Cell-Based Management of Cartilage Defects: A Review

Saif S Aldhuhoori

Department of Orthopedic Surgery, Zayed Military Hospital, Abu Dhabi, UAE

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Abstract

Cartilage injury can be disabling for young and active patients/athletes. These patients often do not regain normal function, and total joint replacement is not the best option for young patients. The current focus is on tissue engineering, scaffolds, and stem cells to form hyaline cartilage that is mechanically functional within cartilage defects. The success of stem cell treatment for animal cartilage defects in vivo is well established. Stem cells have been incorporated for cartilage defect treatment in humans for some time now. Mesenchymal Stem Cells (MSCs) in cartilage repair have shown better outcomes than all available clinical options. Although some issues still need to be addressed, long-term comparative clinical studies of MSCs-based treatments are required before using this technique as the gold standard for cartilage defect treatment.

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Introduction

The real incidence of articular cartilage injury is unclear. Several studies report knee osteochondral defect incidences ranging from 4% -19% in young active patients^1,2,3. The management of hyaline cartilage defects involves procedures that produce fibrocartilage tissue repair (microfractures and abrasion arthroplasty) and those procedures that produce hyaline-like cartilage tissue repair (autologous osteochondral transfer, mosaicplasty and autologous chondrocyte implantation). It was estimated that 250,000 to 300,000 cartilage injuries receive surgical intervention annually in the United States. Of these cartilage injuries, 70% are treated with lavage and debridement, 20% with microfracture, and 10% with other surgical techniques such as autologous chondrocyte implantation, allografts, and autografts⁴.

Chondral articular cartilage injuries (not involving subchondral bone) have a limited chance for spontaneous healing due to its avascularity⁵. However, osteochondral articular cartilage injuries (involving subchondral bone) can undergo cartilage healing that forms fibrocartilage only. Therefore, articular cartilage injury is permanent and may alter joint loading and mechanics. It may lead to clinical symptoms of pain, locking, swelling, and catching^6,7. Articular cartilage defects may fill over time with tissue containing hyaline-like cartilage or fibrocartilage. Fibrocartilage tissue may undergo chondrocyte senescence and death, extracellular matrix (ECM) changes, and fibrillation over time^8-11. If no intervention is undertaken, this process may progress to post-traumatic osteoarthritis¹².

Currently, the focus is to use cell-based tissue engineering and stem cells to produce hyaline cartilage that is mechanically functional within cartilage defects. This review investigates the current concepts and future directions of cell-based management of the articular cartilage defects.

Methods

Search Strategy

A comprehensive review of the literature was conducted. The literature search was based on Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines¹³ (Figure 1). The MEDLINE, Embase, and PubMed databases were searched on March 26^th, 2025. The following search terms were used: Articular cartilage, Cartilage defects, Cartilage regeneration, Stem cells, Mesenchymal stem cells, Chondrocytes, Growth factors. No time limit was given to date of publications.

Figure 1: Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) study selection flow diagram

The title and abstract were screened for all search results and eligible studies received a full-text review. The inclusion criteria were as follows: (1) studies involving animal and human (over the age of 18 years) subjects with cartilage defects (2) clinical or basic science studies assessing MSC-based treatment of cartilage defects, and (3) studies published in English. The exclusion criteria were (1) degenerative cartilage defects, (2) pediatric population, and (3) Opinions, commentaries, and letters to editor.

Cells in Cartilage Regeneration

Various cell types, such as chondrocytes, mesenchymal stem cells, and pluripotent stem cells, can be used for cartilage regeneration.

Chondrocytes

Chondrocytes (cartilage-forming cells) are the initial cells used in cell-based tissue engineering for cartilage lesion repair. They are harvested from the non-weight-bearing area of a joint. They are applied in many evolving cartilage implantation surgical procedures such as Autologous Chondrocyte Implantation (ACI), Characterized Chondrocyte Implantation (CCI), Collagen-covered ACI (CACI) and Matrix-induced ACI (MACI) tissue engineering techniques.

Other sources of differentiated chondrocytes are juvenile/fetal/neonatal chondrocytes. Juvenile chondrocytes grow much faster than adult chondrocytes. They have higher proteoglycan and type II/IX collagen content, resembling cells from native cartilage¹⁴. The disadvantages of adult chondrocytes are donor site morbidity, in-vitro dedifferentiation, and phenotype loss. Both adult and juvenile chondrocytes have limited availability.

Fresh minced juvenile articular cartilage allograft can be delivered with fibrin adhesive monolayer. It is acquired from donors aged from neonates to 13 years old. In this technique, the chondrocytes will migrate from explants and integrate with the native articular cartilage^15,16. The advantages of this technique include single stage procedure, absence of donor site morbidity, does not need subchondral bone violation and low risk of allogeneic response. Fibrin fixation lowers the risk of graft hypertrophy¹⁷. However, this technique has some disadvantages such as graft heterogeneity, graft rejection and disease transmission¹⁸.

Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) are adult tissue-derived cells with high multi-potent differentiation and proliferation capacity and minimal tumorigenicity. The MSCs can be harvested from bone marrow, adipose tissue, synovium, and periosteum¹⁹. They can differentiate into chondrocytes, adipocytes, osteoblasts, and myocytes²⁰.

