MSCs [85]. It was argued aggrecan, and lower expression of type I collagen [26].
that hydrogels lacked proper diffusion of nutrient which ACs seeded on the gel at * 4 kPa also showed the lowest
was vital for sustaining activities of MSCs [85]. On the cell number, indicating chondrocyte commitment to the
other hands, fiber diameters in nanofibers dictated whether secretion activities and not to proliferation [26]. Based on
ACs attached in the flattened or rounded morphologies, tensegrity hypothesis, it is assumed that ACs adjust stiff-
which in turn determined the secretion amounts of GAG ness to match the substrates and subsequently alter physi-
and cartilage-specific ECMs molecules (i.e., type II and IX ological and differentiated phenotype [26].
collagen, aggrecan, and link protein) [21]. Aligned nano- Collagen sponges with low initial stiffness would be
fibers were also shown to be beneficial in promoting the beneficial in secretion activity of cartilage-related mole-
chondrogenic differentiation of MSCs as it caused the cules [83, 84]. Lee et al. [83] obtained collagen sponges
alignment of MSC cytoskeleton reminiscent of matured with different stiffness (145, 346, 369, and 1117 Pa) by
articular ACs [76, 88]. crosslinking with dehydrothermal (DHT), ultraviolet (UV),
In addition to the internal structures, other physical glutaraldehyde (GTA), and carbodiimide (EDC). They
aspects such as stiffness and pore features (e.g., pore size, described that only soft scaffolds (i.e., DHT and UV
porosity, interconnectivity) are of importance for cell fates. crosslinked) exhibited positive staining for type II collagen
Adult stem cells have been found to sense stiffness of their [83]. Vickers et al. [84] also investigated that collagen
surrounding substrates and to adjust their morphologies and sponges crosslinked with DHT exhibited intense staining
activities accordingly (mechano-transduction) [12, 89]. On for proteoglycan and type II collagen compared with
the other hands, well-designed pore features are basic sponges heavily crosslinked with EDC. Such favorable
requirements of the scaffolds [90]. The optimum state for results are mainly attributed to the acute contraction of
these two aspects are rewarding in designing scaffolds scaffolds with low stiffness (reduced down to * 50% of

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original diameter) after several times of culture [84]. At seeded on stiff or soft substrates, indicating the role of
such situation, cell–cell interactions are largely promoted stiffness in diverging the lineage specification of MSCs
and in turn have benefit of the phenotype maintenance. [98].
Interestingly, stiffness in 3-D environment seems to affect Chondro-inductivity of soft substrate was attributed to
ACs in the indirect manner, in contrast with ACs in 2-D the weaker cellular adhesions, which in turn prevent the
environment. extensive formation of stress fiber (a-actin) [98]. Diffused
In the case of hydrogels, effect of stiffness is much organization of a-actin has long been associated with
subtle, because different studies reported conflicting opti- secretion of type II collagen and proteoglycan [99]. Nev-
mal stiffness values. Sanz-Ramos et al. [94] showed that ertheless, when MSC adhesion was largely inhibited,
collagen hydrogels with elastic modulus of * 5 Pa were MSCs failed to differentiate in the chondrogenic pathway
superior in enhancing expression of aggrecan, type II col- despite being cultured in chondro-inductive medium [100].
lagen, and Sox9 than stiffer hydrogels (* 10 and Such result demonstrates the importance of proper cellular
* 20 Pa). On the other hand, Li et al. [27] described that attachment [100].
stiff polyacrylamide gel (29.9 kPa), not the softer gels (3.8 Benefit of low stiffness in promoting initial stage of
or 17.1 kPa), promoted the redifferentiation of ACs and chondrogenic differentiation for MSCs is also observed in
subsequent secretion activities. Schuh et al. [95] investi- 3-D environment. Murphy et al. discovered that MSCs
gated ACs cultured in two agarose gels with different cultured in the non-differentiating medium in the soft col-
stiffness * 3.7 (soft) and * 53 (stiff) kPa) for 2 weeks. lagen-based sponge (compressive modulus of 0.5 kPa) pro-
They discovered indifferent results for type II and I col- moted upregulation of Sox9 (early chondrogenic marker),
lagen staining for both of soft and stiff gels, although higher while stiff collagen-based sponges (1 and 1.5 kPa) favored
ratio of GAG/DNA and greater cell number were observed upregulation of Runx2 (early osteogenic marker) by MSCs
for the soft gel [95]. Further analysis revealed that the soft [31]. However, no significant differences were observed for
gel significantly absorb bovine serum albumin (BSA), any types of scaffolds for terminal differentiation makers
implying the favorable results due to better nutrient per- [i.e., Collagen I, Collagen II, and ALP (alkaline phos-
meability [95]. In addition, RGD-binding motif sometimes phatase)], suggesting the necessity of differentiating-med-
caused complicated results as it effectively induced ded- ium for lineage commitment [31]. Preference of MSCs for
ifferentiation of ACs both in soft and stiff gels [95]. To chondrogenic or osteogenic differentiation was associated
reduce the perplexity of such issues in hydrogel system, it with expression of a-smooth muscle actin (SMA) which was
would be useful to use natural protein-based matrices linearly dependent with compressive modulus [31].
(collagen) and chemically defined medium. Other studies involving hydrogels and nanofibers also
Skotak et al. [96] investigated ACs cultured on gelatin confirmed the advantages of low stiffness in favoring
nanofibers with different stiffness, crosslinked by glu- chondrogenic differentiation [101, 102]. During cell cul-
taraldehyde of various concentrations (0–5 wt%). They ture, soft hydrogels undergo extensive dimensional con-
found that stiffer nanofibers supported higher cell density traction, which in turn promote cellular condensation and
and greater ratio of type II/type I collagen expression the subsequent chondrogenic differentiation of MSCs
compared to softer substrates [96]. Nonetheless, such [103]. Soft nanofibers may provide ‘dynamic environment’,
improvement could be attributed to the improved structural in which flexible fibers are easily deformed by the cultured
stability imparted by extensive crosslinking, since easily cells. Pliable fibers prevent MSCs to develop stress fibers,
degraded nanofibrous caused negative effect for ACs [97]. thus favoring chondrogenic differentiation of MSCs.
Hypertrophic differentiation of MSCs is apparently
3.2 Influence of matrices stiffness to chondrogenic suppressed in low stiffness matrices [36, 104]. Bian et al.
differentiation of MSCs showed that soft hydrogels (* 3 kPa) exhibited signifi-
cantly lower expression of type X collagen (hypertrophic
Stiffness of monolayer substrate affects the differentiation marker) than stiffer hydrogels (* 50 kPa). However, such
lineage of MSCs at initial stage. Park et al. [98] investi- results were not assigned to mechanotransduction since no
gated MSCs on the relatively thick collagen gel (soft sub- blocking of ROCK (Rho-associated protein kinase) and
strate) and thinly collagen-coated dish (stiff substrate). myosin II suppressed matrix calcification at stiffer hydro-
MSCs on soft substrate exhibited lower expression of gels [37]. It was argued that softer hydrogels retain more
smooth muscle markers (a-actin) and significantly higher cartilage-related ECMs (i.e., proteoglycan and type II
type II collagen compared with MSCs seeded on stiff collagen) and these ECMs may curb the hypertrophic dif-
substrate [98]. Subsequent addition of transforming growth ferentiation and matrix calcification [104]. Effect of stiff-
factor beta (TGF-b) significantly promoted either a-actin ness on ACs and MSCs are summarized in Fig. 4.
or type II collagen dependent on whether MSCs were

