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

Abstract

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.

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

“…In the fabrication of human adult chondrocyte sheets, chondrocytes proliferate in the 2D-culture environment, causing the expression of Col2A1 to be reduced and the chondrocyte sheets to be considered dedifferentiated. However, it has been found that they redifferentiate into hyaline cartilage in the transplanted joint for both animal model [12] and human clinical research [9]. Using PD sheets, we have found hyaline cartilage-like tissue in defect areas derived from human cells, indicating that PD sheets also redifferentiate to hyaline cartilage in vivo.…”

“…Efficacy evaluation of PD sheets in rabbits was performed as described previously [12]. A total of 69 female Japanese white rabbits (average weight = 3.0 kg: Tokyo Laboratory Animals Science Co., Tokyo, Japan) was used.…”

“…We have previously reported that the efficacy of human adult knee chondrocyte sheets on osteochondral defects can be directly evaluated via a xenogeneic transplantation model involving immunosuppressant-treated rabbits [12]. In this study, we evaluated the efficacy of PD sheets for osteochondral defects in terms of this xenogeneic model, with gene expression and the secreted proteins of transplanted PD sheets also being evaluated.…”

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

“…Articular cartilage and synovial tissues were enzymatically digested separately to isolate chondrocytes and synoviocytes, respectively. Layered chondrocyte sheets (LC sheets) and SY sheets were made according to the methods described by Kokubo et al (2016) and Takahashi et al (2018), as summarized below. Briefly, articular cartilage or synovial tissues were minced in a petri dish using scissors, and then digested in Dulbecco's modified Eagle's medium-Nutrient Mixture F-12 (DMEM/F12; Gibco, Waltham, MA, USA) containing 5 mg/ml collagenase type I (Worthington, NJ, USA), 20% fetal bovine serum (FBS; Ausgenex, Molendinar, Australia), and 1% antibiotic-antimycotic solution (AA; Gibco).…”

“…In other words, xenogeneic orthotopic transplantation models that approximate the in vivo efficacy of clinical transplantation are required. We previously reported the use of an immunosuppressed rabbit xenogeneic transplantation model to directly evaluate human chondrocyte sheets for articular cartilage repair (Takahashi et al, 2018), but the duration for which immunosuppression was effective limited long-term studies. Ito et al (2012) and Shimizu et al (2015) reported conflicting results concerning the effectiveness of chondrocyte sheets and synoviocyte (SY) sheets using allogeneic transplantation in rabbits and rats, respectively.…”