MSCs can be transplanted alone as an injection or implanted on matrix-associated scaffolds. An enormous amount of MSCs must be transplanted into and retained within the cartilage defect to treat the damaged cartilage. This is because intraarticular injection of MSCs without retaining the MSCs within the defect can result in MSCs dispersion and formation of free bodies in the joint^21,22. This makes the MSCs injection alone a less favorable choice and requires performing subchondral drilling or microfracture procedures as an adjunct to MSCs injections to stimulate the diffusion of bone marrow contents.

MSCs differentiate into chondrocytes through a sequence of stages. These stages include MSCs condensation, chondrocytes proliferation, prehypertrophic and hypertrophic chondrocytes. At the condensation stage, MSCs express Sox9, which is a key chondrogenesis regulatory factor²³. High proliferating chondrocytes undergo maturation and differentiate into prehypertrophic and hypertrophic chondrocytes²⁴. Chondrocytes at the prehypertrophy stage are marked by Indian hedgehog (Ihh) expression and parathyroid hormone 1 receptor (Pth1r). These chondrocytes will develop into early and late hypertrophic chondrocytes. The early hypertrophic chondrocytes show increased expression of collagen, type X, 𝛼1 (Col10a1) as well as decreased expression of Sox5, Sox6, Sox9 and Col2a1. The late hypertrophic chondrocytes express vascular endothelial growth factor A (VEGFA), osteopontin and matrix metalloproteinase 13 (MMP13).

The bone marrow and periosteum MSCs have chondrogenic capacity but are prone to forming bone tissue. The bone marrow source has the advantages of being autologous, multipotential with high Collagen II production, easy collection, long-term safety, and supportive solid evidence. The disadvantages of bone-derived marrow-derived stem cells include hypertrophy after extended culture or implantation. The peripheral blood MSCs can be collected easily, but the clinical evidence is limited. The adipose tissue is abundant, but it has low chondrogenic capacity, low collagen II production, and a lack of clinical evidence. However, the synovial source has high chondrogenic capacity, but synovial-driven stem cells retain fibroblastic features, and clinical evidence is lacking.

Pluripotent Stem Cells (PSCs)

Due to their indefinite self-renewal and ability to differentiate into multiple cell lineages, pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced PSCs (iPSCs), are considered alternative options for cartilage regeneration. ESCs are harvested from the inner cell mass of blastocyst-stage embryos, which raises ethical concerns²⁵.

The iPSCs are ESC-like stem cells derived from a patient’s own skin or blood cells by way of gene transduction employing ESC-specific transcription factors²⁶. PSCs have the risk of tumor development due to their ability to differentiate into multiple cell and tissue lineages and their tendency to grow without restraint²⁷ Direct differentiation of PSCs into chondrocytes is still under investigation, and no clinical trial exists to examine the use of PSCs in managing cartilage defects²⁸.

The derivation of iPSCs from skin or blood cells by gene transduction and direct differentiation of PSCs into chondrocytes is still under investigation. It has been shown that using MSCs in gene therapy does not trigger the immune response²⁹. However, there are some concerns regarding viral transduction³⁰. The most effective way of gene delivery into MSCs is still under investigation.

Table 1 summarizes cell types used in cartilage regeneration and the advantages and disadvantages of each cell type.

Table 1: Cells in Cartilage Regeneration

Type	Cells/Cells source	Advantages	Disadvantages
Chondrocytes	Adult chondrocytes	Autologous cells Differentiated cells	Donor site morbidity In-vitro dedifferentiation & phenotype loss Periosteal donor site morbidity Availability issues
	Juvenile chondrocytes	High collagen II/IX & proteoglycan content Less immunogenic	Availability issues
Mesenchymal stem cells	Bone marrow stem cells	Autologous Multipotentiality High Collagen II production Easy collection Long term safety Strong supportive evidence	Hypertrophy after extended culture or implantation Prone to form bone tissue
	Peripheral blood	Easy collection	Literature paucity
	Adipose-derived stem cells	Autologous Abundant tissue	Low chondrogenic capacity Low collagen II production Lack of clinical evidence
	Synovial-derived stem cells	Autologous High chondrogenic capacity	Retention of fibroblastic features Lack of clinical evidence
	Periosteum	Chondrogenic capacity equivalent to bone marrow	Prone to form bone tissue
Pluripotent stem cells	Embryonic stem cells	Multipotentiality	Ethical issues Tumour development risk
	Induced pluripotent stem cells	Autologous Multipotentiality No ethical issues	Tumour development risk

Signaling Molecules

Growth factors are endogenous and biologically active signaling polypeptide molecules that are used in cartilage regeneration because they stimulate cell growth and enhance chondrogenesis. They include the Fibroblast Growth Factor (FGF) Family, the Transforming Growth Factor-β (TGF-β) Superfamily, the Platelet-derived Growth Factor (PDGF), the Insulin-like Growth Factor (IGF) and exosomes (Table 2).

