It should be noted that the immune response elicited by implants might cause severe clinical symptoms. Although collagen is believed to have low immunogenicity, adverse reactions, such as local hypersensitivity, do occur in the clinic after the use of collagen products. Therefore, it is necessary to investigate the immunological reaction induced by any new collagen product. Here, we studied a type of bovine collagen dermal regeneration matrix. We aimed to thoroughly and comprehensively study the immune response aroused by the material, predicting that it would produce only slight or no host responses to lay the foundation for future clinical trials.
As previously described, multiple collection time points are recommended for anti-drug antibody assessments, and the number of time points should be selected to provide statistically meaningful data for the immunogenicity assessment. Additionally, the highest dose and the most sensitive population are also recommended to achieve the strongest immune response. Similarly, these factors were considered in our scheme to evaluate the immunogenicity of the collagen-based dermal substitute. The low dose was calculated based on the maximum single dose and body surface area ratio of BALB/c mice and humans, and it was one-fourth of the high dose. The longest sampling time (90 days) was the degradation time of DM according to our previous study. Moreover, the experimental animals were 6- to 8-week-old BALB/c female mice. Because BALB/c mice are sensitive to antigens, the females can produce more antibodies than the male, and the younger mice have a greater immune responsive ability than the older and infant mice. We aimed to induce the strongest possible immune response in BALB/c mice with DM-H implants. A benign immune response would be an indication of the extremely low immunogenicity of the DM. Furthermore, this study focused on adaptive immunity, as the innate immune response occurs early after implantation; our study included three implantations, therefore the adaptive immune response was the predominant reaction and was investigated comprehensively. However, the innate immune system can regulate adoptive immunity; thus, innate immunity was also approximated.
According to ISO 10993-20: 2005, predicting the immunotoxicity of new chemicals and materials is difficult, therefore effort and interest need to be focused on the assessment and management of risks. Risk assessment includes hazard identification, dose response assessment, and exposure assessment. Immunological hazards should be identified by assessing exposure to medical device materials to identify the presence of (potentially) immunotoxic agents, and related ex vivo tests were evaluated prior to this study. Dose response assessment and exposure assessments were performed in this study. ISO 10993-20: 2005 also indicates that a medical device implant may induce acute or chronic inflammation, hypersensitivity, immunostimulation, immunosuppression, and autoimmunity. However, immunostimulation and immunosuppression tests should be restricted in general immunotoxic studies, except for those on materials that have been shown to possibly induce immunostimulation and immunosuppression. Bovine collagen is proven nontoxic and can be well tolerated in animal models as well as in the clinic. Therefore, we mainly evaluated the systematic immune response, local inflammation, hypersensitivity, and dose response. Evaluation indexes in this study were set according to the instructions in ISO 10993-20: 2005 and previous literature, though the evaluation scheme also has some weaknesses, such as insufficient functional tests, control selection, and lack of complement detection.
When an antigen enters the body, T lymphocytes and B lymphocytes may be stimulated and start to proliferate rapidly, strongly initiating the acquired immune response. The spleen and lymph nodes are the main locations where lymphocytes settle, as well as the primary regions of the immune response. In accordance with the instructions of ISO 10993-20: 2005, lymphoid organs were chosen to assess the immune response. In this study, the spleen and lymph nodes, as systematic immune response indexes, were studied according to size, cell population, cell phenotype, and activation level to evaluate the immunotoxicology of this collagen-based DM. The cell population of the DM-H group slightly increased in the early time phase (Figure 1C, D). In addition, other two tests, lymphocyte proliferation in vitro following stimulation with PMA and Iono and Ki67 antigen expression in cells, can also reflect the lymphocyte activation level. The former is a functional test, and the latter is not a functional test. A difference in proliferation in vitro between the DM-H group and NC group was also discovered, as indicated by the proportion of splenic lymphocytes in prophase after immunizations (Figure 5). The cell phenotype of the spleen and lymph nodes indicated a weak but evident alteration on day 7 in the DM-H group. These results suggest that DM-H produced some level of immune response in prophase, however over time, the collagen triple-helical structure was destroyed, collagen degraded into small pieces, and immunogenicity declined; thus, the impact on the immune system disappeared over time. However, DM-L did not trigger a noticeable immunological reaction even in the first days after implantation. Though CD11b staining was normal in the present study, it is possible that an innate immune response occurred during the process of the three implantations because our first sampling time was 3 weeks following the first implantation.
