In clinical practice, autologous bone grafts were once favored by orthopedic surgeons because of their good ability of bone induction and bone regeneration; however, they have been plaguing clinicians and patients due to the medical problems such as infection, pain, scar, increased trauma, prolonged operation time and complications after extubation in the donor area [14–16]. Meanwhile, due to the limited donor area, its size and shape are very limited,, which cannot meet the clinical needs, so the allogeneic bone and cartilage transplantation came into being, since the first allogeneic bone transplantation was performed by Macewen in 1878, allogeneic bone transplantation has gone through nearly 150 years of development; during in these 150 years, allogeneic bone grafting has developed significantly, providing a large number of reliable grafts for patients with clinical osteonecrosis, bone tumors, and large bone and cartilage defects. Today, most of the clinically used grafts are decellularized, deantigenized allogeneic bone materials, which are safe and stable, non-immunogenic, and do not cause large immune reactions in the body, However,because of the decellularization process, the transplanted bone and cartilage no longer have biological activity and in the recipient body mainly depends on "crawling substitution" growth, with a slow growth rate. [17].Fresh biologically active allogeneic bone material can be a good solution to this problem, but research in this area has progressed slowly in recent years, due to the difficulty of controlling and correcting the acute immune response of the recipient produced by the graft [18–20], and the poor quality of survival of the implant in the recipient after transplantation.
After allogeneic whole-layer cartilage transplantation,the occurrence of immune rejection is mainly focused on T cell-mediated cytotoxicity, and the pathways of the immune response include: First, the joint action of CD4 + T cells and CD8 + T cells, which promote the proliferation and differentiation of lymphocytes, while mature CD8 + T cells can cause the lysis and death of target cells, and insufficient numbers of CD4 + T cells of CD8 + T cells cannot fully develop and mature, in other words, CD4 + T cells are the activators of the immune response and CD8 + T cells are the killers of the immune response [21–23]. Second, is the direct killing effect of NK on allogeneic cells. When NK cells’ immunoglobulin-like receptors fail to recognize MHC molecules on implant cells, they activate the killing mechanism of NK cells and attack foreign tissues and cells. And the addition of CD8 + T cells during this process leads to further destruction of the grafted cartilage tissue by the receptor [24, 25]. In the observation of the flow cytometry results of this study, it was found that the ratio of CD4 + T and CD8 + T to dendritic cells in the allograft group increased significantly on the 3rd day after operation, reached the peak on the 5th day, some of the data decreased on the 7th day, and other data were also in a stable stage and did not continue to increase significantly. The expression of NK cells in spleen reached its peak at 3 days after transplantation, and then decreased gradually (as shown in Fig. 3), which indicates that the acute immune response after allogeneic whole-layer cartilage transplantation in rats peaked around day 5 and continued to stabilize and slowly decline afterward. As mentioned previously, extracellular inflammatory factors released from major immune cells, such as IL-1β, IL-6, and TNF-α, play an important role in the immune rejection response of the recipient to the graft [26], which is normally found in the human body mainly in the skin, sweat, and urine, while the majority of cells synthesize and secrete IL-1β and need to be stimulated by foreign stimulation by antigens [27]. In terms of mechanism of action, IL-1β causes cartilage matrix degradation, initiates inflammatory regulation, promotes IL-6 and matrix metalloproteinase expression through a cascade reaction, mediates chondrocyte apoptosis, and accelerates matrix degradation by decreasing the ability of cartilage extracellular matrix type II collagen synthesis. TNF-α is also an important factor in contributing to cartilage matrix degradation [28–31]. The previous experiments demonstrated that bFGF can effectively antagonize the degradation of cartilage by inflammatory factors, protect the proliferation of chondrocytes, promote the synthesis and expression of type II collagen and proteoglycan at the genetic level, and promote the repair of chondrocytes [32]. reached a peak at day 5 postoperatively and started to decline at day 7 (as shown in Fig. 6), which may be related to the number of source cells that secrete them. The histological staining results after allogeneic whole cartilage transplantation were more visual, A large number of bone marrow infiltration could be seen at the transplantation-recipient interface 3 days after operation, and the local inflammation was obvious, but there was no damage to the overall state of the implant. On the 5th day after operation, the section showed that the inflammatory manifestations at the interface between the recipient and the graft continued to expand and involved part of the graft, with edema inside the graft, destruction of the cartilage status, and partial fracture of the bone trabeculae inside the cancellous bone. On the 7th day after operation, the implant was completely destroyed, with extensive diffuse inflammatory reaction and necrotic tissue inside, with complete loss of the morphological structure of the total cartilage tissue (shown in Fig. 5). This indicates that the acute immune response after allograft whole-layer cartilage grafting occurs postoperatively, reaches the peak in 5 days, and completes the overall destruction of the graft between 5 and 7 days, with a gradual decrease in the intensity of the response, which corresponds to the results of the flow cytometry and ELISA assays.
In this research,it was also found that the expression of Tregs cells increased with the duration of observation after autologous transplantation, Sakaguchi et al. [33] reported Tregs for the first time in 1995, in recent years, scholars have paid more and more attention to the role of Tregs in the immune process, which stems from their specific biological role - organism Immunosuppression. It is mainly used for immunosuppression by directly inhibiting the activation and proliferation of CD4 + T and CD8 + T cells, which fundamentally reduces the T cell-mediated cytotoxic response [34–36], and then indirectly by inhibiting antigen-presenting cells [37]. Because of this, many scholars in the field deem that Treg has great potential value and have explored its mechanism of action in multiple aspects with certain results [38, 39]. In recent years, with the in-depth study of Treg cells, some scholars reported that Treg can be induced to expand in vitro, such as using IL-2, all-trans retinoic acid, and other drugs [40–43], which provides support for the clinical application of Treg and realizes some clinical translation. T cell-mediated cytotoxicity counter is an important cause of allogeneic whole-layer cartilage transplantation failure, so Treg is considered as a next-generation immunosuppressive drug with great research value in multiple fields [44–49]. This study also found that Treg continued to increase with the extension of time after operation, especially in the autograft group, which increased significantly faster than in other groups, suggesting that it may protect autografts in the direction of suppressing the intensity of immune response, while further specific mechanisms of Treg in allogeneic autologous/allogeneic whole-layer cartilage transplantation need to be further certified in future experiments.
In summary, the results of this experiment suggest that from the first moment after allogeneic whole-layer cartilage transplantation, an acute host anti-graft immune response occurs, as evidenced by an increase in CD4 + T, CD8 + T, NK cells, and various extracellular factors such as IL-1β, IL-6, and TNF-α, which trigger a series of reactions that form a receptor attack on the graft in the absence of external interference, this reaction gradually increased with the progress of time, and began to decline gradually after the peak of the effect on the 5th postoperative day, and the receptor completed the destruction of the implanted bone and cartilage tissue on the 7th postoperative day, and the implant completely lost its original form and formed a large area of necrosis, and the body ended the acute immune response to the implant. The analysis and discussion of the results of this experiment provide important temporal evidence for the acute immune response after the next allogeneic whole-layer cartilage transplantation, and also provide a theoretical basis for the subsequent avoidance of the immune response to the recipient and the development of immunosuppressive drugs.