Chondrocyte Sheets Research Articles

Differentiated and Untreated Juvenile Chondrocyte Sheets Regenerate Cartilage Similarly In Vivo

Differentiated and Untreated Juvenile Chondrocyte Sheets Regenerate Cartilage Similarly <i>In Vivo</i>

Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model.

This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated. (1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining." The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. During 48h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFβ1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFβ pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFβ1 might trigger cartilage protein production through the TGFβ pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

Cryopreservation of Cell Sheets for Regenerative Therapy: Application of Vitrified Hydrogel Membranes.

Organ transplantation is the first and most effective treatment for missing or damaged tissues or organs. However, there is a need to establish an alternative treatment method for organ transplantation due to the shortage of donors and viral infections. Rheinwald and Green et al. established epidermal cell culture technology and successfully transplanted human-cultured skin into severely diseased patients. Eventually, artificial cell sheets of cultured skin were created, targeting various tissues and organs, including epithelial sheets, chondrocyte sheets, and myoblast cell sheets. These sheets have been successfully used for clinical applications. Extracellular matrix hydrogels (collagen, elastin, fibronectin, and laminin), thermoresponsive polymers, and vitrified hydrogel membranes have been used as scaffold materials to prepare cell sheets. Collagen is a major structural component of basement membranes and tissue scaffold proteins. Collagen hydrogel membranes (collagen vitrigel), created from collagen hydrogels through a vitrification process, are composed of high-density collagen fibers and are expected to be used as carriers for transplantation. In this review, the essential technologies for cell sheet implantation are described, including cell sheets, vitrified hydrogel membranes, and their cryopreservation applications in regenerative medicine.

EXOSOMAL MIRNAS INVOLVED IN CARTILAGE REPAIR MECHANISM OF POLYDACTYLY-DERIVED CHONDROCYTE SHEETS

Purpose: From 2016 to 2019, we performed a clinical study of regenerative medicine using allogeneic chondrocyte sheets derived from the surgical remains of young polydactyly patients (PD sheets) with the purpose of establishing a definitive therapy for osteoarthritis of the knee (OAK). To date, 1-year follow-ups have been completed for the ten patients who underwent allogeneic transplantation of PD sheets; no serious adverse events have been detected, and positive results such as improvement in clinical symptoms and cartilage structure have been obtained.

Cartilage Regeneration Characteristics of Human and Goat Auricular Chondrocytes.

Although cartilage regeneration technology has achieved clinical breakthroughs, whether auricular chondrocytes (AUCs) represent optimal seed cells to achieve stable cartilage regeneration is not clear. In this study, we systematically explore biological behaviors of human- and goat-derived AUCs during in vitro expansion as well as cartilage regeneration in vitro and in vivo. To eliminate material interference, a cell sheet model was used to evaluate the feasibility of dedifferentiated AUCs to re-differentiate and regenerate cartilage in vitro and in vivo. We found that the dedifferentiated AUCs could re-differentiate and regenerate cartilage sheets under the chondrogenic medium system, and the generated chondrocyte sheets gradually matured with increased in vitro culture time (2, 4, and 8 weeks). After the implantation of cartilage sheets with different in vitro culture times in nude mice, optimal neocartilage was formed in the group with 2 weeks in vitro cultivation. After in vivo implantation, ossification only occurred in the group with goat-regenerated cartilage sheet of 8 weeks in vitro cultivation. These results, which were confirmed in human and goat AUCs, suggest that AUCs are ideal seed cells for the clinical translation of cartilage regeneration under the appropriate culture system and culture condition.