Immunoglobulins are important effectors mediating humoral immunity, and they are secreted by plasma cells. BALB/c mice are normally used for generating antibodies against globular proteins, and they have been shown to be responsive to collagen. In one study, bovine collagen was found to be nonimmunogenic in the most responsive SJL/J mice, as the total Ig response was minimal. Fish collagen was also tested in BALB/c mice, demonstrating that IgG levels in serum were also marginal on 42 days after three immunizations. In the current study, the total IgG and IgM secretion of BALB/c mice response to bovine collagen DM on 3, 7, 14, 30, 60, and 90 days post-immunization was analyzed by ELISA (Figure 6). IgM and IgG concentrations in mice implanted with DM were comparable to those in normal mice with no implant and much lower than those in mice implanted with bovine tendon, in accordance with previous studies. The similarity in the immunoglobulin concentration between the DM groups and NC group illustrates that a weak humoral immune response was evoked by DM. The fluctuations in the curves can be regarded as experimental errors and individual differences in the mice.
When the body is stimulated by a foreign antigen, chemotactic factors, produced by dendritic cells, will direct inflammatory cells, mainly including neutrophils, lymphocytes, and monocytes, to exit from the bloodstream to the sites of the antigen. Generally, neutrophils play a critical role in the early phase of inflammation, and lymphocytes and monocytes are the major inflammatory cells in the middle and late phases. Inflammatory cytokines, such as TNF-α and IFN-γ, primarily produced by these inflammatory cells are also secreted to regulate inflammation and the immune response. To investigate the local DTH around the implants in the present study, H & E and immunohistochemistry staining of local sites were performed. The initiation of a low level of inflammatory cell infiltration by collagen has been reported in previous studies[36-37]. Here, H & E staining revealed noticeable cell infiltration in the DM groups in the early period (Supplementary Figure S3). Noticeable cell infiltration around DM was present in the first 30 days, and then, cell aggregation subsided with the degradation of collagen, indicating that DM aroused mild acute inflammation. Collagen degradation results in a decrease in antigens, therefore inflammatory cell infiltration decreased from a large area to small dots. Regarding cytokine immunostaining, obvious TNF-αpositive expression areas were observed in local DM implantation sites in the early phase. TNF-α has been reported to promote the immune response and inflammation, and it plays a critical role in inflammation in the skin. However, no obvious IFN-γ staining area was observed from day 7 to day 90 in the DM groups (Supplementary Figure S4). IFN-γ is secreted by Th1 cells and NK cells, and its primary function is to promote the differentiation of Th1 cells, strengthen phagocytosis of phagocytes, and attract macrophages to sites where antigens are present. There was no obvious IFN-γ staining, which may reflect the poor sensitivity of the index, whereas TNF-α was visible in prophase, thus we propose that collagen-based DM induces a benign immune response only in the early period, as its degradation results in a weaker immunological reaction and less TNF-α secretion.
In summary, in the DM-H group, the cell population of the spleen and lymph nodes increased at the early time point, but did not increase in the DM-L group. As shown in Figures 2 and 3, the increase in the whole cell population in the DM-H group was primarily caused by growing T and B lymphocytes on day 7. The Ki67 results further demonstrate that the cell population increase was caused by CD4 T lymphocyte and B lymphocyte activation. A similar conclusion could be drawn from the results of the cell proliferation assay, that is the DM-H group had significantly more cell proliferation in prophase, but the DM-L group did not. Though B lymphocytes were likely slightly activated and proliferated in the above-mentioned tests by DM-H stimulation, no clear increases in immunoglobulins were detected in this study; thus, we conclude that this discrepancy is due to the quite low immunogenicity of the collagen-based DM and some experimental errors. Local cell infiltration and TNF-α secretion indicate that benign inflammation was evoked by DM implantation, however it was temporary. Overall, DM, especially DM-H, may have produced a slight immune response and acute inflammation in the early phase post-implantation, which then subsided gradually with the degradation of collagen. Moreover, the dose reaction evaluation proved that the higher dose induced a more intense immune response. Therefore, the collagen-based DM can be preliminarily regarded as a reliable and safe dermal regeneration matrix with favorable biocompatibility and low immunogenicity, however dose control should not be ignored. Nevertheless, further trials must be conducted in other models and in the clinic to validate its safety. Although the research scheme proposed in this study was based on ISO 10993, there were some shortcomings, and additional studies are needed to formulate a more scientific and meaningful scheme in the future.