Genya Mitani

Cartilage repair in transplanted scaffold-free chondrocyte sheets using a minipig model

Lacking a blood supply and having a low cellular density, articular cartilage has a minimal ability for self-repair. Therefore, wide-ranging cartilage damage rarely resolves spontaneously. Cartilage damage is typically treated by chondrocyte transplantation, mosaicplasty or microfracture. Recent advances in tissue engineering have prompted research on techniques to repair articular cartilage damage using a variety of transplanted cells. We studied the repair and regeneration of cartilage damage using layered chondrocyte sheets prepared on a temperature-responsive culture dish. We previously reported achieving robust tissue repair when covering only the surface layer with layered chondrocyte sheets when researching partial-thickness defects in the articular cartilage of domestic rabbits. The present study was an experiment on the repair and regeneration of articular cartilage in a minipig model of full-thickness defects. Good safranin-O staining and integration with surrounding tissues was achieved in animals transplanted with layered chondrocyte sheets. However, tissue having poor safranin-O staining-not noted in the domestic rabbit experiments-was identified in some of the animals, and the subchondral bone was poorly repaired in these. Thus, although layered chondrocyte sheets facilitate articular cartilage repair, further investigations into appropriate animal models and culture and transplant conditions are required.

The properties of bioengineered chondrocyte sheets for cartilage regeneration.

BackgroundAlthough the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage.ResultsThe expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets.ConclusionThe results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.

Repair of articular cartilage defect with layered chondrocyte sheets and cultured synovial cells

In this study, we investigate the effects of treatment with layered chondrocyte sheets and synovial cell transplantation. An osteochondral defect was created of 48 Japanese white rabbits. In order to determine the effects of treatment, the following 6 groups were produced: (A) synovial cells (1.8 × 10(6) cells), (B)layered chondrocyte sheets (1.7 × 10(6) cells), (C) synovial cells (3.0 × 10(5) cells) + layered chondrocyte sheets, (D)synovial cells (6.0 × 10(5) cells) + layered chondrocyte sheets, (E)synovial cells (1.2 × 10(6) cells) + layered chondrocyte sheets, (F) osteochondral defect. Layered chondrocyte sheets and synovial cells were transplanted, sacrificed four and 12 weeks postoperatively. An incapacitance tester (Linton) was used to find trends in the weight distribution ratio of the damaged limbs after surgery. Sections were stained with Safranin-O. Repair sites were evaluated using ICRS grading system. In groups (A) to (E), the damaged limb weight distribution ratio had improved. The repair tissue stained positively with Safranin-O. Four and 12 weeks after surgery, groups (A) to (E) exhibited significantly higher scores than group (F), and groups (D) and (E) exhibited significantly higher scores than groups (A) and (B). This suggests the efficacy of combining layered chondrocyte sheets with synovial cells.

Studies of the humoral factors produced by layered chondrocyte sheets

The authors aimed to repair and regenerate articular cartilage with layered chondrocyte sheets, produced using temperature‐responsive culture dishes. The purpose of this study was to investigate the humoral factors produced by layered chondrocyte sheets. Articular chondrocytes and synovial cells were harvested during total knee arthroplasty. After co‐culture, the samples were divided into three groups: a monolayer, 7 day culture sheet group (group M); a triple‐layered, 7 day culture sheet group (group L); and a monolayer culture group with a cell count identical to that of group L (group C). The secretion of collagen type 1 (COL1), collagen type 2 (COL2), matrix metalloproteinase‐13 (MMP13), transforming growth factor‐β (TGFβ), melanoma inhibitory activity (MIA) and prostaglandin E2 (PGE2) were measured by enzyme‐linked immunosorbent assay (ELISA). Layered chondrocyte sheets produced the most humoral factors. PGE2 expression declined over time in group C but was significantly higher in groups M and L. TGFβ expression was low in group C but was significantly higher in groups M and L (p < 0.05). Our results suggest that the humoral factors produced by layered chondrocyte sheets may contribute to cartilaginous tissue repair and regeneration. Copyright

Recent technological advancements related to articular cartilage regeneration

Some treatments for full thickness defects of the articular cartilage, such as the transplantation of cultured chondrocytes have already been performed. However, in order to overcome osteoarthritis, we must further study the partial thickness defects of articular cartilage. It is much more difficult to repair a partial thickness defect because few repair cells can address such injured sites. We herein show that bioengineered and layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for the repair of partial thickness defects. We also show that a chondrocyte-plate using a rotational culture system without the use of a scaffold may also be useful as a core cartilage of an articular cartilageous defect. We evaluated the properties of these sheets and plates using histological findings, scanning electrical microscopy, and photoacoustic measurement methods, which we developed to evaluate the biomechanical properties of tissue-engineered cartilage. In conclusion, the layered chondrocyte sheets and chondrocyte-plates were able to maintain the cartilageous phenotype, thus suggesting that they could be a new and potentially effective therapeutic product when attached to the sites of cartilage defects.

Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts.