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Fig. 4 Effect of substrate stiffness (colored boxes) on gene tran- differentiated phenotype of ACs, as reported by Sanz-Ramoz et al.
scription (italic name; Sox9, a-SMA, COL II, Col I, Col X, ACAN) and [94], Schuh et al. [26, 95], Li et al. [27] and Lee et al. [83]. In a
protein synthesize (underlined name; GAG/DNA, COLII) of ACs similar manner, green and yellow boxes are associated with changes
(blue and red colored) and MSCs (green and yellow colored). Blue of chondrogenic markers of MSCs as reported by Bian et al. [37] and
and red boxes respectively indicate positive and negative changes of Murphy et al. [31]. (Color figure online)

3.3 Influence of pore sizes to differentiated speculated to be easier to be filled with cells, which would thus
phenotype of ACs promote cell–cell interactions for maintenance of chondrocyte
phenotype [22, 105, 107]. Pore size might also indirectly affect
Pore sizes smaller than 225 lm in collagen sponges are ACs by altering stiffness of scaffolds [105]. Other factors such
generally beneficial to support ACs to retain the differen- as pore interconnectivity and material composition also sig-
tiated phenotype and to promote the biosynthetic activities nificantly affected the chondrocyte behaviors [86]; thus pru-
of cartilage-related ECMs [22, 105]. Nehrer et al. [23] dence should be necessary in interpreting the pore size effect.
demonstrated that major fraction of ACs retained the In collagen hydrogels, pores or meshes are defined to be
rounded morphology in collagen sponge with small pore spaces between entangled fibers, and the sizes can be
size at 20 lm compared with large one at 80 lm. Signifi- enlarged from 2 to 12 lm by increasing collagen concen-
cantly higher ratio of GAG/DNA was also observed for tration or gelation temperature [82]. Effect of mesh size to
ACs in sponges with smaller pore size at 20 lm than those chondrogenic phenotype of ACs is mainly investigated in
with 80 lm (GAG/DNA: 11.08–4.03) [23]. Nonetheless, it the system of synthetic polymer gels (i.e., poly(ethylene
was argued that scaffolds with pore size in the range of glycol) (PEG) or PEG-based polymer), as their mesh size
tenth micrometer (13–85 lm) limited cellular migration can be finely tuned by modifying the molecular weight of
and medium diffusion, which caused formation of shallow precursor or crosslinker concentration during polymeriza-
depth of cartilaginous film and might be detrimental for tion [108, 109]. It was found that the mesh size around
future clinical application [106]. 70–100 Å could be optimal to enhance the deposition of
To overcome these issues, Zhang et al. [105] investigated type II collagen and aggrecan, and to promote accumula-
pore sizes of collagen sponges in the larger range (150–250, tion and distribution of secreted proteoglycan [108, 109].
250–355, 355–425, and 425–500 lm). Initially, they cultured Mesh sizes rarely influence the distribution of cells in
ACs in the scaffold in vitro for 1 week followed by in vivo hydrogels because cells are usually encapsulated in the hydro-
implantation for 8 weeks [105]. Cells were homogenously gels by mixing with the initial solution [31, 50, 74]; however,
distributed for all scaffolds; however, only ACs seeded in the smaller mesh sizes significantly inhibited cell migration and
scaffold with 150–250 lm of pore size showed improvement circulation of nutrients [50, 82], which in turn might impede the
of differentiated expression of COL2A1 and ACAN, and the interaction with host tissue after implantation. Studies involving
higher ratio of sGAG (sulfated GAG)/DNA, compared with collagen nanofibers rarely detailed the effect of pore size to
ACs in other scaffolds [105]. Other studies employing porous activities of ACs, however it was indicated that ACs readily
sponges of non-collagenous origin also exhibited the advan- penetrated collagen nanofibers of various pore sizes [77, 78].
tages of scaffolds with relatively small pore sizes in supporting
the secretion activities of ACs [106, 107]. Small pore size was

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3.4 Influence of pore sizes to chondrogenic penetration and cell–cell interactions [115, 116]. The effect
differentiation of MSCs of pore size on ACs and MSCs are summarized in Fig. 5.