Table 2: Summary of Signaling Molecules Involved in Cartilage Regeneration

Signaling Molecules	Growth Factor	Chondrogenic/Cartilagenous Effects	References
TGF-β	TGF-β1	Stimulation of ECM production Inhibition of cartilage degradation	49
	BMP-2	Stimulation of ECM production Increase of ECM turnover Increase of aggrecan degradation
	BMP-7	Stimulation of ECM production Inhibition of cartilage degradation
FGF	FGF-2	Inhibition of proteoglycan synthesis Increase of aggrecan degradation Upregulation of MMPs	28
	FGF-18	Stimulation of ECM production Increase of chondrocyte proliferation
IGF	IGF-1	Stimulation of ECM production Decrease of ECM catabolism	51
PDGF	PDGF	MSCs chemotaxis Suppression of IL-1β-induced cartilage degradation	50
Exosomes		Cell-cell communication Promotion of cartilage regeneration Improvement of chondrocyte migration and integration with surrounding tissues	52,53

There are many signaling pathways that contribute to MSCs development into chondrocytes. These signaling pathways include SOX9, Wnt, Indian Hedgehog (Ihh), Bone Morphogenetic Protein (BMP), Fibroblast Growth Factor and Runx family transcription factors. In SOX9 signaling pathway, the transcription factor Sox9 is essential for condensation and maturation of chondrocytes. However, its expression is repressed in hypertrophic chondrocytes³¹. Studies showed that SOX9 promotes chondrogenesis and slows chondrocyte hypertrophy process^32,33,34. Wnt Signaling regulate chondrocyte and osteoblast differentiation. It suppresses chondrocyte differentiation and promotes osteoblast differentiation ^35,36. Wnt-1 class (e.g. Wnt-1, -3a, -7a, and -8) stimulates the canonical Wnt pathway, whereas Wnt-5a class (e.g. Wnt-4, -5a, and -11) stimulates the noncanonical Wnt pathway³⁷. The Canonical Wnt signaling pathway works through 𝛽-catenin to stimulate chondrocyte hypertrophy. Moreover, studies showed that 𝛽-catenin genetic inactivation leads to increased expression of Sox9 expression and chondrocyte differentiation³⁸. On the other hand, the noncanonical Wnt signaling pathway showed that Wnt11 overexpression stimulates MSCs chondrogenic differentiation in synergism with TGF-𝛽³⁹. Furthermore, Wnt5a and Wnt5b coordinate proliferation and differentiation of chondrocytes by regulating expression of chondrocyte-specific Col2a1⁴⁰. Indian Hedgehog (Ihh) signaling is a regulator that is expressed by prehypertrophic and early hypertrophic cells. It promotes chondrocytes proliferation, prevents chondrocyte hypertrophy and controls Parathyroid Hormone-Related Peptide (PTHrP) expression^41,42. Through PTHrP-dependent pathway, Ihh can regulate chondrocyte proliferation and maturation. Furthermore, PTHrP-independent pathway also exists and controls chondrocyte proliferation by the transcription factors of the GLI family^43,44.

Inhibition of Bone Morphogenetic Protein (BMP) Signaling will inhibit chondrocyte formation and lack of chondrocyte maturation⁴⁵. Fibroblast Growth Factor Signaling plays a role in chondrocyte proliferation and chondrocyte hypertrophy. Expression of Fibroblast growth factor receptor 1 (FGFR1) and FGFR2 is evident in condensing mesenchyme that will develop and differentiate into cartilage. FGFR3 is also expressed in proliferating chondrocytes^46,47.

Runx family transcription factors have a role in stimulating chondrocyte hypertrophy. Runx2-deficient and Runx3-deficient animals demonstrated reduction in chondrocyte hypertrophy and maturation⁴⁸.

Fibroblast Growth Factor (FGF) Family

Fibroblast growth factor (FGF), FGF-2 (basic FGF), and FGF-18 exert their effect by binding to cell receptors, anabolic pathways promotion, and minimizing aggrecanase (anabolic enzyme) activity. It was observed in a murine model that FGF-2 administration reduced OA, but OA accelerated in FGF-2 knockout mice⁴⁹. High FGF-2 doses may enhance inflammation and matrix metalloproteinase (MMP) up-regulation.

Transforming Growth Factor-β (TGF-β) Superfamily

The Transforming Growth Factor-β (TGF-β) superfamily includes TGF-β1, TGF-β3, BMP-2, BMP7, and cartilage-derived morphogenetic proteins-1 and -2. They induce differentiation of chondrocytes and stimulate cartilage ECM production⁵⁰. TGF-β is the most common growth factor used to stimulate chondrogenesis. It stimulates the synthesis of ECM and BMSCs chondrogenesis and decreases interleukin-1 (IL-1) catabolic activity. BMP-7 helps to induce ECM synthesis and inhibits some catabolic factors like matrix metalloproteinases (MMP-1, MMP-13, IL-1, IL-6, and IL-8). BMP-2 has the potential to stimulate the synthesis of ECM and reverse the dedifferentiation of chondrocytes. It has also been used to promote fracture healing.

Platelet-derived Growth Factor (PDGF)

Platelet-derived growth factor (PDGF) acts as a chemotactic factor for mesenchymal cells. It promotes cartilage formation with increased production of proteoglycan and cell proliferation⁵¹. Through down-regulation of nuclear factor-κB signaling, PDGF suppresses IL-1β–induced cartilage degradation.

Insulin-like Growth Factor (IGF)

Insulin-like growth factors, e.g., IGF-1, maintain articular cartilage integrity and enhance cartilage repair and anabolic effects. In an equine model, up-regulation of IGF-1 increased type II collagen expression in cartilage repair tissue⁵².

Exosomes

MSC-derived exosomes are extracellular vesicles that are released from MSCs. They involve in cell-cell communication and promotion of cartilage regeneration. They improved chondrocyte migration and integration of hybrid scaffold with surrounding tissues^53,54.

