Cell Co-Implantations With Chondrocytes for Cartilage Repair: A Systematic Review of In Vivo Results

Abstract

Objectives

Damaged articular cartilage cannot regenerate spontaneously. Chondrocyte therapy, the current treatment of choice, requires laboratory expansion, necessitating two surgical procedures. Adding a second cell type to intraoperatively isolated chondrocytes enables single-stage chondrocyte-based repair strategies and has shown promise in vitro. The benefit of this strategy in vivo and in the clinic has not yet been comprehensively assessed. This systematic review assesses the efficacy of cartilage repair by co-implantations of chondrocytes with other cell types over all available in vivo studies.

Design

Medline, Embase and Cochrane databases were searched for studies on co-implantations of chondrocytes with other cell types for hyaline cartilage repair. For each study, extracted data were tabulated and reporting quality and risk of bias were assessed. Studies were categorized based on their control groups for qualitative synthesis.

Results

The search yielded 48 studies across 46 publications: 44 animal studies (26 ectopic and 18 orthotopic) and four clinical trials. Ectopic studies scored poorly on reporting quality and bias, while orthotopic studies were only moderately better. Twenty-seven of 27 experiments with chondrocyte-only controls demonstrated synergistic cartilage formation through co-implantation. Cartilage formation by co-implantations was also found in 15 of 17 experiments without such control and in four clinical trials.

Conclusion

Cartilage repair by articular chondrocytes is improved by adding a second cell type, like mesenchymal stromal cells. This is likely caused by the second cell types’ stimulatory support. Other second cell types and chondrocyte sources have shown promising results too. This is encouraging for further clinical exploration of single-stage co-implantation strategies.

Introduction

Articular cartilage lesions form a significant challenge in orthopedic medicine, often leading to joint degeneration and functional impairment.^1-5 The regenerative capacity of articular cartilage is very limited and its native response to lesions is inadequate, resulting in fibrous tissue formation in osteochondral defects ⁶ and very little repair tissue in chondral lesions. ⁷ Consequently, symptomatic lesions may necessitate surgical intervention to prevent progression toward osteoarthritis (OA).^8-12

An array of treatment options is available, ranging from traditional bone marrow stimulation techniques, like microfracture (MFx), to osteochondral autologous transplantation (OAT). However, cell-based therapies like autologous chondrocyte implantation (ACI) have revolutionized cartilage repair since their clinical introduction in 1994. ¹³ ACI leverages the regenerative potential of an individual’s own (autologous) articular chondrocytes (ACs), the principal cell type responsible for the synthesis and architecture of hyaline articular cartilage. ¹⁴

Despite its efficacy, ACI faces logistical hurdles, including in vitro chondrocyte expansion leading to phenotype shifts, regulatory constraints, and substantial costs associated with two-stage surgical procedures, human tissue transport and Good Manufacturing Practices. To circumvent these issues, recent advancements in rapid chondrocyte isolation have facilitated single-surgery interventions employing non-expanded freshly isolated autologous chondrons (chondrocytes with their pericellular matrix), lower in number but more native in phenotype.^15,16

With the aim of further enhancing the cartilage-producing capacity of non-expanded chondrocytes, co-implantation with stromal cell types has been explored. Cell types researched for this purpose include mesenchymal stromal cells (MSCs), or rapidly obtainable cell fractions like bone marrow-derived mononuclear cells (BM-MNCs) and adipose tissue-derived stromal vascular fraction (SVF). MSCs, also known as mesenchymal stem cells, have alternatively been named medicinal signaling cells due to their trophic role in tissue repair. ¹⁷ They are capable of differentiating into various musculoskeletal lineages in vitro, including chondrocytes, and offer advantages in availability and regenerative capacity.^18,19 Cultured MSCs should not be confused with MNCs; BM-MNCs and SVF are uncultured cell fractions that contain only a low percentage of MSCs. ²⁰

Indeed, co-culture techniques that replace 50% to 90% of chondrocytes by MSCs have showcased similar or superior chondrogenic outcomes compared with 100% chondrocyte monocultures according to a concise review of in vitro co-cultures by De Windt et al. ²¹ Beside, close proximity between the cell types appeared to be pivotal for this synergism. It remained unclear, however, whether the increase in matrix production per chondrocyte was due to differentiation of MSCs to chondrocytes or trophic stimulation by MSCs. Since 2014, in vitro evidence has been published both in favor of MSC differentiation^22,23 and in favor of trophic support.^24-27 Furthermore, since then the need for close proximity has been further confirmed, with a likely role for gap junctions,^28,29 extracellular vesicles (EVs),^30-34 and mitochondrial transfer. ²⁹

These advancements and the pursuit of improved single-stage cartilage repair strategies motivated this systematic review of in vivo co-implantations of chondrocytes with a second cell type. The primary objective was to comprehensively assess the extent of hyaline cartilage repair achieved by such cell combinations and compare that extent with the results obtained by the use of each cell type separately, cell-free carriers, or other treatments. Secondary objectives were to elucidate synergistic mechanisms at play and to compare different cell types, sources, and mixing ratios between co-implantation studies. The outcomes from these objectives may encourage and provide a rationale for future co-implantation studies.

Method

Search Strategy

This study is reported based on the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) 2020 checklist. ³⁵

A literature search was conducted in the electronic databases of Medline, Embase, and Cochrane. First, a search strategy was designed to retrieve all publications mentioning specific words in the title or abstract related to the following aspects: (1) a second cell type, (2) chondrocytes, (3) implantation or in vivo co-culture, and (4) cartilage repair; while filtering out reviews and systematic reviews. Specifically, the following search terms were used: (fibroblast* OR adipocyte* OR macrophage* OR stem cell* OR progenitor cell* OR MSCs OR MSC OR mesenchymal stem OR mesenchymal stromal OR BMSC* OR BMMSC* OR ASCs OR ASC OR SVF OR MNCs OR MNC OR MNF OR BMMNC* OR AMSC* OR ADSC* OR WJMSC* OR SMSC* OR SDSC*) AND (chondrocyte* OR chondron*) AND (implant* OR coimplant* OR transplant* OR (in vivo AND (co-culture OR coculture))) AND (cartilage OR chondrogenesis OR chondrocytic OR chondrogenic OR chondrogeneic OR chondrogenically).

The titles and abstracts of the publications yielded by this search strategy were divided over three separate readers and screened to include only publications in which chondrocytes and a second cell type were implanted together. After this inclusion screening, certain publications were excluded based on the full text, in consultation between the three readers, according to the following exclusion criteria: (1) the study was not focused on hyaline cartilage repair (e.g., those focused on elastic cartilage or bone regeneration); (2) the two cell types were not implanted in close proximity to each other (e.g., zonal constructs wherein the two cell types were physically separated by a material barrier; undigested tissue implants); (3) no cells were co-implanted; (4) no results were presented related to cartilage repair; (5) use of chondrocytes from induced pluripotent stem cell (iPSC) origin; and (6) unavailability of a full English version. The Medline, Embase, and Cochrane searches have been regularly updated for inclusion in this systematic review until September 2, 2024.

Data Extraction and Risk of Bias

The included studies were divided over three readers for in-depth reading and tabulation of the following data (if available): chondrocyte source, second cell-type source, passage and culture conditions, seeding ratio and volume, scaffold type, control groups, group sizes, animal model, and a brief description of the results. For conciseness, not all datapoints collected are included in the results tables of this review and not all datapoints tabulated are included in the text. After tabulation, information regarding reporting quality and risk of bias (RoB) was collected for all animal studies using the CAMARADES reporting quality checklist ³⁶ and the SYRCLE RoB tool. ³⁷ The SYRCLE is based on the Cochrane RoB tool but is adapted for animal interventions, and includes entries related to selection, performance, detection, attrition, reporting, and other biases. For all clinical studies, quality and RoB was scored using an adapted quality appraisal checklist from the Institute of Health Economics (IHE) in Alberta, Canada. ³⁸ This tool was chosen for its completeness and clarity, and for scoring quality and RoB concurrently. This adapted checklist is available upon request. All scoring was performed by two independent readers and consensus was reached for any contradictory findings.

