Osteoarthritis and Cartilage 25 (2017) 1577e1587

Review

Regenerative approaches for cartilage repair in the treatmentof osteoarthritis

M.H. Li, R. Xiao, J.B. Li, Q. Zhu*

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and PharmacologicalEvaluation Study, Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing, China

a r t i c l e i n f o

Article history:Received 8 March 2017Accepted 1 July 2017

Keywords:OsteoarthritisRegenerative therapyCartilageChondrocyteCell homingScaffold

* Address correspondence and reprint requests to: QBioactive Substance and Function of Natural MedicinNew Drug Mechanisms and Pharmacological EvaluPharmacology, Institute of Materia Medica, Chinese Aand Peking Union Medical College, Beijing, China.

E-mail address: zhuq_cams@126.com (Q. Zhu).

http://dx.doi.org/10.1016/j.joca.2017.07.0041063-4584/© 2017 Osteoarthritis Research Society Int

s u m m a r y

Osteoarthritis (OA) as a debilitating affliction of joints currently affects millions of people and remains anunsolved problem. The disease involves multiple cellular and molecular pathways that converge on theprogressive destruction of cartilage. Activation of cartilage regenerative potential and specific targetingpathogenic mediators have been the major focus of research efforts aimed at slowing the progression ofcartilage degeneration and preserve joint function. This review will summarize recent key discoveriestoward better understanding of the complex mechanisms behind OA development and highlight thelatest advances in basic and clinical research in the approach for cartilage regeneration. Prospectively,more potent therapeutic strategies against progressive cartilage deterioration may use a combination ofcytotherapy, pharmacotherapy, and bioscaffoldings for improved chondrogenic differentiation and stem/progenitor cell homing as well as the concomitant reduced enzymatic matrix degradation and inflam-mation. Further, treatments need to be provided with increased preciseness of targeted therapy. Onemight expect that the regenerative therapies could potentially control or even possibly cure OA if per-formed at early stages of the disease.

© 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Introduction

Osteoarthritis (OA) is the most common joint disease with anincidence that increases markedly with advancing age. Accordingto WHO, the symptomatic OA affects approximately 9.6% of menand 18% of women aged over 60 years worldwide. As one of themost leading disabling diseases in developed countries, 80% of OApatients have limitations in movement, among which 25% aredisabled. OA develops through a series of pathological changes dueto injury, aging, inflammatory causes, or genetic factors, resulting inarticular cartilage degeneration in addition to variable degrees ofchondrocyte or synovial hypertrophy, subchondral bone remodel-ing, and chronic joint and systemic inflammation manifested dur-ing disease progression1. The articular cartilage is a specializedhyaline cartilage lining on the articulating surfaces of bone ends in

. Zhu, State Key Laboratory ofes, Beijing Key Laboratory ofation Study, Department ofcademy of Medical Sciences

ernational. Published by Elsevier L

joints, acting as a cushion and reducing friction between bones toallow smooth and painless joint motion. Chondrocytes are the soleresident cells of cartilage accounting for only 1e5% volume of thearticular cartilage, responsible for the production of collagens,proteoglycans and hyaluronan that maintain the extracellular ma-trix (ECM) infrastructure and conferring mechanical properties ofcartilage2. Cartilage degradation is considered a metabolic imbal-ance between catabolic and anabolic factors that constitutes apathological feature of pro-catabolic response leading to focal andprogressive hyaline cartilage loss3. A group of proteolytic enzymes,including a disintegrin and metalloproteinase with thrombo-spondin motifs (ADAMTS), matrix metalloproteinase (MMPs), andcollagenases, collectively contribute to the degradation of proteo-glycan and type II collagen in the ECM4. Due to the sparsity ofchondrocytes and a lack of vascular supply, cartilage has limitedcapacity to combat enzymatic damage or implement autonomicfunctional healing process of the tissue. Current therapeuticsagainst cartilage defects mainly include surgical interventions (e.g.,microfracture, mosaicplasty, tissue engineering including advancedand mimetic biomaterials scaffolds), cell transplantation (stemcell or chondrocyte implants), targeted therapy, and disease

td. All rights reserved.

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modifying therapy (anti-inflammatory agents dampening immuneresponses). In this review, we focus on recently identified patho-genic mechanisms underlying OA cartilage degeneration in pro-gression and highlight the discovery and development of noveltherapeutics that show promising improvement in cartilageregeneration, with discussions on potential extensions for futuretranslational studies and clinical applications.

