Regeneration of Articular Cartilage Surface: Morphogens, Cells, and Extracellular Matrix Scaffolds.

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Tissue Engineering Part B: Reviews Articular Cartilage Regeneration: Challenges and Opportunities (doi: 10.1089/ten.TEB.2014.0661) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

1 Review Article TITLE PAGE Title: Regeneration of Articular Cartilage Surface: Morphogens, Cells and Extracellular Matrix Scaffolds Authors: Ryosuke Sakata, M.D., Ph.D., Takashi Iwakura M.D., Ph.D., and A Hari Reddi, Ph.D* Affiliations: Center for Tissue Regeneration and Repair, Department of Orthopaedic Surgery, University of California, Davis. 4635 Second Avenue, Research Building I, Room 2000, Sacramento, CA, 95817.

TEL: +1-916-734-5749 FAX: +1-916-734-5750

*Corresponding author: A. Hari Reddi, Ph.D Tel: 1-916-734-5749, Fax: 1-916-734-5750 E-mail: [email protected]

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2 Abstract The articular cartilage is a well-organized tissue for smooth and friction-free joint movement for locomotion in animals and humans. Adult articular cartilage has a very low self-regeneration capacity due to its avascular nature. The regeneration of articular cartilage surface is critical for in order to prevent the progression to osteoarthritis. Although various joint resurfacing procedures in experimental articular cartilage defects have been developed, no standardized clinical protocol has yet been established.

The three critical ingredients for tissue regeneration are morphogens and growth factors, cells, and scaffolds. The concepts based on the regeneration triad have been extensively investigated in animal models. However, these studies in animal models have demonstrated variable results and outcomes. An optimal animal model must precisely mimic and model the sequence of events in articular cartilage regeneration in human. In this article, the progress and remaining challenges in articular cartilage regeneration in animal models is reviewed.

The role of individual morphogens and growth factors in cartilage regeneration have been investigated. In normal articular cartilage homeostasis, morphogens and growth

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3 factors function sequentially in tissue regeneration. Mesenchymal stem cells-based repair of articular cartilage defects, performed with or without various growth factors and scaffolds, has been widely attempted in animal models. Stem cells including embryonic and adult stem cells and induced pluripotent stem cells have also been reported as attractive cell sources for articular cartilage surface regeneration. Several studies with regard to scaffolds have been advanced, including recent investigations based on nanomaterials, functional mechanocompatible scaffolds, multi-layered scaffolds, and ECM scaffolds for articular cartilage surface regeneration. Continuous refinement of animal models in chondral and osteochondral defects provide opportunities that support further advances in tissue engineering for the optimal articular cartilage surface regeneration.

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4 Introduction The articular cartilage is a functional tissue for weight-bearing and friction-free locomotion. Unlike most tissues, the articular cartilage is avascular. Because of this intrinsic avascularity, the innate mechanisms of tissue regeneration based on blood supply and recruitment of stem/progenitor cells via vascular system to the site of damage does not occur in the articular cartilage.(1)

The low cell density within cartilage tissue reduces the ability of local chondrocytes to contribute to regeneration.(2, 3) Traumatic and degenerative lesions of the articular cartilage can lead to progression of osteoarthritis (OA). The tremendous clinical and financial burden imposed by OA has motivated basic scientists and clinicians to investigate new strategies for the repair and regeneration of damaged and degenerated articular cartilage. In articular cartilage, the regeneration of superficial zone is critical for maintaining joint movement and minimizing wear by optimizing the lubrication. The superficial zone chondrocytes secrete the superficial zone protein (SZP) also known as lubricin and proteoglycan 4 and encoded by the prg4 gene.(4, 5) SZP plays a central role in maintaining joint homeostasis and minimizing wear as a chondroprotective barrier and as a boundary lubricant against direct solid-to-solid contact.(6) Various joint resurfacing procedures for treating focal articular cartilage defect such as bone marrow stimulation, autologous osteochondral grafts, and 4

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5 transplantation of mesenchymal stem cells (MSCs), chondrocytes, and engineered cartilage have been developed and used in clinical practice.(7-13)

For articular cartilage surface regeneration, the three critical ingredients are morphogens and growth factors, stem cells, and scaffolds and the concepts based on these ingredients have been extensively investigated with in vitro and in vivo experiments. However, experimental studies of articular cartilage regeneration in animal models have yielded variable results, depending on the animal species, the donor age, the area and the depth of a defect, and the techniques used in the study.(2, 14) Despite showing some degree of success, in some aspects, each technique has its limitations and needs to be improved and refined.(15) The animals used in these studies include mice, rats, rabbits, pigs, goats, and horses. The age of the experiment animal is an important factor in determining the outcome and the potential for repair procedures in humans.(16) In experimental models, the weight-bearing area of the knee (e.g., femoral condyle) or the femoral trochlea, which causes maltracking of the patella, were used. Other crucial factors that influence articular cartilage repair are the depth of cartilage defects.(17-19).

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6 Although an optimal animal model would precisely mimic the sequence of events in human, such a model has been difficult to develop.(20) In the present article, we review the results of animal experiments performed on the articular cartilage and consider the future prospects for articular cartilage surface regeneration.

