Various evaluation techniques of newly formed bone in porous hydroxyapatite loaded with human bone marrow cells implanted in an extra-osseous site

The purpose of this work was to develop qualitative methods for in situ ana lysis of bone formation in an osteoconductive hydroxyapatite matrix (ENDOBO N(R)), loaded with human bone marrow cells (HBMSC) implanted subcutaneously in athymic mice. Samples were taken before implantation (T0), 1, 2, 4 and 6 weeks after implantation. Bone-biomaterial interaction were investigated on undecalcified sections by histological, cytochemical, immunological and molecular biology methodologies. Histological observations were performed i n order to observe inflammatory cells, vessels, newly formed bone, woven an d lamellar bone. Enzymohistochemistry was carried out to detect positive ta rtrate resistant acid phosphatase activity (TRAP+). Immunohistochemistry us ing antibodies against type I collagen and osteocalcin permitted us to char acterize the content of the matrix elaborated within the implant. Moreover, in situ hybridization was carried out to discriminate, the implanted human cells from the murine cells, and to evaluate the function of these human c ells in osteogenesis. Results demonstrated an early formation of lamellar b one only in the pores of the studied HAP loaded with HBMSC. This bone conta ined a matrix showing positive reaction for type I collagen and osteocalcin . In situ hybridization identified some of these cells as human cells. At 6 weeks, examination of histological results showed persistance of lamellar bone in the implants. We only found TRAP+ activity in the materials loaded with human bone marrow cells. Molecular hybridization no longer revealed po sitive cells for the human DNA probe. All these results indicate that the v arious evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice. (C) 2000 Elsevier Science Ltd. All rights reserved.