Current understanding of the cellular and molecular events inOA cartilage degradation

Articular chondrocytes in a healthy condition are resistant tohypertrophic differentiation and able to orchestrate fine-tunedgene expressions of ECM products, but as the single cell typewithin the cartilage, they have propensity to switch toward a hy-pertrophic phenotype in osteoarthritic conditions. Chondrocytesfrom OA cartilage have altered signaling activities that inducethemselves to proliferate and differentiate into hypertrophicchondrocytes, demonstrating reduced ability to tolerate mechani-cal stress or maintain cartilage integrity (Fig. 1). Chondrocytes withincreased age decline in their numbers and virtually few of themproduce or respond to anabolic growth factors5,6. Their poorresponse to the growth factor TGF-b1 leads to reduced signalingthrough the Smad3 pathway, thereby exhibiting a disruptivephenotype7. As OA progresses, chondrocytes not only compromisessynthesis of ECM molecules but also respond to proinflammatorystimuli such as IL-1b and TNF-a and produce excessive amounts ofproteolytic enzymes most prominently MMP-13 and ADAMTS-5that cleave collagens, cartilage oligomeric matrix protein, pro-teoglycans, and glycosaminoglycan, leading to disruptive homeo-stasis of ECM in the cartilage8e11.

Subchondral bone osteoblasts (SBOs) in OA conditionscontribute to chondrocyte phenotypic changes at the cellular levelby downregulating the ability of chondrocytes to synthesizeaggrecan but upregulating their capacity in production of cartilageECM-specific enzymes. SBOs exert such effects most likely throughboth direct and indirect cellecell interactions that involve ERK1/2and p38 MAPK12. Reversely, the pathways are also employed bychondrocytes to mediate SBO phenotypic changes in matrixdeposition and osteogenic gene expression13. As a major source ofinflammation, fibroblast-like synoviocytes (FLSs) increase in num-ber in arthritic conditions, playing a critical role in mediating sy-novial recruitment of inflammatory cells, cartilage destruction andpathogenesis. Upon exposure to HIF-2a-induced cytokines bychondrocytes, activated FLSs migrate, attach and invade cartilage/pannus14,15. Collagen released from damaged cartilage furtherstimulates FLSs to upregulate discoidin domain receptor (DDR)-2expression and MMP secretion, exacerbating enzymatic degrada-tion of cartilage16. The bi-directional cell communications betweenarticular chondrocytes and neighboring cells appear to be essentialfor promoting joint inflammation and cartilage destruction duringOA development.

It has long been recognized that cartilage destruction is causedprimarily due to an imbalance between catabolism and anabo-lism17,18. Increased activity of catabolic signaling pathways andECM degrading enzymes leads to chondrocyte hypertrophic dif-ferentiation, matrix degradation and vascular invasion1. Chon-drocytes of aged and osteoarthritic cartilage demonstrate anskewed TGF-b-dependent signaling from the ALK5/Smad2/3 toALK1/Smad1/5/8 pathway in hypertrophic changes and terminaldifferentiation likely induced through activation of the canonicalWnt pathway7,19. The canonical Wnts and WISP1 overexpressed insynoviocytes additionally cause cartilage superficial layer erosionthrough upregulation of MMP and aggrecanase activities20. Acti-vated hedgehog (Hh) drives OA progression by inducing

chondrocyte hypertrophy and upregulating MMP-1321 and possiblyADAMT-5 through cholesterol mediated promoter activation22.

NF-kB as an orchestrator activated from mechanical stress, ag-ing, inflammatory cytokines in disease progression stimulate manycatabolic genes. Activation of the canonical NF-kB signalingpathway by IL-1b inhibits the TGF-b1/Smad signaling mediatedchondrogenesis of articular cartilage stem cells through suppress-ing Sox9, Col2, aggrecan, and hyaluronansynthetase 223. WhereasIL-1-induced aggrecan loss is primarily mediated by JNK-224,excessive mechanical stress upregulate expression of the catabolicfactors MMP-13, TNF-a, and ADAMTS-5 through activation of bothp38 and ERK1/225. Type II collagen released from degraded carti-lage stimulates to further upregulate MMPs through phosphory-lation of the DDR-2-interacting protein annexin A2, therebyinitiating another round of cartilage degeneration16. Takentogether, the above studies indicate that multiple cellular andmolecular pathways converge in the degradation of cartilage dur-ing disease process.

Recent research approaches for cartilage regeneration in OAtreatment

Significant advances have been made in recent years in thedevelopment of regenerative therapies for OA as described below(Fig. 1).

