Divergent Resorbability and Effects on Osteoclast Formation of Commonly Used Bone Substitutes in a Human In Vitro-Assay

Abstract

Bioactive bone substitute materials are a valuable alternative to autologous bone transplantations in the repair of skeletal defects. However, clinical studies have reported varying success rates for many commonly used biomaterials. While osteoblasts have traditionally been regarded as key players mediating osseointegration, increasing evidence suggests that bone-resorbing osteoclasts are of crucial importance for the longevity of applied biomaterials. As no standardized data on the resorbability of biomaterials exists, we applied an in vitro-assay to compare ten commonly used bone substitutes. Human peripheral blood mononuclear cells (PBMCs) were differentiated into osteoclasts in the co-presence of dentin chips and biomaterials or dentin alone (control) for a period of 28 days. Osteoclast maturation was monitored on day 0 and 14 by light microscopy, and material-dependent changes in extracellular pH were assessed twice weekly. Mature osteoclasts were quantified using TRAP stainings on day 28 and their resorptive activity was determined on dentin (toluidin blue staining) and biomaterials (scanning electron microscopy, SEM). The analyzed biomaterials caused specific changes in the pH, which were correlated with osteoclast multinuclearity (r = 0.942; p = 0.034) and activity on biomaterials (r = 0.594; p = 0.041). Perossal led to a significant reduction of pH, nuclei per osteoclast and dentin resorption, whereas Tutogen bovine and Tutobone human strikingly increased all three parameters. Furthermore, natural biomaterials were resorbed more rapidly than synthetic biomaterials leading to differential relative resorption coefficients, which indicate whether bone substitutes lead to a balanced resorption or preferential resorption of either the biomaterial or the surrounding bone. Taken together, this study for the first time compares the effects of widely used biomaterials on osteoclast formation and resorbability in an unbiased approach that may now aid in improving the preclinical evaluation of bone substitute materials.

In vitro effects of particulate bone substitute materials on the resorption activity of human osteoclasts

“…Published work mostly reveals work done by direct cultivation of cells on plates or discs made out of biomaterial with dentin chips serving as control group. The possible comparison of our findings with those of others is limited, but a favorable role of HA can also be deduced (Keller et al, 2012;Schilling et al, 2004;Zhang et al, 2012).…”

“…This indicated a negative impact of BM4 (Cerasorb) on cell differentiation under these conditions. Keller et al (2012) report cultivating human osteoclasts on 10 different biomaterials, using dentin as the control group. In their investigations BM4 (Cerasorb) leads to increased cell adherence, progress of differentiation until day 14, larger numbers of TRAP-positive multinuclear cells on day 28, and positive effects on resorption.…”

“…Keller et al cultivated human osteoclasts on 10 different biomaterials and report that HA-based biomaterials (Tutogen bovin and Tutogen human) increase nuclei count and resorption, but they find no increase formation of mature osteoclasts (Keller et al, 2012). Detsch et al cultured osteoclast-like-cells deriving from a human monocytic leukemia cell line on HA and TCP ceramics.…”

“…Standardized testing of biomaterial degradation patterns is carried out at extreme pH levels (pH 3). However, promising degradation properties retrieved from such tests may emerge as less favorable when analyzed under in vivo conditions (Keller et al, 2012;Zhang et al, 2012). It therefore seems evident that in vivo degradation depends on more factors than merely on solubility.…”

Advanced in vitro systems for efficacy and toxicity testing in nanomedicine

“…Some laboratories opt for scaffolds made in-house, but many groups rely on commercial scaffolds created by standardised procedures and quality-controlled manufacture. There is no accepted standard for NP evaluation (139) and, not unreasonably, pragmatic decisions are made on the basis of convenience and desired functionality (140)(141)(142)(143)(144)(145)(146)(147)(148)(149)(150)(151). For example, Hanada and colleague used an in-vitro rat BBB model (RBT-24H, BBB Kit ™ ; PharmaCo-Cell Company Ltd., Japan) to test the ability of fluorescent silica NPs (30, 100, and 400 nm) to cross the BBB (152).…”