Classification of Articular Cartilage Defects

For clinical application, Brittberg advocated the ICRS (International Cartilage Repair Society) classification, depending on the lesion depth and the area of damaged, to evaluate the hyaline cartilage defect. Briefly, normal cartilage without notable defect is classified as ICRS 0. If the cartilage has an intact surface but fibrillation and/or slight softening is present, it is classified as ICRS 1a, and if there is additional fissures are found, it is classified as ICRS 1b. When the cartilage defect extend up to 50% of the cartilage thickness in depth, it is classified as ICRS 2 and if cartilage defect extend deeper than 50% of the cartilage thickness, it is classified as ICRS 3. ICRS 3 includes four subgroups depending on whether the defect reaches the subchondral bone. In the most severe group, ICRS 4, the cartilage defect extends into subchondral bone.(21)

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7 In contrast, as the cartilage thickness in animal model is thinner than that in human, the cartilage defects in animal models are classified as partial-thickness articular cartilage defects (ICRS 1a to 3) or as full-thickness articular cartilage defects (ICRS 4) based on whether the defect penetrates the marrow spaces of the subchondral bone.(17, 19, 22) Partial-thickness defects, which do not affect the subchondral bone, never repair spontaneously.(18, 23, 24) In contrast, in full-thickness defects penetrating the subchondral bone, the blood supply in the bone starts a healing process in the defect and gives better potential for repair. However, newly regenerated tissue in the full-thickness defects is a type of fibrocartilage, not hyaline cartilage. Although fibrocartilage is able to fill in articular cartilage defect, its structure doesn't withstand the demands of everyday activities as much as hyaline cartilage and repair reaction is limited even in full-thickness cartilage defects.(20)

Partial-Thickness Articular Cartilage Defects Partial-thickness defects of the articular cartilage may resemble the clefts and fissures occurring during the initial stages of OA, and do not penetrate into the subchondral bone (Fig. 1A). Partial-thickness defects in the articular cartilage do not heal spontaneously because of the lack of blood vessels and the absence of blood

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8 clot in the mature articular cartilage and the lack of access to the stem cells from the marrow.(25)

Most human articular cartilage defects do not initially involve the subchondral bone and they lack the spontaneous healing potential. Therefore, the creation of partial-thickness defects in animal experimental model may be of great importance because of its resemblance to the outbreak and initial stages of OA in human joints.(26, 27) However, the creation of partial-thickness defects in the articular cartilage surface in small experimental animals presents great technical challenges, due to the small size.(28)

Full-Thickness Articular Cartilage Defects Full-thickness defects pass through the tidemark layer and the zone of calcified cartilage and penetrate the subchondral bone. (Fig. 1B) Perforation into the subchondral bone allows the defect to be filled with undifferentiated, marrow-derived Mesenchymal Stem Cells (MSCs) that differentiate into new subchondral bone and overlying articular cartilage. However, it has been reported that the regenerated cartilage resemble the original cartilage neither biochemically nor biomechanically

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9 and it eventually deteriorates.(19, 26) During this process, the tissue adjacent to the wound margins becomes necrotic and separates from the sporadic chondrocyte cluster formation, and there is no remodeling. Although, light microscopy revealed continuity between the native tissue and the repair tissue; however, polarized light microscopy revealed lack of true integration of newly differentiated and old extracellular matrices and revealed that the defects in the articular cartilage of full-thickness defect have frequent regions of discontinuity.(19) This review is focused on available animal models and their potential for regeneration.

Animal Models of Articular Cartilage Regeneration and Repair (Table 1) Animal models are useful for investigating the sequential regeneration cascades and development of therapeutic approaches. An ideal animal articular cartilage defect model would imitate the endogenous repair process that occurs in humans. A well-standardized and reproducible defect model would also facilitate a precise comparison of different treatment procedures. However, none of the currently available animal species yield results that can be directly extrapolated to human. Therefore, the choice of the species and the defect model, the age of the animals, individual joint dimensions, joint load and mechanics, and the thickness of the

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10 articular cartilage are critical factors when conducting experiments and in interpretation of the experimental data.(20, 29)

Mouse Rodent models are a cost-effective approach for obtaining proof-of-concept data to serve as a bridge between in vitro experiments and the preclinical studies in large animal models. In mouse models, chondrogenesis has been extensively studied with subcutaneous (30, 31) and intramuscular implantation.(32) However there are few cartilage defect models in mouse (33, 34) due to their small joints and the entire cartilage (3-5 cell layers or less than 0.1 mm).(35-37) Therefore, it is not surprising that there has been no report of partial thickness articular cartilage defect. The principal advantage of using a mouse model is due to the availability of athymic nude mice, and several transgenic or knockout mice.

