Biomaterials and Biosystems 4 (2021) 100030

Contents lists available at ScienceDirect

Biomaterials and Biosystems

journal homepage: www.elsevier.com/locate/bbiosy

Collagen type II: From biosynthesis to advanced biomaterials for cartilage

engineering☆
Z Wu a, SH Korntner a, AM Mullen b, DI Zeugolis a,c,∗
a
Regenerative, Modular & Developmental Engineering Laboratory (REMODEL) and Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM),
National University of Ireland Galway (NUI Galway), Galway, Ireland
b
Teagasc Research Centre, Ashtown, Ireland
c
Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical
Research and School of Mechanical & Materials Engineering, University College Dublin (UCD), Dublin, Ireland

a r t i c l e i n f o a b s t r a c t

Keywords: Collagen type II is the major constituent of cartilage tissue. Yet, cartilage engineering approaches are primarily
Collagen type II based on collagen type I devices that are associated with suboptimal functional therapeutic outcomes. Herein,
Articular cartilage we briefly describe cartilage’s development and cellular and extracellular composition and organisation. We also
Chondrocytes
provide an overview of collagen type II biosynthesis and purification protocols from tissues of terrestrial and
Chondrogenic induction
marine species and recombinant systems. We then advocate the use of collagen type II as a building block in
cartilage engineering approaches, based on safety, efficiency and efficacy data that have been derived over the
years from numerous in vitro and in vivo studies.

1. Introduction degradation products and proinflammatory mediators. Subsequently,

the bone turnover is increased in the subchondral bone and vascular
Articular cartilage is a specialised connective tissue of the joints [1, invasion takes place towards the cartilage region [13]. Clinically, the
2]. Hyaline cartilage provides a smooth and lubricated surface for ar- knee, hip, hand, spine and foot are the most common sites of OA, fol-
ticulation and facilitates the transmission of loads with low frictional lowed by the wrists, shoulders and ankles [14]. OA incidents gradually
coefficient [3, 4]. As articular cartilage lacks blood vessels, lymphat- increase over the years, due to the combined effects of ageing, obe-
ics and nerves, it has limited capacity for intrinsic healing and repair sity and heavy work activities [15–17]. In fact, by 2032, the propor-
[5, 6]. Articular cartilage injuries are frequently caused by sports and tion of the population aged >45 with any doctor-diagnosed OA is es-
recreational activities and, if left untreated, articular cartilage lesions timated to increase from 26.6% to 29.5% [18]. OA is more prevalent
form fibrocartilage and lead to osteoarthritis (OA). in women than in men, with female-to-male ratio ranging from 1.5 to
OA is a whole joint disease, involving structural alterations in the 4.0 [19]. Exercise, weight loss (in the case of overweight patients) and
hyaline articular cartilage, subchondral bone, ligaments, capsule syn- walking aids are widely recommended to improve daily activities of OA
ovium and periarticular muscles [7]. OA affects over 250 million people patients [20, 21]. Pharmacological agents [22–29], surgical procedures
worldwide [8] and financially drains healthcare systems. For example, [30–33] and advanced therapy medicinal products [34–37] have shown
medical costs account for 1.0% to 2.5% of gross domestic product of variable degree of efficiency and effectiveness, considering their com-
high-income countries [9] and in USA alone, the annual insurer spend- plexity, cost of goods and regulatory hurdles. To this end, biomaterial-
ing for OA-related medical care is estimated to be US$ 185.5 billion based therapies are continuously gaining pace, especially for large
[10]. OA, due to the imbalance between the repair and the damage of defects [38–41].
joint cartilage, leads to structural destruction and failure of the syn- Herein, we provide a brief description of cartilage’s development,
ovial joint [11]. The pathogenesis of OA involves compositional changes cellular and extracellular composition and organisation and then focus
and structural / integrity losses of cartilage [12]. Initially, disruption, on collagen type II biosynthesis, extraction protocols, scaffold fabrica-
caused by physical forces, happens at the cartilage surface and is fol- tion and in vitro, in vivo and clinical data, in light of recent studies that
lowed by the expansion of the calcified zone into the radial zone. Then, demonstrate the inability of collagen type I scaffolds to yield functional
the hypertrophic chondrocytes synthesise extracellular matrix (ECM) therapeutic outcomes [42, 43].

☆
This paper was authored by the EiC, but the editorial process of the paper was handled by Prof. Manuela Gomes.
∗
Correspondence author at: REMODEL, NUI Galway & UCD.
E-mail address: [email protected] (D. Zeugolis).

https://doi.org/10.1016/j.bbiosy.2021.100030
Received 31 May 2021; Received in revised form 2 November 2021; Accepted 19 November 2021
2666-5344/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

Fig. 1. Sequential stem cell differentiation to chondrocytes and associated ECM changes (A). Chondrocyte secreted ECM and interactions (B). Chondrocyte cellular
(left) and extracellular (right) organisation (C).

2. Cartilage development, cellular and extracellular composition Articular cartilage contains a highly specialised cell population, the
and organisation chondrocytes, which is responsible for the production, organisation and
maintenance of the cartilage ECM [49, 50]. It also contains a small num-
Cartilage morphogenesis [44–48] is a complex and well-orchestrated ber of mesenchymal progenitor cells, the number of which increases in
sequence of numerous intracellular and extracellular spatiotemporal osteoarthritic articular cartilage [51, 52]. A diverse range of collagen
events, responsible for the tissue composition and structure (Fig. 1). types are encountered in cartilage with distinct functions [53–56]. For
Articular cartilage and long bones are formed by endochondral ossifi- example, collagen type II is the predominant component of the cartilage
cation that is initiated from the lateral growth plate that contains mes- ECM, forms a fibrillar network primarily responsible for the mechanical
enchymal stem cells that secrete hyaluronan and collagen type I. As integrity of the tissue and plays a significant role in chondrocyte dif-
these stem cells move towards the centre of the limb, they aggregate; ferentiation and hypertrophy during normal cartilage development and
stop proliferating and expressing collagen type I; and start expressing OA pathogenesis [57, 58]. Loss of collagen type II has been shown to
N-cadherin, tenascin-C and cell adhesion molecules. Formation of tight accelerate chondrocyte hypertrophy and OA progression, through the
aggregates marks the start of the condensation processes that involves BMP-SMAD1 pathway [59]. Collagen type III is extensively crosslinked
stem cell aggregation and increased hyaluronidase activity that in turn to collagen type II and regulates collagen fibrillar structure and biome-
decreases hyaluronan and cell movement and increases cell-cell inter- chanics in cartilage tissue [60, 61]. Collagen type VI is expressed in both
actions. These increased cell-cell interactions trigger signalling path- healthy and OA cartilage tissues, is the major component of the chondro-
ways responsible for the initiation of chondrogenic differentiation. An cyte pericellular matrix and enhances cartilage regeneration via stimu-
array of small proteoglycans (PGs, e.g. versican, perlecan), growth fac- lation of chondrocyte proliferation [62, 63]. Collagen type IX covalently
tors (e.g. fibroblast growth factors, FGF; bone morphogenetic proteins, crosslinks to collagen type II with the collagenous (called COL3) and the
BMPs; transforming growth factor-𝛽, TGF-𝛽), transcription factors (e.g. non-collagenous (called NC4) domains of the molecules projecting at
Sox5, Sox6, Sox9), signalling molecules (e.g. sonic hedgehog, Indian periodic distances away from the surface of the fibril. These projections
hedgehog) and ECM molecules (e.g. matrilins, fibronectin) contribute allow it to interact with numerous components of cartilage tissue (e.g.
in chondrogenic differentiation and cartilage formation. Subsequently, cartilage oligomeric protein, heparin, fibromodulin), ultimately stabil-
the cells cease the expression of adhesion molecules, resume prolifera- ising and organising the fibrillar collagen network in cartilage [64–68].
tion via action of growth hormone, parathyroid hormone-related pep- Collagen type X is a short chain, non-fibril-forming collagen, primarily
tide and insulin-like growth factor-1 (IGF-1), initiate ECM synthesis (e.g. synthesised by hypertrophic chondrocytes, that enables endochondral
collagen types II, IX, X and XI; aggrecan; decorin; annexin II, V and VI; ossification by regulating matrix mineralisation and is essential for mes-
tenascins; thrombospondins; cartilage oligomeric matrix protein) and enchymal stem cell cartilage formation and endochondral ossification
decrease production of fibronectin. A series of maturation steps then [69–73]. Collagen type XI interacts with various cartilage components
takes place for the differentiation of committed chondrocytes to pre- (e.g. collagen type II, collagen type IX, perlecan, heparan sulphate) to
hypertrophic, hypertrophic and matrix-mineralising chondrocytes. Hy- form a meshwork that provides cartilage matrix stabilisation, mechani-
pertrophic chondrocytes increase in size, start to synthesise calcified ma- cal resilience and homeostasis. The ratio of collagen type XI to collagen
trix rich in collagen type X and alkaline phosphatase, synthesise an array type II regulates fibre diameter, with thick fibres having more collagen
of terminal differentiation molecules (e.g. matrix metalloproteinase-13; type II [74–77]. Articular cartilage also contains a variety of PGs (e.g.
Runx2, Runx3, BMP-6, BMP-2, BMP-7, aggrecan, hyaluronan) and cease aggrecan, decorin, biglycan and fibromodulin) and glycosaminoglycans
to synthesise others (e.g. Sox9, collagen type II). (GAGs, e.g. chondroitin sulphate, keratan sulphate and hyaluronan) that
represent ∼ 10% of tissue dry weight, subject to age and disease state.

