Observations on the microvasculature of bone defects filled with biodegradable nanoparticulate hydroxyapatite.

The microvascularization of metaphyseal bone defects filled with nanoparticulate, biodegradable hydroxyapatite biomaterial with and without platelet factors enrichment was investigated in a minipig model. Results from morphological analysis and PECAM-1 immunohistochemistry showed the formation of new blood vessels into the bone defects by sprouting and intussusception of pre-existing ones. However, no significant differences were observed in the microvascularization of the different biomaterials applied (pure versus platelet factors-enriched hydroxyapatite), concerning the number of vessels and their morphological structure at day 20 after operation. The appearance of VEGFR-2 positive endothelial progenitor cells in the connective tissue between hydroxyapatite particles was also found to be independent from platelet factors enrichment of the hydroxyapatite bone substitute. In both groups formation of lymphatic vessels was detected with a podoplanin antibody. No differences were noted between HA/PLF- and HA/PLF+ implants with respect to the podoplanin expression level, the staining pattern or number of lymphatic vessels. In conclusion, the present study demonstrates different mechanisms of blood and lymphatic vessel formation in hydroxyapatite implants in minipigs.

Effects of platelet factors on biodegradation and osteogenesis in metaphyseal defects filled with nanoparticular hydroxyapatite--an experimental study in minipigs.

There are no studies on the cellular activity in the early phase of biodegradation and bone healing of bone substitutes loaded with platelet factors (PLF). The purpose of this study was to evaluate the cellular effects of PLF in combination with nanoparticulate hydroxyapatite (HA) on the biodegradation and bone formation after 20 days. Autogenous PLFs were obtained by centrifugation of miniature pig blood samples and subsequent degranulation of platelets by calcium and thrombin. A cylindrical bone defect with a diameter of 8.9 mm was created in the distal femoral condyle of 20 miniature pigs. Four of the defects were left empty, 8 were filled with HA with loading and 8 with HA loaded with PLF. The distal femur was harvested after 20 days and TRAP-staining, cathepsin-K and CD44 staining and scanning electron microscopy were performed for cellular assessment of biodegradation was done. Histomorphometry of new bone formation and of biodegradation of HA material was performed. PLF loading of HA led to statistically significant more TRAP-positive cells with enhanced biodegradation of the nanoparticulate HA but no statistically enhanced new bone formation compared to unloaded HA. Furthermore, there was a higher number of CD44 and cathepsin-K positive cells by PLF-loading. In summary, PLF led to stimulation of the cellular process of the biodegradation of HA.

New blood vessel formation and expression of VEGF receptors after implantation of platelet growth factor-enriched biodegradable nanocrystalline hydroxyapatite.

Vascular endothelial growth factor (VEGF) plays a key role for the interaction of osteoblasts and endothelial cells and, therefore, is an important factor for the osteointegration of bone substitutes. The aim of the current work was to study the effects of platelet growth factors (PLF) on new blood vessel formation and VEGF-receptors expression pattern in bone defects filled with nanocrystalline hydroxyapatite (HA) paste in miniature-pigs. Conventional histology, RT-PCR for VEGF and receptors mRNA, Western blot analysis, immunohistochemical staining and quantitative assessment of newly formed vessels was performed. HA enriched with platelet growth factor (HA/PLF+) led to an up-regulation of VEGF-R1 synthesis, a slightly enhanced number of newly formed vessels with higher sprouting activity compared with HA without PLF (HA/PLF-) filling defects. These observation are most likely attributable to a stimulating effect of TGF-ss from the platelet factor on VEGF expression in osteoblasts.