Rat As rats have bigger joints and thicker cartilage thickness compared to the mouse model, (36, 37) it confers ease of handling and surgical feasibility.(38-43) It is now possible to get transgenic rats. However, in rats, the persistence of open growth plates throughout an animal’s lifespan increases the intrinsic healing potential

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11 confounds the interpretation of results.(35) In summary, it is extremely difficult to produce a rodent articular cartilage defect model that is suitable for comparison and translation to the clinic.(44)

Rabbit The rabbit has been widely used for investigating articular cartilage regeneration.(19, 24, 25, 45-52) This is due to the ease of handling, caging, the relatively larger joint and thicker cartilage in comparison with rodents, and the reasonable cost of animal purchase and care.(36) Several analysis of rabbit articular cartilage revealed a mean cartilage thickness of 0.3 to 0.45 mm in thickness of the femoral condyle.(53, 54) Endogenous healing potential in rabbits have been reported to be greater compared to larger animals including human, in which articular cartilage defect show no spontaneous repair without surgical intervention.(19, 20) Moreover, the characteristic mechanical loading condition due to high flexion knee makes it difficult to directly translate the results in rabbits to human clinical practice.(36) Hunziker et al. reported that

partial-thickness

defects

created

in

articular

cartilage

do

not

heal

spontaneously.(24) There are several reports of partial-thickness repair in rabbit models.(52)

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12 Dog The utility of dogs in animal articular cartilage regeneration is defined because of their joint size, thicker cartilage, weight-bearing requirements, low spontaneous healing ability, and the use of arthroscopic assessment. Studies involving the dog have been conducted for articular cartilage regeneration(55-57) The cartilage thickness is 0.9 1.3mm,(35, 37, 58) and a partial-thickness articular cartilage defect can be created in the dog model.(59-61) In addition, their lack of significant intrinsic ability to heal articular cartilage defects is an advantage and is similar to human. They are also easily trainable in postoperative rehabilitation protocol and they are able to accept the braces. As dogs are cared as companion animal status and ethical reasons, the dog model is not widely utilized (44)

Pig and Mini-Pig The pig model represents the human joint more closely than any other species with regard to joint size, weight-bearing characteristics, cartilage thickness, which ranges from 1.5mm(58) to 2.0mm.(62) The choice of the mini-pig alleviates concerns of handling and aggressive demeanor of pigs; therefore, several studies of partial-thickness defects and full-thickness defects have been performed in mini-pigs.(24, 29, 62-64) As juvenile mini-pigs have a relatively high intrinsic healing 12

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13 potential, it is critical to use skeletally mature pigs to reduce the potential for intrinsic cartilage repair.(24, 29)

Sheep and Goat Sheep and goats are commonly used in articular cartilage research because of their large joint size, cartilage thickness, accessibility for arthroscopic procedures, and limited intrinsic healing capacity. The thickness of sheep cartilage ranges from 0.4 mm to 1.6 mm.(65) This variability of cartilage thickness makes the standard deviation larger and makes it difficult for statistical evaluation of the results. Because of this variability and the late skeletal maturity, the reports of articular cartilage regeneration in the sheep model are limited.(65-68)

The thickness of goat cartilage also ranges from 0.8mm to 2mm.(69) This thickness allows researchers to create partial and full-thickness articular cartilage defects, as desired. Furthermore, goats are relatively inexpensive and easier to handle than other large animals. The goat model is a valuable large animal model for articular cartilage defect regeneration.(69-76)

Horse

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14 The horse is the largest animal model available and offers several distinct advantages for articular cartilage regeneration studies.(77-79) The cartilage thickness in horses ranges from 1.7 mm to 2 mm for the medial condyle and this thickness is similar to that in human (2.2 mm).(35, 58, 80) The clinical treatment protocols for cartilage injuries are well developed in horses, largely because of their use in the racing.(81) Therefore, in vitro biochemical, molecular, gene therapeutic, and immunohistochemical assays have been described for the various joint components.(44) Some concerns with using horse models are the high expense, their weight and physiology, and joint loading conditions. It is well accepted that the restriction of joint loading and motion may be a life-threatening health problem in the horse.(36) The horse model is nevertheless highly beneficial and attractive for preclinical evaluation of both partial and full-thickness articular cartilage regeneration.

Tissue engineering in articular cartilage surface regeneration Morphogens and growth factors Regenerative medicine is the discipline based on principles of cellular and molecular developmental biology and morphogenesis. The three key elements for both morphogenesis and regeneration are inductive signals for morphogenesis, stem cells that respond to morphogenetic and growth signals, and the scaffolds of ECM.

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15 Regeneration is essentially a recapitulation of the sequential steps in embryonic development and morphogenesis. Morphogens and morphogenetic proteins are redeployed in the regeneration program of tissues and organs. Cartilage morphogenesis is a multistep cascade that includes factors for initiation, promotion, and maintenance of cartilage and contributes to cartilage surface regeneration. The growing advances in morphogens for the musculoskeletal tissues have identified bone morphogenetic proteins (BMPs), the primary signals for bone morphogenesis and

cartilage-derived

morphogenetic

proteins

(CDMPs),

the

morphogens

homologous to BMPs and derived from articular cartilage.