Grouping and Syntheses

To effectively address the primary objective of this review, the animal studies were grouped into the following categories based on the control group(s) used: (1) empty defect or empty scaffold, (2) second cell-type only, (3) chondrocytes only, and (4) MFx or any other comparative treatment. Each group was then divided into two subsections: orthotopic (here defined as co-implanting chondrocytes into cartilage) and ectopic (here defined as co-implanting chondrocytes into another tissue, typically subcutaneously). Human trials were described separately. For these five groups, the results were synthesized in a qualitative manner. Quantitative or statistical syntheses were not possible due to highly heterogeneous study methods. This factor should be taken into consideration when interpreting the outcomes of this review.

Results

Study Selection

The Medline, Embase, and Cochrane searches yielded 1,045, 723, and 44 publications, respectively. After removal of duplicates and several exclusions rounds, 46 publications were included in this review ( Fig. 1 ). Two publications each contained two separate studies within the inclusion criteria, therefore, 46 publications yielded 48 studies reviewed. Fifty-eight publications were included after screening but excluded when assessed for eligibility. Citations of these publications are available upon request.

Figure 1.

Flow diagram of record identification, inclusion, and exclusion. Adapted from PRIMA 2020 flow diagram.(Page, McKenzie, Bossuyt, Boutron, Hoffmann and Mulrow, 2021).

Reporting Quality and RoB

Reporting quality of animal studies

According to the CAMARADES checklist, the reporting quality of the included studies was generally very poor ( Fig. 2 ). In particular, ectopic studies often lacked descriptions of essential methodological aspects, such as allocation method (D3: 85%), blinding procedures (D4: 100%; D5: 92%), animal selection criteria (D7: 92%), and sample size calculations (D8: 100%). Frequently, even the number of animals was not reported (absent in CAMARADES; Tables 1 – 4 ).

Table 1.

Co-implantation Proof of Concept.

Orthotopic: Rose; Ectopic: Blue; (NE) = non-expanded cells; (RI) = rapidly isolated cells; (e) = evidence for proposed role; ? = data missing or unclear in reviewed study. Cell type and carrier abbreviations are defined in the abbreviations section.

Table 2.

Co-implantation versus Second Cell Type.

Orthotopic: Rose; Ectopic: Blue; (e) = evidence for proposed role; ? = data missing or unclear in reviewed study. Cell type and carrier abbreviations are defined in the abbreviations section.

Table 3.

Co-implantation versus Only Chondrocytes (Orthotopic: Rose).

(e) = evidence for proposed role. Cell type and carrier abbreviations are defined in the abbreviations section.

Table 4.

Co-implantation versus Only Chondrocytes (Ectopic: Blue).

(NE) = non-expanded cells; (RI) = rapidly isolated cells; (e) = evidence for proposed role; ? = data missing or unclear in reviewed study. Cell type and carrier abbreviations are defined in the abbreviations section. LCC (Low-chondrocyte control) groups are highlighted in bold.

Figure 2.

(A) CAMARADES results per ectopic study. (B) CAMARADES results per orthotopic study. (C) CAMARADES summary for ectopic studies. (D) CAMARADES summary for orthotopic studies. Judgments defined by CAMARADES tool criteria, more specific judgment calls are available upon request.

Although slightly better, orthotopic implantation studies demonstrated poor reporting practices too, as 72% did not mention temperature control (D2), 39% did not mention random allocation (D3), 72% did not mention allocation concealment (D4), and only two studies reported a sample size calculation (D8).

Of all studies, 27% did not disclose conflicts of interest (D10). Two studies did not specify whether animal research had received ethics committee approval (D9). The CAMARADES checklist finally revealed that despite these issues, all studies were published in a peer-reviewed journal (D1).

RoB of animal studies

Ectopic studies showed a higher RoB, which was associated with their poor reporting quality ( Fig. 3 ). When allocation sequence (D1), blinding (D3, D5, D7), housing (D4), or random selection for outcome assessment (D6) were not reported, these aspects were rated as unclear RoB, presuming that researchers might adhere to these standards even if not explicitly reported. Baseline characteristics (D2), attrition (D8), reporting (D9), or other biases (D10) could mostly be rated as high or low risk, and not as unclear, since those domains were more clearly extractable.

Figure 3.

(A) SYRCLE results per ectopic study. (B) SYRCLE results per orthotopic study. (C) SYRCLE summary for ectopic studies. (D) SYRCLE summary for orthotopic studies. Judgments defined by SYRCLE tool signaling questions, more specific judgment calls are available upon request.

In ectopic studies, information on housing, blinding, and randomization were rarely reported, with few exceptions. Orthotopic studies scored only slightly better with 17% reporting on allocation concealment (D3), blinding (D5), and random animal selection for outcome assessment (D7). Baseline characteristics (D2) were rarely reported, but 28% of the orthotopic studies had stringent animal inclusion criteria, rendering further baseline characteristics unnecessary. For ectopic studies, a narrow age and/or health range sufficed for a low risk rating in baseline characteristics, whereas orthotopic experiments required at least a narrow weight range for a low risk rating. This explains why 42% of the ectopic studies scored low risk in this domain.

Ectopic studies often failed to report sample sizes or animal numbers, or reported conflicting numbers throughout the publication, leading to an unclear or high risk score for attrition bias (D8) in 58% of these studies. This rate was substantially lower in orthotopic experiments (28%). High risk on selective outcome reporting was mostly due to unrepresentative images lacking any sequence, quantification, or any indication of representativeness, with some studies entirely omitting expected images. High risk of other bias was often attributable to unit of analysis errors, where the number of scaffolds rather than the number of animals was used for statistical analysis. This problem occurred in both orthotopic and ectopic setups. Additional reasons for other bias included the absence of sample sizes or conflict-of-interest statements, the presence of conflicts of interest, and two instances of such poor reporting that the risk of misinterpretation was high.

RoB and reporting quality of clinical trials

All four clinical publications described single-arm case series, a design which can only provide safety and preliminary efficacy data. Without a control group, confounding factors cannot be accounted for and clinical improvements may be caused by a placebo effect.

The IHE quality appraisal for the four clinical studies in this review showed reasonable outcomes with some concerns ( Fig. 4 ). All studies mentioned their objectives, although they could have been clearer (D1). Słynarski et al. ¹⁶ were the only to report a multicenter trial (D3). None of the studies reported consecutive enrollment, which would have removed risk of selection bias (D4). Papakostas et al. ⁷⁷ used very broad inclusion criteria (D6) and performed concomitant surgical procedures in most patients, strongly increasing the risk of confounding (D9). A number of patients in the study of Słynarski et al. ¹⁶ needed additional procedures before their final follow-up, confounding the results. Saris et al. ⁷⁸ separated the results of patients with and without intervention, but only for the final follow-up, while De Windt et al. ¹⁵ did not clearly report the presence of concomitant procedures. Papakostas et al. ⁷⁷ mentioned return to work in the results but not the methods (D10). No issues were found in the studies regarding blinding of outcome measurements (D11) and the availability of measurements before and after intervention (D13). Unfortunately, losses to follow-up were a concern for all four studies (D16). Sometimes, the reasons for loss to follow-up were not reported and none of the studies corrected for those losses in the analysis. Saris et al., however, made the effort to show last observation differences between lost patients and completers. Finally, commercial conflicts of interest were reported in the studies by Słynarski et al. ¹⁶ and Papakostas et al. ⁷⁷ (D20).

Figure 4.

(A) Adapted IHE quality appraisal results per clinical study. (B) Adapted IHE quality appraisal summary for clinical studies. Judgments defined by IHE quality appraisal checklist questions, more specific judgment call available upon request.

All studies were performed prospectively (D2) and clearly described the patient characteristics (D5) as well as the intervention (D8), with patients entering at a similar point in their disease (D7). Appropriate methods (D12), statistics (D14) and follow-up times (D15) were used in all studies and random error measures (D17) and adverse events (D18) reported. Finally, the conclusions were supported by the results in all four studies (D19).

For convenience, total scores for RoB and reporting quality have been added to the review tables for each study. However, these numbers should be interpreted with much caution as bias and reporting quality domains cannot be weighed for their importance.