Anti-degradation

Both biological agents and chemical compounds have beenutilized to inhibit matrix-degrading enzyme activities. Amongseveral mAbs against ADAMTS-5, themost studied 12F4.1H7mAb isshown to specifically suppress ADAMTS-5-induced ARGS-aggrecanrelease and slow cartilage degradation and osteophyte forma-tion26,27. Through pharmacological-based virtual screening, twolead compounds, 409 and 421, have been recently identified aseffective inhibitors for ADAMTS-428. Development of mAbs that areable to recognize both ADAMTS-4 and -5, such as 237-5329, holdinggreat potential for future design of targeted therapy therapeutics.Apart from direct inhibition of matrix-degrading enzymes, inhibi-tion of Smad1/5/8 phosphorylation is shown to effectively reduceMMP-13 expression and prevent chondrocyte terminal differenti-ation30. Either local or systemic administration of anti-TGF-b1neutralizing antibodies reduces uncoupled bone formation andangiogenesis, resulting in decreased subchondral bone sclerosisand delayed cartilage degeneration process31,32. In addition,Wnt/b-catenin signaling inhibitors PKF115-584, PKF118-310 andCGP04909033 and the proteasome inhibitor MG13234 show po-tential in the control of cartilage degeneration.

Anti-inflammation

Based on the fact that cytokines and chemokines are essential instimulating cartilage catabolism, blocking inflammatory mediatorscould substantially prevent OA progression. Treatment with theNF-kB pathway inhibitor BAY11-7082 restores IL-1b-inhibitedchondrogenesis of cartilage stem cells and delays progression ofexperimental OA23. Inhibition of serine proteinases with alpha-1-anti-trypsin-Fc fusion protein markedly suppresses joint inflam-mation by blocking PR3 conversion of the IL-1b precursor to theactive form and inducing IL-1Ra as natural inhibitor of IL-1b35.Additionally, suppression of HIF-2a by gene deletion in damagedchondrocytes and FLSs inhibits production of chemokines involvedin pannus formation and inhibition of cartilage destruction14. Thefebrifugine analog halofuginone has recently been found to inhibitTh17 differentiation in addition to inhibiting TGF-b-mediated

Fig. 1. Convergent pathogenic pathways of cartilage degradation in osteoarthritis and strategies in cartilage regenerative treatment. Chondrocytes, fibroblast-like syno-viocytes (FLSs) and subchondral bone osteoblasts (SBOs) are shown. Mechanical stress and biochemical stimuli on chondrocytes result in increased production of proinflammatorymediators, extracellular matrix (ECM) degrading enzymes, and hypocellularity caused by apoptosis. Activation of NF-kB induces HIF-2a in chondrocytes, which leads to upregulatedproduction of MMP-13 and MMP-3 to degrade ECM and induces expression of RUNX2, Ihh, Col10 and VEGF (not shown) to promote endochondral ossification. Increased expressionof TGF-b is seen in SBO and FLS of OA and contributes to development of osteoarthritis. TGF-b signaling skewing from classical Smad2/3 to Smad1/5/8 leads to hypertrophicdifferentiation of chondrocytes. Inhibition of the inflammatory mediators or interruption of intracellular signaling pathways by mAbs, chemical compounds, and fusion proteinsrepresents recent approaches to the strategies for anti-inflammatory and anti-degradative treatments. Cell-based or cell-homing therapies are a viable means for pro-chondrogenesis facilitating cartilage regeneration, including scaffolding systems containing stem cell homing factors (1, 2) or progenitor cell chemotaxis followed by chondro-genic stimulation (3), autologous or matrix-assisted autologous chondrocyte implantation (ACI or MACI) (4), in vivo generated cartilage from endogenous chondrogenic-differentiated stem cells (5), mesenchymal stem cell implantation (6), and growth factor-induced stem cell expansion and pro-chondrogenic differentiation with augmentedECM production (7). Integrated approach is a combined therapy that utilizes all means of most advanced technologies available targeted, genetic, and cellular therapies to treat andregenerate cartilage faster, safer and more accurately and effectively (8).

M.H. Li et al. / Osteoarthritis and Cartilage 25 (2017) 1577e1587 1579

collagen type I synthesis and antiangiogenesis36. Comprehensiveapproaches show that combined blockade of TNFa and IL-17 withbispecific antibodies exhibits synergistic therapeutic efficacies ininhibiting both cytokines for reduced proinflammatory responsesand cartilage degradation37. Inhibition of TGFa and CCL2 signalingby AG1478 and RS505393, respectively, in chondrocytes reducestheir cooperative effects on elevation of MMP-13 and TNFa inposttraumatic joint degradation38.

