Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria1, which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria. HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month. H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%. The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes.
The purpose of this study is to enhance massive bone allografts osseointegration used to reconstruct large bone defects. These allografts show >50% complication rate requiring surgical revision in 20% cases. A new protocol for total bone decellularisation exploiting the vasculature can offer a reduction of postoperative complication by annihilating immune response and improving cellular colonization/ osseointegration. The nutrient artery of 18 porcine bones - humerus/femur/radius/ulna - was cannulated. The decellularization process involved immersion and sequential perfusion with specific solvents over a course of one week. Perfusion was realized by a peristaltic pump (mean flow rate: 6ml/min). The benefit of arterial perfusion was compared to a control group kept in immersion baths without perfusion. Bone samples were processed for histology (HE, Masson's trichrome and DAPI for cell detection), immunohistochemistry (IHC : Collagen IV/elastin for intraosseous vascular system evaluation, Swine Leukocyte Antigen – SLA for immunogenicity in addition to cellular clearance) and DNA quantification. Sterility and solvent residues in the graft were also evaluated with thioglycolate test and pH test respectively. Compared to native bones, no cells could be detected and residual DNA was <50ng/mg dry weight. Intramedullary spaces were completely cleaned. IHC showed the preservation of intracortical vasculature with channels bounded by Collagen IV and elastin within Haversian systems. IHC also showed a significant decrease in SLA signaling. All grafts were sterile at the last decellularization step and showed no solvent residue. The control group kept in immersion baths, paired with 6 perfused radii/ulnae, showed that the perfusion is mandatory to ensure complete decellularisation. Our results prove the effectiveness of a new concept of total bone decellularisation by perfusion. These promising results could lead to a new technique of Vascularized Composite Allograft transposable to pre-clinical and clinical models.