Proof of Concept

Of the studies that compared co-implantation with implantation of an empty scaffold or empty defect, three were performed in goats, three in rabbits, and two subcutaneously in nude mice ( Table 1 ). These studies can show whether co-implantations lead to improved cartilage repair compared with empty scaffolds or defects but cannot demonstrate any benefit compared with the effect of a single cell type or another treatment.

For each study reviewed in the “Proof of Concept,” “Co-Implantation Versus Second Cell Type Alone,” “Co-Implantation Versus Only Chondrocytes—Orthotopic,” “Co-implantation Versus Only Chondrocytes—Ectopic,” and “Co-Implantation Versus MFx sections, the animal species, cell type, animal model, and scaffold used are logged in Tables 1 – 6 .

Orthotopic

Critchley et al. ³⁹ compared cell-containing biphasic constructs with cell-free multi-layered collagen-hydroxyapatite scaffolds (MaioRegen^©, Fin-ceramica Faenza, Italy) in goat osteochondral defects. The biphasic constructs consisted of a bone phase with BM-MSCs in 3D-printed polycaprolactone (PCL)/alginate, and a cartilage phase with a 1:3 ratio of chondrocytes (source not mentioned) to infrapatellar fat pad (IFP)-derived A-MSCs. After 6 months, the co-implantation group showed larger Safranin-O positive areas, better collagen fiber orientation, and higher International Cartilage Repair Society (ICRS) histology scores compared with cell-free scaffolds. While overall differences were not significant due to high variability, statistically significant improvements were observed in ICRS subcategories matrix staining and cell morphology.

Freshly isolated ACs and IFP-derived SVF were used in a trilayered osteochondral scaffold composed of hydroxyapatite, hyaluronic acid, and type I collagen (Col-I) and type II collagen (Col-II) for goat femoral condyle and trochlea defects. ⁴¹ After 12 months, cartilage repair tissue was similar to cell-free scaffolds, with highly variable macroscopic appearance, Safranin-O and Col-II staining, and ICRS microscopy scores. Only in trochlear defects a trend toward better ICRS scores was found compared with cell-free scaffolds, with significant improvement from the 3-month timepoint.

Col-II positive cartilage repair tissue with smooth articulating surface was observed in goat mandibular cartilage defects when treated with a 3:7 ratio of auricular chondrocytes (AuCs) and BM-MSCs in a hydrogel as evaluated by macroscopy and histology. ⁴³ Cell-free scaffolds were rough and without Col-II. According to histological scores, matrix staining, integration, cell morphology, surface regularity, and thickness of the repair tissue were markedly better in the co-implantation group. Green fluorescent protein (GFP)-labeled MSCs were found back in lacuna-like structures.

In rabbit proof of concept studies, femoral cartilage defects were treated with 1:3 or 1:4 ratios of ACs with either BM-MSCs, A-MSCs or umbilical cord-derived MSCs (UC-MSCs) in various scaffold types. Eight weeks after AC co-implantation with BM-MSCs, macroscopy and Safranin-O staining demonstrated good quality cartilage repair, significantly better than empty scaffolds, which showed thinner repair tissue covered with fibrous tissue. ⁴⁰ Implantation of ACs mixed with UC-MSCs also led to extensive Safranin-O staining and significantly better macroscopic ICRS scores compared with empty scaffolds. ⁴⁴ Finally, ACs/A-MSCs implantation in a transforming growth factor-beta (TGF-β)-containing scaffold resulted in native-like Safranin-O, Alcian Blue and Col-II stains after 16 weeks, whereas cell-free TGF-β-scaffolds showed uncontrolled proteoglycan production and significantly worse integration with native tissue. ⁴²

Ectopic

Long-term (10 months) subcutaneous evaluation of scaffolds with 1:5 ratios of nasoseptal chondrocytes (NCs)/BM-MSCs or NCs/SVF, in contrast to cell-free scaffolds, revealed an abundance of chondrocytes, improved mechanical properties and positive Safranin-O and Alcian Blue staining in both co-implantation scaffolds. ⁴⁵ There was no significant difference in cartilage-forming capacity between NCs/BM-MSCs and NCs/SVF.

NCs and BM-MNCs were rapidly isolated, to resemble a single-stage treatment, and subcutaneously co-implanted in a hydrogel at a 1:5 ratio. This led to a cartilage-like macroscopic appearance and positive Safranin-O and Col-II staining after 8 weeks, in contrast to significantly inferior cell-free hydrogels. ⁴⁶ Also, biochemical GAG content and mechanical properties were significantly better in the co-implantation group.

Altogether, with one exception, ⁴¹ chondrocyte co-implantations with various cell sources and ratios in various animal models resulted in significantly better cartilage repair responses than in cell-free controls groups ( Fig. 5 ).

Figure 5.

Eight studies with an empty defect or empty scaffold control were assessed and divided into three categories. 1) T>C*: Adding cells led to significantly better cartilage repair. 2) T>C insig.: Adding cells led to insignificantly better cartilage repair. 3) T≈C: Adding cells did not lead to better cartilage repair.

Co-Implantation Versus Second Cell Type Alone

In several co-implantation studies it was presupposed that regeneration is driven by differentiation of MSCs into chondrocytes. To determine whether chondrocytes have any role in this process, some of these studies included a second cell type-only control but omitted a chondrocyte-only control ( Table 2 ).

Orthotopic

The role of the implantation site niche on cartilage repair, when mismatched with the scaffold and cell source, was studied by Hou et al. ⁴⁷ To this end, BM-MSCs with or without AuCs in decellularized ear cartilage scaffolds were implanted into pig femoral articular cartilage defects. Conversely, BM-MSCs with or without ACs in decellularized articular cartilage scaffolds were implanted into the auricular cartilage of pig ears. In all four cross-implantation conditions, the repair cartilage tissue resembled the cartilage type of the implantation site niche. Histological scores (Safranin-O, Col-II, Elastin, PRG4, and α-SMA) and gene expression analyses of matching genes with addition of ACAN and SOX9 showed protein levels and gene expression similar to the native implantation site niche. Also, GFP-labeled MSCs were found back in the repair tissue. This study is the only one in this review to suggest that MSCs are capable of almost complete repair on their own, at least when implanted in decellularized cartilage scaffolds. Interestingly, the implantation site niche determined the type of cartilage formed, rather than the scaffold or cell source, which is a hopeful finding in the exploration of novel chondrocyte sources.

Ectopic

In two mouse studies without chondrocyte-only controls, it was shown that adding chondrocytes to MSCs significantly reduced calcium deposition by the MSCs, as evidenced by microcomputed tomography (μCT) ³⁹ and alizarin red staining. ⁴⁸ Co-implantation also reduced vascularization compared with MSC-only controls, although this did not reach significance. ³⁹ These results suggest that MSCs on their own differentiate to hypertrophic chondrocytes, eventually producing bone tissue, a process inhibited by chondrocytes in both studies.

Finally, an ectopic study with embryonic stem cells (ESCs) demonstrated that chondrocytes can stimulate stem cells to differentiate along a chondrogenic pathway. ⁴⁹ Co-implantation of porcine ACs with murine ESCs pre-differentiated to embryonic bodies (EBs) resulted in teratoma formation with pieces of cartilage adjacent to porcine ACs. Such cartilage pieces were rarely found in the embryonic body (EB)-only group. A second co-implantation experiment with EBs grown from fluorescence-activated cell sorting (FACS)-sorted Flk-1⁺ cells confirmed that ACs stimulated the chondrogenic differentiation, since cartilage tissue was found only in the co-implantation group. Although this cartilage may have been produced by only the ACs, some evidence of Flk-1⁺-cell differentiation was shown by co-expression of Col-II and mouse major histocompatibility complex class I (MHC-I) in fluorescent images.

Together the outcomes of these ectopic studies suggest that chondrocytes may enhance stem cell-mediated cartilage repair by guiding differentiation and inhibiting the hypertrophy and calcium deposition that are observed in MSC-only controls ( Fig. 6 ). MSC hypertrophy was not found in the orthotopic study that showed good cartilage repair in the co-implantation group.