Pro-chondrogenesis

Growth factors in combination may exert effective chondro-genic activities. Combined overexpression of IGF-I and FGF-2

within cartilage defects enhances formation of full-thicknessosteochondral cartilage39. FGF2 and Wnt3a signaling protein pro-vide synergistic and prolonged stimulation on the proliferation andchondrogenic potential of mesenchymal stem cells (MSCs)40.Further, prolonged expression of TGF-b3/BMP-6 in adipose-derivedstem cells markedly increases expression of chondrogenic markersSox-9, ACAN, and Col2A1, resulting in suppressed hypertrophy/osteogenesis and formation of a zonal structure neocartilageresembling the articular cartilage41. More sophisticatedly, earlytreatment with FGF2 was shown to maximize MSC expansion po-tential, while the subsequent combinatorial stimulation with TGF-b1 and FGF9/FGF18 enhanced chondrogenic differentiationwith augmented ECM production42. Whereas in combination with

M.H. Li et al. / Osteoarthritis and Cartilage 25 (2017) 1577e15871580

TGF-b1, BMP-7 promotes MSCs to express markers for cartilagesuperficial zone, IGF-1 facilitates upregulation of middle zonemarkers43. It appears that selective combinations and sequence ofdelivery of pro-chondrogenic growth factors are essential forcellular differentiation and structural and functional repair of thecartilage tissue. Current approaches are gravitating toward morestrategic design for growth factor combination as an advancedapproach to cartilage regenerative treatment.

Homing of endogenous cells

Endogenous stem/progenitor cells recruited to injured tissuesare thought to critically contribute in the natural healing process.Arthritic cartilage emits elevated homing signals through chemo-kines and cytokines that bind to their receptors to mediate direc-tional migration and infiltration of reparative cells from distance.TGF-b3 as a chondrogenic growth factor recruits 130% more cells inthe regenerated hyaline cartilage with improved compressive andshear properties44. A 2-fold increase is seen when combined withmechano growth factor for recruitment of MSCs to improve inte-gration of the neo tissue to surrounding cartilage45. Macrophageinflammatory protein (MIP)-3a, IL-846 and stromal cell-derivedfactor-1 (SDF-1)47 also play important roles in MSC recruitment.Homing molecule-loaded bioengineered scaffolds creating afavorable microenvironment to attract and support sustained cellhoming represent advanced techniques for cartilage regeneration.Localization of resident stem/progenitor cells, such as the recentlyidentified and characterized osteochondroreticular (OCR) stemcells48, skeletal stem cells (SSC)49, and Prg4-expressing articularcartilage progenitor cells50, by recruiting them from the vicinitymay hold significant promise in development of regenerativecartilage therapy.

Cell- and scaffold-based cartilage regeneration

Cell therapy for chondrocyte replenishment represents a plau-sible intervention strategy to restore impaired joint cartilage andhypocellularity incurred from chondrocyte loss. Technically,retrieval of autologous donor chondrocytes from cartilage tissue forautologous or matrix-assisted autologous chondrocyte implantation(ACI or MACI) is very limited and, most often, neither starting cellnumbers nor proliferative capacities suffice for implantation over alarge surface area. Bioreactor cultivation has recently been devel-oped to effectively stimulate cellular expansion and ECM expres-sion51. Overlying self-assembled MSCs on top of chondrocyte-ladenhydrogel scaffolds is shown to facilitate cell-mediated regenerationof hyaline-like cartilage52. Interestingly, combination of cell-basedtransplantation, such as MSCs, with autologous mosaicplasty facil-itates the repair of osteochondral defects effectively by promoting acomplete healing of empty spaces between the grafts and sur-rounding tissues53. However, autologous implantation requiresprior open-joint surgical procedures to collect donor chondrocytesfrom usually a non-weight-bearing cartilage area of the joint and isunlikely to apply in the elderly or those with a few other lesions.Importantly, the potential risks of developing long-term donor sitemorbidity after removal of healthy cartilage from intact articularsurface remain undetermined. To avoid explantation of healthycartilage, a novel technique was recently established to generatecartilage for grafting in vivo from endogenous chondrogenic-differentiated stem cells by implanting transcutaneously a collage-nous patch carrying slowly-released BMP-2 sutured onto innersynovial membrane54. The encapsulated stimulating agent pre-sumably recruits synovial stem cells into the outer juxtasynovialspace for chondrogenic differentiation. As this technique involvesarthrotomy only once, it is likely to provide a more acceptable

alternative to in vitro-generated implants for ACI/MACI procedures.Further, the technique may potentially be incorporated into themosaicplasty surgical procedure, which has the advantages ofrepairing large osteochondral defects.