2
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

PGs and GAGs play significant role in both normal tissue function and ular crosslinking enables the formation of insoluble and closely packed
arthritis manifestation and progression [78–89]. Indeed, physiological fibrils of 50 nm in diameter, able to withhold mechanical loads [126,
PGs and GAGs synthesis and composition contribute towards normal 127].
cartilage function and properties; control the release and protect against Over the years, various tissues have been used and extraction proto-
proteolysis of bounded cytokines, chemokines and growth factors; and cols have been proposed to obtain collagen type II, albeit with variable
modulate various signalling cascades that facilitate cell attachment and degree of efficiency with respect to purity, from terrestrial and marine
motility and cell-cell and cell-ECM interactions. For example, under species. From the terrestrial species, bovine [128, 129], porcine [130,
physiological conditions, the major PG found in cartilage is aggrecan 131] and chicken [132, 133] tissues are preferred for collagen type II ex-
that interacts with hyaluronan to occupy the interfibrillar space of the traction. It is interesting to note that it has been suggested that collagen
cartilage ECM and provide cartilage with its osmotic properties to re- type II retains memory of the tissue that is extracted from, with articu-
sist compressive loads [90]. On the other hand, in arthritis, PGs and lar cartilage derived collagen type II to be more effective than auricu-
GAGs are significantly degraded by matrix metalloproteinases and their lar and tracheal cartilage derived collagen type II in inducing chondro-
breakdown products are released into synovial fluid, eliciting an inflam- genic differentiation of human stem cells [131]. With respect to marine
matory response [91–93]. Overall, ECM synthesis and degradation are species, squid [134], jellyfish [135, 136], amur sturgeon [137], hoki
regulated by the change of chondrocyte proliferation and metabolism [138] and chondrichthyes (e.g. sharks [139–141], skates [142], rays
under normal and OA conditions [94–98]; the effect of hormones [99– [143]), a diverse group of cartilaginous fish that lack true bone and ex-
101] and growth factors [102–104]; aging [105–107]; oxygen tension hibit a skeleton solely comprised of unmineralized cartilage, have been
[108–111]; and mechanical loading [112–116]. used for collagen type II extraction. In general, high yield, pure colla-
Structurally speaking, articular cartilage is divided into the super- gen type II preparations are produced by acid solubilisation, pepsin di-
ficial, transitional, radial and calcified zones from the joint surface to gestion and repeated salt precipitation / acid solubilisation and finally
the subchondral bone, with distinct composition and architectural fea- dialysis methods (Fig. 2). Considering though the antibiotic usage in an-
tures [117–120]. The superficial zone is the gliding surface of the joint; imal breeding, religious tenets and the potential for interspecies disease
contains high concentration of collagen and low concentration of PGs; transmission [144–146], recombinant collagen technologies have been
and adjoins a layer of elongated chondrocytes organised parallel to the developed for biomedical applications [147–149]. In this frontier, cells
articular surface. The transitional zone contains collagen fibres larger [150–152], yeast [153] and baculovirus-silkworm [154] systems have
than those in the superficial zone, which are arranged randomly within been used to express procollagen type II. Compared with the yeast ex-
this zone; is composed of spheroid-shaped chondrocytes; and has higher pression system, the insect cell expression system has lower background
concentration of PGs compared to the superficial zone. The radial zone interference and facilitates post-translational processing and modifica-
contains the largest collagen fibres, which are organised in a columnar tion [154, 155]. Nonetheless, recombinant technologies are still of low
pattern, perpendicularly to the joint surface; has the lowest concentra- yield and are primarily utilised for niche biomedicine areas [156].
tion of chondrocytes; and has high PG content. Between the radial and
the calcified zone, there is a wavy and irregular line, termed tidemark, 4. Collagen type II scaffolds in cartilage engineering
that prevents the collagen fibres from being sheared of anchorage to the
calcified zone. The calcified zone separates the radial zone of cartilage Mammalian, marine and recombinant collagen type II-based hydro-
from subchondral bone ensuring a cohesive connection between them; gels and sponges (primary scaffold conformations used in cartilage en-
has no PGs; and contains spheroid-shaped chondrocytes, which present gineering) have been shown to both maintain and induce chondrogenic
a hypertrophic phenotype and synthesise collagen type X. phenotype in vitro (Table 1). To enhance mechanical integrity, crosslink-
ing (e.g. poly(ethylene glycol) ether tetrasuccinimidyl glutarate [157],
3. Collagen type II biosynthesis, extraction and synthesis via carbodiimide [158–160], genipin [161]) and/or blending with other
recombinant technologies polymers (e.g. polyvinyl alcohol [162], poly(L-lactide) and poly(lactide-
co-glycolide) [163, 164], chitosan [165]) is traditionally employed. Al-
Collagen type II exhibits a triple stranded, coiled rod-like struc- though structural configurational differences between collagen type II
ture, is expressed as a homotrimer (i.e. [a1(II)]3 ) and is synthesised hydrogels and sponges have been shown to induce different chondro-
exclusively by chondrocytes [121, 122]. During synthesis, the pro- cyte response (with respect to morphology, proliferation and gene ex-
collagen chains undergo proline and lysine hydroxylation by prolyl- pression), in the end, both scaffolds have been shown to stimulate com-
3-hydroxylase, prolyl-4-hydroxylase and lysyl hydroxylase [123]. The parable chondrogenesis [166]. It is also worth noting that collagen type
modification of proline and lysine hydroxylation requires ascorbic acid, II electrospun scaffolds have also been developed [167–169], but the
iron and 2-oxo-glutarate. These steps occur prior to the formation of unavoidable denaturation of collagen prior or during the process [170,
the triple helical structure as hydroxyproline is critical for the stabili- 171] has restricted their use. Advances in engineering (e.g. bioprinted
sation of the collagen triple helix. Prolyl-4-hydroxylase triggers protein collagen type II hydrogels with cell density gradient [172], alginate /
disulphide-isomerase activity, which leads to the formation of the col- collagen type II microbeads [173], hyaluronic acid / collagen type II
lagen triple helix in the endoplasmic reticulum, as the association of microspheres [174]) and/or functionalisation technologies (e.g. chon-
collagen chains requires correct disulphide bond formation in the C- droitin sulphate [175, 176], hyaluronic acid [158], glycosaminoglycan
propeptide region of procollagen. Lysyl hydroxylase catalyses the hy- [177]) have made available elegant collagen type II scaffolds with en-
droxylation of proline and lysine at both helical and non-helical regions hanced in vitro chondrogenic potential. In preclinical setting, collagen
of procollagen polypeptide chains. Moreover, some of the hydroxylysine type II scaffolds (with / without functional molecules and/or with /
residues then undergo glycosylation mediated by hydroxylysyl galac- without cells) have been shown to stimulate hyaline neocartilage forma-
tosyltransferase and galactosylhydroxylysyl glucosyltransferase [124, tion in chondral and osteochondral defects of a diverse range of animal
125]. Procollagen processing and crosslinking occurs in the extracellu- species (Table 2).
lar space. The C-terminal and N-terminal non-helical propeptides of se- Despite all the available data-to-date that have comprehensively
creted procollagen molecules are removed by procollagen C-proteinases shown the importance of collagen type II in chondrogenic induction or
and members of the ADAMTS (a disintegrin and metalloproteinase maintenance and in cartilage repair and regeneration, numerous stud-
with thrombospondin motifs) family of proteases (N-proteinases). Con- ies still utilise collagen type I in cartilage engineering [178–181]. This is
sequently, the removal of propeptides results in a decreased solubility surprising, as the clear superiority of collagen type II over collagen type
of procollagen molecules, which assemble into a collagen triple helical I in cartilage engineering has been well-documented in the literature,
structure with a higher organisation. Subsequent intra- and inter- molec- possibly due to biochemical signals (i.e. the lack of collagen type I and

3
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

Table 1
Indicative examples of mammalian, marine and recombinant collagen type II scaffolds that have been shown to maintain and/or induce chondrogenic phenotype
in vitro. Abbreviations: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-sulfo-N-hydroxy-succinimide: EDC-NHS; Adipose derived stem cells: ADSCs; Bone marrow
stem cells: BMSCs; Cartilage oligomeric matrix protein: COMP; Chondroitin sulphate: CS; Dehydrothermal: DHT; Extracellular matrix: ECM; Fibroblast growth. factor:
FGF; Glycosaminoglycan: GAG; Hyaluronan: HA; Insulin-like growth factor: IGF; Matrix metalloproteinase: MMP; Proteoglycan: PG; Sex-determining region Y-type
box transcription factor 9: SOX9; Ultraviolet irradiation: UV.

Scaffold conformation Major findings, Reference

Bovine collagen type II, CS and HA sponges Chondrocytes maintained round morphology after 14 days of culture. Increased gene expression of
Genipin crosslinked aggrecan, collagen type II and COMP and greater accumulation of PGs was seen on scaffolds with CS and
Human chondrocytes HA than those without CS and HA [161]
Bovine collagen type II sponges The administration of GAGs to culture medium improved cell differentiation tendency to functional
Genipin crosslinked hyaline cartilage, as evidenced by the upregulation of GAG biosynthesis rate and gene expression of
Human chondrocytes aggrecan and collagen type II after 28 days of culture [201]
Bovine collagen type II and CS sponges The cells produced abundant collagen type II on type II scaffolds and collagen type I on type I scaffolds.
No crosslinker The addition of CS upregulated the gene expression of collagen type II, compared to type I and type II
Human BMSCs alone scaffolds [175]
Bovine collagen type II and CS sponges Chondrocytes maintained round morphology, the cells loaded scaffolds were surfaced with a
EDC-NHS crosslinked cartilaginous-like layer and collagen type II scaffolds contained occasionally clusters of cells inside the
Bovine chondrocytes sponges in contrast to collagen type I sponges after 14 days of culture [202]
Bovine collagen type II sponges The primary chondrocytes in the scaffolds maintained chondrogenic phenotype after 3 weeks of culture.
UV crosslinked The gene expression of collagen type II, collagen type XI, and SOX9 in de-differentiated chondrocytes
Murine chondrocytes cultured in the scaffolds decreased when compared to that in primary chondrocytes after 4 weeks of
culture [203]
Bovine collagen type II coated chitosan fibres The cell number, the matrix production (dry weight, GAG quantifications), and the chondrogenic marker
Polyglycolic acid mesh was used as a reference group gene expression (aggrecan, collagen type II) were upregulated in collagen type II coated chitosan scaffolds
No crosslinker compared to pure chitosan scaffolds and polyglycolic acid scaffolds after 21 days of culture [165]
Murine BMSCs
Porcine collagen type II hydrogels Chondrocytes maintained chondrogenic phenotype and the cell density gradient distribution resulted in a
No crosslinker ECM gradient distribution in the scaffolds after 3 weeks of culture [172]
Rabbit chondrocytes
Porcine collagen type II and CS hydrogels Chondrocytes maintained round morphology, the collagen fibres became thicker and arranged neatly
EDC-NHS crosslinked with the increase of CS in the scaffolds and displayed periodic alternation of light and shade after 7 days
Rabbit chondrocytes of culture [176]
Porcine collagen type II and GAG sheets The addition of 5 ng/ml FGF-2 to the culture medium increased the biosynthetic activity of the cells and
DHT and carbodiimide crosslinked the accumulation of GAGs compared to the addition of 25 ng/ml FGF-2, 100 ng/ml IGF-1, 5 ng/ml FGF-2
Canine chondrocytes plus 100 ng/ml IGF-1 after 2 weeks of culture [177]
Porcine collagen type II sponges Most of the chondrocytes were around the periphery of the sponges, the cells tend to be elongated along
EDC/NHS crosslinked the periphery of the scaffolds and round inside the scaffolds. 𝛼-smooth muscle actin is present in the
Canine chondrocytes cytoplasm of the cells after 4 weeks of culture [200]