Pore sizes larger than 300 lm are advantageous to support

the chondrogenic differentiation of MSCs and their sub- 4 Blending strategy of type I collagen
sequent production of cartilage-related matrices. Matsiko
et al. [33] investigated chondrogenic differentiation of Application of pure type I collagen as a scaffold for CTE
MSCs in collagen-based sponges at different pore sizes has two major issues: (1) weak mechanical robustness
(94, 130, and 300 lm) with relatively similar elastic which heavily impair scaffold handling before and after
modulus. The scaffold with the largest pore size at 300 lm implantation [117], (2) limited chondrogenic properties in
was superior in supporting the chondrogenic differentiation terms of supporting ACs phenotype and chondrogenic
of MSCs compared with the others on the basis of signif- differentiation of MSCs [22, 118]. To overcome the first
icantly higher expression of COL2, SOX9 and lower limitation, collagen crosslinking using carbodiimide or
expression of COL1 for 28 days [33]. Moreover, larger glutaraldehyde has been conducted; however, introduction
amounts of secreted GAG and the gradual increase of of a lot of crosslinking points would consume the cell-
Young’s modulus were observed for the scaffolds with the binding motives (e.g. GFOGER) [119], nullifying the ini-
pore size at 300 lm, indicating further improvement in tial purpose of utilizing collagen. Physical mixing (blend-
biosynthetic activities of cartilage-related ECMs [33]. Such ing) of collagen with other natural polymers, such as silk
favorable results of sponges with large pore sizes fibroin, chitosan, hyaluronic acid is useful to obtain more
([ 300 lm) were also confirmed by other studies which ideal matrices composition without sacrificing desirable
employed non-collagenous materials [32, 110–112]. features of collagen (e.g., cellular adhesion and biocom-
Expression of type X collagen was suppressed with the patibility) [45, 120]. In section IV, we mainly discussed
increasing pore size probably due to the improved chon- investigations for applying the blending strategy of colla-
drogenesis [111]. Chondrogenic differentiation of MSCs gen with several polymers, as described in Fig. 6, to sup-
was strongly inhibited in hypoxia condition [113]; low port phenotypes or activities of ACs and MSCs. Particular
oxygen tension in small pore sized scaffolds may thus instances of materials, such as synthetic polymer or inor-
hamper the chondrogenic differentiation of seeded-MSCs ganic materials, are only discussed shortly because it is
[111]. This is interesting because the trend of optimum size more common to combine them in non-blending strategy,
for collagen-based sponges is completely reversed for ACs as will be given in Sect. 5.
and MSCs.
Unlike sponges, it is difficult to isolate the pore size 4.1 Blending with silk fibroin
effect in hydrogels and nanofibers because it is not possible
to alter their pore size without grossly affecting other Silk fibroin (SF) extracted from Bombyx mori has long
related properties such as stiffness or fiber size [114–116]. been used as a medical suture due to its excellent toughness
Study involving PEG-based hydrogels indicated that pore and structural stability [121, 122]. In terms of mechanical
size effect would not be as influential as hydrogel com- toughness, SF exhibits larger values (ultimate tensile
positions in affecting the chondrogenic differentiation strength (UTS) at 740 MPa, Young’s modulus at 10 GPa,
[114]. On the other hand, nanofibers mat with larger pore elongation at 20%), in contrast with crosslinked collagen
size and fiber diameter apparently supported the change of (UTS at 47–72 MPa, Young’s modulus at 0.4–0.8 GPa,
chondrogenic differentiation of MSCs possibly due to cell elongation at 12–16%) or with mammal bone (UTS at

AC Cells MSC Cells

Scaffold properties:
Low • Oxygen tension High
• Nutrient circulation
• Cell distribution
Small pore size Large pore size
(>250 μm) (<300 μm)
Fig. 5 Schematic illustration of change of scaffold properties with (induce cell aggregation) and low oxygen tension. On the other hand,
evolution of pore or mesh sizes. Small pore sized scaffolds support large pore sized scaffolds enhance the chondrogenic differentiation of
the differentiated phenotype of ACs due to non-even cell distribution MSCs due to higher oxygen tension and better nutrition circulation

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Fig. 6 Chemical structures of A natural polymers [silk fibroin (SF), caprolactone) (PCL), poly(vinyl alcohol) (PVA), poly(D-lactide)
chitosan, hyaluronic acid (HA), chondroitin sulfate-A (CS-A), (PDLA), poly(L-lactide-co-e-caprolactone) (PLA/CL), poly(L-lac-
chondroitin-6 sulfate (CS-C)] commonly blended with collagen tideco-glycolide) (PLGA)] incorporated with collagen matrices in
matrices (sponges, hydrogel, nanofibers) and B synthetic polymers non-blended manner
[poly(lactic acid) (PLA), poly(L-lactide) (PLLA), poly(e-

160 MPa, Young’s modulus at 20 GPa, elongation at 13%) aggregation during freezing, thus yielding mechanically
[122]. SF is environmentally stable fibrous protein owing fragile scaffolds [128]. The addition of collagen would
to its extensive hydrogen bonding, hydrophobic nature, and prevent the aggregation and produce final scaffolds with
high degree of protein crystallinity (b-sheet crystals) [121]. undoubtedly better mechanical properties [128].
Despite the insolubility of SF in mild aqueous solution, Blend of SF with collagen is generally superior in sup-
various methods have been well established to form SF into porting cellular viability in contrast with pure collagen and
sponges, hydrogels, and nanofibers [87, 122–124]. SF scaffolds [125, 128–130]. Wang et al. [125] blended
Nonetheless, the high stability of pure SF might impede its collagen with SF at different ratio in sponge matrices and
application as tissue engineering scaffolds, as it slowly investigated the subsequent MSC proliferation rate. They
degrades after implantation. SF also lacks cell-binding found that ratio of 7:3 (collagen:SF) provided the highest
motives or other mechanism beneficial in supporting cell proliferation rate of MSCs. Similar finding was also con-
activities [122]. Thus, it is advantageous to blend SF with firmed for other type of cells, such as chondrocytes and
collagen [125–129]. HepG2 cells [125, 129]. It was previously described that
Generally, collagen was blended with silk by separately silk provided stabile porous structures in scaffolds, which
adjusting each solution and subsequently mixing them would be important to maintain favorable environment
before next processing (e.g., freeze-drying or electrospin- during culture [124]. No SF apparently possesses chondro-
ning) (Fig. 7A, B) [125, 128, 129]. Chomcalao et al. [129] inductive capabilities, thus disfavoring its application in
synthesized sponges of pure SF and SF-collagen; the cartilage tissue engineering. However, some authors pro-
addition of collagen improved compressive modulus from posed the incorporation of encapsulated TGF-b into SF-
148 to 1532 kPa. The result was less intuitive as why collagen scaffolds to overcome this limitation (Fig. 7C, D)
addition of mechanically weak collagen enhanced rela- [125].
tively stronger silk. It turned out that SF suffered from