Zonal-Layered Chondrocyte Sheets for Repairment of Full-Thickness Articular Cartilage Defect: A Mini-Pig Model

The cell sheet technique is a promising approach for tissue engineering, and the present study is aimed to determine a better configuration of cell sheets for cartilage repair. For stratified chondrocyte sheets (S-CS), articular chondrocytes isolated from superficial, middle, and deep zones were stacked accordingly. Heterogeneous chondrocyte sheets (H-CS) were obtained by mixing zonal chondrocytes. The expressions of chondrocytes, cytokine markers, and glycosaminoglycan (GAG) production were assessed in an in vitro assay. The curative effect was investigated in an in vivo porcine osteochondral defect model. The S-CS showed a higher cell viability, proliferation rate, expression of chondrogenic markers, secretion of tissue inhibitor of metalloproteinase, and GAG production level than the H-CS group. The expressions of ECM destruction enzyme and proinflammatory cytokines were lower in the S-CS group. In the mini-pigs articular cartilage defect model, the S-CS group had a higher International Cartilage Repair Society (ICRS) macroscopic score and displayed a zonal structure that more closely resembled the native cartilage than those implanted with the H-CS. Our study demonstrated that the application of the S-CS increased the hyaline cartilage formation and improved the surgical outcome of chondrocyte implication, offering a better tissue engineering strategy for treating articular cartilage defects.

Preclinical safety and efficacy of juvenile cartilage-derived chondrocyte sheets for treating focal chondral injury

Purpose: Cartilage lacks an innate repair capability after traumatic injury. As such, focal cartilage defects have been implicated as a potential initiator of early osteoarthritic disease, and early treatment of chondral defects may prevent or delay the development of osteoarthritis and improve patient quality of life. Cell sheet technology using temperature responsive cultureware allows production of confluently cultured cells as sheets. Harvested sheets are readily patched on tissue lesions and adhere without suture, fibrin glue or other adhesion techniques.

Development of Injectable Polydactyly-Derived Chondrocyte Sheets.

We are conducting a clinical study of the use of allogeneic polydactyly-derived chondrocyte sheets (PD sheets) for the repair of articular cartilage damage caused by osteoarthritis. However, the transplantation of PD sheets requires highly invasive surgery. To establish a less invasive treatment, we are currently developing injectable fragments of PD sheets (PD sheets-mini). Polydactyly-derived chondrocytes were seeded in RepCell™ or conventional temperature-responsive inserts and cultured. Cell counts and viability, histology, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), and flow cytometry were used to characterize PD sheets-mini and PD sheets collected from each culture. To examine the effects of injection on cell viability, PD sheets-mini were tested in four experimental conditions: non-injection control, 18 gauge (G) needle, 23G needle, and syringe only. PD sheets-mini produced similar amounts of humoral factors as PD sheets. No histological differences were observed between PD sheets and PD sheets-mini. Except for COL2A1, expression of cartilage-related genes did not differ between the two types of PD sheet. No significant differences were observed between injection conditions. PD sheets-mini have characteristics that resemble PD sheets. The cell viability of PD sheets-mini was not significantly affected by needle gauge size. Intra-articular injection may be a feasible, less invasive method to transplant PD sheets-mini.

Evaluation of osteochondral-like tissues using human freeze-dried cancellous bone and chondrocyte sheets to treat osteochondral defects in rabbits.

Human freeze-dried cancellous bone combined with human chondrocyte sheets have recently been used to construct an osteochondral-like tissue, which resembled a cartilage layer on a subchondral bone layer. Nevertheless, the efficacy of these human tissues in a xenogeneic model has been rarely reported. Therefore, this study aimed to evaluate the potential of human freeze-dried cancellous bones combined with human chondrocyte sheets for the treatment of osteochondral defects in rabbits. The key roles of the extracellular matrix (ECM) and released cytokines in these tissues in osteochondral repair were also assessed. Triple-layered chondrocyte sheets were constructed using a temperature-responsive culture surface. Then, they were placed onto cancellous bone to form chondrocyte sheet-cancellous bone tissues. The immunostaining of collagen type II (COL2) and the proteomic analysis of the human tissues were carried out before the transplantation. In our in vitro study, the triple-layered chondrocyte sheets adhered well on the cancellous bone, and the COL2 expression was apparent throughout the tissue structures. From the proteomic analysis results, it was found that the major function of the secreted proteins found in these tissues was protein binding. The distinct pathways were focal adhesion and the ECM-receptor interaction pathways. Among the highly expressed proteins, laminin-alpha 5 (LAMA5) and fibronectin (FN) not only played roles in the protein binding and ECM-receptor interaction, but also were involved in the cytokine-mediated signaling pathway. At 12 weeks after xenogeneic transplantation, compared to the control group, the defects treated with the chondrocyte sheets showed more hyaline-like cartilage tissue, as indicated by the abundance of safranin-O and COL2 with a partial collagen type I (COL1) expression. At 4, 8, and 12 weeks, compared to the defects treated with the cancellous bone, the staining of safranin-O and COL2 was more apparent in the defects treated with the chondrocyte sheet-cancellous bone tissues. Therefore, the human chondrocyte sheets and chondrocyte sheet-cancellous bone tissues provide a potential treatment for rabbit femoral condyle defect. LAMA5 and FN found in these human xenografts and their culture media might play key roles in the ECM-receptor interaction and might be involved in the cytokine-mediated signaling pathway during tissue repair.