Porcine collagen type II sponges Chondrocytes maintained chondrogenic morphology and displayed less shrinkage, higher biosynthetic
UV crosslinked activity and more hyaline cartilage-like tissue formation compared to collagen type I scaffolds after 21
Canine chondrocytes days of culture [204]
Porcine collagen type II and GAG sponges After 4 weeks of culture, a range of pore diameter from 25-257 𝜇m did not affect cell-mediated scaffold
UV crosslinked contraction and 𝛼-smooth muscle actin was present in the cytoplasm of the seeded chondrocytes [205]
Canine chondrocytes
Porcine collagen type II and GAG sheets Scaffolds with low cross-link densities (DHT and low EDC/NHS treatment) enhanced cell proliferation,
DHT and EDC-NHS crosslinked chondrogenic maintenance and collagen type II synthesis and increased the rate of scaffold degradation
Canine chondrocytes compared to scaffolds with high cross-link densities (high EDC/NHS treatment) after 2 weeks of culture
[206]
Porcine collagen type II sheets Static compressions of 50% strain decreased the biosynthetic activity of the chondrocytes (the
DHT and EDC-NHS crosslinked accumulation rate of 3 H-proline-labeled protein and 35 S-sulfate-labeled PG over a 24 h period). Dynamic
Canine chondrocytes compression (3% strain, 0.1 Hz superimposed on 10% strain offset) upregulated protein and PG
biosynthesis compared to statically compressed and uncompressed controls after 7 days of culture [207]
Porcine male and female and articular, tracheal and auricular Articular cartilage derived sponges exhibited significantly higher resistance to enzymatic degradation and
cartilage collagen type II sponges 4-arm polyethylene glycol biomechanical properties in comparison to tracheal and auricular cartilage sponges. Articular cartilage
succinimidyl glutarate sponges induced the highest sulphated GAG synthesis and aggrecan and collagen type II mRNA
Human ADSCs expression [208]
Chicken collagen type II and chondroitin sulphate sponges Chondrocytes maintained round morphology. The cell proliferation, the accumulation of proteoglycans
EDC-NHS crosslinked and collagen type II were enhanced in collagen type II and CS scaffolds compared to pure collagen type II
Rabbit chondrocytes scaffolds after 14 days of cell culture. A cartilaginous-like layer was formed at the periphery of the
scaffolds [160]
Chicken collagen type II hydrogels The cells in collagen type II scaffolds maintained chondrogenic phenotype and displayed increased PGs
No crosslinker synthesis compared to the cells on polystyrene. > 50% of the newly synthesized PGs were recovered from
Rabbit chondrocytes collagen type II scaffolds compared to 13-16% of those recovered from polystyrene [209]
Squid collagen type II coating Chondrocytes cultured in the conditioned medium from collagen type II treated M1 macrophages mostly
No crosslinker maintained round morphology and displayed mild increase in the expression of MMP13, compared with
Murine chondrocytes those in the conditioned medium from untreated M1 macrophages [134]
Lesser spotted dogfish, thorn back ray, cuckoo ray and blonde ray The lesser spotted dogfish sponges induced the highest collagen 𝛼1(I), collagen 𝛼1(III) (in comparison to
collagen type II sponges 4-arm polyethylene glycol succinimidyl thorn back ray and blonde ray), COMP (in comparison to cuckoo ray and blonde ray) and aggrecan (in
glutarate comparison to cuckoo ray) gene expression, indicative of highest chondrogenic induction potential [131]
Human ADSCs
Recombinant human collagen type II hydrogels The cells in the scaffolds displayed similar GAGs deposition and similar chondrogenic marker gene
No crosslinker expression and upregulated gene expression of metallopeptidases compared to the high-density cell pellet
Human BMSCs after 84 days of culture [210]
Recombinant human collagen type II hydrogels Chondrocytes maintained round morphology and the accumulation of GAGs and collagen type II
No crosslinker increased after 4 weeks of culture. The gene expression of aggrecan and collagen type II was increased
Bovine chondrocytes since week 1 [211]
Collagen type II (species is not mentioned) hydrogels The dedifferentiated auricular chondrocytes were converted to articular chondrogenic phenotype in a
No crosslinker collagen type II coated environment after 14 days of culture. The converted auricular chondrocytes
Rabbit chondrocytes 4 histological and biomechanical features as articular chondrocytes in the scaffolds after
expressed similar
28 days of culture [212]
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

Table 2
Indicative examples of mammalian, marine and recombinant collagen type II scaffolds in preclinical models. Abbreviations: 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide-sulfo-N-hydroxy-succinimide: EDC-NHS; Adipose derived stem cells: ADSCs; Chondroitin sulphate: CS; Dehydrothermal: DHT; Bone marrow stem
cells: BMSCs; Glycosaminoglycan: GAG; Osteoarthritis: OA; Ultraviolet irradiation: UV

Scaffold conformation Model, Major findings, Reference

Bovine collagen type II, cadherin 11 and recombinant fibronectin sponges Rabbit chondral defect
Glutaraldehyde crosslinked The cell loaded scaffold induced cartilage formation 12 weeks post-surgery
Rabbit BMSCs [213]
Bovine collagen type II and CS sponges Rabbit chondral defect
Genipin crosslinked Lacuna formation 4 weeks post-surgery and high collagen type II and aggrecan
Rabbit BMSCs and low collagen type I gene expression 24 weeks post-surgery [214]
Bovine collagen type I and collagen type II and CS sponges Rabbit chondral defect
Carbodiimide crosslinked Collagen type I scaffolds attracted progenitor cells into the defect and induced
No cells fibro-cartilage repair, whilst collagen type II scaffolds attracted less cells into the
defected, but the invaded cells adopted a chondrogenic phenotype and increased
the amount of superficial cartilage-like tissue 12 weeks post-surgery [188]
Bovine collagen type II hydrogels Rabbit chondral defect
Pentaerythritol polyethylene glycol ether tetrasuccinimidyl glutarate crosslinked Cartilage repair was improved in cell-scaffold treated groups and collagen type I
Rabbit chondrocytes was not detected 24 weeks post-surgery [215]
Bovine collagen type II sponges Rabbit osteochondral defect
Genipin crosslinked The implanted cells became chondrocytes in the implanted area and cartilage
Rabbit BMSCs structure, same as normal cartilage, was observed in the repair site 24 weeks
post-surgery [216]
Porcine collagen type I, collagen type II and collagen type III blend sponges Ovine chondral defect
No crosslinker Scaffolds with chondrocytes and with microfracture into the subchondral plate
Autologous ovine chondrocytes resulted in hyaline-like cartilage regeneration 16 weeks post-surgery [217]
Porcine collagen type II sponges Canine chondral defect
EDC-NHS crosslinked Scaffolds cultured with chondrocytes for 4 weeks prior implantation increased
Autologous chondrocytes the amount of reparative hyaline cartilage tissue after 15 weeks [200]
Porcine collagen type II or Arg-Gly-Asp sequence with poly(L-lactide) or Rabbit chondral defect
poly(D,L-lactide-co-glycolide) sponges Collagen type II prevented infiltration by host tissue and capsule formation,
Carbodiimide crosslinked showed no inflammation and resulted in partial or full repair with equal
Rabbit chondrocytes cellularity and 75-80% matrix contents of a normal rabbit articular cartilage 8
weeks post-surgery [218]
Porcine collagen type II sponges and films Canine chondral defect
UV crosslinked Total defect filling ranged 56-86%, with the greatest amount found in scaffolds
Autologous canine chondrocytes with cells and microfracture compared to scaffolds alone with microfracture and
microfracture alone 15 weeks post-surgery, the tissue filling the defect was
predominantly fibrocartilage [199]
Porcine collagen type II sponges Rabbit chondral defect
No crosslinker Scaffolds displayed quicker effusion absorption, greater newly formed
No cells cartilage-like areas than the empty group 18 weeks post-surgery, sporadic
cartilage signals first appeared at 6 weeks in the scaffolds [219]
Collagen type II hydrogels Rabbit osteochondral defect
No crosslinker Cells seeded collagen type II hydrogels displayed better cartilage repair
Rabbit chondrocytes compared to sham, cell pellet and scaffolds alone groups [212]
Collagen type II-GAG sponges reconstituted from porcine cartilage and bovine Canine chondral defect
collagen type I sponges with shark CS Both cell-seeded scaffolds exhibited comparable cartilage regeneration potential
DHT and UV crosslinked and increased cartilaginous tissue in chondral defects and adjacent subchondral
Autologous canine chondrocytes bone space compared to empty group 15 weeks post-surgery [189]
Chicken collagen type II and fibrin sealant hydrogels Rabbit chondral defect
No crosslinker Improved overall repair of chondral defects, cellular organisation and collagen
Human ADSCs fibre alignment 12 weeks post-surgery [220]
Chicken collagen type II and rat collagen type I blend hydrogels Rabbit chondral defect
No crosslinker Cell-seeded collagen type I/II scaffolds exhibited better cartilage repair
Autologous rabbit BMSCs outcomes in trochlear groove defects compared to pure collagen type I
hydrogels and empty chondral defects 13 weeks post-surgery [187]
Squid collagen type II intra-articular injection Suppressed pro-inflammatory macrophage phenotype, prevented hypertrophic
No crosslinker chondrocyte phenotype and alleviated inflammation in an OA rat model 6
No cells weeks after OA induction [134]
Shark collagen type II was administered orally Facilitated recovery of articular membranes in the ankle joint and suppressed
No crosslinker rheumatoid arthritis in a complete Freund’s adjuvant-induced rheumatoid
No cells arthritis rat model 2 weeks after rheumatoid arthritis induction [221]
Recombinant collagen type II hydrogels Rabbit osteochondral defect
No crosslinker Cell-scaffold treated group exhibited a slight but insignificant improvement in
Autologous rabbit chondrocytes cartilage repair compared to spontaneous repair group and both groups had
lower modified O’Driscoll’s score than intact cartilage 24 weeks post-surgery
[222]
Recombinant collagen type II and polylactide sponges Porcine chondral defect
Carbodiimide crosslinked Hyaline cartilage formed most frequently in the recombinant collagen type II /
Autologous porcine chondrocytes polylactide / cells group, which also improved biomechanically properties only
over the spontaneous repair group and showed less adverse subchondral
reactions than the Chondro-Gide® (a bilayer collagen type I / collagen type III
membrane) / cells group, but not in comparison to the spontaneous repair group
16 weeks post-surgery [190]

5
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

Fig. 2. Collagen type II extraction flow chart

from cartilage tissues (the detailed protocol can
be found here [131, 143]) and electrophoretic
mobility of collagen type I and collagen type
II (the detailed protocol can be found here
[223]). Notes: As the same protocol is used
to extract collagen type I [224–226], atten-
tion should be paid during dissection to remove
all not cartilaginous tissues. Tissue to acetic
acid / pepsin solution ratio: 1:1 g/l. Tissue to
pepsin ratio: 10:1 w/w. High activity pepsin
(e.g. 3200-4500 units per mg protein) is recom-
mended. Sieve: approximately 1,000 𝜇m in di-
ameter. Filter mesh: 100 𝜇m in diameter. Cen-
trifugation details: 20 min, 8000 rpm, < 8 °C.
After the second NaCl precipitation, dissolution
is conducted in minimum amount of acetic acid
in order to produce a high in concentration col-
lagen type II solution. All experiments are con-
ducted at 4-8 ºC to avoid collagen denaturation.

