Heparin modification of calcium phosphate bone cements for VEGF functionalization.

A promising strategy to promote angiogenesis within an engineered tissue is the local and sustained delivery of an angiogenic factor by the substitute itself. Recently, we reported on functionalization of Biocement D (BioD) and several modifications of this calcium phosphate bone cement with vascular endothelial growth factor (VEGF). Maintenance of biological activity of VEGF after release from the cement was improved by modification of BioD with mineralized collagen type I (BioD/coll). However, BioD/coll composites showed a higher initial burst of VEGF release than do the unmodified BioD. In the present study, VEGF release from BioD/coll composites modified with different amounts of heparin was investigated. We found a distinct reduction of the initial burst of release by adding heparin in a concentration-dependent manner. Moreover, the heparin modification had a positive impact on the biological activity of released VEGF. An advancement of biological properties of BioD/coll by addition of heparin was further shown by improved adhesion of endothelial cells on the cement surface. Characterization of material properties of the heparin-modified BioD/coll composites revealed a finer microstructure with smaller HA-particles and a higher specific surface area than heparin-free BioD/coll. However, higher amounts of heparin resulted in a reduced compressive strength. The rheological properties of these cement pastes have been found to be favorable for good handling particularly with regard to their clinical application.

Calcium phosphate bone cements, functionalized with VEGF: release kinetics and biological activity.

Calcium phosphate bone cements are of great interest for bone replacement since the nanocrystalline structure allows their remodelling into native bone tissue. A strategy to accelerate vascularization of the implant region is the functionalization with vascular endothelial growth factor (VEGF), which is known to mediate angiogenesis in vivo. In this study, the release of recombinant human VEGF (rhVEGF(165)) following physical adsorption to Biocement D (BioD) and several modifications were investigated. Our data demonstrate a high VEGF binding capacity of BioD and a sustained release with a moderate initial burst. A proliferation assay using endothelial cells revealed maintenance of biological activity of VEGF after release from BioD. Release behavior of BioD was not improved by modification with mineralized collagen type I, as well as with a combination of mineralized collagen with O-phospho-L-serine and sodium citrate, respectively. In contrast, a positive impact of these modifications on the activity of released VEGF was observed; in case of the phosphoserine- and sodium citrate-modified cements, the biological efficacy of released VEGF was even higher than that of nonreleased control VEGF. We conclude that the bone implant material BioD and, especially, the phosphoserine modification may support activation of angiogenesis by delivery of VEGF in a local and sustained manner.

Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid.

In this study, the proliferation and differentiation of rat calvarial osteoblasts cultured on either (1) calcium-phosphate bone cement Biocement D, (2) Biocement D with 2.5% (w/w) mineralized collagen type I, or (3) Biocement D with 2.5% (w/w) mineralized collagen type I and 3% (w/w) citric acid were investigated. Incubation of the composites in cell-culture medium resulted in a fast decrease of pH and calcium concentration as well as in an increase of phosphate concentration. Although these effects occurred with all investigated materials, the lowest extent could be observed for the citric-acid-containing composites. As shown by scanning-electron microscopy, osteoblasts adhered to the composite surfaces. Proliferation and differentiation of the cells grown on the composites were found to be reduced compared to cells grown on tissue-culture polystyrene. Cells cultured in the vicinity of the composites but without direct contact also exhibited a reduced rate of proliferation, reduced alkaline phosphatase activity, and reduced mineralization. Simulating the changes in calcium and phosphate concentration occasioned by the composites through exposing cells to EGTA and phosphate gives rise to the same effects of reducing proliferation, ALP activity, and mineralization. No indication for apoptosis in cells exposed to low calcium and high phosphate concentrations was found. The number of necrotic cells, however, increased after incubation with EGTA and phosphate. For assessment of cell-composite interactions and the success of the composites in vivo, as well as for more effective material development, it seems to be important to know how changes in microenvironmental pH and ion composition of the material affect cellular proliferation and differentiation.

Phosphoserine--a convenient compound for modification of calcium phosphate bone cement collagen composites.

Temporary bone replacement materials on the basis of calcium phosphates and hydroxyapatite (HAP) are used in surgery for filling bone defects. Components which are able to control the nucleation and crystal growth of HAP through their functional groups and which can additionally activate bone cells may be helpful in the development of materials with enhanced remodelling in vivo. In this study, the influence of O-phospho-L-serine (PS) on the materials properties of calcium phosphate bone cement composites was investigated. For up to an addition of 25 mg/g PS a strong increase in the stability of the cements under load was determined. The material was studied by scanning electron microscopy and transmission electron microscopy. A more dense microstructure and a plate-like morphology of the HAP-crystals were detected in the modified composites compared with the non-modified samples. By X-ray powder diffraction an inhibition of the dissolution of alpha-tricalcium phosphate (alpha-TCP) and dicalciumphosphate anhydrous (DCPA) particles was found. alpha-TCP and DCPA are the main constituents of the cement precursor. The results of cell culture studies using rat calvaria osteoblasts demonstrate a good viability of the cells on the PS-modified material. Furthermore, the proliferation and differentiation were found to be enhanced on the PS-modified material.

Influence of osteocalcin and collagen I on the mechanical and biological properties of Biocement D.

In order to generate a calcium-phosphate bone cement as a transient replacement for bone defects, we modified Biocement D (Merck Biomaterial GmbH) containing mineralised collagen with osteocalcin, the most abundant non-collageneous protein of bone. Osteocalcin was added to the cement paste during setting in order to control the crystallisation kinetics of hydroxyapatite (HAP) as well as to stimulate the interaction of osteoblasts and osteoclasts with the bone replacement material. Analysis by SEM and AFM shows, that the addition of osteocalcin causes a nanosize microstructure of the calcium cement, which can be explained by inhibited growth of HAP crystals. The fracture strength of the material decreased by incorporation of osteocalcin, pointing onto a higher defect concentration of the crystalline structure. The impact of osteocalcin onto the interaction of bone cells with HAP-Collagen I-cements was studied in a cell culture system using the human osteosarcoma cell line SAOS-2. Results suggest, that osteocalcin might possibly improve the initial adherence of osteoblast-like cells, whereas proliferation of the cells is not effected.