Conventional culture methods using temperature‐responsive culture dishes require 4–5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co‐culture) on temperature‐responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real‐time polymerase chain reaction and immunostaining of the three‐layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan‐1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A‐disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin‐1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co‐cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright

Diffusion tensor imaging can detect the early stages of cartilage damage: a comparison study

BackgroundIn the present study, we measured damaged areas of cartilage with diffusion tensor (DT) imaging and T2 mapping, and investigated the extent to which cartilage damage could be determined using these techniques.MethodsForty-one patients underwent arthroscopic knee surgery for osteoarthritis of the knee, a meniscus injury, or an anterior cruciate ligament injury. Preoperative magnetic resonance imaging of the knee was performed, including T2 mapping and diffusion tensor imaging. The presence of cartilage injury involving the medial and lateral femoral condyles and tibia plateau was assessed during surgery using the Outerbridge scale. The ADC, T2 values and fractional anisotropy of areas of cartilage injury were then retrospectively analysed.ResultsThe ADC results identified significant differences between Outerbridge grades 0 and 2 (P = 0.041); 0 and 3 (P < 0.001); 1 and 2 (P = 0.045); 1 and 3 (P < 0.001); and 2 and 3 (P = 0.028). The FA results identified significant differences between grades 0 and 1 (P < 0.001); 0 and 2 (P < 0.001); and 0 and 3 (P < 0.001). T2 mapping identified significant differences between Outerbridge grades 0 and 2 (P = 0.032); 0 and 3 (P < 0.001); 1 and 3 (P < 0.001); and 2 and 3 (P < 0.001). Both the T2 mapping (R2 = 0.7883) and the ADC (R2 = 0.9184) correlated significantly with the Outerbridge grade. The FA (R2 = 0.6616) correlated slightly with the Outerbridge grade.ConclusionsT2 mapping can be useful for detecting moderate or severe cartilage damage, and the ADC can be used to detect early stage cartilage damage. The FA can also distinguish normal from damaged cartilage.

Potential utility of cell sheets derived from the anterior cruciate ligament and synovium fabricated in temperature‐responsive culture dishes

Development of tissue-engineered materials to treat anterior cruciate ligament (ACL) injury has been limited by the lack of phenotypic markers. We investigated the feasibility of inducing ACL regeneration using cell sheet technology based on the expression of tenomodulin (TNMD) as an early phenotypic marker of ligaments. ACL remnants, the synovium surrounding cruciate ligaments (SCL), the synovium surrounding the infrapatellar fat pads (SIF), and subcutaneous fat tissue (SCF) were obtained from patients undergoing ACL reconstruction or total knee arthroplasty. ACL cell sheets and SCL-derived cell sheets were fabricated successfully A three-dimensional bioengineered ACL was generated by combining triple-layered ACL cell sheets with a bioabsorbable mesh composite. Immunohistochemical examination showed that TNMD was expressed in human ACL fibers, triple-layered ACL cell sheets, ACL remnants, SCL, and SIF, but not in SCF. Real-time PCR showed that TNMD mRNA was expressed at substantially higher levels in the ACL, SCL, and SIF than in the SCF. These results suggest that TNMD is a specific marker of the human ACL and that ACL sheets have a phenotype similar to that of the ACL. The greater expression of TNMD in the SCL- and SIF- suggests that the synovium is a potential cell source for ACL regeneration.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential.

BackgroundLike all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span.MethodsWe transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo.ResultsOur results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components.ConclusionsFocal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

Usefulness of the photoacoustic measurement method for monitoring the regenerative process of full-thickness defects in articular cartilage using tissue-engineering technology

We demonstrated the capability of photoacoustic measurement for viscoelastic characterization. Since tissue viscoelasticity affects the propagation and attenuation of photoacoustic waves generated in the tissue, the relaxation times of the photoacoustic waves give the viscosity-elasticity ratio of the tissue. The relaxation times of photoacoustic waves of articular cartilage tissues engineered under various culture conditions were closely correlated with intrinsic viscosity-elasticity ratios measured by using a conventional viscoelastic analyzer (R > 0.98). In order to apply the photoacoustic measurement method to evaluation of the regeneration of articular cartilage as a method to validate the surgery, the method should enable not only evaluation of engineered tissue during cultivation in vitro but also evaluation after transplantation of engineered tissue in vivo. The aim of this study was to verify the usefulness of the photoacoustic method for repeated measurement of viscoelastic properties in order to evaluate the process of regeneration of a full-thickness defect in rabbit articular cartilage using allografted tissue-engineered cartilage. Photoacoustic waves were induced by 266- and 355-nm, 5-7 ns, light pulses delivered through an optical silica fiber from an Q-switched Nd:YAG laser and were detected by a piezoelectric transducer, which we had designed. About a 40% difference between the viscosity-elasticity ratio of allografted cartilage that of tissue surrounding the defect was shown just after surgery. The difference was significantly reduced at 4 and 12 postoperative weeks. Therefore, since the photoacoustic measurement method enables assessment of the progress of restoration of the viscoelasticity of articular cartilage, its main function, this method would be useful as an evaluation method in regenerative medicine.