the presence of collagen type II and other bounded cartilage-specific In the commercial arena, it is interesting to note that, to the best of
constituents) [131]. In in vitro setting, for example, collagen type II, our knowledge, only a handful of companies provide high purity col-
as opposed to collagen type I, scaffolds have been shown to maintain lagen type II (e.g. porcine articular cartilage derived collagen type II,
round chondrocyte morphology and to significantly increase DNA and Symatese) and that no single collagen type II-based device is available,
collagen type II and GAG synthesis [182, 183]. Collagen type II, as op- whilst numerous collagen type I devices are available for cartilage en-
posed to collagen type I, scaffolds have also been shown to more ef- gineering (e.g. Chondro-Gide®, Geistlich Pharma AG; Novocart® Basic,
fectively induce chondrogenic induction of adipose derived stem cells TETEC AG; MeRG®, Bioteck Srl), despite the fact that collagen type I
[184] and bone marrow stem cells [175, 185], as judged by round cell scaffolds have failed to demonstrate efficiency in healing of human os-
morphology (via the integrin 𝛽1-mediated Rho A/Rock signalling path- teochondral [43] and large cartilage [42] defects. This limited technol-
way [184]), upregulation of chondrogenic genes (e.g. collagen type II, ogy transfer of collagen type II can be attributed to two main reasons.
collagen type X, aggrecan, COMP, SOX6, SOX9) and increased synthesis Firstly, we believe that commercialisation of collagen type II-based de-
of cartilage matrix (e.g. collagen type II, PG, GAG). It is also worth not- vices has been compromised by early studies that showed native col-
ing that increased collagen type II, as opposed to collagen type I, scaf- lagen type II from human, chick, murine and bovine cartilage to in-
folds induced differentiation of adipose derived stem cells to nucleus duce inflammatory arthritis in rats [191–193] and in non-human pri-
pulposus cells, as judged by increased SOX9, aggrecan and collagen mates [194]; and antibodies of native and denatured collagen type II
type II gene expression; increased sulphated PG synthesis; expression of to be present in patients with early rheumatoid arthritis and chronic
KRT19 marker; and increased phosphorylated Smad3 expression [186]. gouty arthritis [195–197]. It is worth noting though that effectively
These in vitro observations were also verified in preclinical models. For crosslinked collagen type II does not induce arthritis in rats [198] and
example, in cartilage defects in the femurs of rabbits, 13 weeks post studies have demonstrated collagen type II devices to promote efficient
implantation, collagen type I / collagen type II hydrogels showed sta- defect filling and hyaline neocartilage formation. For example, 15 weeks
tistically higher cartilage repair score than either collagen type I alone post operation, defects in trochlear grooves of adult dogs (that were
hydrogels (both loaded with bone marrows stem cells) or empty defect treated with microfracture, microfracture with collagen type II scaffold
controls (pure collagen type II hydrogels were not used) [187]. In full- and collagen type II scaffold loaded chondrocytes) resulted in 56% to
thickness defects in the femoral trochlea of adolescent rabbits, although 86% total defect filling, with the microfracture with collagen type II scaf-
collagen type I scaffolds induced higher than collagen type II scaffolds fold treatment group inducing the highest defect filling capacity [199].
cell migration into the defect, the collagen type II scaffolds more ef- When collagen type II scaffold were seeded for 4 weeks with chondro-
fectively than collagen type I scaffolds directed invaded cells towards cytes (after 3 weeks of monolayer expansion) and then implanted in
chondrocyte phenotype and 12 weeks post implantation, the cartilage a canine trochlear groove defect model, 15 weeks post implantation,
contours in defects with collagen type I scaffolds were repaired with although the repaired tissue formed had significantly lower compres-
fibro-cartilage tissue, whilst defects treated with collagen type II scaf- sive stiffness than the native cartilage, the total defect filling ranged
folds, although the original contour was not completely restored in all from 70% to 100%, with hyaline cartilage accounting for 42 ± 10%
animals, showed an increase in the amount of superficial cartilage-like of the defect area [200]. The second issue that may be responsible for
tissue [188]. In the trochlea grooves of the knees of dogs, 15 weeks post the limited use of collagen type II in medical device development is the
implantation, groups treated with collagen type II scaffolds and chon- difficulty in producing high amounts of high purity and high yield, all
drocytes showed the greatest total amount of reparative tissue and the in comparison to collagen type I, collagen type II preparations. Obvi-
tissue at subchondral region of defects was positive for collagen type ously, the main reason behind this is the articular cartilage tissue avail-
II, GAGs and PGs [189]. In a 4-month-old domestic pig full-thickness ability, in comparison to skin, for example, tissue. Having said that, a
cartilage lesion model, 4 months post operation, a human recombinant typical cartilage defect is a lot smaller than a typical skin defect and
collagen type II / polylactide scaffold most frequently formed hyaline therefore extraction of collagen type II constitutes a value for money
cartilage than the spontaneous healing group and a collagen type I / proposition.
collagen type III scaffold [190].

6
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

5. Conclusions [15] Coggon D, Reading I, Croft P, McLaren M, Barrett D, Cooper C. Knee osteoarthritis
and obesity. Int J Obes Relat Metab Disord 2001;25:622–7.
[16] Silverwood V, Blagojevic-Bucknall M, Jinks C, Jordan J, Protheroe J, Jordan K.
Cartilage injuries and pathophysiologies continuously increase and Current evidence on risk factors for knee osteoarthritis in older adults: a systematic
financially drain healthcare systems worldwide. In the quest of the op- review and meta-analysis. Osteoarthritis Cartilage 2015;23:507–15.
timal building block for cartilage engineering scaffolds, collagen type II [17] Harris EC, Coggon D. Hip osteoarthritis and work. Best Prac Res Clin Rheumatol
2015;29:462–82.
has been overlooked due to either outdated data or economic drivers, [18] Turkiewicz A, Petersson IF, Björk J, Hawker G, Dahlberg LE, Lohmander LS,
despite being the most abundant extracellular matrix component of car- Englund M. Current and future impact of osteoarthritis on health care: A
tilage. This review clearly illustrates the beneficial effects of collagen population-based study with projections to year 2032. Osteoarthritis Cartilage
2014;22:1826–32.
type II in cartilage engineering and urges the adaptation of a more ra-
[19] Arden N, Nevitt MC. Osteoarthritis: Epidemiology. Best Prac Res Clin Rheumatol
tional and biomimetic approach in designing biomaterial-based thera- 2006;20:3–25.
peutic strategies for functional cartilage repair and regeneration. [20] Kolasinski SL, Neogi T, Hochberg MC, Oatis C, Guyatt G, Block J, Callahan L, Copen-
haver C, Dodge C, Felson D. American College of Rheumatology/Arthritis Founda-
tion guideline for the management of osteoarthritis of the hand, hip, and knee.
Conflict of Interest Arthritis Care Res 2019;72(2020):149–62.
[21] Nelson AE, Allen KD, Golightly YM, Goode AP, Jordan JM. A systematic review of
recommendations and guidelines for the management of osteoarthritis: The chronic
The authors declare no conflict of interest. osteoarthritis management initiative of the US bone and joint initiative. Semin