Construction and Evaluation of Osteochondral-Like Tissue Using Chondrocyte Sheet and Cancellous Bone.

The manipulation of human chondrocyte sheets in target areas frequently results in their tearing because they are thin and fragile. In this study, human cancellous bones were used as a supporting material to create chondrocyte sheet-cancellous bone tissues, and their properties were evaluated. Using cell sheet technology, human chondrocytes were constructed into triple-layered chondrocyte sheets that displayed chondrogenic properties. After transferring the chondrocyte sheets onto cancellous bones, the top area of the chondrocyte sheet-cancellous bone tissues exhibited a smooth surface topography without cell sheet floating within 7 days of culture. The immunofluorescence staining of collagen type II (COL2A1) and fibronectin (FN1) was also performed and examined. Using the shotgun proteomic analysis, the proteins associated with cell adhesion, extracellular matrix (ECM) organization, cell-substrate junction assembly, and cell adhesion mediated by integrin were observed in the chondrocyte sheets, cancellous bones, and chondrocyte sheet-cancellous bone tissues. Three integrin members, including integrin β4 (ITGB4), ITGB6, and ITGB8, were found in the chondrocyte sheets. Only ITGB8 was found in the chondrocyte sheets and chondrocyte sheet-cancellous bone tissues. During 48 h, the mean velocity of the individual cell migration was low, which did not affect the structure and chondrogenic properties of the chondrocyte sheets. Staining of the filamentous actin (F-actin) cytoskeleton in the migratory cells also provided a better understanding of the dynamic communication between the cell cytoskeleton and adhesion molecules through ITGB8, which may play a key role in the attachment of the chondrocyte sheets and the synthesis of the cartilage ECM. Therefore, we suggest that cancellous bone could be used as a supporting material to construct chondrocyte sheet-cancellous bone tissues for potential treatment of osteochondral lesions. Impact Statement We proposed a method to construct an osteochondral-like tissue by placing human chondrocyte sheets onto cancellous bone. The stationary chondrocyte sheets and the low mean velocity of the individual cell migration on the cancellous bone with the expression of COL2A1 indicated that the cancellous bone served as an appropriate supporting material. Moreover, the cellular mechanism for the adhesion of the chondrocyte sheets on the cancellous bone based on ITGB8-mediated adhesion through the rearrangement of filamentous actin provided a better understanding to improve the construction of osteochondral-like tissues, and to predict the repair mechanism in osteoarthritis therapy.

Regenerative effects of human chondrocyte sheets in a xenogeneic transplantation model using immune-deficient rats.

