Immunogenicity of unprocessed and photooxidized bovine and human osteochondral grafts in collagen-sensitive mice.

BACKGROUND: Autologous and allogeneic osteochondral grafts have been used to repair damaged or diseased cartilage. There are drawbacks to both of these methods, however. Another possible source for osteochondral grafting is photooxidized xenograft scaffolds. The purpose of this study was to evaluate the adaptive immune response to unprocessed and photooxidized xenogeneic osteochondral grafts in a collagen-sensitive mouse model. METHODS: Unprocessed and photooxidized bovine and human osteochondral grafts were used. The grafts were implanted subcutaneously in collagen-sensitive DBA/1LacJ mice for four or twelve weeks. ELISPOT assays were conducted with spleen cells to evaluate the number of collagen-specific T cells that produce IL-2, IL-4, IL-5 or IFN-gamma. Serum was collected and ELISA assays were performed to determine the titers of collagen-specific and total IgG, IgG1, IgG2a, or IgM antibodies. Histology was conducted on the retrieved osteochondral grafts. RESULTS: Results indicated that, with respect to adaptive T cell immunity, the photooxidized bovine grafts, unprocessed human grafts and photooxidized human grafts did not induce a significant response to collagen. The unprocessed bovine grafts, however, were slightly more immunogenic, inducing a weak immune response. With respect to antibody production, the bovine grafts were less immunogenic than the human grafts. Bovine collagen-specific IgG antibodies were not induced by these grafts, but production of IgM after twelve weeks was observed with both the unprocessed and photooxidized bovine grafts. In contrast, photooxidized human osteochondral grafts induced IgG1 and IgG2a antibodies, while the unprocessed human grafts did not. Pre-existing human collagen-specific IgM antibodies were present in all mice, including sham-operated negative controls that did not receive an implant. Histological analysis revealed some degree of fibrous encapsulation and inflammatory infiltrations in both bovine and human implants, whether unprocessed or photooxidized. CONCLUSION: Both bovine and human cartilage grafts showed weak, but clear immunogenicity in the DBA/1LacJ mice, indicating that immunogenic collagen was still contained in the grafts, even after cleaning and photooxidation. The process of photooxidation is still important in osteochondral grafting, since it stabilizes the surface of the cartilage by cross-linking the collagen fibers, and allows for immediate load bearing and joint resurfacing.

Immunologic testing of xeno-derived osteochondral grafts using peripheral blood mononuclear cells from healthy human donors.

BACKGROUND: One means of treating osteoarthritis is with autologous or allogeneic osteochondral grafts. The purpose of this study was to evaluate the innate immunological response in humans toward xeno-derived osteochondral grafts that have been partially or entirely treated by the photooxidation process. METHODS: The antigens tested included bovine, porcine, ovine and equine osteochondral samples that have been treated in successive steps of photooxidation. ELISPOT assays were used to evaluate the production of IL-1, IL-4, IL-6, IL-10, IL-12 and TNF-alpha by human monocytes in response to the antigens. RESULTS: Results indicated vigorous production of IL-1, IL-6, IL-10 and TNF-alpha in response to untreated bovine, porcine and equine specimens. This indicates that these samples are perceived as foreign, or stimulatory, by the human monocytes. There was no induction of IL-4 or IL-12, which is required for Th2 and Th1 immunity, respectively. In contrast, the processed bovine, porcine and equine samples did not induce significant activation of cells of the innate immune system. This occurred after the first step in processing (after cleaning in increasing strengths of ethanol). This suggests that the processing steps dramatically, if not completely, negated the immunostimulatory properties of the test sample. The results for the ovine samples indicate a reverse response. CONCLUSION: The findings of the study suggest that photooxidized bovine, porcine or equine samples have the potential to be used as an osteochondral graft. Although the first step in processing reduced the immunological response, photooxidation is still necessary to retain the structure and mechanical integrity of the cartilage, which would allow for immediate joint resurfacing.

Effect of a diode laser on wound healing by using diabetic and nondiabetic mice.

The purpose of this study was to evaluate a 980-nm gallium-aluminum-arsenide diode laser for wound healing. Using genetically diabetic and nondiabetic mice, two 6-mm wounds were created on the back of each mouse by using a punch biopsy. The mice were assigned to 1 of 4 subgroups for laser treatment at different fluence and frequency of treatment: 5 W (18 J/cm2) every 2 days, 5 W (18 J/cm2) every 4 days, 10 W (36 J/cm2) every 2 days, and 10 W (36 J/cm2) every 4 days. In addition, control mice were used and the wounds were allowed to heal naturally. Wound healing was evaluated on days 5, 12, and 19 by percentage of wounds healed and percent wound closure. A maximum of 5 mice per subgroup were killed at days 7, 14, and 21, and histology was conducted on the wound sites. For diabetic mice receiving 5 W every 2 days, the percentage of wounds healed after 19 days was 100% versus 40% in the control group. Only 20% of wounds in the 10-W diabetic subgroups achieved healing during the same period. For the subgroups whose wounds did not completely heal, all but the 10 W every 2 days subgroup had average closure of >90%. The 100% closure for the 5 W every 2 days subgroup was significantly greater than the other subgroups. For nondiabetic mice, 100% of the wounds in the 5 W every 4 days and control subgroups were completely healed, whereas 90% of the wounds from the 5 W every 2 days and the 10 W every 4 days subgroups were completely healed. In the latter 2 subgroups, wound closure was 99.4% and 98.8%, respectively. These differences were not significant. The histologic results confirmed these findings. In conclusion, treatment at 18 J/cm2 shows a beneficial effect on wound healing in diabetic mice and does not have a detrimental effect in nondiabetic mice.