Introduction

Infection of endoprostheses is a serious complication in orthopedic surgery. As silver is known for its antibactierial effects, silver-coated endoprostheses have gained increased attention to decrease infection rates. However, cytotoxic effects of silver on bone cells have not been investigated in detail. We aimed to investigate whether silver nano-/microparticles and ionic silver exert cytotoxic effects on osteoblasts and osteoclasts in vitro and to correlate potential effects with the antibacterial effect on Staph. epidermidis.

Aim

Synovial sarcoma (SS) is a malignant soft tissue sarcoma with a poor prognosis because of late local recurrence and distant metastases. To our knowledge, no studies have minimum follow-up of 10 years that evaluate long-term outcomes for survivors.

Sufficient vascularization is essential for osseointegration of biomaterials and their substitution by new bone. Angiogenic growth factors such as VEGF are promising agents to promote the vascularization of bone substitutes. To optimize the efficacy of VEGF delivery a continuous administration of low concentrations of VEGF seems to be beneficial. We hypothesized that a long-term release of VEGF from calcium phosphate ceramics may induce a sustained angiogenic response and sufficiently promote biomaterial vascularization in vivo.

Vascular endothelial growth factor (VEGF, Genentech Inc., South San Francisco, USA.) was co-precipitated onto biphasic calcium phosphate ceramics (BCP, 80% HA, 20% β-TCP) at a concentration of 1μg/ml and 5μg/ml. The passive release and the cell-mediated release of VEGF were analyzed over 19 days by ELISA. For in vivo investigations BCP ceramics were implanted into a cranial window preparation in Balb/c mice. Angiogenesis and vascularization were investigated over 28 days by means of intravital microscopy. Functional capillary density (FCD, mm/mm²) served as parameter of biomaterial vascularization.

Co-precipitation of VEGF onto BCP ceramics resulted in a significant improvement of protein retention as compared to conventional adsorption of the growth factor [Cumulative VEGF release: Adsorption: 320 ± 2.6 ng/ml, Co-precipitation 116 ± 14.6 ng/ml (p< 0.05)]. Murine bone marrow cells differentiated towards osteoclasts mediated a sustained release of co-precipitated VEGF. Preliminary in vivo results showed a significant increase of functional capillary density after implantation of BCP ceramics co-precipitated with VEGF as compared to negative controls [day 7: 1.7 ± 0.2 mm/mm² vs. 0.9 ± 0.5 mm/mm²; day 14: 6.1 ± 0.3 mm/mm² vs. 2.1 ± 0.6 mm/mm²; day 28: 8.7 ± 0.3 mm/mm² vs. 3.9 ± 0.7 mm/mm², p< 0.05]. At 14 and 28 days after implantation, FCD induced by BCP ceramics co-precipitated with VEGF was significantly higher as compared to FCD induced by ceramics adsorbed with the VEGF [day 14: 6.1 ± 0.3 mm/mm² vs. 4.0 ± 1.4 mm/mm²; day 28: 8.7 ± 0.3 mm/mm² vs. 5.9 ± 0.7 mm/mm², p< 0.05].

The release kinetics critically influences the efficacy and the risks of local VEGF administration. By applying a co-precipitation technique the initial high liberation rate of VEGF was reduced and a sustained cell-mediated release at low concentrations was achieved. In vivo, VEGF promoted angiogenesis and vascularization of BCP ceramics. Vessel formation was more pronounced if VEGF was co-precipitated onto ceramics as compared to superficial adsorption of the growth factor, indicating that VEGF delivery at later stages of the healing process is beneficial. The present study provides evidence that, by delivering VEGF in a sustained manner at low local concentrations biomaterial vascularization can be markedly enhanced.

The reconstruction of bone defects with biomaterials represents a potential alternative to the transplantation of autologous and allogenic bone. Ceramic materials can be combined with growth factors (i.e. BMPs) to render them osteoinductive. Coating of biomaterials with growth factors has mostly been attempted by adsorption onto the material’s surface. The superficial deposition usually results in an immediate passive release of the proteins, thus restricting their temporal availability during bone healing. It was hypothesized that a co-precipitation of proteins onto calcium phosphate ceramics may provide the possibility to achieve a prolonged release of proteins from the material without impairing the biologic activity of growth factors.

Tritium labelled bovine serum albumin ([3H]BSA) and recombinant human BMP2 (rhBMP2) were coated onto biphasic calcium phosphate (BCP) ceramics using a coprecipitation technique of proteins together with calcium phosphate (Liu Y et al. 2001). The co-precipitation was compared to conventional adsorption of proteins to ceramic materials. The passive and cell-mediated release of [3H]BSA was investigated during 19 days. To analyze the cell-mediated protein release, murine bone marrow cells were seeded onto ceramics and differentiated to osteoclasts or to monocytes/macrophages. To assess whether rhBMP2 co-precipitated to BCP ceramics retained its biologic activity the growth factor’s ability to induce the differentiation of primary murine osteoblasts was studied.

After 19 days 71.7±5.3% of the adsorbed [3H]BSA was passively released (63.0±6.0% within 4 days). The passive liberation of [3H]BSA was effectively reduced using the coprecipitation technique (12.5±2.0% within 19 days, 10.1±2.3% within 4 days, p< 0.001). Further analysis demonstrated a sustained, osteoclast-mediated release of coprecipitated [3H]BSA from calcium phosphate ceramics which was blocked by the addition of calcitonin. Passive release of adsorbed and co-precipitated BMP2 led to a temporally restricted stimulation of murine osteoblasts. Cell-mediated liberation of co-precipitated BMP2 induced a sustained stimulation of the differentiation of osteoblasts.

The successful application of exogenously added growth factors depends critically on the mode of delivery. It has been shown that a sustained availability of BMP2 is beneficial for bone healing. Application of the co-precipitation technique resulted in a long-term release of proteins from BCP ceramics mediated by active resorbing osteoclasts without impairing the biologic activity of rhBMP2. Co-precipitating growth factors onto BCP ceramics provides a potential to shift the initial extensive liberation to a sustained release of bioactive proteins. This method of protein delivery may represent a possibility to achieve a more physiological availability of growth factors during bone regeneration.