Figure 6.

Four studies with a second cell type-only control were assessed and divided into three categories. 1) T>C*: Replacing a fraction of a second cell type by chondrocytes led to significantly better cartilage repair. 2) T>C insig.: Replacing a fraction of a second cell type by chondrocytes led to insignificantly better cartilage repair. 3) T≈C: Replacing a fraction of a second cell type by chondrocytes led to similar cartilage repair.

Co-Implantation Versus Only Chondrocytes—Orthotopic

Of all reviewed co-implantation studies, six employed a chondrocyte-only control in an orthotopic setup ( Table 3 ). The studies primarily used small mammal models (one rabbit, four rat), apart from one study that used goats. ⁵³ Four studies used ACs and two assessed rat costal chondrocytes (CCs). Co-implantation ratios were primarily 1:1, with two exceptions of 1:3.^51,52 In all studies, chondrocytes were cultured before implantation and chondrocyte-only controls contained the same total number of cells as the co-implantation groups.

Articular chondrocytes

The chondrogenic potential of co-implanting human umbilical cord Wharton’s jelly-derived MSCs (UC-MSCs) with ACs was studied by Zhang et al. ⁵³ using a decellularized pig cartilage scaffold in a goat femoral cartilage defect model. The cell combination at a ratio of 1:1 was compared with each cell type alone and to empty scaffold and empty defect controls at 6 and 9 months post-implantation. The co-implantation and MSC-only groups had almost completely filled the cartilage defects with smooth, homogeneous repair tissue similar to native cartilage on magnetic resonance imaging (MRI) scans and macroscopic images. This outcome was significantly superior to the AC-only group. The co-implantation group displayed native cartilage-like Safranin-O and Col-I and Col-II stain results, whereas the MSC-only group showed positive Col-I staining and the AC-only group exhibited thinner repair tissue with fewer lacunae. Furthermore, the co-implantation group demonstrated a significantly higher Young’s modulus and GAG content compared with the other groups. Immunofluorescence for human leukocyte antigen (HLA)-ABC confirmed the presence of UC-MSCs in the regenerated tissue.

Studying the effects of two different second cell types, Ba et al. ⁵⁰ implanted scaffolds with a 1:1 ratio of ACs and either SVF cells or A-MSCs in rabbit femoral cartilage defects. These groups, as well as an AC-only and empty scaffold control, all received concurrent MFx. At 10 weeks, histological evaluation revealed mainly fibrous tissue with non-degraded scaffold remnants in the cell-free scaffolds. The AC-only group exhibited limited cartilage formation, while the A-MSC/AC group displayed moderate repair with irregular extracellular matrix (ECM) deposition and sporadic lacuna-like structures. The AC/SVF scaffolds demonstrated superior cartilage repair, with intense stain results for Safranin-O and Alcian Blue, along with lacunae, which was reflected by a significantly higher ICRS score. This study illustrates a clear benefit of substituting half of the ACs, with SVF outperforming A-MSCs as the second cell type.

Dahlin et al. ⁵¹ co-implanted bovine ACs and rat BM-MSCs, seeded at 1:3 on PCL scaffolds, in cartilage defects in rat knees. At 12 weeks, histological analysis of the co-implantation and AC-only groups showed similar levels of cartilage repair quality, but significantly superior to MSC-only and empty scaffold control groups which primarily formed fibrocartilage. μCT results indicated significantly less mineralized bone formation in the co-implantation and AC groups compared with the controls.

Zhao et al. ⁵⁴ traced the fate of co-implanted cells using fluorescence. Neonatal BM-MSCs and ACs were tagged with fluorescent markers and co-implanted at 1:1 or implanted alone. After 4 weeks, a greater number of GFP-labeled chondrocytes was evident in the co-implantation group compared with the 100% AC group. On the other hand, almost no MSCs were found back in the lesion area. Despite limited MSC survival, histological analysis demonstrated significantly enhanced GAG and collagen deposition compared with the chondrocyte-only control group, suggesting that ACs were responsible for matrix production, while stimulated by disappearing MSCs.

Costal chondrocytes

Alternative sources of MSCs and chondrocytes were assessed by Zheng et al. ⁵⁵ Rat CCs were co-cultured with UC-MSCs at 1:1. The co-culture pellets, alongside single cell type control groups, were implanted in rat cartilage defects and evaluated over 12 weeks. Safranin-O and Col-II staining revealed that the co-culture group exhibited significantly superior cartilage production and better integration with surrounding native cartilage than the UC-MSC only control. Compared with the CC-only group cartilage repair was improved too, but this did not reach significance. The co-culture group also showed delayed and mitigated hypertrophy compared with the CC-only group, which exhibited signs of hypertrophic differentiation and ossification.

In another study, CCs were co-implanted with S-MSCs to determine whether S-MSCs could mitigate the hypertrophy and calcification tendencies of CCs. ⁵² Cells were co-cultured at 1:3 and pellets were implanted for 4 weeks in rat cartilage defects. Both the co-implantation and CC-only groups showed cartilage restoration, but the CC-only group exhibited significantly higher levels of Col-X, suggesting hypertrophic differentiation and calcification. GAG deposition was similar between the co-implantation and CC-only groups, however, the co-implantation group displayed a more uniform distribution of Col-II. In a Transwell® co-culture system, MSCs enhanced CC proliferation, suggesting a stimulatory role for the MSCs.

Co-implantation Versus Only Chondrocytes—Ectopic

A total of 21 studies included a chondrocyte-only control in ectopic (subcutaneous) co-implantation experiments ( Table 4 ). Among these, BM-MSCs were investigated in 15 studies, A-MSCs in eight, and SVF in one. In three studies, multiple second cell types were investigated for co-implantation. ACs were investigated in 15 studies, NCs in four studies, AuCs in three, and costal and tracheal cartilage in one study each. Ectopic studies with AuCs were only included for review if they were compared with a chondrocyte type sourced from hyaline cartilage, as AuCs natively produce elastic cartilage, which is outside the focus of this systematic review.

Various co-implantation ratios were tested in subcutaneous models with the majority involving a higher proportion of a second cell type relative to the chondrocytes, ranging from 1:1 up to 1:19 (chondrocytes: second cell type). In only one study, MSCs represented the smaller portion of the co-implantation ratio (3:1). ⁶⁰ Four studies tested multiple ratios.^58,67,73,75 Three studies tested freshly isolated non-expanded chondrocytes.^58,69,70 Four studies included a low chondrocyte control (LCC) to compare co-implantations with implantation of the same absolute number of chondrocytes.^58,64,69,70 Unless mentioned otherwise, chondrocytes were cultured in plastic flasks before implantation, and chondrocyte-only controls contained the same total number of cells as the co-implantation groups.

ACs with A-MSCs

Gelatin-chondroitin sulfate gels with ACs and A-MSCs in a 1:3 ratio displayed a superior Young’s modulus and significantly better Safranin-O and Col-II stain results over the single cell type controls at 3 and 8 weeks post-implantation. ⁷² However, Col-I was also most intensely stained in the cell combination group. Col-X was minimally deposited in all groups.

The ratio of 1:1 co-implantation of ACs with A-MSCs in elastic cryogels showed Safranin-O, Alcian Blue, and Col-II stain results equal to AC-only gels after 2 weeks of in vitro culture and 8 subsequent weeks in vivo. ⁵⁹ In addition, gels that had undergone dynamic loading during in vitro pre-culture performed better than static-culture gels.

Nürnberger et al. ⁶⁸ seeded decellularized bovine ear cartilage scaffolds (Auriscaff, Ludwig Boltzmann Institute, Austria) with aged human ACs together with immortalized human A-MSCs in a 1:3 ratio, and compared this with the same cell combination on the Chondro-Gide® (Geistlich, Switzerland) scaffold. Both conditions were placed in osteochondral plugs and then implanted in nude mice. This subcutaneous plug model should translate better to orthotopic models compared with regular subcutaneous implantation studies. After 6 weeks, the Auriscaff scaffold contained many cells and newly produced Col-I and Col-II, which was also observed in the Chondro-Gide® scaffold but only in restricted areas. A subsequent experiment with young bovine ACs co-implanted with A-MSCs in the Auriscaff scaffold showed 80% Col-II staining, compared with 90% in the AC-only group. Although the co-implantation group was reported to form less cartilage matrix than chondrocytes alone, cartilage matrix production per implanted chondrocyte was higher. However, the relevance of this study is trivial with no report of sample size nor statistical significance.