Morphogens

and

growth

factors

can

stimulate

cell

proliferation,

growth,

morphogenesis, and differentiation. In articular cartilage regeneration, numerous growth factors have been investigated for their stimulatory effect on synthesis of cartilage markers, proteoglycan, aggrecan, and type II collagen by chondrocytes, synoviocytes and MSCs proliferation and chondrogenic differentiation. In cartilage regeneration, transforming growth factor (TGF)-β superfamily members including TGF-β isoforms, TGF-β1, 2, and 3(82, 83) and BMP family members BMP-2,(40, 47) BMP-4,(41, 84) and BMP-7(85, 86) are most thoroughly investigated and they stimulate chondrocyte gene expression, chondrogenic differentiation of MSCs, and 15

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16 suppress the action of inflammatory cytokines such as IL1β and TNFα. In contrast, Hulth showed the risk that TGF-β1 could induce synovitis and sometimes induce localized necrosis of cartilage.(87) Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) were also well investigated.(88, 89) Recently, CDMPs have been studied of its effect on cartilage regeneration both in vitro and in vivo. (90, 91)

Sellers et al. reported that recombinant human BMP-2 (rhBMP-2) impregnated collagen sponges implanted in rabbit full-thickness articular cartilage defect enhanced repair process in comparison with the repair achieved by the use of the sponge only.(47) Nawata et al. reported that rhBMP-2 encouraged the repair of articular cartilage defects in rats in a dose-dependent manner.(40) Jiang et al. reported that the delivery of BMP-4 in a bilayer collagen scaffold stimulated the formation of cartilage tissue.(84) Kuroda et al. showed that the local delivery of BMP-4 by genetically engineered muscle-derived stem cells improved the articular cartilage repair.(41) BMP-7 was studied for its capacity to regenerate articular cartilage.(86). The repair of articular cartilage defects is strongly enhanced by TGF-β1;(83) TGF-β also has been reported to have a critical anabolic regulator of SZP synthesis, which can play a central role in maintaining joint homeostasis and lubrication. On the other hand, undesirable adverse effects such as synovial inflammation occur when TGF-β1 16

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17 is injected into the joint in mouse and rabbit models.(92, 93) In animal studies, FGF-2 and FGF-18 have attracted attention for articular cartilage repair, although the intra-articular administration of FGF-2 and -18 results in inflammation and osteophyte formation and shows less healing capacity than expected.(89) Singh et al. determined the influence of IGF-1 in articular cartilage repair with and without autografting; the outcome was that cartilage formation apparently declined and appeared to converge to osseous tissue.(88)

Although extensive studies of the effect of growth factors in cartilage regeneration have been conducted, most of them have been evaluated one at a time. Because, in normal cartilage homeostasis, numerous growth factors are needed to function both simultaneously and sequentially, more than a single growth factor may work in combinatorial mode to initiate, promote and maintain articular cartilage regeneration. BMP-7 acts synergistically with other growth factors such as TGF-β and IGF-1.(85) Based on these concepts, the increasing application of autologous mixture and concentration of and growth factors for cartilage regeneration must be investigated using composite biologics such as platelet-rich plasma (PRP) and bone marrow concentrate.

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18

Platelet rich plasma (PRP) Recently, Platelet-rich plasma (PRP) has received considerable attention for tissue regeneration in variety of tissues including articular cartilage. PRP can be prepared by a safe and simple method that permits collection of natural concentrate of autologous growth factors from platelets and has a potential to play at least two of three critical ingredients in tissue engineering; growth factors and scaffold.(94). Platelets are small cytoplasmic organelles released by budding from megakaryocytes in the bone marrow. They contain two types of granules, α-granules and dense granules.

α-granules

contain

various

types

of

growth

factors,

including

platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), FGF, IGF, VEGF and so on.(94, 95) BMP-2, -4, and -7 have the remarkable ability to induce cartilage formation, are present in PRP.(96, 97) In recent work, we identified the presence of SZP/lubricin in PRP, which is the primary lubricant in articular cartilage and functions as a boundary lubricant and may play a critical role for resurfacing osteochondral defects in PRP preparations. In our study, PRP stimulated SZP synthesis from chondrocytes and synoviocytes. PRP was also found to be an optimal lubricant for the cartilage and may provide an autologous and renewable source for replenishing the diminished boundary lubricating ability of synovial fluid in arthritic 18

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19 knees.(98) Activation of PRP may release numerous bioactive growth factors. Several studies have been conducted about PRP use in osteochondral repair and regeneration in vivo animal model. Milano et al. reported that PRP treatment improved the cartilage repair and restoration macroscopically and histologically in a sheep model.(99) Smyth et al. demonstrated that PRP treatment adjunct to autologous osteochondral transplantation in rabbit osteochondral defect model had positive effect on graft integration and clinical outcomes.(100) Betsch et al. showed similar result in mini pig model.(97) Xie et al. showed that, combined with adipose and bone marrow derived MSCs, PRP could have a potential to repair and regenerate osteochondral defect in a the rabbit knee.(101) Lee et al. implanted PRP gel, activated with thrombin, with synovium membrane-derived MSCs into rabbit osteochondral defect. They indicated that stem cells embedded PRP gel could stimulate resurfacing the osteochondral defect.(102)

Stem Cells (Table. 2)

Mesenchymal Stem Cells In animal models, MSCs-based repair of full-thickness articular cartilage defects, used with various growth factors and carrier matrixes, has been widely attempted. In 19