Although cell transplantation has attracted much attention in regenerative medicine, animal models continue to be used in translational research to evaluate safety and efficacy because cell sources and transplantation modalities are so diverse. In the present study, we investigated the regenerative effects of human chondrocyte sheets on articular cartilage in a xenogeneic transplantation model using immune‐deficient rats. Osteochondral defects were created in the knee joints of immune‐deficient rats that were treated as Group A, untreated (without transplantation); Group B, transplantation of a layered chondrocyte sheet containing 5.0 × 105 cells (layered chondrocyte sheet transplantation); Group C, transplantation of a synoviocyte sheet containing 5.0 × 105 cells (synoviocyte sheet transplantation); or Group D, transplantation of both a synoviocyte sheet plus a layered chondrocyte sheet, each containing 5.0 × 105 cells (synoviocyte sheet plus layered chondrocyte sheet transplantation). Histological evaluation demonstrated that Group B showed cartilage regeneration with hyaline cartilage and fibrocartilage. In Groups C and D, the defect was filled with fibrous tissue but no hyaline cartilage. Transplanted cells were detected at 4 and 12 weeks after transplantation, but the number of cells had decreased at 12 weeks. Our results indicate that layered chondrocyte sheet transplantation contributes to articular cartilage regeneration; this model proved useful for evaluating these regenerative effects.

Development of an efficient vitrification method for chondrocyte sheets for clinical application.

IntroductionRegenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinical application of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible cryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice.MethodsWe developed a “circulating vitrification bag” as a container to process cell sheets for vitrification in an efficient and sanitary fashion. Moreover, we invented the “vitrification storage box”, which is useful for the vitrification of cell sheets, long-term preservation, and transportation. These devices were used to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics and the viability of the component cells after rewarming.ResultsIn all cell sheet samples (n = 7) vitrified by the circulating vitrification bag method, the integrity of the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrified samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitrification similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrification storage box, the cell sheets maintained normal structure and cell viability (survival rate: 81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet.ConclusionOur results indicate that the circulating vitrification bag method is an effective approach for realizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is also useful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of the clinical application of cryopreserved chondrocyte sheets.

Transcriptomic and Proteomic Analyses Reveal the Potential Mode of Action of Chondrocyte Sheets in Hyaline Cartilage Regeneration.

Chondrocyte sheet transplantation is a novel and promising approach to treating patients who have cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration by autologous chondrocyte sheets has already been demonstrated in clinical research. In this study, the efficacy of polydactyly-derived chondrocyte sheets (PD sheets) as an allogeneic alternative to standard chondrocyte sheets was examined using an orthotopic xenogeneic transplantation model. In addition, the expression of genes and the secreted proteins in the PD sheets was analyzed using a microarray and a DNA aptamer array. The efficacy of PD sheets with respect to cartilage defects was assessed using histological scores, after which the expressions of genes and proteins exhibiting a correlation to efficacy were identified. Enrichment analysis of efficacy-correlated genes and proteins showed that they were associated with extracellular matrices, skeletal development, and angiogenesis. Eight genes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA) exhibited a positive correlation with the efficacy of PD sheets, and three genes (RARRES2, APOE, and PGF) showed a negative correlation for both transcriptomic and proteomic analyses. Among these, MIA, DKK1, and GREM1 involved in skeletal development pathways and ESM1 involved in the angiogenesis pathway exhibited a correlation between the amount of secretion and efficacy. These results suggest that these secreted factors may prove useful for predicting PD sheet efficacy and may therefore contribute to hyaline cartilage regeneration via PD sheets.

Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis

Current cartilage regenerative therapies are not fully effective in treating osteoarthritis of the knee (OAK). We have developed chondrocyte sheets for autologous transplantation and tested these in in vitro and in vivo preclinical studies, and have reported that the transplantation of chondrocyte sheets promoted hyaline cartilage repair in rat, rabbit, and minipig models. However, autologous transplantation of chondrocyte sheets has yet to be reported in humans. Here, we report our combination therapy in which conventional surgical treatment for OAK, is followed by autologous chondrocyte sheet transplantation for cartilage repair. Eight patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV receive the therapy. Patients are thoroughly assessed by preoperative and postoperative X-rays, magnetic resonance imaging (MRI), arthroscopy, Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm Knee Score (LKS), and a laser-induced photoacoustic method to assess cartilage viscoelasticity. Arthroscopic biopsies of all patients are performed 12 months after transplantation for histological evaluation. The properties of the chondrocyte sheets are evaluated using gene expression analysis to investigate the ability to predict the clinical and structural outcomes of the therapy. For this small initial longitudinal series, combination therapy is effective, as assessed by MRI, arthroscopy, viscoelasticity, histology, and the clinical outcomes of KOOS and LKS. Gene marker sets identified in autologous chondrocyte sheets may be predictive of the overall KOOS, LKS, and histological scores after therapy. These predictive gene sets may be potential alternative markers for evaluating OAK treatment.

