Histological integration of allogeneic cancellous bone tissue treated by supercritical CO2 implanted in sheep bones

Abstract

Different chemical or physical methods of bone processing have been developed to decrease the antigenicity of allogeneic bone which may delay or prevent graft integration. We have developed a method based on delipidation and deproteination of the bone with a supercritical fluid and hydrogen peroxide. Cylinders of cancellous allogeneic bone treated in this way were implanted for four weeks, four months or eight months in holes drilled in sheep condyles or tibial plateau. Histological sections were then processed and analysed qualitatively and quantitatively using an image analysis software coupled to a light microscope. Measurements were made of the trabecular bone surface (BS/TS), the relative osteoid surface (OS/BS), the active osteoid surface (OS/BS), active resorption surface (Oc.S/BS) and the relative surface of newly formed bone. After four weeks, the control cylinders (non-treated allogeneic bone) had been invaded by cellular tissue composed of lymphocytes and plasmocytes surrounding remnants of the donor bone marrow tissue. The processed cylinders showed osteoid apposition at the surface of their external trabeculae. The trabecular bone and osteoid surfaces were significantly higher in the processed bone sections than in the control bone sections. After four months, most of the control material had been osteolysed and replaced by connective tissue containing lymphocyte islets, while the processed materials showed a large amount of bone synthesized at the surface of implant trabeculae which appeared fragmented and disseminated within the newly formed bone. All the histomorphometric parameters measured were significantly different from those of the control.By eight months, most of the control material had been totally osteolysed with very little bone ingrown in the implantation site. Only one control implant had been integrated. The processed cylinders were difficult to discern from the bone in which they were implanted. The parameters measured on the processed cylinders were significantly higher than those measured on the control sections.In conclusion: the treatment applied to the bone enhanced allogeneic bone integration and could provide a new kind of tissue treatment for bone banking.

The influence of processes for the purification of human bone allografts on the matrix surface and cytocompatibility

“…2. process etOH: dehydration for 3 h in 95% ethanol (etOH) and 2 h in pure etOH. This dehydration method is obligatory and served as control for the other processes and is part of almost all reported process [10,16,20,21]. 3. process NaHCO 3 : soaking in NaHCO 3 (5% in distilled water) at 50 1C for 24 h (Sigma-Aldrich) [10].…”

“…After three washes in distilled water, slices or cubes were extensively defatted in two baths of pure acetone (8 h) followed by two baths of 401 etOH (2 h each). 4. process H 2 O 2 : soaking in a 35% H 2 O 2 solution (Acros Organics, New Jersey, USA) at 40 1C for 2 h with sonication as reported elsewhere [16] and dehydration as described above in process etOH. This group evaluated the specific effects of H 2 O 2 on bone.…”

“…That remodeling and osseointegration of allografts treated with strong oxidizing reagents could be compromise is questionable. Frayssinet et al [16] have found that cancellous bone tissue defatted with supercritical CO 2 and treated with H 2 O 2 +NaOH presented a better integration at the histological level than non-defatted, non-processed allografts in sheep bones after 8 month. However, their study did not include a defatted non-processed group.…”

“…Viral and prion inactivation has been recommended by immersion in 1 N NaOH [14,15]. A study combining these different steps on sheep allogenic cancellous bone has reported satisfactory results in vivo [16]. Another process combining osmolysis, H 2 O 2 , NaOH and acetone is used to prepare the patented bone graft Tutoplast s (Tutogen medicals GmbH, Erlangen, Germany) [17].…”

Recommendations and Considerations for the Use of Biologics in Orthopedic Surgery

“…The biocompatibility of apatitic CaP cements is excellent, and their biodegradation is much larger than that of hydroxyapatite, although incomplete and slow. [52] …”