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20 1999, Pittenger et al. reported that bone marrow derived MSCs had a self-renewal and multi-lineage differentiation potential, including chondrogenesis.(103) Since then, MSCs have been identified in adipose tissue,(104) synovium,(105) periosteum,(106) skeletal muscle,(107) umbilical cord,(108) and skin.(109) MSCs from various sources have extensive proliferation potential and may be expanded without compromising their multi-lineage differentiation potential.(110) Meanwhile, some studies have indicated that each source of MSCs has a specific differentiation lineage.(111-113) Several animal studies have been conducted concerning articular cartilage defect regeneration. Park et al. showed that, when transplanted into articular cartilage defect in rats, MSCs from bone marrow and periosteum were superior to cells isolated from fat with respect to forming hyaline cartilaginous tissue.(114) Koga et al. compared the in vivo chondrogenic potential among various types of MSCs in rabbits and demonstrated that MSCs from synovium and bone marrow had a higher potential to repair articular cartilage defects than MSCs from skeletal muscle and adipose tissue.(115) Although MSCs could be an attractive cell source for articular cartilage defect regeneration, some concerns have suggested that, when implanted subcutaneously

or

intramuscularly,

MSCs-derived

chondrocytes

express

hypertrophy-related genes, thereby leading to cell death or calcification, followed by

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21 vascularization.(116) In addition, the best cell source, culture condition, and combination of scaffolds, and growth factors remain under consideration.

Embryonic Stem cells and induced Pluripotent Stem cells Embryonic stem cells (ESCs) and embryonic-like stem cells, such as induced pluripotent stem cells (iPSCs), have been considered for regenerative medicine applications

because

of

their

pluripotency

and

continuously

self-renewal

capacity.(117)

Evans and Kaufman established ESCs in 1981 from a mouse embryo.(118) Thomson later isolated lines of human ESCs and established their pluripotency.(119) The ability of ESCs to form cartilage is well known because cartilage tissue has been detected in teratomas on transplantation into nude mice. In vitro well-controlled differentiation of ESCs along the chondrogenic lineage is still under investigation.(120) Many approaches such as using embryoid body (EB)(120) or co-culture methods incorporating growth factors have been assessed.(121, 122) ESCs or ESCs-derived cells have been used in repair of articular cartilage. Wakitani et al. observed that the transplantation of ESCs for rat articular cartilage defect in an immunosuppressed rat

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22 has a positive effect on articular cartilage repair.(123) Toh et al. reported ESC-derived chondrocyte-engineered cartilage showed better histologic repair without tumorigenicity in rat full-thickness cartilage defect.(124) Dattena et al. also showed that ESCs transplantation embedded in fibrin glue encouraged articular cartilage defect regeneration in a sheep model.(125) However, human ESCs need to be studied further before they can be applied in the clinical use because of challenges such as teratoma formation, chondrocyte hypertrophy and calcification of ESCs-derived chondrocytes, and continuing ethical issues.

In the initial landmark publication on iPSCs, Takahashi and Yamanaka reported the reprogramming of mouse somatic cells to iPSCs through the forced expression of 4 transcription factors: Oct3/4, Sox2, Klf4 and C-Myc. The iPSCs exhibited the morphology and growth properties of ESCs, including their ability to form teratomas.(126) Hyaline cartilage is present in teratomas, suggesting that iPSCs have the potential for hyaline cartilage regeneration.(127) In 2007, human iPSCs were generated by using the Yamanaka factors (128) In addition to mouse and human iPSCs, iPSCs have already established from other species such as monkey, rat, pig and horse.(129) Important features of iPSCs are their unlimited proliferation ability with maintaining their pluripotency and their ability to be induced by 22

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23 patient-specific cells.(130) Thus, iPSCs can be used for transplantation therapy, drug discovery, and pathogenesis research with minimal ethical and immunological concerns. Few groups have examined chondrogenic differentiation of iPSCs. Teramura et al. induced chondrogenic differentiation of mouse iPSCs by treating cells in a micro-mass culture.(131) A primary challenge in using iPSCs for therapeutic applications is to achieve uniform differentiation to the cell type of interest. Diekman et al. purified the mouse iPSCs by type II collagen-driven green fluorescent protein expression; the purified iPSCs showed integrative repair in the in vitro cartilage defect model.(132) In vivo cartilage defect model, Ko et al. demonstrated that chondrocyte induced iPSCs by TGF-β showed better quality of cartilage repair in a rat full-thickness cartilage defect.(133) Recently, Yamashita and Tsumaki showed successful hyaline chondrogenesis from human iPSCs with scaffoldless suspension culture method. They transplanted newly generated hyaline cartilage particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs and they showed that neo-cartilage survived and had potential for integration into native cartilage. (134)

Direct conversion of the cells without an intervening stage of the iPSC generation using cell-reprogramming technique has also been studied. Tsumaki et al. 23

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24 demonstrated that misexpression of two reprogramming factors (c-Myc and Klf4) and the key chondrogenic transcription factor (Sox9) in mouse and human dermal fibroblasts demonstrated the induction of chondrocytes.(127, 135, 136) However, in vivo experiments including efficacy and safety in cartilage defect model are necessary for application in articular cartilage surface regeneration.