Scaffold techniques emerged for years as a novel approach to theregenerative treatment and many sophisticated devices arecurrently being developed. Biphasic scaffold platforms, which arecomposed of chitosan thermogel and demineralized bone matrixcontaining affinity peptide E7 specific for cell homing, are shown toeffectively promote recruitment of bone-derived MSCs from thesubchondral marrow or peripheral blood superior to microfractureprocedures55. Encapsulated in hydrogels containing collagen-mimetic proteins, MSCs exhibit improved viability and signifi-cantly enhanced chondrogenic differentiation56. A recently devel-oped scaffolding system applies a chitosan adhesive patch withattached platelet-derived growth factor-AA contained fibrin gel,capable of recruiting autologous bone-marrow derived MSCs formigrating to the site of cartilage defect57. This novel controlledrelease scaffold also retains migratory MSCs on the affected area bypreventing them from dispersion, thus significantly improvingcartilage repair. In order to ensure endogenous cells executeregeneration after being recruited into the scaffold, a two-stepstrategy seeded polymer network hydrogel with SDF-1a for chon-drocyte progenitor cell (CPC) chemotaxis, followed by chondro-genic stimulation58. This maneuver enabled rapid chondrocyterepopulation in the full-thickness chondral defects and near-complete restoration of the cartilage matrix. As CPCs can sensecell death and display phagocytic propensity to clear cell debris indamaged cartilage59, their scavenging property may additionallyaccount for the improved outcomes achieved by this strategy.

Recent clinical approaches for cartilage regeneration in OAtreatment

It is worthy to note that current regenerative therapies for OAthat have been clinically investigated mainly focus on the treat-ment of moderate and advanced stages of the disease60e79. Types oftreatment and clinical outcomes are summarized in Table I.

MSC-based therapy

MSCs are capable of reducing and/or eliminating the need forrepetitive administration and proved to be safe. Matrix-inducedautologous MSC implantation showed tendency for earlier clinicalimprovements as compared to implantation of chondrocytes70.Recent results of a phase IeII clinical trial show a long-lastingamelioration of pain and quality of life, and improved MRI T2relaxation times at 12 months post-BMSC treatment of knee OA76.Intraarticular injection of adipose tissue derived MSCs also inducedregeneration of hyaline-like articular cartilage of severe OA kneejoint with greatly improved function and pain67,79. Therapeuticeffects for affected ankles or hips of OA patients were also observedin a long-term follow-up clinical trial73. Due to the immune privi-lege trait, intraarticular administration of allogeneic MSCs hasshown no to minor adverse effects, and intraarticular trans-plantation of allogeneic BM-derived MSCs was shown to promotecartilage regeneration in OA knees with decreased MRI T2 relaxa-tion times, reduced pain and disability66,72.

Scaffold-based therapy

The US FDA recently approved the first MACI product usingporcine-derived collagen membrane for the repair of cartilage de-fects of the knee. A 2-year clinical trial demonstrated reduced painand improved function in comparison to microfracture80. As the

Table IRecent clinical studies on the regenerative treatment for OA

Types oftreatment

Therapeutic agents Cases Follow-up(month)

Affectedjoint

Diseasestage

Clinical outcomes Reference

MSCimplantation

Autologous BMSC 15 12 Knee KL 2-3 Cartilage regeneration with decreased MRI T2relaxation times in all patients, improvedbodily pain and physical functioning

76

Autologous BMSC 12 12 Knee KL 2-4 Partial articular cartilage healing in 91%patients with decreased MRI T2 relaxationtimes, significant recovery of functional loss

62

Autologous BMSC 50 12 Knee KL 2-4 Cartilage repair in 74% patients with decreasedMRI T2 relaxation times and improvedsymptoms

61

Autologous BMSC 18 30 Knee, ankle, hip KL 3-4 Some patients with reduced subchondraledema and repaired articular cartilage, anddecreased VAS and WOMAC scores

73

Autologous ASC 18 6 Knee KL 2e4ICRS 3

No changes in KL grade, but improved ICRS inpartial defected cartilage in the high-dosegroup, hyaline-like cartilage regeneration

