Induced Microcracking Affects Osteoblast Attachment on Hydroxyapatite Scaffolds for Tissue Engineering

Abstract

Bone microdamage caused by routine activity plays an important role in triggering targeted and nontargeted bone remodeling. Targeted remodeling occurs near localized areas of microdamage^[1-4]. We hypothesize that bone remodeling may be directly and positively influenced by inducing microcracks in hydroxyapatite (HA) scaffolds for bone tissue engineering. A study by Case et al. showed microcracking in HA discs having >98% theoretical density^[5]. These microcracks occurred without the application of external stress, likely as a result of thermal expansion anisotropy (TEA). TEA generates microcracks only when the specimen grain size exceeds a critical value (G_CR). Due to conflicting data in the literature on the thermal expansion along the crystallographic axes of HA, it is difficult to estimate G_CR precisely for HA, but G_CR likely ranges from a few tenths of microns to several microns^[6]. As the grain size of a polycrystalline specimen increases above the critical grain size, the number of microcracks also increases but the number density of microcracks is difficult to control. Therefore, in this study we used Vickers microindentation to induce controlled microcracks in >99% dense HA discs. The effect of microcracking on the osteoblast (OB) attachment was then quantified. Control HA discs and microcracked HA were seeded with MC3T3-E1 OBs and cultured for 4 hours. The OBs were then stained with Rhodamine-Phalloidin and Hoechst fluorescent dyes to identify the actin fibers and nuclei, respectively. OB attachment was quantified using fluorescent light microscopy. OB attachment at 4 hours was 29% ± 7.6% of the initial seeded OBs for the microcracked HA specimens and 18% ± 6.1% of initial seeded OBs for the non-microcracked control HA specimens. The difference in these OB attachment values was statistically significant as determined via the Student t-test (p = 0.004, with p < 0.05 taken to indicate significance).