Scaffolds It is widely accepted that scaffolds function as an artificial and sometimes temporary ECM, mimicking the physiological and mechanical properties, structure, and functions of the native ECM. In addition, an optimal scaffold can attract cell attachment, proliferation, and differentiation based on physical and chemical properties. Polymer scaffolds serve as a carrier of cultured cells and a mechanical substrate, and they interact with cells, bioactive molecules, and mechanical signals in a dynamic and synergistic manner to direct and organize the process of regeneration.(106) The scaffold should be sterilizable, biocompatible, and biodegradable and possesses sufficient mechanical strength; and support cell differentiation and cartilage matrix production.

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25 Many different types of polymeric matrixes such as naturally derived and synthetic polymers with and without cells have been tested in vitro and experimental animals for their ability to promote articular cartilage repair.(137) Natural polymers, including hydrogels such as alginate, agarose, and gelatin;(138) collagen sponge-like matrixes(48) and gels;(46) hyaluronan;(139) fibrin;(78) and chitosan;(140) are comparatively weaker and softer materials than ceramics, but promote flexibility and operability. In addition, natural polymers usually contain specific molecular domains, which can stimulate cells at various stages at their development.(141)

Biodegradable synthetic polymers including polylactic acid (PLA);(142) polyglycolic acid

(PGA),(143)

co-polymer

of

PLA

and

PGA

(PLGA),(144,

145)

and

polycaprolactone (PCL).(146) are one of the most well studied fields in tissue engineering. They may be obtained with controlled distribution of molecular weight and have good biomechanical strength.(141) In addition, fabrication of synthetic polymers can easily scaled up to industrial scale, which can meet the potential needs. Nevertheless, they have some drawbacks in tissue engineering because of their hydrophobic surface and degradation products. Hydrophobic surface may be obstacle for cell attachment and migration and it is hard to elucidate actual degradation pathway and functions of each degradation products. 25

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26

Recent

studies

focused

nanomaterials,(147)

functional

scaffolds

including

mechanocompatible scaffolds,(148) multi-layered scaffolds,(149) and ECM scaffolds for cartilage and bone regeneration. The benefits of using multi-layered scaffolds include the potential to recreate the zone dependent morphology and characteristic of native cartilage and the potential to imitate osteochondral constructs consisting a layer of cartilage integrally bound to a layer of underlying bone.(149) As the ECM of the cartilage is a structurally complex 3-dimensional environment composed of various types of collagens and proteoglycans with multiple bioactive factors, use of biomaterials and scaffolds based on autologous ECMs attract attention for tissue engineering in cartilage and bone regeneration．ECM based tissue engineering strategies have already been used clinically in other tissue, including heart valves,(150) muscle and tendons.(151) For treating full-thickness cartilage defect, non-decellularized and decellularized cartilage particles, cartilage matrix, and osteochondral graft have been investigated. The ECM scaffolds created from cultured cells are also developed. ECM based scaffold can be a promising option for tissue engineering based on following advantages: i) to retain the growth factors including TGF-β, FGF, and IGF, ii) to provide appropriate environment for cell attachment, proliferation, and differentiation, and iii) to support biomechanical properties at the 26

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27 defect site.(152, 153) Wang et al. demonstrated that biphasic constructs, composed of decellularized cartilage and bone, cultured with MSCs before implantation to osteochondral defect in canine knee led to full repair after 6 months. Marmotti et al. showed that cartilage fragment loaded scaffold led to formation of functional hyaline-like repair in goat osteochondral defect model. Despite increasing interest and great advance in scaffold based tissue engineering for cartilage and bone, a truly optimal scaffold still needs to be examined. Researchers conduct the studies to seek optimal materials, shapes, combinations, and administration methods to attract the cells, to encourage articular cartilage surface regeneration, and to overcome some shortcomings.(154)

Perspectives There have been numerous approaches to relieve pain, to prevent OA, and to repair damages in articular cartilage defect. Recently, tissue engineering technique has provided several promising possibilities in animal experiments for healing articular cartilage damage. It combines stem cells and chondrocytes, chondrogenic signals, and scaffolds to prepare a biological and biomechanical substitute of hyaline cartilage tissue. Although excellent recent advances has made in each tissue engineering approach, each approach has its own shortcomings and problem to solve as we have 27

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28 focused in this review. In general, native articular cartilage is elegantly organized and truly outstanding tissue in the joint with durability, strength, and lubrication. To produce newly engineered cartilage with physiological function and to translate into human clinical practice, there are several major hurdles to overcome. The paramount challenge is the three-layered construct with superficial zone, middle zone, and the deep calcified zone interdigitating with finger-like projections into the subchondral bone. Next, the integration of regenerated cartilage with the adjacent native cartilage is also a critical challenge. Finally, the optimal biomechanical function with well endowed lubrication and minimal wear. Although the integration between native and newly regenerated cartilage is critical as it provides stable fixation and load distribution, the well-integrated cartilage is rarely achieved in animal experiments possibly due to the abundance of ECM and relatively reduced cellularity. Currently, no other materials can be obtained with the compressive and tensile properties of native cartilage with the optimal friction coefficient. Further study for well-organized combination of approaches should be needed such as effective practical use of PRP to overcome these tough obstacles such as integration, biomechanical properties, and lubrication for better cartilage surface regeneration.

