Rapid quantitative bioassay of osteoinduction

Abstract

We developed a reproducible, relatively rapid bioassay that quantitatively correlates with the osteoinductive capacity of demineralized bone matrix obtained from human long bones. We have found that Saos human osteosarcoma cells proliferate in response to incubation with demineralized bone matrix and that an index of this proliferative activity correlates with demineralized bone matrix-induced osteogenesis in vivo. The bioassay (Saos cell proliferation) had an interassay coefficient of variation of 23 +/- 2% and an intra-assay coefficient of 11 +/- 1%. Cell proliferation was normalized to a standard sample of demineralized bone matrix with a clinically high osteoinductive capacity, which was assigned a value of one. The Saos cell proliferation for each sample was related to the standard and assigned a value placing it into the low (0.00-0.39), intermediate (0.40-0.69), or high (0.70-1.49) osteoinductive index group. Osteoinduction of human demineralized bone matrix was quantitated by expressing new bone formation as a function of the total bone volume (new bone plus the demineralized bone powder). The demineralized bone matrix was placed in pouches formed in the rectus abdominis muscles of athymic rats, and endochondral bone formation was assessed at 35 days following implantation, when marrow spaces in the ossicles were formed by new bone bridging the spaces between demineralized bone matrix particles. The proliferative index correlated with the area of new bone formation in histological sections of the newly formed ossicles. When the proliferative index (the osteoinductive index) was divided into low, intermediate, and high groups, the correlation between it and new bone formation (osteoinduction) was 0.850 (p < 0.0005) in 25 samples of demineralized bone matrix. There was no overlap in the osteoinduction stimulated between the samples with low and high osteoinductive indices. We conclude that the proliferation assay is useful for the routine screening of bone allograft donors for osteoinductive potential. Furthermore, the two-dimensional area of new bone formation, as it relates to total new bone area, is a quantitative measure of osteoinduction.

BMP depletion occurs during prolonged acid demineralization of bone: characterization and implications for graft preparation

“…Finally, it is possible that prolonged exposure of BMP-7 to HCl could have degraded a portion of it. However, 0.5 N and 0.6 N HCl have been the preferred demineralization medium for over 40 years due to the ability to remove bone mineral efficiently while leaving the graft osteoinductive (Adkisson et al 2000;Gendler 1986;Harakas 1984;Jazayeri et al 1994;Nilsson et al 1990;Pietrzak 2006;Reddi and Huggins 1972;Shigeyama et al 1995;Urist 1965;Yazdi et al 1991;Zhang et al 1997). Demineralization cycles as long as several days have been used.…”

“…As such, the demineralization process should maximize BMP retention by the graft. Although most protocols utilize HCl (0.5-0.6 N) for demineralization, little standardization exists (Adkisson et al 2000;Gendler 1986;Harakas 1984;Jazayeri et al 1994;Mizutani and Urist 1982;Nilsson et al 1990;Pietrzak 2006; Reddi and Huggins 1972;Shigeyama et al 1995;Swenson and Arnoczky 2003;Urist 1965;Urist et al 1968;Yazdi et al 1991;Zhang et al 1997). For instance, HCl exposures of 30 min to 30 days have been reported but there are no studies of time, as an independent variable, on retained BMP levels.…”

Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms

“…Adkisson et al developed a "rapid quantitative bioassay of osteoinduction" by using SaOS-2 osteosarcoma cells and studied cell proliferation rates under the infl uence of DBM. However, correlation between cell proliferation and osteoinduction was not strong (Adkisson et al, 2000). Zhang et al and Wolfi nbarger and Zheng used human periosteal cells and human dermal fibroblasts to relate cellular ALP activity to DBM osteoinductivity (Wolfi nbarger and Zheng, 1993;Zhang et al, 1997).…”

A review of osteoinductive testing methods and sterilization processes for demineralized bone

“…Interassay and intra-assay variations are a crucial concern with such heterogeneous preparations. Using 25 samples of human DBP, Adkisson et al (2000) reported that DBP stimulated DNA synthesis by human SAOS osteosarcoma cells in serum-free medium, and that the magnitude of stimulation was positively correlated (r 2 = 0.72) to the histomorphometric measure of bone area induced in athymic rats. It seems curious to identify the magnitude of stimulation of DNA synthesis as the 'osteoinductive index' when serum would have a similar effect.…”