Study of Cell Viability and Etiology of Contamination in Decalcified Bone Allograft: A Pilot Study

Bone allografts can elicit immune responses which is correlated with the presence of Human Leukocyte Antigen (HLA) and cellular DNA. It also has risk of causing occult infection arising out of contamination during its processing and storage. The presence of immunogenic materials like cells, cellular remnants and DNA in a decalcified bone allograft during different phases of processing has never been studied. Present study was conducted to explore- the cell viability using routine Hematoxylin and Eosin, presence of DNA using Feulgen staining and etiology of contamination in decalcified bone allograft during procurement, demineralization and ethanol preservation.

The harvested bones from patients undergoing hemireplacement/THR/TKR were processed to prepare decalcified bone allografts. The samples during procurement (A), HCL treatment (B) and ethanol preservation (C) were sent for histopathological analysis (number of osteocytes in the maximum density field under 40x and the cells demonstrating presence of DNA on feulgen stain) and microbiological assessment (aerobic/anaerobic/fungal cultures).

Histopathological study demonstrated the presence of osteocytes and other cells like bone marrow, adipocytes, endothelial cells in the decal bone allograft. The average number of osteocytes gradually decreased from 55.47, 9.6, 0.86 in sample A, B, C, respectively. Feulgen staining confirmed the presence of DNA in osteocytes and other cells which decreased both qualitatively and quantitatively in subsequent stages of processing. Rate of contamination demonstrated at the procurement was 6.67% (Staphylococcus aureus). After treatment with HCl (demineralisation), 7.14% of non-contaminated allografts were found contaminated (Staphylococcus epidermidis). None of the remaining 13 non-contaminated allografts showed contamination after storage in ethanol. Overall 13% of the patients had positive cultures on microbiological assessment.

The population of osteocytes in the harvested bone reduced significantly after processing with HCl and ethanol preservation. Presence of DNA, demonstrated by using Feulgen staining, was observed in bone marrow cells, adipocytes along with osteocytes which showed quantitative reduction on processing. Hence, antigenicity, conferred by cells and their DNA, reduced significantly after processing of decal bone. Contamination rate of banked decalcified allograft was 13%. Thus, culture and sensitivity tests should be carried out at each step of processing of decal bone allograft.