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29 To develop or improve better articular cartilage surface regeneration technique, animal experiments and evaluation methods should be refined following 3R rule; replacement, reduction, and refinement.(155) No animal model has been established as ideal model for every type of project in articular cartilage research. A comprehensive analysis of each available species needs to be considered when planning an animal study. Even in animal experiments, regenerated cartilage should be evaluated noninvasively and evaluated extensively such as surface lubrication. Most of the concepts have been initially investigated in in vitro experiments and in smaller animal models. There are practical challenges to in vivo scale-up, which is critical step to reliably assess new approaches or concepts prior to clinical application can take place. The challenges include the establishment of appropriate large animal models for testing clinical hypothesis and the preparation of larger scale of biomaterials and culture cells than in usual experiments in the laboratory. Therefore, establishment and refinement of animal models with chondral and osteochondral defects could make a great contribution to the advances of tissue engineering in cartilage surface regeneration. We are optimistic that in the coming years the domain of articular cartilage surface regeneration and homeostasis will have major quantum strides and successful translational progress in the amelioration of horrendous pain in patients with osteoarthritis. .

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Frenkel, S.R., Clancy, R.M., Ricci, J.L., Di Cesare, P.E., Rediske, J.J., and Abramson, S.B. Effects

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52

Figure Legends

52

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53

Figure 1. Classification of articular cartilage defect (A) Partial-thickness defects of the articular cartilage do not penetrate into subchondral bone. The lack of blood supply and the lack of access to the stem cells are found in this kind of defect, and the only cell source for regeneration is synovial fluid or synovial membrane. This impedes self-repair ability in partial thickness cartilage defect. (B) Full-thickness articular cartilage defect pass through the tidemark layer and penetrate into subchondral bone. The access to the bone marrow-derived stem cells support spontaneous repair in full-thickness cartilage defect.

53


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54

54

Tissue Engineering Part B: Reviews Articular Cartilage Regeneration: Challenges and Opportunities (doi: 10.1089/ten.TEB.2014.0661) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f