Cartilaginous extracellular matrix derived from decellularized chondrocyte sheets for the reconstruction of osteochondral defects in rabbits

Cartilaginous extracellular matrix (ECM) materials derived from decellularized native articular cartilage are widely used in cartilage regeneration. However, it is difficult for endogenous cells to migrate into ECM derived from native cartilage owing to its nonporous structure and dense nature. Moreover, current decellularization approaches frequently lead to architectural breakdown and potential loss of surface composition of ECM. To solve this problem, we aimed to establish a novel biological ECM scaffold from chondrocyte sheets for cartilage regeneration. We cultured chondrocytes harvested from the auricular cartilage of 4-week-old New Zealand rabbits and enabled them to form cell sheets. These sheets were decellularized using sodium dodecyl sulfate (SDS) with three different concentrations, namely, 1%, 5%, and 10%, followed by 1% Triton X-100 and deoxyribonuclease enzyme solution. In vitro microstructural examination and mechanical tests demonstrated that 1% SDS not only removed chondrocytes completely but also maintained the native architecture and composition of ECM, thus avoiding the use of high-concentration SDS. Application of decellularized chondrocyte sheets for osteochondral defects in rabbits resulted in substantial host remodeling and variant regeneration of osteochondral tissues. One percent SDS-treated decellularized chondrocyte sheets contributed to the superior reconstruction of osteochondral defects as compared with 5% and 10% SDS groups, which includes vascularized subchondral bone, articular cartilage with adequate thickness, and integration with host tissues. Furthermore, ECM from 1% SDS significantly increased the migrating potential of bone marrow mesenchymal stem cells (BMSCs) in vitro. RT-PCR and western blot also revealed that ECM increased the expression of SOX-9 in BMSCs, whereas it decreased COL-X expression. In conclusion, our results suggested that the chondrocyte sheets decellularized with 1% SDS preserved the integrity and bioactivity, which favored cell recruitment and enabled osteochondral regeneration in the knee joints of rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. Statement of SignificanceAlthough biological extracellular matrix (ECM) derived from decellularized native cartilage has been widely used in cartilage regeneration, it is difficult for endogenous cells to migrate into ECM owing to its dense nature. Moreover, current decellularization approaches lead to architectural breakdown of ECM. This study established a novel biological ECM from decellularized chondrocyte sheets for cartilage regeneration. Our results suggested that cartilaginous ECM favored cell recruitment and enabled osteochondral regeneration in rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. SDS 1% adequately decellularized the chondrocytes in cell sheets, whereas it maintained the native architecture and composition of ECM, thereby avoiding the use of high-concentration SDS and providing a new way to acquire cartilaginous ECM.

Intra-articular administration of EP2 enhances the articular cartilage repair in a rabbit model