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Fig. 7 Structure and properties of collagen/silk fibroin (Col/SF) SF). D Histological evaluation of Col/SF scaffolds after implantation
scaffolds. A Microstructure of freeze-dried SF scaffold. B Microstruc- in vivo for 12 weeks. COL/SF and no scaffold as a control group.
ture of freeze-dried Col/SF scaffolds with 20 wt% Col and 4 wt% SF. (Parts of figure are adapted with the permission from [128] A, B and
C Microstructure of Col/SF scaffolds incorporated with TGF-b1 [125] C and D)
containing poly(lactic-co-glycolic-acid) microsphere (TGF-b1- COL/

4.2 Blending with chitosan [133, 135, 136]. Moderate addition of chitosan is beneficial
to enhance the overall mechanical properties and structural
Chitosan is a linear polysaccharide of glucosamine and stability of scaffolds (Fig. 8A–C) [131, 133–137]. Chen
N-acetyl glucosamine mainly derived from deacetylation et al. blended chitosan into collagen nanofibers and
of chitin which commonly finds in crustacean (crabs, reported the increase of UTS, strain, and Young’s modulus
shrimp, and lobster) shells or cell wall of fungi [131]. Long with contents of chitosan up to 20% (w/v). Further addition
chain of amine endows chitosan with various unique of chitosan embrittled the scaffold as UTS and Young’s
properties, such as antibacterial, cationic nature, and modulus grossly increased without improvement of tensile
resistance to enzymatic degradation [131, 132]. Chitosan is strain [136, 138]. Analysis of Fourier transform infrared
widely used as biomaterial or drug-delivery agent due to its (FTIR) spectra assigned the increase of mechanical prop-
non-toxicity, low immunogenicity, and antibacterial erties to the formation of hydrogen bond of collagen and
activities. Several studies have also indicated the capacity chitosan, possibly between –C=O and –NH2 groups of
of chitosan in supporting synthetic activity of chondro- collagen with –OH and –NH2 groups of chitosan [136]. At
cytes, suggesting its potential in CTE [131–134]. Never- higher contents of chitosan, stronger ionic bond could form
theless, chitosan resists enzymatic hydrolysis and hardly and subsequently contribute to the brittleness of scaffold
degrades in vivo [131]. Thus it might be useful to combine [135, 136]. Excessive amounts of chitosan blend formed
chitosan with easily degraded biopolymer to ensure clumps, which would block the pores of scaffolds and
appropriate tissue remodeling. inhibit penetration of cells into the scaffolds [135]. Chi-
Chitosan can be easily mixed with collagen to obtain the tosan-collagen sponges retained * 40% of their original
final forms of sponges, hydrogels, and nanofibers weight after enzyme treatment in contrast with fully

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Fig. 8 Structure and properties of collagen/chitosan scaffolds. **p \ 0.01; ***p \ 0.001. D, E Determination of sGAG deposition
A Microstructure of freeze-dried collagen/chitosan scaffold. of collagen (collagen:chitosan at 100:0) versus collagen/chitosan
B Change of mechanical properties of collagen/chitosan scaffold (collagen:chitosan at 75:25) scaffolds, by Safranin O staining
caused by the variation of chitosan concentration (0–50 wt%). (D) and sGAG quantification (E). Data (n = 3) is plotted in
C Remaining of collagen/chitosan scaffold after incubation for mean ± standard deviation. Significance is indicated by *p \ 0.05;
28 days in serum containing media. Data (n = 3) is plotted in **p \ 0.01; ***p \ 0.001. (Parts of figure are adapted, with
mean ± standard deviation. Significance is indicated by *p \ 0.05; permission, from [137] A, B and [139] C, D, E)

degraded pure collagen matrices, indicating improvement commonly found in native articular cartilage tissues. A lot
of in vitro structural stability [134]. of CS and other sulfated GAG are attached with the core
Biosynthetically active ACs mainly produced anionic protein, which forms gigantic and bottlebrush-like mole-
GAG (e.g., chondroitin sulfate, heparan sulfate) during cell cules called aggrecan [141]. Due to the anionic nature of
culture, and the presence of chitosan could be useful to CS, aggrecan attracts huge amounts of water and provide
retain such GAG secretion, since the cationic nature of osmotic resistance of articular cartilage during compres-
chitosan allows interaction with negatively charged GAG sion [141]. Aggrecan associates with a link protein and
or other anionic proteoglycan [131]. Yan et al. [134] cul- binds to a single molecule HA to form larger proteoglycan
tured AC in a scaffold of chitosan-blended collagen and complexes, which together with type II collagen provide
described higher amounts of GAG content compared with tensile and compressive resilience of articular cartilage [6].
pure collagen scaffolds. The bioactive signals of chitosan Roles of blending HA with collagen in modulating
can be attributed to its structural similarity with GAG phenotype and bioactivities of ACs and MSCs have been
molecules [131, 133]. Build-up of GAG in scaffolds is investigated [109, 142–147]. HA-collagen composites
highly desirable as it may contribute further to the chondro- supported higher fractions of ACs to retain rounded shapes
inductive environment and gradual improvements of and to promote the secretion of cartilage-related ECMs,
mechanical properties of final structures (Fig. 8D, E) much better than pure collagen counterpart (Fig. 9A, B)
[36, 139, 140]. [35]. In the case of MSCs, addition of HA into collagen
sponges apparently promoted the cellular infiltration and
4.3 Blending with hyaluronic acid and chondroitin favored expression of chondrogenic markers (i.e. Sox9 and
sulfate COLII) in the short term culture period (B 14 days)
[30, 35, 148]. CD44 is a main receptor for HA and their
Hyaluronic acid (HA) and chondroitin sulfate (CS) are corresponding bindings have been shown to be crucial to
members of long unbranched polysaccharides (GAG)