Arthritis Rheum 2014;43:701–12.
[22] da Costa BR, Reichenbach S, Keller N, Nartey L, Wandel S, Jüni P, Trelle S. Effec-
Declaration of interests
tiveness of non-steroidal anti-inflammatory drugs for the treatment of pain in knee
and hip osteoarthritis: a network meta-analysis. Lancet 2017;390:e21–33.
The authors declare that they have no known competing financial [23] Zeng C, Wei J, Persson MS, Sarmanova A, Doherty M, Xie D, Wang Y, Li X,
interests or personal relationships that could have appeared to influence Li J, Long H. Relative efficacy and safety of topical non-steroidal anti-inflam-
matory drugs for osteoarthritis: a systematic review and network meta-analy-
the work reported in this paper. sis of randomised controlled trials and observational studies. Br J Sports Med
2018;52:642–50.
[24] Wang ZY, Shi SY, Li SJ, Chen F, Chen H, Lin HZ, Lin JM. Efficacy and safety of
Acknowledgements
duloxetine on osteoarthritis knee pain: a meta-analysis of randomized controlled
trials. Pain Med 2015;16:1373–85.
This work forms part of the Teagasc Walsh Fellowship (grant agree- [25] Lohmander LS, Hellot S, Dreher D, Krantz EF, Kruger DS, Guermazi A, Eck-
stein F. Intraarticular sprifermin (recombinant human fibroblast growth factor 18)
ment No. 2014045) and the ReValueProtein Research Project (grant
in knee osteoarthritis: a randomized, double-blind, placebo-controlled trial. Arthri-
agreement No. 11/F/043) supported by the Department of Agriculture, tis Rheumatol 2014;66:1820–31.
Food and the Marine under the National Development Plan 2007-2013 [26] Deshmukh V, Hu H, Barroga C, Bossard C, Kc S, Dellamary L, Stewart J, Chiu K,
funded by the Irish Government. This publication has emanated from Ibanez M, Pedraza M. A small-molecule inhibitor of the Wnt pathway (SM04690)
as a potential disease modifying agent for the treatment of osteoarthritis of the
research supported by grants from Science Foundation Ireland (SFI) un- knee. Osteoarthritis Cartilage 2018;26:18–27.
der grant numbers 15/CDA/3629 and 19/FFP/6982 and Science Foun- [27] Sampson ER, Hilton MJ, Tian Y, Chen D, Schwarz EM, Mooney RA, Bukata SV,
dation Ireland (SFI) and European Regional Development Fund (ERDF) O’Keefe RJ, Awad H, Puzas JE. Teriparatide as a chondroregenerative therapy for
injury-induced osteoarthritis. Sci Transl Med 2011;3:101ra193.
under grant number 13/RC/2073_2. This work has also received funding [28] Kosloski MP, Goss S, Wang SX, Liu J, Loebbert R, Medema JK, Liu W, Dutta S. Phar-
from the European Research Council (ERC) under the European Union’s macokinetics and tolerability of a dual variable domain immunoglobulin ABT-981
Horizon 2020 research and innovation programme, grant agreement No. against IL-1𝛼 and IL-1𝛽 in healthy subjects and patients with osteoarthritis of the
knee. J Clin Pharmacol 2016;56:1582–90.
866126. [29] Huang Z, Ding C, Li T, Yu SP-C. Current status and future prospects for disease
modification in osteoarthritis. Rheumatology 2018;57 iv108-iv123.
References [30] Kraeutler MJ, Aliberti GM, Scillia AJ, McCarty EC, Mulcahey MK. Microfracture
versus drilling of articular cartilage defects: a systematic review of the basic science
[1] Kazemnejad S, Khanmohammadi M, Baheiraei N, Arasteh S. Current state of carti- evidence. Orthop J Sports Med 2020;8:2325967120945313.
lage tissue engineering using nanofibrous scaffolds and stem cells. Avicenna J Med [31] Gao L, Goebel LK, Orth P, Cucchiarini M, Madry H. Subchondral drilling for articu-
Biotechnol 2017;9:50–65. lar cartilage repair: a systematic review of translational research. Dis Model Mech
[2] Goldberg A, Mitchell K, Soans J, Kim L, Zaidi R. The use of mesenchymal stem 2018;11 dmm034280.
cells for cartilage repair and regeneration: a systematic review. J Orthop Surg Res [32] Gowd AK, Cvetanovich GL, Liu JN, Christian DR, Cabarcas BC, Redondo ML,
2017;12:39. Verma NN, Yanke AB, Cole BJ. Management of chondral lesions of the knee: anal-
[3] Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: structure, ysis of trends and short-term complications using the national surgical quality im-
composition, and function. Sports Health 2009;1:461–8. provement program database. Arthroscopy 2019;35:138–46.
[4] Kelly P, O’Connor J. Transmission of rapidly applied loads through articular carti- [33] DeFroda SF, Bokshan SL, Yang DS, Daniels AH, Owens BD. Trends in the sur-
lage Part 1: Uncracked cartilage. Proc Inst Mech Eng H 1996;210:27–37. gical treatment of articular cartilage lesions in the United States from 2007
[5] Pickett AM, Hensley Jr DT. Knee cell-based cartilage restoration. J Knee Surg to 2016. J Knee Surg 2020 May 29. Epub ahead of print. PMID: 32483798.
2019;32:127–33. doi:10.1055/s-0040-1712946.
[6] Reissis D, Tang QO, Cooper NC, Carasco CF, Gamie Z, Mantalaris A, Tsiridis E. Cur- [34] Graceffa V, Vinatier C, Guicheux J, Stoddart M, Alini M, Zeugolis DI.
rent clinical evidence for the use of mesenchymal stem cells in articular cartilage Chasing chimeras–the elusive stable chondrogenic phenotype. Biomaterials
repair. Expert Opin Biol Ther 2016;16:535–57. 2019;192:199–225.
[7] Poole AR. Osteoarthritis as a whole joint disease. HSS J 2012;8:4–6. [35] Graceffa V, Vinatier C, Guicheux J, Evans CH, Stoddart M, Alini M, Zeugolis DI.
[8] Carlson AK, Rawle RA, Adams E, Greenwood MC, Bothner B, June RK. Applica- State of art and limitations in genetic engineering to induce stable chondrogenic
tion of global metabolomic profiling of synovial fluid for osteoarthritis biomarkers. phenotype. Biotechnol Adv 2018;36:1855–69.
Biochem Biophys Res Commun 2018;499:182–8. [36] Pourakbari R, Khodadadi M, Aghebati-Maleki A, Aghebati-Maleki L, Yousefi M.
[9] Hunter DJ, Schofield D, Callander E. The individual and socioeconomic impact of The potential of exosomes in the therapy of the cartilage and bone complications;
osteoarthritis. Nat Rev Rheumatol 2014;10:437–41. Emphasis on osteoarthritis. Life Sci 2019;236:116861.
[10] Kotlarz H, Gunnarsson CL, Fang H, Rizzo JA. Insurer and out-of-pocket costs of [37] Confalonieri D, Schwab A, Walles H, Ehlicke F. Advanced therapy medicinal prod-
osteoarthritis in the US: evidence from national survey data. Arthritis Rheum ucts: a guide for bone marrow-derived MSC application in bone and cartilage tissue
2009;60:3546–53. engineering. Tissue Eng Part B 2018;24:155–69.
[11] Fu K, Robbins SR, McDougall JJ. Osteoarthritis: the genesis of pain. Rheumatology [38] Wei W, Ma Y, Yao X, Zhou W, Wang X, Li C, Lin J, He Q, Leptihn S, Ouyang H.
2018;57 iv43-iv50. Advanced hydrogels for the repair of cartilage defects and regeneration. Bioact
[12] Horton W, Bennion P, Yang L. Cellular, molecular, and matrix changes in cartilage Mater 2020;6:998–1011.
during aging and osteoarthritis. J Musculoskelet Neuronal Interact 2006;6:379–81. [39] Yilmaz EN, Zeugolis DI. Electrospun polymers in cartilage engineering—state of
[13] Loeser RF, Collins JA, Diekman BO. Ageing and the pathogenesis of play. Front Bioeng Biotechnol 2020;8:77.
osteoarthritis. Nat Rev Rheumatol 2016;12:412–20 PMID: 32483798. [40] Armiento A, Stoddart M, Alini M, Eglin D. Biomaterials for articular cartilage tissue
doi:10.1055/s-0040-1712946. engineering: learning from biology. Acta Biomater 2018;65:1–20.
[14] Newman AB, Haggerty CL, Goodpaster B, Harris T, Kritchevsky S, Nevitt M, [41] Campos Y, Almirall A, Fuentes G, Bloem HL, Kaijzel EL, Cruz LJ. Tissue engineering:
Miles TP, Visser M. Strength and muscle quality in a well-functioning cohort of an alternative to repair cartilage. Tissue Eng Part B 2019;25:357–73.
older adults: the health, aging and body composition study. J Am Geriatr Soc [42] Schüttler K-F, Götschenberg A, Klasan A, Stein T, Pehl A, Roessler P, Figiel J,
2003;51:323–30. Heyse T, Efe T. Cell-free cartilage repair in large defects of the knee: increased