Pre-Clinical and Clinical studies

Animal studies involving sheeps, goats, horses and rabbits, yielded positive results in cartilage regeneration and provided preclinical assessment of MSC-based treatment of articular cartilage defects^55-65. MSCs implanted into cartilage defect produced cartilage-like tissue similar to the native tissue. When MSCs are implanted with hyaluronic acid (HA), they also produce hyaline-like cartilage⁶⁶. These pre-clinical studies^67-95 evaluating cell-based management of cartilage defects are summarized in Table 3.

Table 3: Pre-clinical studies evaluating cell-based management of cartilage defects

Authors	Year	Model	Defect Site	Defect Type	Defect Diameter	Procedure/Technique	Follow Up (Months)	Findings
Guo et al.67	2004	28 sheep	Medial femoral condyle	Osteochondral	8 mm	Implantation of BM-derived MSCs seeded on tricalcium phosphate (TCP) scaffold vs cell-free scaffolds & empty defects	6	Histology: Type II collagen & Proteoglycan consistent with hyaline cartilage in MSC group, compared with fibrocartilage in cell-free group; Biochemistry: GAG quantity in MSC group 89% of native cartilage
Wayne et al.68	2005	10 dogs	Medial & lateral femoral condyle osteochondral	Osteochondral	6 mm	BM-derived MSCs implantation on a PLA scaffold	1.5	Histology: hyaline & fibrocartilage integration with surrounding tissue
Ando et al.69	2007	9 piglets	Medial femoral condyle chondral defects; cylindrical	Chondral	8.5 mm	Implantation of allogeneic synovial MSCs & cultured in 3D tissue-engineered construct (TEC)	6	Histology: integrated tissue containing collagen II & proteoglycans TEC group; higher ICRS scores in the TEC group. Mechanical: viscoelastic properties are similar in native cartilage & TEC
Lee et al.70	2007	27 minipigs	Medial femoral condyle	Chondral	8.5 mm	BM-derived MSCs with HA injection followed by HA weekly for 2 weeks	3	Histology: hyaline-like cartilage in MSC; improved score in Wakitani histologic score with MSCs
Saw et al. 71	2009	15 goats	Femoral trochlea	Chondral	4 mm	BMDC Injection with hyaluronic acid (HA) weekly for 3 wk after subchondral drilling vs drilling with or without HA	6	Histology: BMDC + HA group had superior type II collagen & proteoglycan content. HA group had type II collagen mixed with type I collagen & proteoglycans
Zscharnack et al.72	2010	10 sheep	Medial femoral condyle	Osteochondral	7 mm	BM-derived MSCs implantation in type I collagen gel either immediately or after precultivation (2 weeks)	6	Histology: Better O’Driscoll & ICRS scores in precultivation group vs nonprecultivated group.
Shimomura et al.73	2010	6 piglets 7 pigs	Medial femoral condyle	Chondral	8.5 mm	Synovial MSCs implantation derived from piglets & cultured in 3D TEC	6	Histology: Good integration of tissue & higher ICRS scores in the TEC group.
Wegener et al.74	2010	9 sheep	Medial femoral condyle	Chondral	8 mm	BM cells implantation in fibrin glue on PGA scaffold; fixed to subchondral bone by PLGA darts	3	Histology: Fibrous tissue and hyaline-like cartilage found in BM cell-seeded scaffolds; Similar O’Driscoll score in both cell-free and cell-seeded scaffolds
Marquass et al.75	2011	9 sheep	Medial femoral condyle	Osteochondral	7 mm	BM-derived MSCs implantation in type I collagen gel implanted immediately or 2 weeks post precultivation; Comparison to MACI	12	Histology: Better O’Driscoll & ICRS scores in precultivation vs nonprecultivated & MACI. MRI: MOCART score better in precultivated MSCs is similar to MACI but better than non-precultivated MSCs
McIlwraith et al.76	2011	10 horses	Medial femoral condyle	Chondral	1 cm2	Injection of BM-derived MSCs Injection with hyaluronic acid (HA) into knee joint 1 month after microfracture; compared to HA alone injection & MFX	12	Histology: No difference in structure, cellular architecture & subchondral regeneration; Biochemistry: GAG equivalent; MRI: no difference
Ando et al.77	2012	6 piglets	Medial femoral condyle	Chondral	8.5 mm	Allogeneic synovial MSCs implantation & cultured in 3D TEC	6	Histology: tissue containing proteoglycans & higher O’Driscoll scores in TEC group; in the TEC group. Mechanics: TEC & native cartilage have similar features
Zhang et al.78	2012	12 minipigs	Femoral trochlea	Chondral	6 mm	BMDCs implantation in type II collagen hydrogel	2	Macroscopy: Good defect filling with BMDCs; Histology: hyaline-like cartilage with BMDCs; O’Driscoll score equivalent in BMDC & MSC groups