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Fig. 9 A, B Histological sections of scaffolds of collagen (A) and in mean ± standard deviation. *indicates insignificant differences.  
collagen/HA (B). Red color indicates sGAG staining by Safranin-O. indicates p \ 0.01. (Parts of figure are adapted from [35] A, B and
C Effect of HA concentration of the collagen/HA scaffolds on the [144] C). (Color figure online)
total amount of deposited chondroitin sulfate. Data (n = 6) is plotted

maintain chondrogenic states and cellular motility collagen culture, possibly due to the binding with specific
[142, 143, 148]. cell receptor. Other reasons would be the gradual release of
HA seems to require optimum concentration in order for added-CS into the medium throughout the period of cell
its addition to elicit the expected benefits. Kawasaki et al. cultures [34].
[144] incorporated HA at different concentrations (0, 0.01, Incorporation of CS apparently benefit the chondrogenic
0.1, 1.0 mg/ml) in type I collagen gel, and they found that differentiation of MSCs in the low stiffness environment
HA at 0.1 mg/ml was optimal for enhancing chondrocyte (Fig. 10A) [30, 38]. In the absence of differentiating
proliferation and CS secretion (Fig. 9C). In contrast, HA medium, blending of CS into collagen sponges with a low
caused no change at higher or lower concentration (0.01 or stiffness (* 0.5 kPa) promoted early upregulation of Sox9.
1.0 mg/ml). Further analysis also revealed that only Meanwhile, CS caused a favorable expression of osteo-
0.1 mg/ml HA-collagen hydrogel altered the ratio of genic marker (Runx2) in the high stiffness sponges (* 1
secreted CS in favor of higher production of cartilage-re- and 1.5 kPa) [31]. Furthermore, CS addition seems to
lated chondroitin-6 sulfate (CS-C), instead of ligament- suppress hypertrophic differentiation of MSCs, as evi-
related chondroitin-4 sulfate (CS-A). Other study reported denced by the downregulation of type X collagen and
similar findings, in which excessive addition of HA Alizarin staining [33, 37, 38].
([ 2% w/w) suppressed the secreted amounts of cartilage- Cautions are necessary to interpret synergistic effects of
related by ACs [149]. In the aqueous solution, HA is HA and CS in a single collagen scaffold. Final composi-
known to form polyion complexes with collagen mole- tions of trimeric scaffold (HA/CS/collagen) are easily
cules, thus preventing the formation of homogeneous altered during preparation of scaffolds or cell-culture
scaffolds [150]. Furthermore, HA in high concentration stages [153, 154]. HA strongly interacts with CS when
may entangle and form hydrophobic patches which are mixed in aqueous solution, preventing considerable
detrimental for secretion of cartilage-related ECMs [151]. amounts of CS to be incorporated in collagen hydrogel
Collagen-CS scaffolds are frequently employed for [153]. CS are also easily washed away during cell culture
culturing chondrocyte aiming to maintain the round mor- due to their high water-solubility (Fig. 10B) [34, 154].
phology and its biosynthetic activities [13, 22, 38, 83, 84]. Addition of HA or CS would hardly cause any change to
Surprisingly, studies aimed to scrutinize chondrogenic mechanical properties of collagen matrices [35], thus sev-
benefits of CS addition showed mixed results [18, 146]. eral authors have proposed additional blending with more
Van Susante et al. [146] crosslinked CS into type I collagen robust polymer (e.g. chitosan, methacrylic anhydride)
sponges and observed no difference in chondrogenic [35, 134, 155].
expression (i.e., aggrecan, biglycan, decorin), despite the
improved amount of secreted GAG. In similar study, Pieper 4.4 Blending with synthetic polymer and inorganic
et al. [19] described that no major difference was caused by materials
the absence or presence of CS in type I collagen scaffold.
These contradictory results would be explained by differ- Organic solvents are generally required to process bio-
ence of CS types used. Nishimoto et al. [152] investigated compatible synthetic polymers, such as poly-e-caprolac-
that CS-C, and not CS-A, upregulated chondrogenic tone (PCL), poly-L-lactide-co-glycolide (PLGA), and
expression (i.e., type II collagen and aggrecan) in 3-D polylactic acid (PLA), and thus blending collagen in such