We have reported the usefulness of chondrocyte sheets on articular cartilage repair in animal experiments. Here, we investigated the regenerative effects of EP2 signalling with or without chondrocyte sheets. Forty-five rabbits were used, with six rabbits in each of the six groups and nine rabbits for chondrocytes and synovial cells harvesting to fabricate triple-layered chondrocyte sheets: osteochondral defect only (control, Group A), EP2 agonist (Group B), EP2 antagonist (Group C), chondrocyte sheets (Group D), EP2 agonist and chondrocyte sheets (Group E), and EP2 antagonist and chondrocyte sheets (Group F). After surgery, the weight distribution ratio was measured as an indicator of pain alleviation. Injections of the EP2 agonist or EP2 antagonist were given from 4weeks after surgery. The rabbits were sacrificed at 12weeks, and the repaired tissues were evaluated for histology. The weight distribution ratio and International Cartilage Repair Society grading were as follows: Group A: 40.5% ± 0.2%, 14.8±0.5; Group B: 43.4%±0.7%, 25.4±0.8; Group C: 38.7%±0.7%, 13.7±0.3; Group D: 48.6%±0.6%, 40.2±0.5; Group E: 49.1%±0.3%, 40.5±0.4; and Group F; 46.8%±0.4%, 38.7±0.5. Significant differences in histology and pain alleviation were observed between groups except between Groups A and C, between Groups D and E, and between Groups D and F. These findings show that the intra-articular administration of an EP2 agonist achieved pain alleviation and tissue repair. However, no synergistic effect with chondrocyte sheets was observed.

Cartilage repair and inhibition of the progression of cartilage degeneration after transplantation of allogeneic chondrocyte sheets ina nontraumatic early arthritis model.

IntroductionUsing a rat model of nontraumatic early arthritis induced by intra-articular administration of low-dose monoiodoacetic acid (MIA), we transplanted allogeneic chondrocyte sheets and examined the effects on tissue repair.MethodsMIA (0.2 mg/50 μl) was injected into the right knee of 20 male Wistar rats. Four weeks later, rats were randomly allocated into three groups: Group A was examined 4 weeks after administration of MIA; Group B, 8 weeks after MIA injection and chondrocyte sheet transplantation, and Group C, 8 weeks after MIA injection but without chondrocyte sheet transplantation. Allogeneic chondrocyte sheets were transplanted into the right knee of Group B rats. Pain was assessed as the weight distribution ratio of the damaged to undamaged limb. The OARSI score was used for histological scoring.ResultsThe limb weight distribution ratio indicated significantly less pain in Group B. Histological scoring showed significant differences in cartilage repair and inhibition of the progression of cartilage degeneration between Groups B and C, but not between Groups A and B, or Groups A and C.ConclusionsThese findings suggest that, in this rat model of nontraumatic early arthritis induced by low-dose MIA injection, allogeneic chondrocyte sheet transplantation induces cartilage repair and suppresses cartilage degeneration.

Rabbit xenogeneic transplantation model for evaluating human chondrocyte sheets used in articular cartilage repair.

Research on cartilage regeneration has developed novel sources for human chondrocytes and new regenerative therapies, but appropriate animal models for translational research are needed. Although rabbit models are frequently used in such studies, the availability of immunocompromised rabbits is limited. Here, we investigated the usefulness of an immunosuppressed rabbit model to evaluate directly the efficacy of human chondrocyte sheets through xenogeneic transplantation. Human chondrocyte sheets were transplanted into knee osteochondral defects in Japanese white rabbits administered with immunosuppressant tacrolimus at a dosage of 0.8 or 1.6 mg/kg/day for 4 weeks. Histological evaluation at 4 weeks after transplantation in rabbits administered 1.6 mg/kg/day showed successful engraftment of human chondrocytes and cartilage regeneration involving a mixture of hyaline cartilage and fibrocartilage. No human chondrocytes were detected in rabbits administered 0.8 mg/kg/day, although regeneration of hyaline cartilage was confirmed. Histological evaluation at 12 weeks after transplantation (i.e., 8 weeks after termination of immunosuppression) showed strong immune rejection of human chondrocytes, which indicated that, even after engraftment, articular cartilage is not particularly immune privileged in xenogeneic transplantation. Our results suggest that Japanese white rabbits administered tacrolimus at 1.6 mg/kg/day and evaluated at 4 weeks may be useful as a preclinical model for the direct evaluation of human cell‐based therapies.

Analysis of micro-RNAs in exosomes secreted by human polydactyly-derived chondrocyte sheets

Analysis of micro-RNAs in exosomes secreted by human polydactyly-derived chondrocyte sheets