7
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

failure rate 5 years after implantation of a collagen type I scaffold. Arch Orthop staal M, van de Peppel J, Wolvius E, Narcisi R. Collagen type X is essential for
Trauma Surg 2019;139:99–106. successful mesenchymal stem cell-mediated cartilage formation and subsequent
[43] Christensen BB, Foldager CB, Jensen J, Jensen NC, Lind M. Poor osteochondral endochondral ossification. Eur Cell Mater 2019;38:106–22.
repair by a biomimetic collagen scaffold: 1-to 3-year clinical and radiological fol- [74] Rodriguez R, Seegmiller R, Stark M, Bridgewater L. A type XI collagen mutation
low-up. Knee Surg Sports Traumatol Arthrosc 2016;24:2380–7. leads to increased degradation of type II collagen in articular cartilage. Osteoarthri-
[44] Goldring M, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem tis Cartilage 2004;12:314–20.
2006;97:33–44. [75] Smith SM, Melrose J. Type XI collagen–perlecan–HS interactions stabilise the peri-
[45] Mackie E, Ahmed Y, Tatarczuch L, Chen K, Mirams M. Endochondral ossification: cellular matrix of annulus fibrosus cells and chondrocytes providing matrix stabil-
how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell isation and homeostasis. J Mol Histol 2019;50:285–94.
Biol 2008;40:46–62. [76] Lawrence EA, Kague E, Aggleton JA, Harniman RL, Roddy KA, Hammond CL.
[46] Gadjanski I, Spiller K, Vunjak-Novakovic G. Time-dependent processes in stem cel- The mechanical impact of col11a2 loss on joints; Col11a2 mutant zebrafish show
l-based tissue engineering of articular cartilage. Stem Cell Rev Rep 2012;8:863–81. changes to joint development and function, which leads to early-onset osteoarthri-
[47] Tsang K, Tsang S, Chan D, Cheah K. The chondrocytic journey in endochondral tis. Philos Trans R Soc Lond B 2018;373:20170335.
bone growth and skeletal dysplasia. Birth Defects Res C 2014;102:52–73. [77] Garofalo S, Metsäranta M, Ellard J, Smith C, Horton W, Vuorio E, de Crombrug-
[48] Chijimatsu R, Saito T. Mechanisms of synovial joint and articular cartilage devel- ghe B. Assembly of cartilage collagen fibrils is disrupted by overexpression of nor-
opment. Cell Mol Life Sci 2019;76:3939–52. mal type II collagen in transgenic mice. Proc Natl Acad Sci U S A 1993;90:3825–9.
[49] Lotz M, Loeser RF. Effects of aging on articular cartilage homeostasis. Bone [78] Eyre D. Articular cartilage and changes in arthritis: collagen of articular cartilage.
2012;51:241–8. Arthritis Res Ther 2001;4:30–5.
[50] Akkiraju H, Nohe A. Role of chondrocytes in cartilage formation, progression of [79] Kuiper N, Sharma A. A detailed quantitative outcome measure of glycosaminogly-
osteoarthritis and cartilage regeneration. J Dev Biol 2015;3:177–92. cans in human articular cartilage for cell therapy and tissue engineering strategies.
[51] Fickert S, Fiedler J, Brenner RE. Identification of subpopulations with character- Osteoarthritis Cartilage 2015;23:2233–41.
istics of mesenchymal progenitor cells from human osteoarthritic cartilage using [80] Watanabe H, Cheung SC, Itano N, Kimata K, Yamada Y. Identification of hyaluro-
triple staining for cell surface markers. Arthritis Res Ther 2004;6:1–11. nan-binding domains of aggrecan. J Biol Chem 1997;272:28057–65.
[52] Alsalameh S, Amin R, Gemba T, Lotz M. Identification of mesenchymal progeni- [81] Horkay F, Basser PJ, Hecht AM, Geissler E. Structure and properties of cartilage
tor cells in normal and osteoarthritic human articular cartilage. Arthritis Rheum proteoglycans. Macromol Symp 2017;372:43–50.
2004;50:1522–32. [82] Vynios DH. Metabolism of cartilage proteoglycans in health and disease. Biomed
[53] Eyre DR, Weis MA, Wu J-J. Articular cartilage collagen: an irreplaceable frame- Res Int 2014;2014:1–9.
work. Eur Cell Mater 2006;12:57–63. [83] Knudson CB, Knudson W. Cartilage proteoglycans. Semin Cell Dev Biol
[54] Aigner T, Stöve J. Collagens—major component of the physiological cartilage ma- 2001;12:69–78.
trix, major target of cartilage degeneration, major tool in cartilage repair. Adv Drug [84] Roughley PJ, Lee ER. Cartilage proteoglycans: Structure and potential functions.
Deliv Rev 2003;55:1569–93. Microsc Res Tech 1994;28:385–97.
[55] Luo Y, Sinkeviciute D, He Y, Karsdal M, Henrotin Y, Mobasheri A, Önner- [85] J. Bertrand, A. Held, Role of proteoglycans in osteoarthritis, Cartilage, Springer
fjord P, Bay-Jensen A. The minor collagens in articular cartilage. Protein Cell 2017, pp. 63-80.
2017;8:560–72. [86] Muir H. Proteoglycans of cartilage. J Clin Pathol Suppl 1978;12:67–81.
[56] Bielajew BJ, Hu JC, Athanasiou KA. Collagen: Quantification, biomechanics and [87] Carney S, Muir H. The structure and function of cartilage proteoglycans. Physiol
role of minor subtypes in cartilage. Nat Rev Mater 2020;5:730–47. Rev 1988;68:858–910.
[57] Yang C, Rui H, Mosler S, Notbohm H, Sawaryn A, Müller P. Collagen II from ar- [88] Mankin HJ, Lippiello L. The glycosaminoglycans of normal and arthritic cartilage.
ticular cartilage and annulus fibrosus: structural land functional implication of tis- J Clin Invest 1971;50:1712–19.
sue specific posttranslational modifications of collagen molecules. Eur J Biochem [89] Hjertquist S-O, Lemperg R. Identification and concentration of the glycosaminogly-
1993;213:1297–302. cans of human articular cartilage in relation to age and osteoarthritis. Calcif Tissue
[58] Nah HD, Swoboda B, Birk DE, Kirsch T. Type IIA procollagen: expression in devel- Res 1972;10:223–37.
oping chicken limb cartilage and human osteoarthritic articular cartilage. Dev Dyn [90] Han E, Chen S, Klisch S, Sah R. Contribution of proteoglycan osmotic
2001;220:307–22. swelling pressure to the compressive properties of articular cartilage. Biophys J
[59] Lian C, Wang X, Qiu X, Wu Z, Gao B, Liu L, Liang G, Zhou H, Yang X, Peng Y. 2011;101:916–24.
Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis [91] Roughley P. Articular cartilage and changes in arthritis: noncollagenous pro-
progression by promoting integrin 𝛽1−SMAD1 interaction. Bone Res 2019;7:1–15. teins and proteoglycans in the extracellular matrix of cartilage. Arthritis Res
[60] Wang C, Brisson BK, Terajima M, Li Q, Han B, Goldberg AM, Liu XS, Marco- 2001;3:342–7.
longo MS, Enomoto-Iwamoto M, Yamauchi M. Type III collagen is a key regula- [92] Ghosh P, Smith M. Osteoarthritis, genetic and molecular mechanisms. Biogerontol-
tor of the collagen fibrillar structure and biomechanics of articular cartilage and ogy 2002;3:85–8.
meniscus. Matrix Biol 2020;85:47–67. [93] Fosang A, Last K, Maciewicz R. Aggrecan is degraded by matrix metalloproteinases
[61] Wu J-J, Weis MA, Kim LS, Eyre DR. Type III collagen, a fibril network modifier in in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activ-
articular cartilage. J Biol Chem 2010;285:18537–44. ities can be independent. J Clin Invest 1996;98:2292–9.
[62] Smeriglio P, Dhulipala L, Lai JH, Goodman SB, Dragoo JL, Smith RL, Maloney WJ, [94] Lippiello L, Hall D, Mankin H. Collagen synthesis in normal and osteoarthritic hu-
Yang F, Bhutani N. Collagen VI enhances cartilage tissue generation by stimulating man cartilage. J Clin Invest 1977;59:593–600.
chondrocyte proliferation. Tissue Eng Part A 2015;21:840–9. [95] Nelson F, Dahlberg L, Laverty S, Reiner A, Pidoux I, Ionescu M, Fraser GL, Brooks E,
[63] Pullig O, Weseloh G, Swoboda B. Expression of type VI collagen in normal and Tanzer M, Rosenberg LC. Evidence for altered synthesis of type II collagen in pa-
osteoarthritic human cartilage. Osteoarthritis Cartilage 1999;7:191–202. tients with osteoarthritis. J Clin Invest 1998;102:2115–25.
[64] Müller-Glauser W, Humbel B, Glatt M, Sträuli P, Winterhalter KH, Bruckner P. On [96] Maldonado M, Nam J. The role of changes in extracellular matrix of cartilage in
the role of type IX collagen in the extracellular matrix of cartilage: Type IX collagen the presence of inflammation on the pathology of osteoarthritis. Biomed Res Int
is localized to intersections of collagen fibrils. J Cell Biol 1986;102:1931–9. 2013:1–10 2013.
[65] Diab M, Wu J, Eyre D. Collagen type IX from human cartilage: a structural profile [97] Goldring MB. Chondrogenesis, chondrocyte differentiation, and articular car-
of intermolecular cross-linking sites. Biochem J 1996;314:327–32. tilage metabolism in health and osteoarthritis. Ther Adv Musculoskelet Dis
[66] Parsons P, Gilbert SJ, Vaughan-Thomas A, Sorrell DA, Notman R, Bishop M, 2012;4:269–85.
Hayes AJ, Mason DJ, Duance VC. Type IX collagen interacts with fibronectin [98] Rim YA, Nam Y, Ju JH. The role of chondrocyte hypertrophy and senescence in
providing an important molecular bridge in articular cartilage. J Biol Chem osteoarthritis initiation and progression. Int J Mol Sci 2020;21:2358.
2011;286:34986–97. [99] Talwar RM, Wong BS, Svoboda K, Harper RP. Effects of estrogen on chondrocyte
[67] Brewton R, Wright D, Mayne R. Structural and functional comparison of type IX proliferation and collagen synthesis in skeletally mature articular cartilage. J Oral
collagen-proteoglycan from chicken cartilage and vitreous humor. J Biol Chem Maxillofac Surg 2006;64:600–9.
1991;266:4752–7. [100] Stevens DA, Williams GR. Hormone regulation of chondrocyte differentiation and
[68] Blumbach K, Bastiaansen-Jenniskens Y, DeGroot J, Paulsson M, Van Osch G, Za- endochondral bone formation. Mol Cell Endocrinol 1999;151:195–204.
ucke F. Combined role of type IX collagen and cartilage oligomeric matrix protein [101] He H, Wang C, Tang Q, Yang F, Xu Y. Elucidation of possible molecular mechanisms
in cartilage matrix assembly: cartilage oligomeric matrix protein counteracts type underlying the estrogen-induced disruption of cartilage development in zebrafish
IX collagen–induced limitation of cartilage collagen fibril growth in mouse chon- larvae. Toxicol Lett 2018;289:22–7.
drocyte cultures. Arthritis Rheum 2009;60:3676–85. [102] Fortier LA, Barker JU, Strauss EJ, McCarrel TM, Cole BJ. The role of growth factors
[69] Von der Mark K, Kirsch T, Nerlich A, Kuss A, Weseloh G, Glückert K, Stöss H. Type in cartilage repair. Clin Orthop Relat Res 2011;469:2706–15.
X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte [103] Ellman M, Yan D, Ahmadinia K, Chen D, An H, Im H. Fibroblast growth factor
hypertrophy. Arthritis Rheum 1992;35:806–11. control of cartilage homeostasis. J Cell Biochem 2013;114:735–42.
[70] Shen G. The role of type X collagen in facilitating and regulating endochondral [104] Patil A, Sable R, Kothari R. An update on transforming growth factor-𝛽 (TGF-𝛽):
ossification of articular cartilage. Orthod Craniofac Res 2005;8:11–17. sources, types, functions and clinical applicability for cartilage/bone healing. J Cell
[71] Aigner T, Reichenberger E, Bertling W, Kirsch T, Stöss H, Von der Mark K. Type X Physiol 2011;226:3094–103.
collagen expression in osteoarthritic and rheumatoid articular cartilage. Virchows [105] DeGroot J, Verzijl N, Bank RA, Lafeber FP, Bijlsma JW, TeKoppele JM. Age-related
Arch B 1993;63:205–11. decrease in proteoglycan synthesis of human articular chondrocytes: The role of
[72] von der Mark K, Frischholz S, Aigner T, Beier F, Belke J, Erdmann S, Burkhardt H. nonenzymatic glycation. Arthritis Rheum 1999;42:1003–9.
Upregulation of type X collagen expression in osteoarthritic cartilage. Acta Orthop [106] Madej W, van Caam A, Davidson EB, Hannink G, Buma P, van der Kraan P. Age-
Scand Suppl 1995;66:125–9. ing is associated with reduction of mechanically-induced activation of Smad2/3P
[73] Knuth C, Sastre EAndres, Fahy N, Witte-Bouma J, Ridwan Y, Strabbing E, Koud- signaling in articular cartilage. Osteoarthritis Cartilage 2016;24:146–57.