Nam et al. 79	2013	18 goats	Medial femoral condyle	Chondral	5 mm	BM-derived MSCs Injection weekly for 3 weeks after subchondral drilling vs drilling alone	6	Histology: High O’Driscoll score, improved type II collagen & proteoglycan in the MSC group; Biochemistry: high GAG quantity with MSCs
Bekkers et al.80	2013	8 goats	Medial femoral condyle	Chondral	5 mm	Chondrons & BM derived MSCs in fibrin glue	6	Macroscopy: Improvement in defect filling with MSC + chondrons. Histology: high O’Driscoll score in the MSC + chondron group. Biochemistry: High GAG content & GAG/DNA in MSC + chondron group
Kamei et al.81	2013	16 minipigs	Patella	Chondral	6 mm	Injection of ferumoxide labeled MSCs	3	Arthroscopy: improved smoothness & integration Histology: High type II collagen content & improved Wakitani scale
Fu et al.82	2014	30 rabbits	Trochlear groove distal femur	Osteochondral	5 mm	Peripheral blood MSCs & bone marrow MSCs implantation into trochlear groove defects	6	Histology: histological scores significantly better in peripheral blood MSCs. Abundant cartilage matrices
Broeckx et al.83	2014	50 Horses	Fetlock joint	Surgically induced OA	NA	Single IA injection of PB-MSCs with or without PRP injection	12	Improved short & long-term clinical scores; increases in Collagen II, aggrecan & cartilage oligomeric matrix protein levels
Broeckx et al.84	2014	165 Horses	Fetlock, Stifle, coffin, pastern joints	OA	NA	Single IA injection of PB-MSCs with or without PRP injection	4.5	Improved short- and long-term clinical evolution scores*; Relief from locomotor disorder
Zhang et al.85	2018	6 goats	Lateral & medial femoral condyle	Chondral	6.5 mm	Implantation of human umbilical cord MSCs combined with extracellular matrix scaffold	9	Histology: High extracellular cartilage, cartilage lacuna & collagen type II MRI: high quality cartilage
Feng et al.86	2018	30 sheep	Knee joint	NA	NA	Adipose derived MSCs + HA Intra-articular injection	4.5	MRI: Smooth, intact cartilage layer; high MOCART & ICRS scores
Chu et al.87	2018	8 horses	Mid-lateral trochlea	Chondral	15 mm	Arthroscopic application of bone marrow concentrate clot	12	Arthroscopy: Defect filling at least 50% of the defect MRI: heterogeneous repair tissue Histology: fibrous to fibrocartilaginous tissues
Li et al.88	2018	24 canines	Knee joint	NA	NA	Intra-articular injection of MSCs + HA	28	MRI: New cartilage-like tissue formation Histology: Neocartilage covering defect site, chondrocytes formed & normal matrix staining
Wei et al.89	2019	24 goats	Femoral head	Osteochondral	10 mm	BMSCs +porous tantalum + 3D collagen scaffold implantation	4	Smooth & continuous cartilage surface High modified O’Driscoll score
Broeckx et al.90	2019	75 Horses	Fetlock joint	Early staged fetlock OA	NA	Articular injection of chondrogenic induced PBMSCs	12	Improved American association of equine practitioners (AAEP) score, Flexion score and Pain score
Broeckx et al.91	2019	12 Horses	Metacarpophalangeal OA	Surgically induced OA	NA	Articular injection of chondrogenic induced PBMSCs	2.75	Higher viscosity score, less wear lines, higher collagen type II & glycosaminoglycans in the intervention group
Henson et al.92	2021	40 sheep	Medial femoral condyle	Chondral	8 mm	Apheresis-derived cells & HA intra-articular injection to augment microdrilling	6	MRI: SPION labeled cells not detected within defects Histology: modified O'Driscoll score greater in apheresis-derived cells, HA & Microdrilling group than microdrilling alone; repaired tissue was fibrocartilagenous in nature rather than hyaline cartilage
Kim et al.93	2022	27 goats	Knee joint	Condral	NA	MSCs + Cartilage acellular matrix intraarticular injection	6	Improvement in lameness score & K&L score
Murata et al.94	2022	3 ponies	Medial femoral condyle	Osteochondral	6.8 mm	Synovial MSCs + 3D scaffold implantation	6	Lower radiolucent volume percentages; better MOCART scores, higher average histological scores.
Zhang et al.95	2022	12 micripigs	Medial femoral condyle	Osteochondral	6 mm	Intra-articular injection of MSCs exosomes + HA	4	MRI: better MRI scores; Micro-CT showed higher bone volume & trabecular thickness. Histology: functional cartilage & subchondral bone repair; better macroscopic & histological scores & biomechanical properties (Young modulus and stiffness).

