This study evaluated the tissue reaction to porous hydroxyapatite (HA) granules in a critical sized tibial-defect of New Zealand white rabbits for a period of 2, 6, 12 and 24 weeks. Physicochemical characterizations of the granules were done using transmission electron microscopy, scanning electron microscopy, X-ray diffraction, and fourier transform infrared spectroscopy to analyse the microstrucutre, composition, phase purity crystallinity and functional groups of HA. Prior to in vivo testing, the HA granules had proved to be biocompatible and cytocompatible en route in vitro studies using L929 mouse fibroblast cells. In the histologic evaluation, as early as 2 weeks, bone ingrowth was observed in the pores and interstices of the granules forming a network of bony trabeculae and over 6, 12 and 24 weeks, it was seen that the granules assisted in bone formation. Fluorochrome multilabels of yellow, red and orange lines showed active sites of bone mineralization in progress in the pores and periphery of the granules. Good osteointegration of the granules with the host bone was observed. There was neither inflammation nor fibreous tissue interposition while resorption of the material was in effect a slow process, since the HA granules still persisted after 24 weeks.

This study investigates quantitatively and qualitatively the sol–gel derived bioactive glass–ceramic system (BGS)-apatite–wollastonite (AW) type granules in the size range of 0.5–1 mm, as an effective graft material for bone augmentation and restoration. Scanning electron micrographs (SEM) of the sintered granules revealed the rough material surface with micropores in the range 10–30 𝜇m. X-ray diffraction (XRD) pattern of the granules revealed the presence of crystalline phases of the hydroxyapatite and wollastonite, and the functional groups of the silicate and phosphates were identified by Fourier transform infrared spectroscopy (FT-IR). The in vitro cell culture studies with L929 mouse fibroblast cell line showed very few cells adhered on the BGS disc after 24 h. This could be due to the highly reactive surface of the disc concomitant with the crystallization but not due to the cytotoxicity of the material, since the cellular viability (MTT assay) with the material was 80%. Cytotoxicity and cytocompatibility studies proved that the material was non-toxic and biocompatible. After 12 weeks of implantation of the BGS granules in the tibia bone of New Zealand white rabbits, the granules were found to be well osteointegrated, as observed in the radiographs. Angiogram with barium sulphate and Indian ink after 12 weeks showed the presence of microcapillaries in the vicinity of the implant site implicating high vascularity. Gross observation of the implant site did not show any inflammation or necrosis. SEM of the implanted site after 24 weeks revealed good osteointegration of the material with the newly formed bone and host bone. New bone was also observed within the material, which was degrading. Histological evaluation of the bone healing with the BGS granules in the tibial defect at all time intervals was without inflammation or fibrous tissue encapsulation. After 2 weeks the new bone was observed as a trabeculae network around the granules, and by 6 weeks the defect was completely closed with immature woven bone. By 12 weeks mature woven bone was observed, and new immature woven bone was seen within the cracks of the granules. After 24 weeks the defect was completely healed with lamellar bone and the size of the granules decreased. Histomorphometrically the area percentage of new bone formed was 67.77% after 12 weeks and 63.37% after 24 weeks. Less bone formation after 24 weeks was due to an increased implant surface area contributed by the material degradation and active bone remodeling. The osteostimulative and osteoconductive potential of the BGS granules was established by tetracycline labelling of the mineralizing areas by 2 and 6 weeks. This sol–gel derived BGS granules proved to be bioactive and resorbable which in turn encouraged active bone formation.

Tissue-engineered bone regeneration has attracted much attention because of its high clinical demand for restoration of injured tissues. In the present study, we have evaluated the capability of bare (without cells) and tissue-engineered (with osteogenic-induced rat Mesenchymal Stem Cells (MSCs)) bioactive ceramics such as hydroxyapatite (HA) and triphasic ceramic-coated hydroxyapatite (HASi) to mediate vascularisation and osteoinduction at an extraskeletal site of rat model. The viability, proliferation and osteogenic differentiation of MSCs on the scaffolds were assessed in vitro and thereby established the capability of HASi in providing a better structural habitat than HA. The vascular invasion was relatively low in bare and tissueengineered HA at 2 and 4 weeks. Interestingly, the implantation site was well vascularised with profuse ingrowth of blood capillaries in HASi groups, with preference for tissue-engineered HASi groups. Similarly, neo-osteogenesis studies were shown only by tissue-engineered HASi groups. The ingrowth of numerous osteoblast-like cells was seen around and within the pores of the material in bare HASi and tissue-engineered HASi groups (very low cellular infiltration in bare HA groups), but there was no osteoid deposition. The positive impact in forming bone in tissue-engineered HASi groups is attributable to the scaffold and to the cells, with the first choice for scaffold because both HA and HASi were engineered simultaneously with the cells from same source and same passage. Thus, highly porous interconnected porous structure and appropriate chemistry provided by HASi in combination with osteogenic-induced MSCs facilitated better vascularisation that lead to neo-osteogenesis.