8
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

[107] Rahmati M, Nalesso G, Mobasheri A, Mozafari M. Aging and osteoarthritis: central [139] Jeevithan E, Bao B, Bu Y, Zhou Y, Zhao Q, Wu W. Type II collagen and gelatin
role of the extracellular matrix. Ageing Res Rev 2017;40:20–30. from silvertip shark (Carcharhinus albimarginatus) cartilage: Isolation, purifi-
[108] Fermor B, Christensen S, Youn I, Cernanec J, Davies C, Weinberg J. Oxygen, nitric cation, physicochemical and antioxidant properties. Mar Drugs 2014;12:3852–
oxide and articular cartilage. Eur Cell Mater 2007;13:56–65. 3873.
[109] Buckley CT, Vinardell T, Kelly DJ. Oxygen tension differentially regulates the func- [140] Kittiphattanabawon P, Benjakul S, Visessanguan W, Shahidi F. Isolation and charac-
tional properties of cartilaginous tissues engineered from infrapatellar fat pad de- terization of collagen from the cartilages of brownbanded bamboo shark (Chiloscyl-
rived MSCs and articular chondrocytes. Osteoarthritis Cartilage 2010;18:1345– lium punctatum) and blacktip shark (Carcharhinus limbatus). LWT-Food Sci Tech-
1354. nol 2010;43:792–800.
[110] Qu C, Lindeberg H, Ylärinne JH, Lammi MJ. Five percent oxygen tension is not [141] Merly L, Smith SL. Collagen type II, alpha 1 protein: a bioactive component of shark
beneficial for neocartilage formation in scaffold-free cell cultures. Cell Tissue Res cartilage. Int Immunopharmacol 2013;15:309–15.
2012;348:109–17. [142] Mizuta S, Hwang J, Yoshinaka R. Molecular species of collagen in pectoral fin car-
[111] Shi Y, Ma J, Zhang X, Li H, Jiang L, Qin J. Hypoxia combined with spheroid culture tilage of skate (Raja kenojei). Food Chem 2003;80:1–7.
improves cartilage specific function in chondrocytes. Integr Biol 2015;7:289–97. [143] Wu Z, Korntner S, Mullen A, Zeugolis D. In the quest of the optimal chondrichthyan
[112] Bleuel J, Zaucke F, Brüggemann G-P, Niehoff A. Effects of cyclic tensile strain on for the development of collagen sponges for articular cartilage. J Sci-Adv Mater Dev
chondrocyte metabolism: a systematic review. PLoS One 2015;10:e0119816. (In Press) 2021.
[113] Jørgensen AEM, Kjær M, Heinemeier KM. The effect of aging and mechanical load- [144] Hashim P, Ridzwan MM, Bakar J, Hashim MD. Collagen in food and beverage in-
ing on the metabolism of articular cartilage. J Rheumatol 2017;44:410–17. dustries. Int Food Res J 2015;22:1–8.
[114] Meinert C, Schrobback K, Hutmacher DW, Klein TJ. A novel bioreactor system for [145] Avila Rodríguez MI, Rodriguez Barroso LG, Sánchez ML. Collagen: a review on its
biaxial mechanical loading enhances the properties of tissue-engineered human sources and potential cosmetic applications. J Cosmet Dermatol 2018;17:20–6.
cartilage. Sci Rep 2017;7:1–14. [146] Silvipriya K, Kumar KK, Bhat A, Kumar BD, John A, Lakshmanan P. Collagen: ani-
[115] Bian L, Fong JV, Lima EG, Stoker AM, Ateshian GA, Cook JL, Hung CT. Dynamic mal sources and biomedical application. J Appl Pharm Sci 2015;5:123–7.
mechanical loading enhances functional properties of tissue-engineered cartilage [147] Chen Z, Fan D, Shang L. Exploring the potential of the recombinant human col-
using mature canine chondrocytes. Tissue Eng Part A 2010;16:1781–90. lagens for biomedical and clinical applications: a short review. Biomed Mater
[116] Mauck R, Byers B, Yuan X, Tuan R. Regulation of cartilaginous ECM gene tran- 2020;16:012001.
scription by chondrocytes and MSCs in 3D culture in response to dynamic loading. [148] Olsen D, Yang C, Bodo M, Chang R, Leigh S, Baez J, Carmichael D,
Biomech Model Mechanobiol 2007;6:113–25. Perälä M, Hämäläinen E-R, Jarvinen M. Recombinant collagen
[117] Bhosale AM, Richardson JB. Articular cartilage: Structure, injuries and review of and gelatin for drug delivery. Adv Drug Deliv Rev 2003;55:1547–
management. Br Med Bull 2008;87:77–95. 1567.
[118] Muir H, Bullough P, Maroudas A. The distribution of collagen in human artic- [149] Fertala A. Three decades of research on recombinant collagens: reinventing the
ular cartilage with some of its physiological implications. J Bone Joint Surg Br wheel or developing new biomedical products? Bioengineering 2020;7:155.
1970;52:554–63. [150] Fertala A, Holmes DF, Kadler KE, Sieron AL, Prockop DJ. Assembly in vitro of thin
[119] Chen R, Chen S, Chen X, Long X. Study of the tidemark in human mandibular and thick fibrils of collagen II from recombinant procollagen II. The monomers in
condylar cartilage. Arch Oral Biol 2011;56:1390–7. the tips of thick fibrils have the opposite orientation from monomers in the growing
[120] James C-B, Uhl TL. A review of articular cartilage pathology and the use of glu- tips of collagen I fibrils. J Biol Chem 1996;271:14864–9.
cosamine sulfate. J Athl Train 2001;36:413–19. [151] Fertala A, Sieron AL, Ganguly A, Li S-W, Ala-Kokko L, Anumula KR, Prockop D.
[121] Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem Synthesis of recombinant human procollagen II in a stably transfected tumour cell
2009;78:929–58. line (HT1080). Biochem J 1994;298:31–7.
[122] Frazer A, Bunning RA, Thavarajah M, Seid JM, Russell RGG. Studies on type II [152] Ala-Kokko L, Hyland J, Smith C, Kivirikko KI, Jimenez SA, Prockop DJ. Expression
collagen and aggrecan production in human articular chondrocytes in vitro and of a human cartilage procollagen gene (COL2A1) in mouse 3T3 cells. J Biol Chem
effects of transforming growth factor-𝛽 and interleukin-1𝛽. Osteoarthritis Cartilage 1991;266:14175–8.
1994;2:235–45. [153] Xi C, Liu N, Liang F, Zhao X, Long J, Yuan F, Yun S, Sun Y, Xi Y. Molecular assembly
[123] Kivirikko KI, Myllylä R, Pihlajaniemi T. Protein hydroxylation: Prolyl 4-hydrox- of recombinant chicken type II collagen in the yeast Pichia pastoris. Sci China Life
ylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J Sci 2018;61:815–25.
1989;3:1609–17. [154] Qi Q, Yao L, Liang Z, Yan D, Li Z, Huang Y, Sun J. Production of human type II
[124] Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The biosynthesis of collagen collagen using an efficient baculovirus-silkworm multigene expression system. Mol
and its disorders. N Engl J Med 1979;301:77–85. Genet Genomics 2016;291:2189–98.
[125] Myllyharju J. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis. Ma- [155] Ikonomou L, Schneider Y-J, Agathos S. Insect cell culture for industrial production
trix Biol 2003;22:15–24. of recombinant proteins. Appl Microbiol Biotechnol 2003;62:1–20.
[126] Siegel RC. Biosynthesis of collagen crosslinks: Increased activity of purified [156] Browne S, Zeugolis DI, Pandit A. Collagen: Finding a solution for the source. Tissue
lysyl oxidase with reconstituted collagen fibrils. Proc Natl Acad Sci U S A Eng Part A 2013;19:1491–4.
1974;71:4826–30. [157] Collin EC, Grad S, Zeugolis DI, Vinatier CS, Clouet JR, Guicheux JJ, Weiss P,
[127] Eyre DR, Paz MA, Gallop PM. Cross-linking in collagen and elastin. Annu Rev Alini M, Pandit AS. An injectable vehicle for nucleus pulposus cell-based therapy.
Biochem 1984;53:717–48. Biomaterials 2011;32:2862–70.
[128] Herbage D, Bouillet J, Bernengo J. Biochemical and physiochemical characteriza- [158] Taguchi T, Ikoma T, Tanaka J. An improved method to prepare hyaluronic acid
tion of pepsin-solubilized type-II collagen from bovine articular cartilage. Biochem and type II collagen composite matrices. J Biomed Mater Res 2002;61:330–6.
J 1977;161:303–12. [159] Yang K, Sun J, Wei D, Yuan L, Yang J, Guo L, Fan H, Zhang X. Photo-crosslinked
[129] Bishop P, Crossman M, McLeod D, Ayad S. Extraction and characterization of mono-component type II collagen hydrogel as a matrix to induce chondro-
the tissue forms of collagen types II and IX from bovine vitreous. Biochem J genic differentiation of bone marrow mesenchymal stem cells. J Mater Chem B
1994;299:497–505. 2017;5:8707–18.
[130] Eyre D, Muir H. The distribution of different molecular species of collagen in fi- [160] Cao H, Xu S-Y. EDC/NHS-crosslinked type II collagen-chondroitin sulfate scaffold:
brous, elastic and hyaline cartilages of the pig. Biochem J 1975;151:595–602. characterization and in vitro evaluation. J Mater Sci Mater Med 2008;19:567–75.
[131] Wu Z, Korntner S, Mullen A, Skoufos I, Tzora A, Zeugolis D. In the quest of the [161] Ko C-S, Huang J-P, Huang C-W, Chu I-M. Type II collagen-chondroitin sul-
optimal tissue source (porcine male and female articular, tracheal and auricular fate-hyaluronan scaffold cross-linked by genipin for cartilage tissue engineering.
cartilage) for the development of collagen sponges for articular cartilage. Biomed J Biosci Bioeng 2009;107:177–82.
Eng Adv 2021;1:100002. [162] Lan W, Xu M, Zhang X, Zhao L, Huang D, Wei X, Chen W. Biomimetic polyvinyl
[132] Cao H, Xu S-Y. Purification and characterization of type II collagen from chick alcohol/type II collagen hydrogels for cartilage tissue engineering. J Biomater Sci
sternal cartilage. Food Chem 2008;108:439–45. Polym Ed 2020;31:1179–98.
[133] Akram A, Zhang C. Extraction of collagen-II with pepsin and ultrasound treatment [163] Hsu Sh, Tsai CL, Tang CM. Evaluation of cellular affinity and compatibility to
from chicken sternal cartilage; physicochemical and functional properties. Ultrason biodegradable polyesters and type-II collagen-modified scaffolds using immortal-
Sonochem 2020;64:105053. ized rat chondrocytes. Artif Organs 2002;26:647–58.
[134] Dai M, Liu X, Wang N, Sun J. Squid type II collagen as a novel biomaterial: Isolation, [164] Yen H-J, Tseng C-S, Hsu S-h, Tsai C-L. Evaluation of chondrocyte growth in the
characterization, immunogenicity and relieving effect on degenerative osteoarthri- highly porous scaffolds made by fused deposition manufacturing (FDM) filled with
tis via inhibiting stat1 signaling in pro-inflammatory macrophages. Mater Sci Eng type II collagen. Biomed Microdev 2009;11:615–24.
C 2018;89:283–94. [165] Ragetly G, Griffon DJ, Chung YS. The effect of type II collagen coating of chitosan
[135] Rigogliuso S, Salamone M, Barbarino E, Barbarino M, Nicosia A, Ghersi G. Produc- fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis. Acta
tion of injectable marine collagen-based hydrogel for the maintenance of differen- Biomater 2010;6:3988–97.
tiated chondrocytes in tissue engineering applications. Int J Mol Sci 2020;21:5798. [166] Zhang J, Yang Z, Li C, Dou Y, Li Y, Thote T, Wang D-a, Ge Z. Cells behave distinctly
[136] Sewing J, Klinger M, Notbohm H. Jellyfish collagen matrices conserve the chon- within sponges and hydrogels due to differences of internal structure. Tissue Eng
drogenic phenotype in two- and three-dimensional collagen matrices. J Tissue Eng Part A 2013;19:2166–75.
Regen Med 2017;11:916–25. [167] Shields KJ, Beckman MJ, Bowlin GL, Wayne JS. Mechanical properties and cellular
[137] Zhang X, Adachi S, Ura K, Takagi Y. Properties of collagen extracted from Amur proliferation of electrospun collagen type II. Tissue Eng 2004;10:1510–17.
sturgeon Acipenser schrenckii and assessment of collagen fibrils in vitro. Int J Biol [168] Barnes CP, Pemble IV CW, Brand DD, Simpson DG, Bowlin GL. Cross-linking elec-
Macromol 2019;137:809–20. trospun type II collagen tissue engineering scaffolds with carbodiimide in ethanol.
[138] Cumming M, Hall B, Hofman K. Isolation and characterisation of major and minor Tissue Eng 2007;13:1593–605.
collagens from hyaline cartilage of hoki (Macruronus novaezelandiae). Mar Drugs [169] Matthews JA, Boland ED, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of
2019;17:223. collagen type II: a feasibility study. J Bioact Compat Polym 2003;18:125–34.