ACs with BM-MSCs

Prasadam et al. ⁷¹ placed 1:1 pellets (AC/BM-MSC) covered in Matrigel in a human osteochondral articular cartilage defect plug and subcutaneously implanted the plugs into mice, like Nürnberger et al. ⁶⁸ Compared with BM-MSC- and AC-only defects, the co-implantation group showed the most intense Safranin-O stain after 3 weeks in vitro and 3 weeks in vivo. This was reflected by a significantly higher Bern score. Furthermore, both the AC-only and co-implantation group displayed a white and smooth defect surface, while the BM-MSC group exhibited blood vessel invasion.

The ratio of 3:1 subcutaneous co-implantation of ACs and BM-MSCs in silk fibrin showed significantly increased Safranin-O and Col-II stains and a less intense Col-I stain compared with the AC-only control 4 weeks post-implantation. ⁶⁰ Overexpression of Kindlin-2, a regulator of ECM synthesis, further enhanced this effect.

Likewise, the Col-II stain 4 weeks after subcutaneous co-implantation of hydrogels with 1:4 ACs and BM-MSCs was significantly more intense compared with AC- or MSC-only hydrogels. ⁶¹

The ratio of 1:1 co-implantation of BM-MSCs and ACs in alginate for 8 weeks resulted in low GAG and Col-II deposition, similar to AC-only implantation, as evaluated by biochemical assays and Safranin-O staining. ⁷⁶ Pre-culture of the two constructs in FGF18 improved all results with the co-implantation group significantly outperforming the AC-only control in GAG and Col-II assays.

Liu et al. ⁶³ compared 1:3 co-implantations of ACs and BM-MSCs in different collagen hydrogels against AC- and MSC-only gels. Safranin-O, Col-II, and Aggrecan staining in the co-implantation groups were much more intense than in MSC-only gels, although they did not reach the levels of AC-only gels. GAG assays showed a similar trend. Alizarin Red staining indicated that MSC-mediated calcification was absent in the co-implantation group.

Yang et al. ⁷⁵ implanted hydrogels with ACs and BM-MSCs in a 1:1 or 1:3 ratio or with the separate cell types alone. Reverse transcription polymerase chain reaction (RT-PCR) for Col-II and Aggrecan, along with GAG analysis, generally revealed similar chondrogenic expression in the AC-only and 1:1 co-implantation groups. Alcian Blue, Safranin-O, Col-II, Aggrecan, and Sox9 stainings were more intense in the 1:1 group than in the AC-only group. The 1:3 cell combination results were weaker than ACs alone but generally better than only MSCs. GFP-labeled MSCs were found back in the tissue after 6 weeks.

Sabatino et al. ⁷³ tested scaffolds with ACs and BM-MSCs in 1:19 and 1:4 ratios, comparing these with only ACs or MSCs. Surprisingly, the 1:19 group exhibited the most extensive Safranin-O staining and GAG content, significantly more than the AC group, but only in scaffolds that were precultured for 2 weeks. Without pre-culture, the AC-only group performed best. MSCs on their own were never able to produce many GAGs. Demonstrating improvement over 100% ACs, these results surpass those of Bekkers et al. ⁵⁸ that demonstrated the benefit of up to 1:19 co-implantations compared with LCCs.

Liu et al. ⁶⁴ co-implanted Acs and BM-MSCs in a hydrogel at a 3:7 ratio, comparing this with 100% Acs, 100% BM-MSCs, and an LCC group with the same absolute number of chondrocytes (30% Acs). After 8 weeks in vivo, the GAG content in the co-implantation group was similar to the 100% Acs group and significantly higher than both the 30% AC and the 100% MSC groups. The co-implantation group showed positive staining for Col-II and Safranin-O. GFP-labeled MSCs were found back in the regenerated cartilage, suggesting potential MSC differentiation to the chondrocyte lineage.

Bekkers et al. ⁵⁸ used a rapid isolation protocol to isolate articular chondrons which were subcutaneously implanted with BM-MSCs in several ratios (1:4, 1:9 and 1:19) in fibrin glue. As a control, like Liu et al., ⁶⁴ Acs were implanted using the same absolute chondrocyte numbers. After 4 weeks, GAG levels and Safranin-O staining were improved in the co-implantation groups compared with the AC groups but the GAG improvement was not significant in the 1:19 group.

ACs—direct second cell-type comparisons

Wu et al. ⁷⁴ compared the effect of adding A-MSCs versus SVF to Acs, with Acs only as a control. After 8 weeks in alginate in vivo, the AC/SVF group showed the most intense Col-II staining, followed by the AC/A-MSC and then the AC-only group. GAG/DNA was significantly higher in the AC/SVF co-implantation group compared with the AC/A-MSC and AC-only groups. This result aligns with Ba et al., ⁵⁰ who demonstrated the benefit of SVF over A-MSCs orthotopically. Using short tandem repeat (STR) analysis in vitro, Wu et al. also found that SVF cells disappeared in co-culture, with the AC/SVF ratio evolving from 1:4 to 3:2 in 4 weeks. This finding may support the theory of altruistic cell death by the stimulatory cell type.

Mesallati et al. ⁶⁵ tested Acs in a 1:4 ratio with either BM-MSCs or IFP-derived A-MSCs using two types of osteochondral scaffolds. After 6 weeks in vitro and 6 weeks in vivo, all BM-MSC- and A-MSC-only scaffolds showed weak cartilage, with mineralization and Col-I and Col-X deposition. Mineralization was absent in both co-implantation groups, which formed cartilage similar to AC-only constructs, based on Alcian blue and Col-X stains. The AC-only and AC/A-MSC groups displayed native Col-I and Col-II staining, while the AC/BM-MSC group showed more Col-I and less Col-II compared with Acs only. This suggests an advantage of A-MSCs over BM-MSCs, where Pleumeekers et al. ⁶⁹ did not show a clear difference between the two cell types and a donor-dependent effect of A-MSCs.

Using 1:4 ratios in alginate disks, Pleumeekers et al. ⁶⁹ also compared BM-MSCs with A-MSCs but, like Bekkers et al., ⁵⁸ they co-implanted the cell types with non-expanded Acs that hold promise for single-stage therapies. The co-implantations were compared with 100% Acs and 100% of either second cell type, along with an LCC group (20% Acs) and a group of empty alginate disks. Eight weeks after implantation, both co-implantation groups contained GAG and collagen amounts similar to the AC-only group and all AC-containing groups significantly outperformed the LCC group. Both in histology and biochemical analyses, the AC/A-MSC group was more donor-dependent than the AC/BM-MSC group. Species-specific gene expression analysis in vitro indicated that aggrecan and collagen II were produced by bovine Acs and not by human MSCs. Moreover, MSCs greatly enhanced chondrogenic gene expression in Acs.

Nasoseptal chondrocytes

The same group previously tested other non-expanded chondrocyte sources too. They conducted an experiment with non-expanded bovine AuCs or NCs co-implanted with human BM-MSCs in the same 1:4 setup in alginate disks. ⁷⁰ These groups were compared with 100% AuCs or 100% NCs, and 100% MSCs. All AuC-containing groups and the MSC-only group produced fragile tissue. GAG and collagen content per construct was similar between 100% NCs and the NC/BM-MSC co-implantation group, indicating synergism. In vitro, an additional LCC group exhibited significantly lower GAG and collagen, highlighting the additive effect of MSCs. Again, species-specific gene expression analysis in vitro provided evidence for the stimulatory role of MSCs, enhancing aggrecan production by NCs.

