This study is the first large animal experiment that proves the proliferation ability of juvenile cartilage is stronger than that of adult cartilage, and that juvenile cartilage has strong proliferation potential. It also proposes that the proliferation ability of juvenile cartilage may be due to the activation of Lin28. Lin 28, by combining with several metabolic enzymes to enhance its functions, promotes oxidative metabolism (glycolysis and oxidative phosphorylation) and the bioenergy characteristics of chondrocytes to facilitate the repair and proliferation of juvenile cartilage. We hope that this study can provide some reference for future clinical application of PJAC technology. At present, PJAC has been proven to be one of the safest and most effective treatments for focal articular cartilage defects. Compared with ACC transplantation that can only produce better results in younger people, PJAC has been successfully applied in all age groups with high-quality repaired tissues. In 2013, Tompkins et al. applied PJAC to treat 16 patients with patellar cartilage lesions; they found that at 28 months postoperatively, not only the International Knee Documentation Committee (IKDC) scores of the patients were significantly improved, but also 89% and 73% of their MRI results showed that the defect area appeared to be “normal or near-normal” . In 2014, Farr et al. followed 25 patients after PJAC surgery for 24 months; they found that the scores of IKDC, Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analogue scale (VAS) were all improved, and biopsy of the repair tissue of 8 patients showed that the tissue was composed pf hyaline change and fibrous cartilage. The success of PJAC surgery is based on the proliferation ability of juvenile cartilage in vitro[7, 8]. Previous in vitro studies have proven that juvenile cartilage has a strong proliferation ability compared to adult cartilage. This may be the reason why PJAC’s clinical efficacy is better than ACC. The most important limiting factor of ACC is the low proliferation ability of adult cartilage[3–6], leading to poor clinical symptoms and imaging data, and unstable repair tissue.
We used immunohistochemistry to analyze the cartilage proliferation through antigen and antibody reactions. By analyzing the Ki-67 positive rate, we found an interesting phenomenon: the expression of Ki-67 in the PJAC group was stronger than that of ACC group within 3 months. This may be the reason why the repair ability and proliferation ability of juvenile cartilage continue to increase. Recent studies have shown that Ki-67 protein was originally defined by the prototype monoclonal antibody Ki-67 (Gerdes et al. 1983), which was produced by immunizing mice with the nucleus of Hodgkin’s lymphoma cell line L428. The name is derived from the city of origin and the number of original clones on 96-well plates. Since the antigen was not identified initially, it was mainly called the Ki-67 antigen. When the antigen was found to be a protein whose primary structure could be inferred from the corresponding cDNA, it is found that it has no homology with any known polypeptide. The prototype antibody and antigen are called Ki-67 antibody and Ki-67 protein, respectively. Ki-67 protein is expressed in the G1, G2, S, and M cycles of active cell proliferation, but not in the resting G0 cell phase. Based on this feature, immunostaining of Ki-67 is often used to determine the ability of cell proliferation in a tissue, not only in the field of tumor cell proliferation, but also the proliferation ability of cartilage[18–22]. This study investigated the expression of proliferation-related antigen Ki-67 in transplanted cartilage, and showed the immunohistochemical positive rate of juvenile cartilage was more than that of adult cartilage. This result proved that the proliferation ability of juvenile cartilage is stronger than that of adult cartilage. Studies have found that Lin28 is a highly conserved RNA-binding protein that is expressed during embryogenesis and plays a role in the development, pluripotency and metabolism. In order to determine whether Lin28 affects adult tissue repair, researchers have used several injured tissue models to reactivate the expression of Lin28. They found that Lin28 combined with several metabolic enzymes and enhanced its function, promoted oxidative metabolism (glycolysis and oxidative phosphorylation) and the bioenergy characteristics of embryonic cells to facilitate tissue repair. Finally, it was found that the re-expression of Lin28 promoted the repair and regeneration of cartilage, bone and interstitial tissue after ear and finger injuries. Lin28 may be the key gene that make the proliferation ability of juvenile cartilage stronger than that of adult one. We further verified this hypothesis through related experiments and found that the expression of Lin28 protein in juvenile cartilage was stronger than that in adult one. Maybe the activation of Lin28 has caused the increase of Lin28 gene expression. The mechanism of Lin28 facilitating tissue repair and proliferation of juvenile cartilage needs further research.
Recent studies have found that PJAC surgery is very effective in clinical application, and the clinical symptoms and imaging data of patients have improved significantly. However, there has been no animal study to verify whether the proliferation ability of juvenile cartilage is stronger. The purpose of our large animal experiment is to confirm the proliferation ability and potential of PJAC by immunohistochemical technique to promote its further application. The results of this study are similar to previous researches. Goss et al. created oral mucosal, skin and cartilage wounds of the fetus in the rat's uterus; they observed the wounds within 72 hours, and the cartilage wounds healed rapidly without inflammation during the healing process. Cherukupally et al. studied the staining of proliferation markers after cricoid cartilage injury in rabbits of 1.5, 4, and 8 weeks; they found that the proliferation markers were progressively attenuated with age. Wagner et al. found that compared with adult cartilage, neonatal rat cartilage had the ability to rapidly regenerate without scarring after full-thickness incision. Namba et al. found that in the injury model of fetal sheep, the partial-thickness defect of articular cartilage was completely and spontaneously repaired after 28 days. By culturing juvenile cartilage and adult cells in vitro, Bonasia et al. found that the proliferation of juvenile cartilage was significantly better than that of adult cells and mixed-cultured cells while there was no significant difference in the proliferation of mixed-cultured cells and adult cells. Marmotti et al. combined the juvenile cartilage particles and mature cartilage particles with hyaluronic acid scaffolds cultured in vitro and found that the proliferation ability of juvenile cartilage was significantly stronger than that of mature cartilage, and the regeneration ability would decrease with age. These studies have confirmed the strong proliferation ability of fetal and juvenile cartilage. To our knowledge, this study is the first to have proven this phenomenon in large animals. We hope it can provide a scientific basis for producing better therapeutic effects in the clinical application of PJAC.
Adult cartilage has very weak proliferation ability due to few blood vessels and lymphoid tissue. Besides, due to limited tissue sources, ACC transplantation requires two operations, which is more traumatic and has unstable phenotype. In recent years, it has been found that the long-term efficacy of microfractures is not good. The better proliferation ability of juvenile cartilage has made it a very potential treatment plan for cartilage injuries. Although some PJACs have been applied to the repair of cartilage defects and have achieved certain clinical efficacy, whether the donor cells are involved in the process of repairing cartilage defect from the histological point of view remains unknown. To explore the repair mechanism of juvenile cartilage, we chose Guizhou minipigs as the large animal model to simulate the process of cartilage transplantation in PJAC. Compared with dogs and other smaller animal models, the size, weight requirements and cartilage thickness of pigs are much closer to those of humans, and they are physiologically and physically similar to us, and the cartilage defect repair process of pigs may be closer to that of humans. Therefore, this study is of high clinical reference value[29–32].
This study also has limitations, such as the number of animal samples is relatively small; the observation period is limited to less than 3 months, instead of a longer period of time; has only investigated the proliferation ability without further in-depth study, etc. More long-term in-depth research will be conducted in the future.