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Fig. 10 A Sox9 expression of MSCs seeded in scaffold of colla- cell culture media. 2af-CS and 2rf CS indicate afibrillar type II
gen/chondroitin sulfate (CCS) with different compressive modulus collagen-CS and fibrillar type II collagen-CS scaffolds, respectively.
(stiffness). B Amount of CS (chondroitin sulfate) in two different type (Parts of figure are adapted from [31] A with permission and [34]
of collagen scaffolds at initial time and after 3 days of incubation in B under a Creative Commons Attribution License)

solvents is not feasible due to denaturation of collagen. 4.5 Collagen denaturation in blending strategy
Lack of common solvent limits the feasible option to
hydrophilic synthetic polymers [156]. Collagen is denatured by physical stress [167], temperature
Poly(vinyl alcohol) (PVA) is one of the synthetic and increase [168], or exposure to strong organic solvent [169].
biocompatible polymers feasible to be blended with col- Denatured collagen losses the native helical structure (tri-
lagen [157, 158]. Mechanical properties of PVA can be ple helix) and subsequently forms a hydrolyzed product
altered by several processing methods (e.g., freeze thaw- (gelatin) [169]. Gelatin still supports the cellular activities
ing, or crosslinking) [159, 160]. Since PVA incapable of (cell attachment and cell proliferation) [96]; however
supporting cell adhesion, it gains benefit by mixing with physical properties of gelatin are different from collagen,
collagen [157]. Moreover, PVA also provides cheaper in which it shows rapid degradation and inferior mechan-
alternative to fill the bulk volume of collagen matrices. ical properties [170]. Loss of native structure of collagen
Mehrasa et al. [157] electrospun pure PVA and colla- scaffolds is associated with the altered attachment behavior
gen-mixed PVA, and discovered that the addition of col- of ACs [171] and the diminished capacity of type II col-
lagen increased the tensile strength, Young’s modulus, and lagen to stimulate chondrogenic differentiation of MSCs
elasticity of the final scaffolds. PVA and collagen com- [118]. In other words, denaturation of collagen may
posites form strong hydrogen bonding in the composites, diminish the mechanical and biological performance of
which improves thermal stability and mechanical tough- scaffolds in CTE.
ness. Nonetheless, PVA-collagen composites apparently Denaturation limits the options of materials able to be
showed no improvement of bioactivities for chondrocytes processed by blending strategy. Attempts to achieve the
[157]. homogeneous blending of collagens requires exposure of
Inorganic materials are rarely used for the application of collagen to high temperature or strong inorganic solvent,
articular cartilage regeneration, because its excessive inadvertently lead to the collagen denaturation
stiffness might not be suitable for chondrocytes and most [170, 172–175]. Silk fibroin is a hydrophobic polymer
of composites involving collagen and inorganic materials [122], thus silk shows limited miscibility with collagen.
are commonly intended for osteochondral applications Several investigators attempted to blend silk and collagen
[161]. Nevertheless, Ohyabu et al. [162] demonstrated that solution at high temperature (60 °C) [128, 172], inadver-
hydroxyapatite (HAp) nanoparticles of moderate amounts tently damaged the native structure of collagen in the
were beneficial to sensibly improve mechanical properties process. Other investigators used fluoroalcohol-based sol-
of collagen sponges. HAp nanoparticles also increased vent [(1,1,1,3,3,3-hexafluoro-2-propanol (HFP)) to electro-
significantly surface area of the collagen sponges spin the blend of collagen/synthetic polymers PCL [175] or
(6.52–34.5 m2/g), thus enhancing cells (MSCs) distribution poly(L-lactic acid) (PLLA)] [176]. HFP was previously
and adhesion [163]. showed to be detrimental for helical conformation of col-
Aforementioned effects of collagen blending to final lagen, effectively transforming the collagen in final scaf-
mechanical properties were summarized in Table 4. folds into gelatin [169]. The final polymer/collagen
scaffolds exhibit no negative effects on the biological
activities of cells [175, 176]; however, collagen scaffolds

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Table 4 The effect of blending collagen on mechanical properties of final matrices

Matrices composition [form] Initial strength Initial elasticity References

SF (6 wt%); SF/collagen (25:75) [Sponges] Not mentioned 148 ± 12 kPa; 1532 ± 697 kPac [129]
SF (4 wt%); SF/collagen (10, 20 wt%) 20 ± 1 kPa; 310 ± 10, 0.43 ± 0.055 MPa; 10 ± 0.1, [128]
[Sponges] 354 ± 25 kPab 30 ± 0.1 MPac
Collagen (0.6%w/v); collagen/chitosan 0.37 ± 0.05 MPa; 0.51 ± 0.08 MPaa Not mentioned [134]
(1%w/v with 80:20 w/w) [Sponges]
Collagen (2.10 mg/ml); collagen/chitosan Not mentioned * 10 kPa; * 15, 20 kPac [133]
(2:1, 1:1 w/w) [Hydrogels]
Collagen/chitosan (100/0; 80/20; 60/40; 23.7 ± 5.8; 21.7 ± 19.3; 62.8 ± 14.1; 1371 ± 225; 1611 ± 793; 5966 ± 2137; [136]
50/50; 40/60; 20/80% content in complex) 61.8 ± 26.0, 47.0 ± 19.1; 6801 ± 3256; 4159 ± 1195;
[Nanofibers] 10.5 ± 8.0 MPaa 3601 ± 485 MPac
Collagen (2 wt%); collagen/HA (9:1 v/v) Not mentioned * 15 kPa; * 15 kPac [164]
[Sponges]
Collagen; collagen/HA (0.5, 1.0 mg/ml) Not mentioned 24.3 ± 5.2 Pa; 19.8 ± 3.1, [165]
[Hydrogel] 15.8 ± 3.4 Pad
4.8 ± 2.0 kPa; 11.6 ± 8.3,
41.6 ± 20.9 kPac
Collagen (0.5%w/v); collagen/CS (0.05%w/ Not mentioned * 0.2 kPa; 0.2 kPa [35]
v) [Sponges]
PVA; collagen/PVA (60:40 v/v) 0.98 ± 0.75 MPa; 1.32 ± 0.33 MPaa 3.12 ± 0.78 MPa; 5.40 ± 1.32 MPac [157]
b
Collagen; collagen/hydroxyapatite * 0.6 kPa; * 2.8 kPa * 1 kPa; * 4.9 kPac [166]
nanoparticles
a
Tensile strength
b
Compression strength
c
Elastic modulus
d
Storage modulus