Anderson-Baron et al. ⁵⁶ implanted cell pellets of the NC/BM-MSC combination in a 1:3 ratio or pellets containing only NCs or BM-MSCs. Co-implantation pellets were tested with or without parathyroid hormone-related peptide (PTHrP) hormone. After 3 weeks in vitro and 3 weeks in vivo, co-implantation pellets showed better Safranin-O staining than the NC-only group, but only when PTHrP was added. On the contrary, the co-implantation group without PTHrP displayed low Safranin-O staining, increased Col-X expression and calcium deposition, suggesting NC-MSC synergy only when hypertrophic differentiation of MSCs is inhibited by PTHrP.

The same NC/BM-MSC cell combination was bio-printed in a 1:4 ratio onto a nanofibrillated cellulose/alginate grid and implanted into nude mice.^57,66 After 30 and 60 days in vivo, the number of cells surrounded by GAGs and the GAG-positive area in the co-implantation group were lower than in the NC-only group but higher than expected based on the initial number of NCs, indicating quicker NC proliferation. Also, the co-implantation group stained positive for Alcian Blue and Col-II, ⁵⁷ with more pronounced Alcian Blue staining than the AC-only group in the second study. ⁶⁶ Interestingly, using male NCs and female MSCs and FISH (fluorescence in situ hybridization) analysis of the X and Y chromosomes, both studies showed that the cells in the repair tissue were derived from NCs, supporting the hypothesis that MSCs have a trophic role.

Tracheal and costal chondrocytes

Morrison et al. ⁶⁷ tested AuCs and/or tracheal chondrocytes (not clearly specified) in combination with A-MSCs in ratios of 1:1, 1:2, 1:5, and 1:10, and compared with a 100% chondrocytes control. After 4 weeks of pre-culture and 4 weeks subcutaneously in nude mice, all groups formed cartilage, with positive Safranin-O and Col-II stains and no significant differences in GAG content. Safranin-O staining was more intense in the co-implantation groups compared with chondrocytes alone.

Landau et al. ⁶² tested several 1:1 co-implantation groups using different chondrocyte sources in PCL scaffolds, constructs were cultured subcutaneously in mice for 12 weeks. Microtia AuCs with auricular A-MSCs, CCs with costal A-MSCs, and CCs with microtia AuCs were compared with CC- and AuC-only control groups. No significant differences in stains were found between any of the five groups, nor in the capacity of cells to form typical lacunae. Although these results might be considered inconclusive, they do support the hypothesis that a portion of chondrocytes can be replaced by a second cell type without losing chondrogenic matrix production.

Together, the 27 publications that studied co-implantations of chondrocytes with various second cell types in various ratios compared with chondrocytes alone most often reported similar or better cartilage repair in the co-implantation group ( Fig. 7 ). In four studies, such synergy was only seen under specific conditions.^56,65,73,75 Four other studies demonstrated reduced repair compared with chondrocytes only, while still demonstrating improved repair per chondrocyte.^57,63,64,68

Figure 7.

27 studies with a chondrocyte-only control were assessed and divided into four categories. 1) T>C: Replacing a fraction of chondrocytes by another cell type led to better cartilage repair than chondrocytes alone. 2) T≈C: Replacing a fraction of chondrocytes by another cell type led to similar cartilage repair to chondrocytes alone. 3) T.

Co-Implantation Versus MFx

As an intermediate step before clinical trials, five pre-clinical studies have compared orthotopic co-implantations with MFx ( Table 5 ). Bekkers et al.^58,79 published two studies assessing single-stage co-implantations in goat knee cartilage defects against MFx in defects of the contralateral knee. Both studies utilized ACs harvested using a rapid isolation technique, producing chondrons, which exhibited elevated GAG production during in vitro co-culture with BM-MSCs compared with chondrocytes without pericellular matrix. ⁵⁸ In one study, non-expanded ACs combined with allogeneic BM-MSCs demonstrated significantly superior microscopic, macroscopic, and GAG regeneration compared with MFx after 6 months. ⁵⁸ The other study tested freshly isolated autologous BM-MNCs instead of allogeneic MSCs. ⁷⁹ 6 months post-implantation, co-implantations showed significantly higher macroscopic repair scores and less cartilage degeneration compared with MFx. Although microscopic scores were also improved, the differences were not statistically significant and GAG content was comparable between the treatments.

Table 5.

Co-implantation versus Microfracture (Orthotopic: Rose).

(NE) = non-expanded cells; (RI) = rapidly isolated cells. Cell type and carrier abbreviations are defined in the abbreviations section.

BM-MNCs were also tested in combination with non-expanded growth plate-derived chondrocytes. ⁸² Unknown numbers of this cell combination were seeded into a hyaluronic acid matrix (Hyalofast^©, Anika Therapeutics, USA) and implanted in pig knee cartilage defects, with Hyalofast^© + MFx in contralateral knees as control. Positive indications of cartilage repair were found in both groups, but the co-implantation group performed significantly better in all measured macroscopic, microscopic and histological parameters.

Less positive results were found 12 weeks after 1:4 AC/BM-MSC implantation in pig cartilage defects. ⁸⁰ ICRS II scores based on Safranin-O staining and mechanical analysis revealed no significant differences between the MFx and co-implantation groups. However, the repair tissues from both treatments were significantly worse than the healthy control.

Finally, 35 days after implantation of hydrogels seeded with ACs and BM-MSCs in a 1:2 ratio in rat cartilage defects, significantly superior cartilage repair was demonstrated compared with MFx + hydrogel or MFx alone in both macroscopic and microscopic evaluations. ⁸¹

Overall, four of five pre-clinical studies demonstrated that co-implantations of chondrocytes in combination with either MSCs or MNCs provided superior cartilage regeneration compared with MFx ( Fig. 8 ). These pre-clinical results support the exploration of co-implantations in clinical trials.

Figure 8.

Five studies with an MFx control were assessed and divided into two categories. 1) T>C*: Coimplantation led to significantly better cartilage repair than MFx. 2) T≈C: Co-implantation led to similar cartilage repair to MFx.

Clinical Co-Implantations

Promising results with co-cultures and co-implantations have led to four clinical publications to date ( Table 6 ). These studies represent the results from 91 patients who received single-surgery treatments from two single-armed clinical trials: IMPACT (35 patients, 1.5- and 5-year follow-up)^15,78 and INSTRUCT (40 patients, 2-year follow-up), ¹⁶ and one single-arm prospective case series (16 patients, 5-year follow-up). ⁷⁷ All four publications implanted chondrons, chondrocytes surrounded by pericellular matrix, derived by rapid cell isolations in a single surgery.

Table 6.

Clinical Co-implantations (Orthotopic: Rose).

(NE) = non-expanded cells; (RI) = rapidly isolated cells; (e) = evidence for proposed role. Cell type and carrier abbreviations are defined in the abbreviations section.

In the IMPACT study, 35 patients presenting with knee cartilage defects were treated in a single operation with rapidly isolated autologous articular chondrons mixed with allogeneic BM-MSCs at a ratio of 1:4 or 1:9 in fibrin glue.^15,78 After 3 months, patient-reported outcome measures (PROMs) immediately showed improvements that persisted up to the 18-month timepoint, reaching statistical significance over baseline in all Knee Injury and Osteoarthritis Outcome Score (KOOS) subscores and in the Visual Analogue Scale (VAS) for pain. ¹⁵ Biochemical MRI values displayed no significant difference between healthy and regenerated cartilage while both arthroscopy in 33 patients and Safranin-O and Col-I and Col-II collagen staining in 32 patients showed hyaline-like repair tissue (not clearly defined in this study) that had integrated successfully with native tissue. Two biopsies exhibited mainly Col-I (fibrocartilage). STR analysis at 12 months demonstrated that no allogeneic cells were present, indicating that MSCs had not differentiated to chondrocytes. After 5 years, five patients were identified as treatment failures. ⁷⁸ 7 patients were lost to follow-up. Despite a slight decline compared with the 18-month timepoint, the remaining 28 patients’ PROMs at the 5-year follow-up still demonstrated statistically significant and clinically relevant improvements compared with baseline. The patients lost to follow-up had significantly higher visual analogue scale (VAS) scores and insignificantly lower KOOS scores at their final follow-up compared with the patients who continued to the 5-year mark. This indicates that the 5-year follow-up cohort’s outcomes may be overestimated.

