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.

Purpose: Only very partial integration of massive allografts is generally achieved, affecting bone-graft junctions and the peripheral cortical. In clinical practice, this is not a major problem for massive reconstructions with a sleeve prosthesis but can be a handicap for junctional grafts or osteoarticular grafts where weak recolonisation can be a source of complications.

Material and methods: Extraperiosteal resection measuring 5 cm in length was made in the mid shaft region and bridged by a cyropreserved non-irradiated allograft before stabilisation with a static locked nail. Three groups of ten sheep were studied. The first group received a simple allograft without perforation; the allograft was perforated in the second group (1.1 mm drill bit); and the perforations in the allograft in the third group were lined with decalcified bone powder with assumed potential for inducing bone growth. The implantation was studied after a delay of six months. There were three infections so the analysis was made on 27 grafts. Plain x-rays (consolidation of the graft-bone junctions), histomorphometrics (porosity, new peripheral and endomedullary bone deposit, cortical thickness), and bone density were studied.

Results: Rate of bone-graft consolidation was not significantly different in the three groups. The callus was more endosteal in groups 2 and 3 (p< 0.02) and endomedullary bone deposit was greater (p=0.0001) than in group 1 without perforation. There was approximately three times more bone deposit in the perforated allografts than in the non-perforated allografts; Adjunction of demineralised bone around the perforated grafts did not lead to any significant difference compared with the perforated allografts (group 2).

Discussion: Significantly more bone deposit observed with perforated allografts should lead to better biomechanical behaviour. This is being tested in further work.

Conclusion: Perforations induce a significant increase in new bone deposit in massive cortical allografts, remodelling is much more active and extensive than with non-perforated allografts. It would be logical to propose perforated allografts for junctional or osteochondral massive cortical grafts.