VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo.

Bone formation and osseointegration of biomaterials are dependent on angiogenesis and vascularization. Angiogenic growth factors such as vascular endothelial growth factor (VEGF) were shown to promote biomaterial vascularization and enhance bone formation. However, high local concentrations of VEGF induce the formation of malformed, nonfunctional vessels. We hypothesized that a continuous delivery of low concentrations of VEGF from calcium phosphate ceramics may increase the efficacy of VEGF administration.VEGF was co-precipitated onto biphasic calcium phosphate (BCP) ceramics to achieve a sustained release of the growth factor. The co-precipitation efficacy and the release kinetics of the protein were investigated in vitro. For in vivo investigations BCP ceramics were implanted into critical size cranial defects in Balb/c mice. Angiogenesis and microvascularization were investigated over 28 days by means of intravital microscopy. The formation of new bone was determined histomorphometrically. Co-precipitation reduced the burst release of VEGF. Furthermore, a sustained, cell-mediated release of low concentrations of VEGF from BCP ceramics was mediated by resorbing osteoclasts. In vivo, sustained delivery of VEGF achieved by protein co-precipitation promoted biomaterial vascularization, osseointegration, and bone formation. Short-term release of VEGF following superficial adsorption resulted in a temporally restricted promotion of angiogenesis and did not enhance bone formation. The release kinetics of VEGF appears to be an important factor in the promotion of biomaterial vascularization and bone formation. Sustained release of VEGF increased the efficacy of VEGF delivery demonstrating that a prolonged bioavailability of low concentrations of VEGF is beneficial for bone regeneration.

Bioresorbable composites prepared by supercritical fluid foaming.

Bone is a natural composite construct, with a gradient structure going from a loose interconnected cellular core to an outer dense wall, thus minimizing bone weight while keeping a high mechanical resistance. Due to this unique and complex structure, bone defects are difficult to replace or repair. Tissue engineering aims at providing artificial bone grafts. Several techniques have been proposed to produce porous structures or scaffolds, but, as yet, with no optimal solutions. This article focuses on bioresorbable ceramic-polymer composite foams obtained by supercritical fluid foaming. This flexible technique enables an adequate morphology and suitable properties for bone tissue engineering to be obtained. Composite scaffolds are biocompatible, allowing cell proliferation and differentiation.

Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist.

Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [125I]RANTES and [125I]IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.

Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected in HEK 293 cells and the recombinant ligands.

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully characterised the recombinant proteins expressed in E. coli. They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells. THP-1 cells express three of the known CC chemokine receptors. In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay. RANTES competes for [125I]RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants. These affinities are the same as those measured on the THP-1 cell line. The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells. This indicates that this cellular response can be mediated through the CC CKR-1 receptor.