67

Autologous ASC 18 6 Knee KL 3-4 Improved pain and cartilage condition 79

Allogeneic BMSC 55 24 Knee PMM Increased meniscal volume, reduced pain 66

Allogeneic BMSC 30 12 Knee KL 2-4 Improved cartilage quality, reduced pain anddisability, 77% of the patients satisfied

72

Scaffold Type I collagen-hydroxyapatite 10 30 Knee, ankle KL 1-2 No improved MOCART, incomplete cartilagerepair, poor subchondral bone repair, butimproved KOOS pain subscale

71

Type I/III collagen membrane þsynovial-derived MSC orautologous chondrocyte

14 24 Knee KL 1-2 Improved MOCART and KOOS, regeneratedcartilage filled the defects, early clinical andradiological improvement for MSC treatment

70

Chitosan þ microfracture 80 13 Knee ICRS 3-4 >90% lesions filled with repair tissue,decreased MRI T2 relaxation times, wellsurfaced cartilage architecture, improved cellviability and cell distribution

63,77

Type I collagen sponge þautologouschondrocyte

23 24 Knee ICRS 3-4 Increased MOCART, graft integrated toadjacent bone and cartilage, but irregularitywithin the cartilage superior zone

69

type I collagen-hydroxyapatite

27 60 Knee ICRS 3-4 Significant improvement in MOCART andsubchondral bone status, 78% lesionscompletely filled of cartilage

64

Disease-modifyingdrugs

ABT-981 36 2 Knee KL 1-3 Reduced high-sensitivity C-reactive protein,decreased MMP degradation products type Iand III collagen

60,78

Strontium ranelate 330 36 Knee KL 2-3 Decreased cartilage volume loss on theplateaus and bonemarrow lesions progressionin the medial compartment

65

Sprifermin 168 12 Knee KL 2-3 Reduced cartilage loss and reduced joint spacewidth narrowing, increased cartilagethickness and WOMAC pain scores

68,74

Gene therapy Genetically engineeredallogeneic chondrocyteexpressing TGF-b1

102 13 Knee KL 3 Hyaline cartilage regeneration with improvedIKDC and VAS scores

75

Abbreviations: OA, osteoarthritis; MSC, mesenchymal stem cell; ASC, adipose-derived stem cell; BMSC, bone marrow-derived mesenchymal stem cell; PMM, partial medialmeniscectomy; KL, KellgreneLawrence; ICRS, International Cartilage Repair Society; IKDC, International Knee Documentation Committee Subjective Knee Form; VAS, visual analogscale; WOMAC, Western Ontario and McMaster Universities Osteoarthritis; KOOS, Knee Injury and Osteoarthritis Outcome Score; TGF-b1, transforming growth factor-b1.

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cellular sheet is trimmable, such autologous cellularized scaffoldcan be tailored to patients depending on the number and size ofcartilage defect. A phase III trial using MACI 3D scaffold withsimplified surgical procedures showed significant improvement inclinical and radiological outcomes for 2 years in treatment of largefocal chondral and osteochondral lesions69. A different approachtaken with multilayered collagen scaffolds composed of collagentype I and magnesium-hydroxyapatite implanted onto the articularchondral and osteochondral lesions produced satisfactory out-comes in patients with chondral lesion or osteochondritis dis-secans64,81. Studies on layered cell-free porous 3D biomimeticscaffolds, however, suggested incomplete regeneration in cartilageand subchondral bone although clinical improvements occurred71.

Symptomatic and disease-modifying therapies

Pain-alleviating medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs), are the most commonly prescribed

agents for OA and demonstrate some therapeutic effects forsymptomatic treatment. The slow-acting drugs for osteoarthritis(SADOAs), including glucosamine and chondroitin sulfate, havecomparable efficacy on pain relief and functional improvement inOA patients with moderate-to-severe pain82,83. Whereas combinedglucosamine and chondroitin sulfate did not demonstrate superi-ority over placebo in reducing pain and function impairment84,these medications together with NSAIDS did show significantlyreduced cartilage loss in advanced stages of OA85. Further explo-ration is clearly warranted to determine the clinical significance ofsuch combined therapy.