without native structures might not be suitable for CTE processing by water-based solvent at pH * 3 and below
applications. room temperature.
Chen et al. [177] pioneered the first generation of hybrid
scaffolds, in which they formed type I collagen webs at the
5 Non-blending strategy (hybrid scaffolds) of type interstices of highly porous woven PLGA (Fig. 11B). By
I collagen this configuration, PLGA played a role of housing frame-
work, while collagen provided 3-D environment for
Non-blending strategy involves combination of different PLGA-collagen structure was used to produce tissue
forms of matrices (i.e., sponges, hydrogels, and nanofibers) engineered cartilage by initially culturing chondrocytes in
into a single scaffold without altering the chemical com- the scaffolds in vitro for 1 week, then subcutaneously
position of matrices (Fig. 11A). Components of hybrid implanted the scaffolds in the dorsum of athymic nude
scaffolds were classified into two parts: (1) external mice for 12 weeks [177]. During the in vitro culture,
housing framework and (2) internal bioactive filler. Hous- chondrocytes were mainly suspended on the collagen web
ing framework, as the name suggested, mainly function to and gradually occupied the openings of scaffolds
shelter the bioactive filler and to provide necessary (Fig. 12A–C) [180]. Stabilization of chondrocytes pheno-
mechanical integrity. Meanwhile, the bioactive filler is type was indicated by northern blot analysis in which type
composed of collagen or blended-collagen matrices II collagen and aggrecan were upregulated while type I
including ACs or MSCs. In this way, conventional collagen collagen was diminished over the course of 12 weeks
matrices may receive benefits from recent breakthroughs in (Fig. 12D) [180]. Such favorable result originated from
synthetic polymer forming process (e.g., 3-D printing, etc.). accumulation of ACs on the web like-collagen, which in
Moreover, collagen denaturation can be minimized as turn helps to maintain its differentiated phenotype [180].
collagen is handled under appropriate conditions, such as After implantation in vivo, glistening white appearance of
scaffolds was observed, suggesting large secretion of

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Fig. 11 A Conceptual illustration of hybrid scaffolds in which it is figure) was used as housing framework for collagen sponges to obtain
composed of two different components: housing framework and hybrid scaffold (right figure). D Appearance of hybrid scaffold of
bioactive filler. B–D are the examples of collagen-based hybrid nanofibers type I/II collagen (left figure) and the corresponding
scaffolds. B PLGA mesh (left figure) was furnished with collagen internal structure (right figure). (Parts of figure are adapted, with
web to form hybrid scaffold (right figure). C 3D printed PLGA (left permission, from [177] B, [178] C and [179] D)

Fig. 12 Hybrid scaffold of collagen PLGA mesh/collagen web. respectively. D Northern blot analyses of chondrocytes seeded in
A Initial microstructure of PLGA mesh/collagen web, B, C mi- PLGA mesh/collagen web scaffolds for the gene encoding ColI,
crostructure of PLGA mesh/collagen web after 1 and 4 weeks of COLII, and ACAN for the period of 0, 2, 4, 12 weeks. (Parts of
chondrocyte incorporation. Chondrocyte was attached and suspended figure are adapted from [180] A, B, C, and D with permission)
on the collagen web. P and C indicate PLGA mesh and collagen web,

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proteoglycan and GAG. These findings were further sup- modifying the spacing of PLGA fibers or stacking angles
ported by staining with Safranin-O and toluidine blue (Figs. 11C, 13A). These parameters can be further adjusted
[177]. Secretion of type II collagen was also positive for to optimize the proliferation and bioactivities of chondro-
the implanted scaffolds, implying the formation of hyaline- cytes (Fig. 13B, C) [178].
like tissues [177]. Housing frameworks determined biocompatibility of the
Usage of knitted meshes may be limited by pre-fixed hybrid scaffolds (Fig. 14A). Tanaka et al. investigated the
microstructures and macroscopic shapes, thus fabrication immunogenicity of AC-laden hybrid scaffolds with hous-
of housing framework by 3D printing may provide better ing framework made by five different synthetic polymers:
alternatives. Chen et al. employed selective laser deposi- PLGA with low molecular weight (PLGA-L), PLGA with
tion method to fabricate PCL with unique microstructures. high molecular weight (PLGA-H), poly(D-lactide)
They vertically stacked layers of PCL struts at angles of 0°/ (PDLA), poly(L-lactide-co-e-caprolactone) (PLA/CL), and
90°/0°/90° between each layer [181]. The structures poly(L-lactide) (PLLA) [182]. Two months post-implan-
obtained were then immersed in type I collagen solution tation in the back of nude mice showed PLGA-L and
containing AC suspension, then subsequently gelled to PLGA-H were deemed to be biocompatible compared with
form the hybrid scaffolds [181]. Compared with single other polymers, since these two matrices largely degraded
PCL matrices, hybrid scaffolds of PCL/collagen exhibited without attracting large number of macrophages (Fig. 14B)
significantly higher cell proliferation and secretion of GAG [182]. Macrophages secrete several cytokines [e.g., TNF-a
and type II collagen for a period of 28 days [181]. Yen (tumor necrosis factor-a), IL-1b (interleukin-1)] capable of
et al. constructed hybrid scaffolds of PLGA/type II colla- inducing degradation of healthy cartilage matrices, thus
gen and easily controlled the physical features of hybrid correct selection of housing framework material is crucial
scaffolds (i.e., compressive modulus and porosity) by to avoid unnecessary complication.