Page 55 of 62

55 TABLE 1. CARTILAGE DEFECT MODEL Duration Species

Age

Defect

of

Technique Location

treatment Mice Rat

Rabbit

Dog

Dimension (mm) Type

Study Size

Depth

8W

12W

Groove

FD

Needle

ø0.6-0.8

~BM

30

8W

16W

Groove

FD

Needle

ø0.6-0.8

~BM

31

8.5M

8W

MFC

FD

Drilling

ø0.85

~BM

35

12W

24W

MFC

FD

Needle

ø1.5

3

36

8.5M

24W

Groove

FD

Drilling

ø2

2

37

12W

24W

Groove

FD

Punch

ø1.5

2

38

12W

5W

Groove

FD

Drilling

ø1.5

1.5

39

6W

24W

Groove

FD

Custom-built device

0.7×4

0.8

40

7/12W

48W

MFC/LFC/Groove

FD

Drilling

ø3

0.5

42

30W

24W

Groove

FD

Drilling

ø4

4

43

4-6/7-12W

48W

Groove

FD

Drilling

ø3

~BM

17

16W

24W

MFC

FD

Drilling

6×3

3

22

Adult

48W

MFC/Groove

PD

Custom-built device

1×4-6

0.2/0.25

21

Adult

24W

Groove

FD

Drilling

ø3

~BM

45

8M

24W

Groove

FD

Drilling

ø3

3

44

4M

4W

MFC

FD

Drilling

3×6

3

46

Adolescent

4W

Groove

FD

Drilling

ø3

4

47

Adult

24W

Groove

FD

Punch

ø7

7

48

15W

24W

Groove

FD

Curette

2×7

~SB

49

N/A

12W

MFC

FD

N/A

10×15

5

52

Adult

78W

Groove

FD

Punch/curette

ø4

0.3-0.5

53

55


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56 Adult

52W

MFC

FD

Drilling

ø5

6

54

Adult

15W

Groove

FD

Punch

Ø4

10

56

Adult

30W

Groove

FD

Punch

Ø2.7 - 6.5

4.5

57

Adult

15W

LFC

PD

Dermal punch

Ø4.5

N/A

58

0.5×7-9

0.6

21

ø5.4

8

26

0.5×15

0.5

59

TABLE 1. (To be continued) Minipig

2-4Y

7W

MFC

PD

Custom-built device

26M

52W

Groove

FD

Trephine

4M

8W

Groove

PD

gouge

2-3Y

24W

MFC

FD

T-handle instrument

ø7

10

60

Pig

8-9M

52W

Groove

FD

Punch/knife/curette

ø6

SB

61

Sheep

2-4Y

24W

Groove

PD

Custom-built device

5×10

0.25

62

Adult

12W

MFC

FD

N/A

ø15

14

63

Adolescent

26W

MFC/LFC

FD

N/A

ø6.3

8-10

64

Adult

12W

MFC/LFC

FD

Punch

ø9

8

65

3Y

8W

Groove

FD

Drilling

ø6

0.8

66

6Y

52W

MFC

FD

Drilling

ø10

~BM

67

2-3Y

16W,

MFC/Groove

FD

Drilling

ø3

4

68

Adult

52W

MFC

FD

Custom-built device

ø6

6

69

Adult

104W

MFC

FD

Drilling

ø12

15

70

1.5Y

14W

LFC

FP

N/A

ø6

0.8

71

2-4Y

12W

MFC

PD

Punch

ø6

N/A

72

2.5-3.5Y

16W

MFC/Groove

FD

Punch

ø7

BM

73

N/A

9M

Groove

FD

Drilling

5×5/15×15

~BM

74

2-6Y

6M

Groove

FD

Drilling

ø15

~SB

75

4-6Y

22M

Groove

PD

Abrading tool

20×20

0.5

76

Goat

Horse

56


Page 57 of 62

57 2-7Y

16W

MFC

FD

Drilling

ø10

10

77

D, day; W, week; M, month; Y, year; N/A, not available; MFC, medial femoral condyle; LFC, lateral femoral condyle; OCD, osteochondral defect; PD, partial thickness cartilage defect; FD, full thickness cartilage defect; BM, bone marrow; SB, subchondral bone.

57


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58

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Tissue Engineering Part B: Reviews Articular Cartilage Regeneration: Challenges and Opportunities (doi: 10.1089/ten.TEB.2014.0661) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f Page 59 of 62

59

TABLE 2, Cell Source Accessibility and

CELL FOR

CARTILAGE REPAIR

59

Differentiation Method References

STEM


Page 60 of 62

60 Tissue Source Bone Marrow (BMMSC)

Chondrogenic ability  Invasive harvest technique

98

 Widely used stem cell source  Abundunt source and less invasive

Adipose Tissue (AMSC)

 Micromass

technique  Less chondrogenic potential

Adult Human Stem Cells

Synovium (SMSC)

Periosteum (PMSC)

 Great chondrogenic potential

or

3D

pellet

culture

99, 106

system  Chondrogenic

medium

including

 Less invasive harvest technique

insulin, transferrin, selenous acid,

 Readily accessible cell source

ascorbic acid, sodium pyluvate, and

 Invasive harvest technique

dexamethasone.

100, 111

 Growth factors

 Donor-site Morbidity

101

 Similar chondrogenic ability to bone marrow

Muscle (MDMSC)

 Controversial chondrogenic ability  Unlimited self-renewal and pluripotency

()

Embryonic Stem (ES) cells

102  Co-culture with mature chondrogenic cells

 Immortal cell source

 Embryoid body (EB) formation system

 Risk of teratoma formation

 Culture EB under hypoxia, with

 Donor-host rejection

growth factors such as BMP2, 4,

 Ethical concerns

and TGFß-1

113-120

 Patient specific therapy Induced Pluripotent Stem (iPS) Cells

 Establishment of disease model for

No established technique for articular cartilage differentiation

pathological therapy  Risk of initiating cancer

60

121-126

Tissue Engineering Part B: Reviews Articular Cartilage Regeneration: Challenges and Opportunities (doi: 10.1089/ten.TEB.2014.0661) been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ f Page 61 of 62

61

BMP, Bone morphogenetic protein; TGF, Transforming growth factor.

61


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62

62

The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.

Highly Porous Gelatin Reinforced 3D Scaffolds for Articular Cartilage Regeneration.

Human stem cells and articular cartilage regeneration.

Stem cells in articular cartilage regeneration.

Development of hybrid scaffolds using ceramic and hydrogel for articular cartilage tissue regeneration.

Naturally Occurring Extracellular Matrix Scaffolds for Dermal Regeneration: Do They Really Need Cells?

Multipotent Stromal Cells Outperform Chondrocytes on Cartilage-Derived Matrix Scaffolds.

Extracellular osmolarity regulates matrix homeostasis in the intervertebral disc and articular cartilage: evolving role of TonEBP.

ECM scaffolds for cartilage regeneration.

Articular cartilage regeneration using cell sheet technology.

Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes.

Extracellular Vesicle-functionalized Decalcified Bone Matrix Scaffolds with Enhanced Pro-angiogenic and Pro-bone Regeneration Activities.

Superficial Zone Extracellular Matrix Extracts Enhance Boundary Lubrication of Self-Assembled Articular Cartilage.

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

Electrospun cartilage-derived matrix scaffolds for cartilage tissue engineering.

cartilage extracellular matrix scaffolds with sequential delivery of TGF-β3 for chondrogenic differentiation of adipose-derived stem cells.

Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix.

Regeneration of articular cartilage in healer and non-healer mice.

Preparation of Extracellular Matrix Developed Using Porcine Articular Cartilage and In Vitro Feasibility Study of Porcine Articular Cartilage as an Anti-Adhesive Film.

Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

Mesenchymal stem cells and nanofibers as scaffolds for the regeneration of thyroid cartilage.

Pore size effect of collagen scaffolds on cartilage regeneration.

Integrins and chondrocyte-matrix interactions in articular cartilage.

Flow-independent viscoelastic properties of articular cartilage matrix.