Clinical studies on different stages of OA have demonstrated thatcertain disease-modifying drugs (DMDs) targeting inflammatorymediators can halt or delay disease progression and preserve thestructure and function of damaged cartilage. ABT-981, a potentbispecific antibody recently developed against both IL-1a and IL-1b,is capable of decreasing inflammation and slowing OA progressionmore effectively than either isoform alone60,86. The Phase II study of

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this biological agent is currently underway. Strontium ranelate as aDMD for osteoporosis to inhibit bone resorption and promote boneformation has recently been of particular interest in OA treatmentbecause of its inhibitory effects on the production of IL-1b andMMPs87. Treatment with strontium ranelate at a dose of 2 g/day forknee OA in a Phase III clinical trial resulted in a significant reductionof cartilage volume loss65,88. The synthetic anabolic drug sprifermin(rhFGF18) signaling through FGFR3 is shown to promote chon-drogenesis and cartilage matrix production in double-blind pla-cebo-controlled randomized trial. Intraarticular injections ofsprifermin in symptomatic knee OA patients reduced the loss ofcartilage thickness and volume68,74. The first-in-human, double-blind, randomized, placebo-controlled, dose ascending (3e300 mg)study of intraarticular sprifermin conducted in patients withadvanced knee OA revealed no serious safety concerns89. Anypositive effects on cartilage regeneration are expected to see infuture investigation.

Gene therapy

Gene therapy is envisaged as a promising clinical approach fordelivering genes encoding for cartilage growth factors, pro-regenerative mediators, and inflammatory inhibitors specificallyto the site of damage. This strategy aims to reduce the frequency of

Fig. 2. Current and emerging principles of regenerative therapies for OA treatment. Cudisease at moderate and advanced stages primarily using autologous mesenchymal stemalleviate their symptoms with various degrees of improved clinical outcomes. Specificchemotactic agents or activation of resident cells with regenerative potential chemically orhaving early sign of disease. MSC may be genetically modified or optimized for improvemeninvasive treatments for later stages of OA. As the prevalence of OA is increasing and continueoffer the possibility of halting disease progression more effectively but also increase the lik

invasive modes of delivery but provide sustained continuoustherapeutic effects in contrast to daily injection of recombinantproteins or chemical compounds. Cell-mediated gene therapieswith allogeneic chondrocytes genetically engineered to produceTGF-b1 have shown significant symptomatic and joint functionimprovements after intraarticular implantation in severe late-stageOA phase I and II trials75,90,91. A most recent randomized doubleblind, multi-center, placebo-controlled phase III trial with 1 yearfollow-up shows significant improvement in IKDC, VAS, WOMACand KOOS after single administration of chondrocytes retrovirallytransduced TGFb192.

Future prospects for the regenerative treatment of OA

MSCs have been one of the most attractive candidates for clin-ical application of cell-based therapy. Their regenerative andimmunomodulatory characteristics have made themselves highlyefficient on attenuation of harmful inflammatory responses anddifferentiation into a variety of appropriate tissue-specific cell typesfor tissue repair93. However, it is suggested that choosing the righttype of cells for chondrogenesis lead to greater therapeutic efficacy.Depending on culture conditions, MSCs may express a high basallevel of the transcription factor Slug that inhibits chondrogenicdifferentiation even in the presence of TGF-b and elevated levels of

rrent clinical approaches in regenerative treatment of OA concentrate mainly on thecells (MSCs) and a variety of bioscaffolds, separately or in combination. Patients canhoming of tissue-derived stem/progenitor cells to the site of cartilage defect usingbiologically is a nearly noninvasive approach, potentially more acceptable for patientst in regenerative and reparative capabilities. Scaffolds and microfracture techniques ares to rise markedly with age, earlier and less invasive therapeutic regimens may not onlyelihood of cure for OA at earlier stages.

mailto:Image of Fig. 2|tif

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Sox994. Removal of such cell population or modification to silencethe gene appears to be essential before use. MSCs exhibit differ-ential secretion patterns of TIMPs to regulate pericellular MMPactivities for cell migration and BM-derived MSCs secrete moreTIMPs than fat and traumatized muscle-derived MSCs95. Theinhibitory proteolytic environment provided by BM-MSCs mayprohibit themselves from reaching defect area for tissue repair.Therefore, use of tissue-derived specific MSCs seems sounder andmay be prioritized in consideration of regenerative treatment. OCRand SSCs as newly identified group of tissue-specific stem cells arevital for early postnatal skeletogenesis48,49. The capability of suchendogenous OCR/SSC inducible to proliferate and differentiate intochondrocytes may indicate a huge therapeutic potential.