9
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

[170] Yang L, Fitié CF, van der Werf KO, Bennink ML, Dijkstra PJ, Feijen J. Me- [197] Kim HA, Seo Y-I, Lee J, Jung YO. Detection of anti-type II collagen antibodies in
chanical properties of single electrospun collagen type I fibers. Biomaterials patients with chronic gouty arthritis: findings from a pilot study. J Clin Rheumatol
2008;29:955–62. 2016;22:360–3.
[171] Zeugolis DI, Khew ST, Yew ES, Ekaputra AK, Tong YW, Yung L-YL, Hutmacher DW, [198] Thompson H, Henderson B, Spencer J, Hobbs S, Peppard J, Staines N. Tolerogenic
Sheppard C, Raghunath M. Electro-spinning of pure collagen nano-fibres–just an activity of polymerized type II collagen in preventing collagen-induced arthritis in
expensive way to make gelatin? Biomaterials 2008;29:2293–305. rats. Clin Exp Immunol 1988;72:20–5.
[172] Ren X, Wang F, Chen C, Gong X, Yin L, Yang L. Engineering zonal cartilage through [199] Breinan HA, Martin SD, Hsu HP, Spector M. Healing of canine articular cartilage
bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte den- defects treated with microfracture, a type-II collagen matrix, or cultured autologous
sity gradient. BMC Musculoskelet Disord 2016;17:1–10. chondrocytes. J Orthop Res 2000;18:781–9.
[173] Annamalai RT, Mertz DR, Daley EL, Stegemann JP. Collagen Type II enhances [200] Lee C, Grodzinsky A, Hsu HP, Spector M. Effects of a cultured autologous chondro-
chondrogenic differentiation in agarose-based modular microtissues. Cytotherapy cyte-seeded type II collagen scaffold on the healing of a chondral defect in a canine
2016;18:263–77. model. J Orthop Res 2003;21:272–81.
[174] Chang SJ, Kuo SM, Manousakas I, Niu GC-C, Chen JP. Preparation and character- [201] Wu C-H, Ko C-S, Huang J-W, Huang H-J, Chu I-M. Effects of exogenous gly-
ization of hyaluronan/collagen II microspheres under an electrostatic field system cosaminoglycans on human chondrocytes cultivated on type II collagen scaffolds.
with disc electrodes. Acta Biomater 2009;5:101–14. J Mater Sci Mater Med 2010;21:725–9.
[175] Tamaddon M, Burrows M, Ferreira S, Dazzi F, Apperley J, Bradshaw A, Brand D, [202] Pieper J, Van Der Kraan P, Hafmans T, Kamp J, Buma P, Van Susante J, Van
Czernuszka J, Gentleman E. Monomeric, porous type II collagen scaffolds promote Den Berg W, Veerkamp J, Van Kuppevelt T. Crosslinked type II collagen matrices:
chondrogenic differentiation of human bone marrow mesenchymal stem cells in preparation, characterization, and potential for cartilage engineering. Biomaterials
vitro. Sci Rep 2017;7:43519. 2002;23:3183–92.
[176] Gao Y, Li B, Kong W, Yuan L, Guo L, Li C, Fan H, Fan Y, Zhang X. Injectable and [203] Mukaida T, Urabe K, Naruse K, Aikawa J, Katano M, Hyon S-H, Itoman M. Influence
self-crosslinkable hydrogels based on collagen type II and activated chondroitin of three-dimensional culture in a type II collagen sponge on primary cultured and
sulfate for cell delivery. Int J Biol Macromol 2018;118:2014–20. dedifferentiated chondrocytes. J Orthop Sci 2005;10:521–8.
[177] Veilleux N, Spector M. Effects of FGF-2 and IGF-1 on adult canine articular chon- [204] Nehrer S, Breinan HA, Ramappa A, Young G, Shortkroff S, Louie LK, Sledge CB,
drocytes in type II collagen–glycosaminoglycan scaffolds in vitro. Osteoarthritis Yannas IV, Spector M. Matrix collagen type and pore size influence behaviour of
Cartilage 2005;13:278–86. seeded canine chondrocytes. Biomaterials 1997;18:769–76.
[178] Szychlinska MA, Calabrese G, Ravalli S, Dolcimascolo A, Castrogiovanni P, Fabbi C, [205] Lee CR, Breinan HA, Nehrer S, Spector M. Articular cartilage chondrocytes in type
Puglisi C, Lauretta G, Di Rosa M, Castorina A. Evaluation of a cell-free collagen I and type II collagen-GAG matrices exhibit contractile behavior in vitro. Tissue
type I-based scaffold for articular cartilage regeneration in an orthotopic rat model. Eng 2000;6:555–65.
Materials 2020;13:2369. [206] Vickers SM, Squitieri LS, Spector M. Effects of cross-linking type II collagen-GAG
[179] Calabrese G, Gulino R, Giuffrida R, Forte S, Figallo E, Fabbi C, Salvatorelli L, scaffolds on chondrogenesis in vitro: dynamic pore reduction promotes cartilage
Memeo L, Gulisano M, Parenti R. In vivo evaluation of biocompatibility and chon- formation. Tissue Eng 2006;12:1345–55.
drogenic potential of a cell-free collagen-based scaffold. Front Physiol 2017;8:984. [207] Lee C, Grodzinsky A, Spector M. Biosynthetic response of passaged chondrocytes
[180] Yang J, Chen X, Yuan T, Yang X, Fan Y, Zhang X. Regulation of the secretion of in a type II collagen scaffold to mechanical compression. J Biomed Mater Res A
immunoregulatory factors of mesenchymal stem cells (MSCs) by collagen-based 2003;64:560–9.
scaffolds during chondrogenesis. Mater Sci Eng C 2017;70:983–91. [208] Wu Z, Korntner S, Mullen A, Skoufos I, Tzora A, Zeugolis D. In the quest of the
[181] Cao C, Zhang Y, Ye Y, Sun T. Effects of cell phenotype and seeding density on optimal tissue source (porcine male and female articular, tracheal and auricular
the chondrogenic capacity of human osteoarthritic chondrocytes in type I collagen cartilage) for the development of collagen sponges for articular cartilage. Biomed
scaffolds. J Orthop Surg Res 2020;15:1–11. Eng Adv 2021;1.
[182] Nehrer S, Breinan HA, Ramappa A, Shortkroff S, Young G, Minas T, Sledge CB, [209] Malemud CJ, Stevenson S, Mehraban F, Papay RS, Purchio AF, Goldberg VM. The
Yannas IV, Spector M. Canine chondrocytes seeded in type I and type II collagen proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II col-
implants investigated in vitro. J Biomed Mater Res 1997;38:95–104. lagen gels. Osteoarthritis Cartilage 1994;2:29–41.
[183] Veilleux N, Yannas I, Spector M. Effect of passage number and collagen type on [210] Muhonen V, Narcisi R, Nystedt J, Korhonen M, van Osch GJ, Kiviranta I. Recombi-
the proliferative, biosynthetic, and contractile activity of adult canine articular nant human type II collagen hydrogel provides a xeno-free 3D micro-environment
chondrocytes in type I and II collagen-glycosaminoglycan matrices in vitro. Tissue for chondrogenesis of human bone marrow-derived mesenchymal stromal cells. J
Eng 2004;10:119–27. Tissue Eng Regen Med 2017;11:843–54.
[184] Lu Z, Doulabi B, Huang C, Bank R, Helder M. Collagen type II enhances chondro- [211] Pulkkinen H, Tiitu V, Valonen P, Hamalainen E, Lammi M, Kiviranta I. Recombi-
genesis in adipose tissue–derived stem cells by affecting cell shape. Tissue Eng Part nant human type II collagen as a material for cartilage tissue engineering. Int J
A 2010;16:81–90. Artif Organs 2008;31:960–9.
[185] Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T. Chondrogenic [212] Wong C-C, Chen C-H, Chiu L-H, Tsuang Y-H, Bai M-Y, Chung R-J, Lin Y-H, Hsieh F-J,
differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different Chen Y-T, Yang T-L. Facilitating in vivo articular cartilage repair by tissue-engi-
hydrogels: Influence of collagen type II extracellular matrix on MSC chondrogene- neered cartilage grafts produced from auricular chondrocytes. Am J Sports Med
sis. Biotechnol Bioeng 2006;93:1152–63. 2018;46:713–27.
[186] Tao Y, Zhou X, Liu D, Li H, Liang C, Li F, Chen Q. Proportion of collagen type II [213] Dong S, Guo H, Zhang Y, Li Z, Kang F, Yang B, Kang X, Wen C, Yan Y, Jiang B.
in the extracellular matrix promotes the differentiation of human adipose-derived rFN/Cad-11-modified collagen type II biomimetic interface promotes the adhesion
mesenchymal stem cells into nucleus pulposus cells. Biofactors 2016;42:212– and chondrogenic differentiation of mesenchymal stem cells. Tissue Eng Part A
223. 2013;19:2464–77.
[187] Kilmer CE, Battistoni CM, Cox A, Breur G, Panitch A, Liu JC. Collagen type I and II [214] Chen WC, Wei YH, Chu I, Yao CL. Effect of chondroitin sulphate C on the in vitro
blend hydrogel with autologous mesenchymal stem cells as a scaffold for articular and in vivo chondrogenesis of mesenchymal stem cells in crosslinked type II colla-
cartilage defect repair. ACS Biomater Sci Eng 2020;6:3464–76. gen scaffolds. J Tissue Eng Regen Med 2013;7:665–72.
[188] Buma P, Pieper JS, van Tienen T, van Susante JL, van der Kraan PM, Veerkamp JH, [215] Funayama A, Niki Y, Matsumoto H, Maeno S, Yatabe T, Morioka H, Yanagimoto S,
van den Berg WB, Veth RP, van Kuppevelt TH. Cross-linked type I and type II Taguchi T, Tanaka J, Toyama Y. Repair of full-thickness articular cartilage defects
collagenous matrices for the repair of full-thickness articular cartilage defects—A using injectable type II collagen gel embedded with cultured chondrocytes in a
study in rabbits. Biomaterials 2003;24:3255–63. rabbit model. J Orthop Sci 2008;13:225–32.
[189] Nehrer S, Breinan H, Ramappa A, Hsu H, Minas T, Shortkroff S, Sledge C, Yannas I, [216] Chen W-C, Yao C-L, Wei Y-H, Chu I-M. Evaluating osteochondral defect repair po-
Spector M. Chondrocyte-seeded collagen matrices implanted in a chondral defect tential of autologous rabbit bone marrow cells on type II collagen scaffold. Cy-
in a canine model. Biomaterials 1998;19:2313–28. totechnology 2011;63:13–23.
[190] Muhonen V, Salonius E, Haaparanta AM, Järvinen E, Paatela T, Meller A, Han- [217] Dorotka R, Windberger U, Macfelda K, Bindreiter U, Toma C, Nehrer S. Repair of ar-
nula M, Björkman M, Pyhältö T, Ellä V. Articular cartilage repair with recombi- ticular cartilage defects treated by microfracture and a three-dimensional collagen
nant human type II collagen/polylactide scaffold in a preliminary porcine study. J matrix. Biomaterials 2005;26:3617–29.
Orthop Res 2016;34:745–53. [218] Hsu Sh, Chang SH, Yen HJ, Whu SW, Tsai CL, Chen DC. Evaluation of biodegrad-
[191] Trentham DE, Townes AS, Kang AH. Autoimmunity to type II collagen an experi- able polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering
mental model of arthritis. J Exp Med 1977;146:857–68. scaffolding materials for cartilage regeneration. Artif Organs 2006;30:42–55.
[192] Holmdahl R, Vingsbo C, Malmström V, Jansson L, Holmdahl M. Chronicity of arthri- [219] Chen H, Yang X, Liao Y, Zeng X, Liang P, Kang N, et al. MRI and histologic analysis
tis induced with homologous type II collagen (CII) in rats is associated with anti-CII of collagen type II sponge on repairing the cartilage defects of rabbit knee joints. J
B-cell activation. J Autoimmun 1994;7:739–52. Biomed Mater Res B 2011;96:267–75.
[193] Ofosu-Appiah W, Morgan K, Holt PLennox. Native type II collagen-induced arthritis [220] Lazarini M, Bordeaux-Rego P, Giardini-Rosa R, Duarte AS, Baratti MO, Zorzi AR,
in the rat. III. Relationship between the cellular immune response to native type II de Miranda JB, Lenz Cesar C, Luzo Â, Olalla Saad ST. Natural type II collagen
collagen and arthritis. Ann Rheum Dis 1983;42:331–7. hydrogel, fibrin sealant, and adipose-derived stem cells as a promising combination
[194] Cathcart E, Hayes K, Gonnerman W, Lazzari A, Franzblau C. Experimental arthri- for articular cartilage repair. Cartilage 2017;8:439–43.
tis in a nonhuman primate. I. Induction by bovine type II collagen. Lab Invest [221] Chen L, Bao B, Wang N, Xie J, Wu W. Oral administration of shark type II colla-
1986;54:26–31. gen suppresses complete Freund’s adjuvant-induced rheumatoid arthritis in rats.
[195] Cook AD, Rowley MJ, Mackay IR, Gough A, Emery P. Antibodies to type II collagen Pharmaceuticals 2012;5:339–52.
in early rheumatoid arthritis. Correlation with disease progression. Arthritis Rheum [222] Pulkkinen H, Tiitu V, Valonen P, Jurvelin J, Rieppo L, Töyräs J, Silvast T,
1996;39:1720–7. Lammi M, Kiviranta I. Repair of osteochondral defects with recombinant human
[196] Stuart JM, Townes AS, Kang AH. Collagen autoimmune arthritis. Ann Rev Immunol type II collagen gel and autologous chondrocytes in rabbit. Osteoarthritis Cartilage
1984;2:199–218. 2013;21:481–90.

10
Z. Wu, S. Korntner, A. Mullen et al. Biomaterials and Biosystems 4 (2021) 100030

[223] Capella-Monsonís H, Coentro JQ, Graceffa V, Wu Z, Zeugolis DI. An experimental [225] Zeugolis D, Paul R, Attenburrow G. Factors influencing the properties of reconsti-
toolbox for characterization of mammalian collagen type I in biological specimens. tuted collagen fibers prior to self-assembly: animal species and collagen extraction
Nat Protoc 2018;13:507–29. method. J Biomed Mater Res A 2008;86:892–904.
[224] Sorushanova A, Skoufos I, Tzora A, Mullen AM, Zeugolis DI. The influence of animal [226] Delgado LM, Shologu N, Fuller K, Zeugolis DI. Acetic acid and pepsin result in high
species, gender and tissue on the structural, biophysical, biochemical and biological yield, high purity and low macrophage response collagen for biomedical applica-
properties of collagen sponges. J Mater Sci Mater Med 2021;32:1–12. tions. Biomed Mater 2017;12:065009.

11