In a similar single-stage strategy, 40 patients were treated with INSTRUCT: an osteochondral biodegradable load-bearing scaffold seeded with rapidly isolated autologous articular chondrons and autologous BM-MNCs. ¹⁶ All isolated chondrons were used and MNCs were added up to a fixed total cell number, leading to a variety of ratios in different patients with a mean of 1:22. Scaffold delamination and post-operative adhesions resulted in the removal of the scaffold in two patients. Two additional patients were lost to follow-up at the 2-year timepoint. Like IMPACT, PROM improvements compared with baseline were achieved from the 3-month timepoint on and reached significance in all timepoints from 1 to 2 years as evaluated by KOOS, VAS, and International Knee Documentation Committee (IKDC) scores. Second-look arthroscopy revealed an improvement of ICRS grade from III-IV to I-II in 23 of 27 patients and histology demonstrated the presence of hyaline-like repair tissue in 22 of 31 biopsy specimens (positive for Col-II, Aggrecan and Safranin-O, negative for Col-I, and no birefringence of polarized light), although most specimens contained predominantly fibrous tissue or undifferentiated mesenchyme. Of the other nine specimens, six showed fibrocartilage and three showed only fibrous tissue. MRIs revealed complete defect filling in all patients and good integration with the surrounding native tissue for the majority of the patients.

Furthermore, additional prospective clinical data for single-stage autologous AC/BM-MNC implantation has recently been published. ⁷⁷ This rapidly isolated cell mixture, similar to the INSTRUCT cell combination (CartiONE™, Cartilage Repair Systems, LLC, USA), was seeded on Hyalofast^© and implanted in articular cartilage defects. Like INSTRUCT, all isolated chondrons were implanted with MNCs added to a fixed total cell number. This approach led to a mean ratio of 1:39. 17 knees treated across 16 patients revealed significant improvement over baseline of IKDC and KOOS scores at 2 years. With 6 patients lost to follow-up, those significant improvements persisted up to 5 years after implantation in 11 knees across 10 patients.

Together with the INSTRUCT and IMPACT trials, this clinical study successfully translated the single-stage co-implantation strategy using rapidly isolated chondrons and provided promising initial clinical data.

Discussion

Altogether, of the 48 studies reviewed across 46 publications, a combination of 15 orthotopic and 26 ectopic studies consistently demonstrated the superior efficacy of co-implantation strategies compared with controls, while three orthotopic studies did not demonstrate improvements over controls. Four clinical publications of single-arm human trials reported significant improvement over baseline for co-implantation treatments at short- term and midterm. More specifically, of the animal studies, seven of eight demonstrated better repair than scaffolds or defects without cells; three of four studies reported better repair than implantation of only the non-chondrocyte cell type, while the fourth study showed extensive cartilage repair in both conditions; and, most importantly, 27 of 27 studies showed a synergistic benefit of adding a second cell type to chondrocytes, although some studies only showed a benefit under specific experimental conditions ( Fig. 7 ; Tables 3 and 4 ). In addition, four of five studies demonstrated that co-implantation outperforms MFx in animals.

These results should be interpreted with caution due to the generally low reporting quality of the studies, the high RoB, and potential publication bias that may have prevented the publication of negative results. Furthermore, cell types, animal models, co-implantation ratios, scaffolds, and implantation methods varied greatly across the studies, making quantitative synthesis challenging.

When using MSCs for cartilage repair it is important to consider the risk of calcification caused by MSCs following their native bone-forming pathway and differentiating to hypertrophic chondrocytes. For instance, hypertrophic differentiation and calcification was observed in subcutaneously implanted NC/BM-MSC cell pellets by Anderson-Baron et al. ⁵⁶ Various other rodent studies showed that the presence of chondrocytes reduced hypertrophy and calcification observed in MSC-only groups.^39,48,63,71 Orthotopic and clinical setups in this review did not report the occurrence of calcification, potentially because MSCs played a more stimulatory role in orthotopic settings

Since previous in vitro data were ambiguous about the role of MSCs in cartilage repair strategies, several studies in this review aimed to clarify whether MSCs have a trophic role or differentiate into chondrocytes. While five studies suggested MSC differentiation after observing the incorporation of fluorescently labeled MSCs in the regenerated cartilage tissue,^{43,47,53,64,75} more convincing evidence supports a trophic role. For instance, STR analysis in IMPACT patients revealed that MSCs had disappeared over time, ¹⁵ which was corroborated by STR analysis in vitro. ⁷⁴ This suggests that MSCs were not responsible for generating tissue but may either have undergone “altruistic” cell death or did not graft after having instructed the chondrocytes. This is also supported by Zhao et al., ⁵⁴ who observed a consistent increase in GFP-chondrocytes and a decrease in red fluorescent protein (RFP)-MSCs in implanted pellets. Apelgren et al. ⁵⁷ and Möller et al. ⁶⁶ further confirmed that chondrocytes, not MSCs, were found back in the repair tissue using gender-specific cell types and FISH analysis for the X and Y chromosomes.

Moreover, several co-implantation studies included in vitro evidence for a trophic role of the second cell type. For example, MSCs in a Transwell® culture system have shown to enhance chondrocyte proliferation ⁵² and chondrogenic expression. ⁷⁶ Finally, Pleumeekers et al.^69,70 and Zheng et al. ⁵⁵ convincingly demonstrated, using species-specific RT-PCR primers, that chondrogenic gene expression by low fractions of chondrocytes in co-cultures was higher than in 100% chondrocyte cultures, while MSCs hardly expressed these genes in the Pleumeekers studies. Thus, although MSC differentiation might play a role and some MSCs may remain in the repair tissue, it is most likely that they exert a trophic effect. This aligns with the consensus, previously advocated by Arnold Caplan, that MSCs are not stem cells but Medicinal Signaling Cells. ¹⁷

The comprehensive overview of all co-implantation studies for hyaline cartilage repair allows for a closer examination of potential trends related to specific methods, such as chondrocyte source, second cell-type source or mixing ratio.

The majority of the co-implantation studies implanted chondrocytes with MSCs derived from either bone marrow or adipose tissue, showing positive results. Several studies directly compared different second cell types in vivo. For instance, Mesallati et al. ⁶⁵ demonstrated a benefit of A-MSCs over BM-MSCs, while Pleumeekers et al. ⁶⁹ found no clear difference between the two cell types. In addition, both Ba et al. ⁵⁰ and Wu et al. ⁷⁴ showed superior results achieved by SVF compared with A-MSCs. Apelgren et al. ⁴⁵ compared SVF and BM-MSCs showing no clear difference. Despite the promising results with SVF, Levingstone et al. ⁴¹ combined ACs and SVF and did not show a clear improvement compared with empty scaffolds in goat defects. As the results from Ba et al. ⁵⁰ and Wu et al. ⁷⁴ favored the use of SVF, more research into this cell fraction would be of high value, holding great potential for clinical applicability as a primary cell fraction that can be autologously obtained within a single operation.

MNCs from bone marrow offer the same benefit and have shown promising pre-clinical data in combination with ACs in mice, ⁴⁶ pigs ⁸² and goats, ⁷⁹ and clinical data in the INSTRUCT trial ¹⁶ and CartiONE™/Hyalofast^© case series. ⁷⁷ This fully autologous single-stage cell combination may therefore be closer to clinical application than ACs/SVF. Contrary to common assumptions, the BM-MNC fraction contains only 0.001% to 0.01% MSCs and the reported chondrogenic effects are therefore most likely due to a combination of other cell types within the fraction. SVF, although less researched, contains at least 500-fold more MSCs than the MNC fraction, is less invasive to harvest than bone marrow and may therefore be interesting to explore clinically too. ²⁰

S-MSCs were tested in a single study showing similar cartilage repair in a 1:3 ratio compared with 100% chondrocytes. ⁵² Finally, UC-MSCs were tested in three studies, showing synergistic cartilage production in goat ⁵³ and rat ⁵⁵ defects, similar to chondrocyte-only groups, and successful regeneration in rabbit defects, outperforming empty scaffolds. ⁴⁴ Further research is necessary to validate the potential of UC-MSCs and S-MSCs as second cell types, to compare their efficacy with other cell types, and to rule out the effects of poor reporting and high RoB observed in the UC-MSC studies.