Although MSCs provide ideal building blocks to restoredamaged tissues, current commercially available therapies have yetto show more complete morphological formation and functionalrecovery of native-like cartilage tissue. The complexity of cartilagestructure indeed highlights the need for more comprehensive andeffective strategies for regeneration of various types of focal lesions(Fig. 1). 3D printings with biocompatible polymers enable layered,mechanically enhanced and biomimetically accurate scaffolds withimprove attachment with surrounding cartilage tissues. Use of 3Dcell printing technologies to fabricate implants has shown togreatly improve cell viability and functionality of chondrocytes96.Small molecule compounds that promote MSCs aggregation andchondrogenesis could potentially further enhance cartilage repairwhen loaded in scaffolds for controlled release97. Manufacturing ofhigh-resolution constructs to more closely imitate native cartilageECM and mechanical property for complete tissue regrowth couldbe considered a favorable option for future therapy development.

Cartilage regeneration process that involves recruitment ofreparative cells can be negatively affected by antichondrogenic orproinflammatory cells chemoattracted concomitantly to thedamaged area. The SDF-1/CXCR4 pathway is known to recruit andactivate MSCs in chondrogenic differentiation47,98; however,blocking this pathway to some extent attenuates cartilage patho-genesis by reducing MMP and aggrecanase production stimulatedfrom chondrocytes99,100. A recent study shows that engineeringchondrocytes to overexpress HMGB1 A-box significantly decreasesthe IL-1b-stimulated production of MMPs and ADAMTs and sup-pression of HMGB1/TLR4/NF-kB pathway improves chondro-genesis101. Thus, the molecular pathways that are involved in eitherdestroying or rebuilding process of cartilage regeneration need tobe identified in order to increase the precision of targeted therapy.Effective application of new promising biologically active com-pounds or natural products by incorporating advanced deliverytechnology such as nanoformulations, microencapsulation, nano-micelle, and live vectors, may likely make a further step to improvetargeted therapy102e106. Genetic transfer with powerful and dura-ble delivery of therapeutic candidates would probably enhancecartilage regenerative process, and integration of targeted and/orgene therapy to cellular therapy with MSCs could probably achieveeven safer and more effective clinical outcomes.

Based on the fact that OA pathogenesis becomes more compli-cated and destructive as disease progresses, treatments can beconsiderably difficult in reversing disease progression at advancedstages. Patients with an early stage of OA are often in denial andaccurate diagnostic criteria for earlier stages OA are not wellestablished. Medications taken usually depend on the symptoms toa great extent for alleviation or palliation of developed disease.Before articular radiographic appearance or worsening of clinicalsymptoms, few take an immediate action to receive treatment for asooner protection against cartilage deterioration. Sensitive labora-tory tests and advanced high-resolution imaging technologies thusneed to be developed for effective evaluation of early metabolic

imbalance signs and initial microscopic changes107e109. Both bio-markers and molecular imaging may potentially be useful for earlyOA detection and distinguishing different OA stages110e114 althoughthe correlation between laboratory findings and a series of clinicalsteps in OA development is yet to be well established. One mightexpect that a combined biomarker and molecular imagingapproach may help increase accuracy of prognostic diagnosis whilethe difficulties in OA treatment may readily be overcome by anearlier intervention with regenerative therapies (Fig. 2).

Conclusion

Treatment of cartilage defects is complicated by several signif-icant medical conditions, ranging from lack of self-repair andunique biomechanical properties to homeostatic imbalance andchronic joint inflammation. More comprehensive therapeuticstrategies need to be considered in tackling these difficulties.Synergistic effects on cartilage healing and restoration of jointfunctionality are expected to achieve by combined therapiespossibly with variable biological and/or non-biological agents foranti-inflammation and anti-degradation, pro-chondrogenesis, andhoming of endogenous stem/progenitor cells. Such integrativetherapeutics may hopefully lead to more effective treatment mo-dalities with substantially improved patient outcomes through alldisease stages. To take a further step, regenerative therapies infuture clinical approach may be also designed to treat OA at earlierstages in instead of leaving patients almost untreated until thedisease becomes symptomatic or severe. A combination of mini-mally invasive regenerative therapies may possibly lead toward amore successful control and even cure of OA in its infancy. Suchstrategy would probably help significantly reduce the globalburden of the disease.

Author contributionsML and QZ contributed to the conception and design of this study.ML, RX and JBL performed searches, analyses and interpretations.ML drafted the paper, and all authors participated in the writingprocess and QZ gave final approval of the version to be submitted.

Conflicts of interestThe authors declare no conflict of interest.

Acknowledgment

This work was supported in part by the State Key Laboratory ofBioactive Substance and Function of Natural Medicines, NationalNatural Science Foundation of China (31170872, 31370922), BeijingNatural Science Foundation (5131002), CAMS Major CollaborativeInnovation Project (2016-I2M-1-011), and National Health andFamily Planning Commission Fund (201402001).

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