There is evolving clinical evidence supporting using MSC-based treatment for cartilage regeneration in humans. Clinical studies assessing MSC-based treatment for cartilage defects have demonstrated a successful option to treat osteochondral lesions. These clinical studies^96-118 evaluating cell-based management of cartilage defects are summarized in Table 4.

Table 4: Clinical studies evaluating cell-based management of cartilage defects

Authors	Year	Study Type	Evidence Level	Subjects	Defect Location & Type	Defect Diameter	Procedure/Technique	Follow Up (Months)	Findings
Wakitani et al.96	2002	Prospective comparative study	III	24	Medial femoral condyle	4.9 cm2	Autologous BMSCs transplantation into cartilage defect covered with autologous periosteum	6	Arthroscopy: defect covered with hyaline-like cartilage Better histological grading score in cell-transplanted group Clinical improvement in Hospital for Special Surgery knee rating scale
Wakitani et al.97	2004	Case report	IV	2	Patella	4.5 cm2, 12 cm2	Autologous BMSCs transplantation into articular cartilage defect of patellae; covered with autologous periosteum	6	Arthroscopy: Defects covered with fibrocartilage Clinical: Improvement in clinical symptoms
Kuroda et al.98	2007	Case report	IV	1	Medial femoral condyle chondral	6.0 cm2	BM-derived MSCs implantation within type I collagen gel on collagen scaffold & periosteal flap coverage	12	Arthroscopy: Smooth, stable repair tissue. Histology: Hyaline-like cartilage. MRI: Chondral & subchondral irregularities. Clinical: Return to previous activity level
Wakitani et al.99	2007	Case series	IV	3	Femoral trochlea, patella chondral	0.7 – 4.2 cm2	BM-derived MSCs implantation within type I collagen gel on a collagen scaffold; periosteal flap or synovium coverage; subchondral drilling	18	Arthroscopy: Smooth, stable tissue. Histology: Atypical cartilage. MRI: Defects completely covered. Clinical: Symptoms & IKDC improvement & return to work
Giannini et al.100	2009	Case series	IV	48	Talar dome osteochondral	2.07 ± 0.48 cm2	BMDCs seeded on HA scaffold or implantation within collagen/platelet paste	24-35	Arthroscopy:  all integrated with native cartilage. Histology: Some hyaline quality. MRI: new tissue formation in all lesions. Clinical: AOFAS scores improvement
Buda et al.101	2010	Case series	IV	20	Medial femoral condyle & lateral condyle, osteochondral	NR	BMDCs Seeded on a HA scaffold supplemented with platelet-rich fibrin	29 ± 4.1	Histology: collagen II & proteoglycan content consistent with hyaline-like cartilage. MRI: variable signal correlated with KOOS score. Clinical: IKDC & KOOS scores improvement
Nejadnik et al.102	2010	Prospective comparative study	III	72	Patella, femoral trochlea, femoral condyle, chondral	4.6 ± 3.5 cm2	BM-derived MSCs covered by a periosteal flap	24	Arthroscopy: smooth tissue. Histology: collagen II & aggrecan content consistent with hyaline cartilage. Clinical: SF-36 Improvement Physical Role Functioning greater in MSCs versus chondrocytes; IKDC, Tegner & Lysholm scores equivalent improvement following MSC & chondrocyte implantation; Outcomes superior in males vs females
Giannini et al.103	2010	Prospective comparative study	III	81	Talar dome osteochondral	>1.5 cm2	BMDCs Implantation seeded on a HA scaffold with platelet-rich fibrin	59.5 ± 26.5	Arthroscopy: Defect coverage. Histology: Hyaline-like cartilage. MRI: Integration in 76% & homogenous tissue in 82% of cases. Clinical: AOFAS scores improvement.
Haleem et al.104	2010	Case series	IV	5	Femoral condyle chondral	3 – 12 cm2	BM-derived MSCs implantation with platelet-rich fibrin glue; Periosteal flap coverage	12	Arthroscopy: smooth tissue. MRI: Defect filling with good congruity. Clinical: Lysholm & RHSSK scores improvement
Saw et al.105	2011	Case series	IV	5	Lateral femoral condyle, patella, femoral trochlea	0.5 – 8.8 cm2	Peripheral blood-derived MSCs injection with HA weekly; subchondral drilling; pre-injection GCSF; lateral patellar release or adjunctive HTO	10 - 26	Arthroscopy: Good defect filling Histology: proteoglycan staining; type I collagen, type II collagen in deep area
Kasemkijwa-ttana et al.106	2011	Case series	IV	2	Lateral femoral condyle, chondral	2.2 – 2.5 cm2	BM-derived MSCs implantation seeded on type I collagen scaffold + fibrin glue; periosteal flap coverage; adjunctive ACL reconstruction, meniscal repair		Arthroscopy: Good defect fill & integration. Clinical: IKDC & KOOS scores improvement
Gobbi et al.107	2011	Case series	IV	15	Patella, femoral trochlea, medial tibial plateau, medial femoral condyle & lateral condyle chondral	(9.2 ± 6.3 cm2 )	BMDCs mixed with batroxobin covered by a type I/III collagen matrix	24-38	Arthroscopy: Smooth, integrated tissue; no hypertrophic tissue. Histology: various features of hyaline & fibrocartilage. MRI: 93% integration & 80% complete defect filling Clinical: VAS, KOOS, Tegner, Marx, IKDC and Lysholm scores improvement; single & smaller lesions showed better outcomes
Gigante et al.108	2012	Case report	IV	1	Medial femoral condyle, Chondral	3 cm2	BMDCs implantation within fibrin glue & collagen membrane coverage post microfracture	24	MRI: Good defect filling with tissue, no bone edema. Clinical: Return to activity
Saw et al.109	2013	RCT	II	49	Patella, trochlea, femoral condyle & tibial plateau, chondral	NR	Peripheral blood-derived MSCs injection & HA 5-weekly after subchondral drilling then 3-weekly at 6 months	24	Arthroscopy: Smooth filling of defect. Histology: ICRS II score  better in MSC + HA group. MRI: improved integration, articular morphology & defect filling in MSC + HA group. Clinical: IKDC scores improvement
Enea et al.110	2013	Case series	IV	9	Medial femoral condyle & lateral condyle chondral	2.6 ± 0.5 cm2	BMDCs implantation within fibrin glue with PGA-HA membrane coverage post microfracture	22 ± 2	Arthroscopy: ICRS CRA: 1 normal, 3 nearly normal & 1 abnormal. Histology: hyaline-like cartilage tissue. MRI: Defect filling in all cases; hypertrophy in 1 case. Clinical: IKDC and Lysholm scores improvement; No change in Tegner score.
Giannini et al.111	2013	Case series	IV	49	talar dome, osteochondral	2.2 ± 1.2 cm2	BMDCs implantation within collagen/platelet paste or seeded on HA scaffold with platelet gel	48	MRI: Defect filling in 45%, integration in 65%, subchondral disruption in 65% & hypertrophy in 45% of patients; 78% of repair area had hyaline quality. Clinical: AOFAS scores improvement; 78% of patients returned to pre-injury sports
Koh et al.112	2014	Case series	IV	37	Knee joint	5.4 ± 2.9cm2	MSCs implantation into cartilage defects	26.5	Arthroscopy: 76% had repair rated as abnormal or severely abnormal by ICRS standards Improved IKDC & Tegner activity scale
Sekiya et al.113	2015	Case series	IV	10	Femoral condyle	2 cm2	Arthroscopic transplantation of synovial stem cells	48	Increased MRI score post treatment. Histology: hyaline cartilage. Increased Lysholm score.
Kim et al.114	2016	Prospective cohort study	III	20	Knee joint	NA	Arthroscopic implantation of MSCs into cartilage lesions	24	MRI Cartilage lesion grades better than preoperative values Clinical outcomes improved & correlated with the MOAKS and MOCART score
Saw et al.115	2021	RCT	I	69	Knee, chondral	≥3 cm2	Arthroscopic subchondral drilling followed by PBSC & HA injection	24	MRI: significant improvement in MOCART score (P < 0.0001) Clinical: significant improvement in IKDC, KOOS & NRS (P < 0.0001)
Lim et al.116	2021	RCT	I	114	Knee joint	4.9 cm2	Umbilical cord blood derived MSCs + HA implantation	60	Improvement by ≥1 ICRS grade seen in 97.7% Improvement in VAS pain, WOMAC & IKDC scores
Zhou et al.117	2021	RCT	I	30	Knee joint	NA	Arthroscopic injection of knee infrapatellar fat pad cell concentrates containing MSCs into cartilage lesion	12	Lower WOMAC & VAS scores High MRI MOCART scores
Monckeberg et al.118	2024	Prospective cohort study	III	25	Hip, chondral	Zone 2: 12.4 ± 3.1 mm Zone 3: 13.5 ± 2.8 mm Zone 4: 11.4 ± 1.9 mm	Hip arthroscopy microdrilling & Intra‐articular injection of PBSC with HA‐based scaffold	31.8 ± 9.6	MRI: ICRS MSS increased > 3 points (p < 0.001) Clinical: HOS increased (p < 0.001); VAS-Pain decreased (p < 0.001); 92% patient improved outcomes

