Cell sheet engineering was developed as an advanced approach, designed to avoid the shortcomings of traditional tissue engineering. Studies have shown that cell sheets have biological scaffolds composed of collagen type I and can be transplanted with intact cell–cell junctions and undamaged ECM16,28. Therefore, they increase survival of cells29, enable the delivery of large number of cells15, and enhance the stemness and transdifferentiation capability30. Presently, several improvements have been made to harvest the living cell sheet more easily, such as a temperature-responsive culture dish31, the coating of dishes with a thermo-responsive hydrogel32, and so forth. However, the entire grafting process remains relatively complicated, time-consuming, and requires special materials. In our study, a simple and inexpensive Vc-mediated procedure was used to obtain ADSC sheets20.
There are several reports on the characteristics of Vc-induced cell sheets. Ando et al.33 reported that Vc-induced cell sheets formation derived from synovial MSCs possessed sufficiently self-supporting mechanical properties despite not containing artificial scaffolding. Additionally, they revealed that the sheets contained collagen I and III, fibronectin, and vitronectin, and exhibited stable adhesion to the surface of cartilage matrix. We investigated the optimal condition of Vc-induced ADSC sheets formation and indicated osteogenic potential of the sheets20. However, the properties of Vc-induced cell sheets have many unclear parts such as strength, species, incubation period, and quantity of Vc. The Vc-induced ADSC sheets used in this study had the property of transplanting many ADSCs alive and was effective in suppressing articular cartilage degeneration. However, there may be better conditions for fabrication of them. Therefore, further studies are needed to decide an appropriate dosage of Vc that could make the cell sheets reach the balance point of transdifferentiation capability and mechanical strength.
Stem-cell therapy is an emerging treatment option for OA. Inspection by animal experiments is required to establish the new treatment, but it is necessary to use animal models with good reproducibility that are suitable for an experiment on this occasion. We evaluated the efficacy of intra-articular injection of autologous ADSC sheets to treat OA in an experimental rabbit model. We chose a rabbit OA model induced by ACLT surgery, because the ACLT model is similar to the pathology of human OA and has been widely validated for investigating OA5,17,23. Additionally, the mouse and rat models are too small to inject the cell sheets we made, and making the OA knee is difficult in the pig or large animals. Furthermore, it is difficult to secure a population. The rabbit was the most appropriate for this experiment due to its experimental ease. However, the rabbit does not meet the characteristic of the human knee in that it is not bipedal. Inspection in bipedal animals, such as a chicken or monkey, is desirable in the future.
Previous studies have evaluated the safety and efficacy of ADSCs for OA treatment. Desando et al.26 found that autologous ADSCs attenuated inflammation in synovial membranes and prevented damage to cartilage and menisci in a rabbit ACLT model. Kuroda et al.5 revealed that a few DiI-ADSCs homed to intra-articular soft tissue and showed the trophic effects in a rabbit OA model. In this study, a large quantity of DiI-ADSCs became clumped and survived in the subintimal layers of the synovium and protected chondrocytes from inflammatory factor-induced damage. There was no evidence of local inflammation. We did not find the formation of neocartilage in both groups, but the OA progression was significantly milder in the ADSC sheets group. After ACLT, as time progressed, more serious OA developed in both groups. However, the cartilage was thicker in the ADSC sheets group. To the best of our knowledge, our study is the first to prove the role of autologous ADSC sheets in the treatment of OA, and further research is needed to improve the effectiveness of ADSC sheets.
Some possible mechanisms of action underlie the effectiveness of MSCs in the OA treatment. First, injected MSCs differentiate into chondrocytes and fill articular cartilage lesions34. Second, injected MSCs could influence the microenvironment via trophic mechanism of action by the release of chondroprotective growth factors and cytokines5. Our data showed that MMP–1, MMP–13, and ADAMTS–4 were less expressive in the ADSC sheets group. MMPs comprise a large group of zinc-dependent proteases that can degrade components of the extracellular matrix such as collagen, elastin gelatin, and casein35. ADAMTS proteases are multidomain extracellular protease enzymes. Their functions include processing of procollagens as well as cleavage of aggrecan, versican, brevican and neurocan. MMPs and ADAMTS family have crucial roles in the initiation and progression of cartilage damage during OA. MMP–1 and MMP–13 degrade type II collagen, which is the main component of the cartilage matrix and is responsible for the degradation of native collagen fibers36. ADAMTS–4 is a major enzyme responsible for aggrecan degradation37. Therefore, we suggested that the ADSC sheets inhibited progression of articular cartilage degeneration by secreting liquid factors having chondroprotective effects. In short, we agree with the second mechanism.
At the early stage of ADSC sheets OA treatment, we suggested that the secretion of trophic factors by autologous ADSCs is the main mechanism responsible for their chondroprotective effect. Injection of ADSCs may be advantageous for secreting various factors to mediate anti-inflammatory, anti-fibrotic, and anti-apoptotic functions38. However, as time progresses, the trophic effect of them will become weaker. The OA knee model used in this study was induced by the instability of the joint caused by the dissection of the ACL. Since ADSCs could not suppress the mechanical factor, overt degeneration occurred in both groups at 12 weeks in this study. To maintain the chondroprotective effect, we suggested periodic injection of large number of autologous ADSCs, namely, weekly injection of ADSC sheets are needed. Still, complete suppression of OA progression would be impossible. Stem cell therapy of OA caused by the mechanical factor may play only an adjunct role in treatment. Further studies are necessary to prove this hypothesis.
Our study has some limitations. First, the long-term culture may have changed the properties of the ADSCs sheet at late phase injection. As mentioned above, there are many unclear points about the characteristics of the ADSC sheets, and the change with the culturing period and the optimal amount of Vc are the subject of future research. Second, the details of the ability of ADSCs to differentiate into chondrocytes are not known. The possible mechanism by which stem cells differentiate into chondrocytes and accumulate in lesions was discussed above. Although this study did not mention the mechanism, we believe that if ADSCs can be induced to differentiate into chondrocytes, further therapeutic effects can be expected. Third, we have not verified the effects of cell sheets. Specifically, the progress of OA was not evaluated between the ADSC sheets transplantation and the only ADSCs transplantation. However, it is technically difficult to transplant and retain the same number of cells as the ADSC sheets without creating the sheet. In the present study, as shown by the DiI labeling data, the cell sheets transplantation clearly had a greater amount of cell colonization in the joint than the cells alone transplantation. From this point, the cell sheet approach is considered to have great significance.