Altogether, although most co-implantation studies implanted MSCs from adipose tissue or bone marrow, various other second cell types have shown promising results, with BM-MNCs and SVF being of particular interest as potential rapidly isolated autologous fractions.

CC,^52,55,62 NC,^{45,46,56,57,66,70} and tracheal ⁶⁷ chondrocyte have been proposed as potential alternatives to ACs for hyaline cartilage regeneration. Although these studies used various experimental setups and outcome measurements, all demonstrated the formation of hyaline-like cartilage (tissue resembling native hyaline cartilage). CCs on their own had hypertrophic and calcifying tendencies in rat cartilage defects but this was removed by the addition of MSCs.^52,55 In a direct comparison in a subcutaneous setting, Pleumeekers et al. ⁷⁰ found that NCs produced better hyaline-like cartilage than elastic cartilage-derived AuCs. However, orthotopic studies suggest that the implantation site niche rather than the chondrocyte source is the determining factor for the type of cartilage produced. AuCs implanted with MSCs in mandibular cartilage defects resulted in smooth collagen type II-rich repair tissue, although elastin presence was not tested. ⁴³ Similarly, AuCs with MSCs in femoral cartilage defects produced Safranin-O/Col-II-positive and elastin-negative repair tissue. ⁴⁷ By implanting ACs/MSCs in the ear and showing elastin-positive cartilage repair, the same study further confirmed that the implantation niche determined the type of repair tissue formed. Although only two co-implantations with AuCs in articular cartilage defects have been performed to date, these studies suggest, along with evidence from other hyaline chondrocyte sources, that hyaline articular cartilage repair can be induced by any chondrocyte type, provided it is implanted in a hyaline cartilage niche. However, donor-site morbidity must be taken into account when using alternative chondrocyte sources.

The importance of the implantation site niche directly illustrates the limitation of the ectopic studies that lack such a cartilage niche. Therefore, results obtained from ectopic studies cannot reliably predict cell behavior in cartilage defects. The low translational value in combination with the general low quality of ectopic study reports may be seen as an unnecessary expenditure of animal suffering associated with small mammal research. Despite the limited value of these studies, ectopic experiments can still provide initial indications of cellular interactions in a more systemic in vivo environment as opposed to in vitro cultures, which are even less relevant to cartilage regeneration.

The systematic review has revealed a high degree of variability in reporting quality and RoB, which were especially poor for the ectopic studies reviewed. Poor comprehension of these metrics by researchers may lead to invalid and unreliable results, which in the long term may be wrongly accepted as definitive by readers and reviewers. A greater emphasis on the quality, reproducibility and translatability of in vivo studies will provide more valuable results for the research community. This is particularly applicable to future small mammal research which, if conducted at all, should aim to minimize bias and improve reporting quality by adhering to recognized animal research guidelines and checklists.

Generally, implantation ratios did not clearly influence chondrogenic results. Although Yang et al. ⁷⁵ demonstrated co-implantation synergism only for a 1:1 ratio and not for a 1:3 ratio, Bekkers et al. ⁵⁸ showed a benefit of co-implanting at ratios up to 1:19 compared with LCCs, with 1:4 and 1:9 being the most favorable. Sabatino et al. ⁷³ even observed the most extensive cartilage repair at a 1:19 ratio. Morrison et al. ⁶⁷ did not show any difference between several ratios from 1:1 to 1:10, although all co-implantations outperformed 100% chondrocytes in Safranin-O staining. Strikingly, these studies suggest that a wide range of implantation ratios is feasible for cartilage repair and that large portions of chondrocytes might be replaced by second cell types without compromising cartilage repair quality.

Beside implantation ratios, it is likely that absolute chondrocyte numbers and total cell numbers will affect cartilage repair. However, these conditions are too complex to compare between studies due to the highly varied use of animal models, defect sizes, and co-implantation ratios. Likewise, no representative results for any specific type of cell-carrying scaffold or hydrogel can be synthesized due to the large variation in experimental setups. Nevertheless, the comprehensive tables presented in this review may still inform readers who are interested in specific carriers or other experimental variables.

The ratio comparison study results are advantageous for the clinical application of single-stage co-implantations as only a relatively low number of non-expanded chondrocytes can be harvested through clinical rapid isolation techniques. ⁸³ In fact, these studies, along with positive results from numerous other 1:3 to 1:9 ratio studies in this review, suggest that low chondrocyte numbers, in combination with an abundant second cell type, may be sufficient for cartilage repair without the need for complex in vitro chondrocyte expansions. This, along with the benefit of using non-expanded over dedifferentiated chondrocytes, has been the rationale for the INSTRUCT, IMPACT and CartiONE™ clinical studies. In these clinical studies an abundance of a second cell type was implanted with a low number of non-expanded chondrocytes, either at varying ratios up to a fixed cell number,^16,77 or at 1:4 or 1:9 ratios, ¹⁵ respectively. The outcomes of these single-armed studies were promising but require further confirmation in controlled trials.

No large controlled clinical trial has been initiated to date. However, a follow-up IMPACT trial is currently ongoing, which includes a conservative treatment control group with a crossover option at 9 months. ⁸⁴ In addition, results are pending for the similar RECLAIM trial, in which MSCs derived from adipose tissue instead of bone marrow are used. ⁷⁸ For the CartiONE™ therapy, the retrospective ICONIC trial is currently ongoing, focused on 1- to 13-year follow-up MRI and PROM data. The Robert Jones and Agnes Hunt Orthopedic Hospital is currently working on the ASCOT clinical trial where a co-implantation of autologous ACs and BM-MSCs is compared with single cell-type control groups. ⁸⁵ However, this trial uses cultured ACs, rather than chondrons, necessitating two separate surgeries.

The completed and ongoing clinical trials have made different choices for autologous or allogeneic stimulatory cells. Allogeneic MSCs (IMPACT, RECLAIM) are needed if MSCs are preferred in a single-stage setting. MSCs are more controllable and characterizable than autologous single-stage fractions like SVF or BM-MNCs but require a GMP facility for the expansion and storage of allogeneic cells. Also, despite their reported hypo-immunogenicity, ⁸⁶ allogeneic cells could theoretically lead to immune rejection, ⁸⁶ but there is no such evidence the IMPACT trial ¹⁵ and MSC injection trials.^87,88 Alternatively, autologous BM-MNCs (INSTRUCT, CartiONE™) are preferred to reduce risk of rejection, to reduce costs associated with GMP facilities, and to achieve a geographically independent treatment option. Disadvantages are that the MNC fraction is less elaborately researched and that a bone marrow biopsy is needed. Autologous MSCs and a larger number of expanded chondrocytes were preferred in the ASCOT trial, requiring two surgeries and GMP cell culture facilities.

In addition to co-implantations, minced cartilage implantation (MCI) is another emerging technology that achieves single-stage cartilage repair and is less affected by regulatory requirements than cell therapies. Instead of fully isolating chondrocytes or chondrons, autologous cartilage is minced and placed back in the cartilage defect. In vitro, chondrocytes have been shown to migrate out of the minced cartilage and to start producing new matrix. ⁸⁹ First clinical results with up to 2-year follow-up are promising. ⁹⁰ Like isolated chondrocytes, minced cartilage may benefit from the addition of factors like platelet-rich plasma (PRP), which is commonly used in MCI, or even a second cell type like MSCs, MNCs, or SVF. However, adding a second cell type would complicate the regulatory status of MCI. MCI with second cell-type studies—if available at all—were not included in this review due to the exclusion criterion of cell types not being in close proximity.

Based on the comprehensive and consistent positive findings across 45 of 48 studies, it is reasonable to conclude that chondrocytes in combination with additional cell types, such as MSCs, could be successful in inducing articular cartilage repair and likely more successful than either cell type alone. These results provide a framework for the design of future co-implantation studies to help guide choices of cell types and ratios. Furthermore, they encourage the exploration of co-implantation strategies as adequate clinical alternatives to more expensive two-stage treatments like first- to third-generation ACI, or biologically inferior treatments, such as MFx.