In these clinical studies MSCs have been utilized to treat cartilage defects of the femoral condyle, femoral trochlea, tibial plateau, patella, talus, and hip. In addition, MRI and arthroscopy-based studies illustrated that the hyaline-like cartilage (derived from MSCs transplantation) integrates with the native tissue. However, hypertrophic cartilage, incongruent resurfacing, defect incomplete filling, and separated osteochondral interfaces were noted in some cases. Histological studies demonstrated that biopsies of the repaired cartilage have moderate to larger amounts of collagen II, less collagen I, and differentiated chondrocytes with intense proteoglycan staining. Comparative clinical studies of MSC/BMDC transplantation showed similar clinical outcomes. Furthermore, the MSCs group in studies showed better physical role functioning on clinical outcome scores such as the Short Form SF-36 (SF-36) scale than control group. Similar findings were observed in arthroscopy, MRI, and histology.

Discussion

This review illustrates that cell-based treatment is a potential future option for treating human cartilage defects. Until now, cartilage regeneration techniques have not produced histologically comparable cartilage that resembles native hyaline cartilage architecture.

Many future strategies for cell-based cartilage defect management can be developed. These strategies include synovial or adipose tissue MSCs, gene therapy, and the use of a magnetic cell delivery system.

Change of MSCs source to synovial or adipose tissue MSCs can be considered in future instead of autologous bone marrow. Synovial MSCs show high chondrogenic capacity, and adipose tissue is widely available. Embryologically, the synovial tissue originates from the mesenchymal layer, providing the origin of bone, muscle, cartilage, and ligaments. Type A (tissue macrophages) and type B (synovial fluid forming) synoviocytes are two cell types in the synovium¹¹⁹. MSCs were isolated from the synovium (connective tissue layer lining the joint surfaces)¹²⁰. In-vitro studies demonstrated that synovial MSCs have a high potential to differentiate into chondrocytes when compared to MSCs originating from other sites. Moreover, synovial MSCs transplantation in animal studies have illustrated cartilage formation /regeneration and chondrocyte formation, especially at the superficial zones closer to the synovium^121,122. It was noted that synovial MSCs increase after cartilage degeneration, joint osteoarthritis and intra-articular ligament injury¹²³. However, it’s unclear if this increased number of synovial MSCs is related to the amount of cartilage damage or part of a normal healing process. Number of stem cells required for cartilage repair is not clear. More current analysis about MSCs is that they don’t function only to turn into differentiated cells such as chondrocytes, but they recruit other stem cells, secrete bioactive factors and function to modulate the local environment and decrease inflammation¹²⁴.

In-vitro and in-vivo studies support synovial MSCs as an alternative cell-based treatment option. Further studies should focus on synovial MSCs isolation, expansion, and interaction with the external environment before the application of these types of cells into clinical practice.

Gene therapy is a new treatment approach that involves enhancement of the expression of growth factors like TGF-β, BMPs and FGF-2, which promotes cartilage regeneration¹²⁵.