Fig. 13 Hybrid scaffolds of type II collagen/PLGA obtained by fused C deposited amount of GAG by chondrocytes seeded on hybrid
deposition manufacturing (FDM). A Illustration of PLGA scaffolds scaffold of different parameters. Sample naming is 4D/dh and 8D/dh.
obtained by stacking four layers of extruded polymer fiber in different FD is freeze-dried type II collagen. n = 3, p \ 0.05, * and # are
angle (4D:0°, 45°, 90°, 135°). dh and Un indicate fiber distance and significantly higher and lower from other scaffolds. (Parts of figure are
nozzle aperture, respectively. B Cell number of chondrocytes and adapted from [178] A, B, C with permission)

Fig. 14 A Hybrid scaffolds of PLLA obtained by fused-deposition polymer (PLLA, PLGA(L), PLGA(H), PLA/CL, and PDLA after
method (FDM) with different aperture size (1, 1.5, 2 mm) and 2 months of subcutaneous implantations in mice. (Parts of figure are
collagen. Control is a collagen gel. B Number of macrophages adapted from [182] with permission)
accumulated in the hybrid scaffolds of collagen and various synthetic

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Internal bioactive filler may reap benefits from previ- 6 Conclusion

ously optimized collagen-blended composition. Liao et al.
[151] mixed HA with type I collagen solution, then filled Type I collagen is a compelling platform for next devel-
the 3D printed poly(propylene fumarate). ACs cultured on opment of CTE scaffolds, considering its proven short- and
HA-collagen hybrid scaffolds exhibited strong staining of mid-term clinical efficacy and the potentially smooth
GAG, type II collagen and weak staining of type I collagen, access to enter the market of health products. Nonetheless,
compared with pure collagen hybrid scaffolds [151]. Fibrin long-term clinical performance of type I collagen scaffolds
gels can be formed by adding thrombin into fibrinogen may be hampered by its limited chondrogenic capacity,
solution and gelation can be conducted quickly without weak mechanical strength, and significant shrinkage.
harming cells; however, similar with other hydrogels, If one considers chondrogenic performance, type II
mechanical strength of fibrin gels is weak. To overcome collagen-based scaffolds would be a promising alternative
this problem, Deponti et al. [183] proposed a hybrid scaf- of type I collagen; however additional studies are necessary
fold of fibrin and collagen. Initially, they suspended ACs to confirm safety issues of type II collagen. Therefore, in
into fibrinogen solution, dropped the mixture into collagen the foreseeable future, type I collagen is the more feasible
sponges, and subsequently added thrombin to harden fib- option of CTE scaffold in clinical setting.
rinogen. After 3 weeks of culture, ACs in the hybrid In designing scaffold of CTE, physical (i.e., internal
scaffolds showed homogeneous cell distribution and higher structure, pore size, stiffness) and chemical features of
production of GAG and type II collagen compared with scaffold (i.e., blend composition) are crucial in influencing
ACs cultured in sponges without fibrin glue. Interestingly, the differentiation fate and synthetic activities of ACs and
the hybrid scaffolds seem to rescue chondrocyte phenotype MSCs.
during 3 weeks culture, as evidenced in the gradual Different forms of 3-D collagen scaffolds (i.e., sponges,
increasing of GAG/DNA ratio and gene upregulation of hydrogel, nanofibers) possess unique internal structure,
COLII, ACAN, and Sox9 [183]. which may affect chondrogenic activities of ACs and
Xu et al. [184] developed nanofibers hybrid scaffolds by MSCs in the particular ways. For examples, ACs are sus-
employing PCL nanofiber as a housing framework and pended on the fiber network of hydrogel, while ACs
chondrocyte/fibrinogen/collagen gels as a bioactive layer. assume flat appearance on cell wall of sponges. Physical
Electrospinning was used to deposit the fibrous PCL on the features of scaffolds also influence phenotype and activities
1st, 3rd, and 5th layers, while inkjet printing was used to of ACs/MSCs. Scaffolds with low stiffness favor chon-
dispense the solution of Col-layer at 2nd and 4th layers, drogenic phenotype of ACs and chondrogenic differentia-
together with thrombin to allow the gelation of fibrinogen tion of MSCs by discouraging the formation of organized
[184]. The multilayered constructs showed higher ultimate stress fibers or promoting scaffold shrinkage. Strikingly,
tensile strength and Young’s modulus (1.1 and 1.8 MPa) positive influence of pore size apparently depends on cell
compared with isolated component; PCL (0.9 and types. Small pore size (\ 225 lm) is desirable for ACs,
0.8 MPa) or collagen/fibrinogen matrix (incapable for whereas large pore size ([ 300 lm) is suitable for MSCs.
testing) [184]. It indicated that the hybrid scaffolds did not Therefore, the matrices in CTE should be optimized by
affect viability and bioactivity of ACs, as evidenced by the considering type of scaffold and cells used.
live/dead assay (82%) and positive staining for type II Composition of collagen scaffold can be altered by
collagen and GAG after in vitro and in vivo experiments. blending with other molecules (e.g., chitosan, HA, CS) to
On the other hand, Reboredo et al. [179] used electro- improve mechanical properties and biological activities of
spinning to obtain hybrid scaffolds with the structure final scaffold. Despite the versatility of collagen bending,
mimicking native cartilage tissues; the scaffolds had five options of feasible additive materials are limited, as type I
layers, 1st and 5th layers included type I collagen nanofi- collagen easily denature at harsh processing condition.
bers aligned randomly, 2nd and 4th layers consisted of Synthetic polymer is a structurally robust and easy-to-be
mixture of type I and II collagen nanofibers, and 3rd layer fabricated material. However, physical and chemical nature
of aligned type II collagen nanofibers (Fig. 10D). The of synthetic polymer do not allow direct blending with type
hybrid scaffolds showed average elastic modulus and ten- I collagen. To overcome these problems, the concept of
sile strength of * 1046 and * 32 MPa, respectively. hybrid scaffold was introduced. Hybrid scaffolds consist of
MSCs seeded on the hybrid scaffolds synthesized proteo- two parts: (1) housing framework and (2) bioactive filler.
glycan and type II collagen in contrast with human cell Mechanically robust and easy-to-be processed polymer
pellets culture, indicating the layer-by-layer scaffolds may constitute the housing framework. Additionally,
would induce chondrogenic differentiation of MSCs [179]. physically and compositionally optimized type I collagen
may serve as the bioactive filler. Configuration of hybrid

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