The general strategies of gene therapy involves a target gene (growth factor or cytokine), a vector and delivery method (in-vivo or ex-vivo). Viral vectors are most commonly used and different vectors vary in duration of effect, efficiency and safety. Adenoviral vectors have short-term transgene expression and high transduction efficiency but it triggers strong immune response. However, Adeno-associated viral vectors have good safety profile, long-term expression and trigger weak immune response. On the other hand, non-viral vectors like naked DNA and liposomes have low immunogenicity and are significantly less efficient than viral vectors^126,127.

In-vivo gene therapy is one of the delivery strategies in which genetic material is injected directly or through a vector into the joint/cartilage defect. Moreover, ex-vivo gene therapy involves chondrocytes or MSCs harvested from a patient, genetic modification in the lab, expansion in 3D scaffold and implantation into cartilage defect¹²⁸.

Gene therapy has some potential undesired risks related to safety and toxicity. It can trigger undesirable immune response and cause liver toxicity due to segregated vectors in the liver¹²⁹. Moreover, genotoxicity risk can occur from integrating vector that causes mutations and lead to malignancies. Thrombotic microangiopathy (TMA) is one of the most prevalent adverse events of gene therapy, which occurs secondary to complement recongnition of the adeno-associated virus (AAV) vector^130,131.

The magnetic cell delivery system also known as magnetic targeting is used to retain MSCs at the cartilage defect site. It involves MSCs labeling with magnetic nanoparticles (MNPs) e.g. iron oxide nanoparticles (Ferumoxides), labeled MSCs intra-articular injection, targeting through external magnet and MSCs retention and cartilage regeneration¹³². Studies demonstrated successful tracking, targeting and bioactivity of MNP-labeled MSCs^133,134. Many studies showed improved MSCs retention and delivery at the defect sites^135,136.

The advantages of this technique include minimal invasiveness, increased MSCs retention and high efficiency and precision.

The challenges related to this technique include the uncertainty of chondrogenic capacity of labeled MSCs compared to nonlabeled MSCs, unknown long term fate of the iron oxide nanoparticles in the body and unknown appropriate magnitude of the magnetic field to control the labeled MSCs. The location of the osteochondral defect can have an impact on the application of the magnetic cell delivery systems. Although this system is not difficult to apply in patellar osteochondral defects, it is difficult to apply in femoral condyle or tibial plateau osteochondral defects¹³⁷.

There are clinical challenges for MSCs therapies. Limited number of implanted MSCs and their diminished proliferation capacity are some of these challenges. The proliferation capacity of adult MSCs is limited compared to embryonic stem cells. This is because embryonic stem cells have telomerase activity, whereas telomerase activity in adult MSCs is undetectable¹³⁸. To overcome this obstacle, MSCs life span is extended and multilineage potential maintained by use of human telomerase reverse transcriptase (hTERT) and actin disrupting agents e.g. FGF-2¹³⁹.

Chondrocytes respond to mechanical stress throughout life. Mechanical stimuli include mechanical compression and hydrostatic pressure. Mechanical compression can affect matrix production, collagen synthesis and proteoglycans synthesis^140,141. Hydrostatic pressure can lead to homogenous stress on the cell without deformation of the cell. Mechanical stress induces chondrogenesis and promotes maturation. Cartilage underloading can result in cartilage thinning, softening and decreased content of proteoglycan.

Automated bioreactors facilitate metabolite exchange and nutrient supply to mimic the physiological conditions for cartilage tissue development. The bioreactors increase reproducibility, lower contamination and are capable of mechanobiologic activation of scaffolds¹⁴².

There are hydrostatic, dynamic loading and hydrodynamic types of bioreactors for construction of cartilage tissue. Hydrostatic bioreactors can enhance MSCs chondrogenesis by administering hydrostatic pressure to resemble joints hydrostatic load. Dynamic-loading bioreactors provide mechanical loading to cells constructs at specified magnitudes and frequencies of strain. These Dynamic-loading bioreactors mimic joint physiologic weight bearing to enhance MSC chondrogenesis and cartilage construct mechanical features. Hydrodynamic bioreactors have instruments that rotate to encourage gas exchange, nutrient transport and removal of metabolite. These bioreactors enhance cartilage construct compressive properties due to enhanced production of matrix proteoglycan¹⁴³.

This review has some limitations. The review was restricted to studies published in English and may have missed relevant studies as the review was limited to indexed studies in major databases. Other limitation is the lack of long-term clinical studies to enhance persuasiveness of the research.

Conclusion

MSCs implantation techniques produced hyaline-like cartilage incorporated into native tissue. Further research is required to identify the most appropriate method of acquiring MSCs, their differentiation, growth factors, and delivery to the defect site within a scaffold. Before utilizing MSCs in cartilage repair clinically, some issues need to be addressed. These issues include the failure of MSCs integration with native tissue, the degradation of implanted matrices, and the loss of transplanted MSCs. Long-term comparative clinical studies of MSCs-based treatments should be implemented to clarify the use of this technique as the gold standard option for cartilage defects treatment.

Gene therapy can be considered a one-step minimally invasive procedure that provides continuous therapeutic protein at the cartilage defect site and allows delivery of multiple genes· Challenges of gene therapy application include immune response to viral vectors, determining the optimal vector, regulation of transgene expression and conduction of large scale clinical trials. More research is needed in the field of magnetic targeting that may become a strong tool in regenerative orthopedics.

Ethics Approval

No ethics approval was required.

Conflicts of Interest

The author reports no conflicts of interest in this work.

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