Maria Norgård

Resveratrol-conjugated poly-ε-caprolactone facilitates in vitro mineralization and in vivo bone regeneration

Incorporation of osteoinductive factors in a suitable scaffold is considered a promising strategy for generating osteogenic biomaterials. Resveratrol is a polyphenol found in parts of certain plants, including nuts, berries and grapes. It is known to increase DNA synthesis and alkaline phosphatase (ALP) activity in osteoblasts and to prevent femoral bone loss in ovariectomized (OVX) rats. In the present study resveratrol was coupled through a hydrolysable covalent bond with the carboxylic acid groups in porous poly-ε-caprolactone (PCL) surface grafted with acrylic acid (AA). The osteogenic effect of this new scaffold was evaluated in mesenchymal cell culture and in the rat calvarial defect model. We found that the incorporation of resveratrol caused increased ALP activity of rat bone marrow stromal cells and enhanced mineralization of the cell-scaffold composites in vitro. After 8 weeks the calvarial defects implanted with resveratrol-conjugated PCL displayed a higher X-ray density than the defects implanted with control PCL. Bone-like structures, positively immunostained for bone sialoprotein, were shown to be more extensively formed in the resveratrol-conjugated PCL. These results show that incorporation of resveratrol into the AA-functionalized porous PCL scaffold led to a significant increase in osteogenesis.

Isolation of rat intestinal microsomes: Partial characterization of mucosal cytochrome P-450

A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.

Osteoclast polarization is not required for degradation of bone matrix in rachitic FGF23 transgenic mice.

Hypophosphatemic transgenic (tg) mice overexpressing FGF23 in osteoblasts display disorganized growth plates and reduced bone mineral density characteristic of rickets/osteomalacia. These FGF23 tg mice were used as an in vivo model to examine the relation between osteoclast polarization, secretion of proteolytic enzymes and resorptive activity. Tg mice had increased mRNA expression levels of the osteoblast differentiation marker Runx2 and mineralization-promoting proteins alkaline phosphatase and bone sialoprotein in the long bones compared to wild type (wt) mice. In contrast, expression of alpha1(I) collagen, osteocalcin, dentin matrix protein 1 and osteopontin was unchanged, indicating selective activation of osteoblasts promoting mineralization. The number of osteoclasts was unchanged in tg compared to wt mice, as determined by histomorphometry, serum levels of TRAP 5b activity as well as mRNA expression levels of TRAP and cathepsin K. However, tg mice displayed elevated serum concentrations of C-terminal telopeptide of collagen I (CTX) indicative of increased bone matrix degradation. The majority of osteoclasts in FGF23 tg mice lacked ultrastructural morphological signs of proper polarization. However, they secreted both cathepsin K and MMP-9 at levels comparable to osteoclasts with ruffled borders. Mineralization of bone matrix thus appears essential for inducing osteoclast polarization but not for secretion of osteoclast proteases. Finally, release of CTX by freshly isolated osteoclasts was increased on demineralized compared to mineralized bovine bone slices, indicating that the mineral component limits collagen degradation. We conclude that ruffled borders are implicated in acidification and subsequent demineralization of the bone matrix, however not required for matrix degradation. The data collectively provide evidence that osteoclasts, despite absence of ruffled borders, effectively participate in the degradation of hypomineralized bone matrix in rachitic FGF23 tg mice.

Fluid pressure induces osteoclast differentiation comparably to titanium particles but through a molecular pathway only partly involving TNFα

In contrast to the well‐understood inflammatory pathway driven by TNFα, by which implant‐derived particles induce bone resorption, little is known about the process in which loosening is generated as a result of force‐induced mechanical stimulus at the bone–implant interface. Specifically, there is no knowledge as to what cells or signaling pathways couple mechanical stimuli to bone resorption in context of loosening. We hypothesized that different stimuli, i.e., fluid flow versus wear particles, act through different cytokine networks for activation and localization of osteoclasts. By using an animal model in which osteoclasts and bone resorption were induced by fluid pressure or particles, we were able to detect distinct differences in osteoclast localization and inflammatory gene expression between fluid pressure and titanium particles. Fluid pressure recruits and activates osteoclasts with bone marrow contact away from the fluid pressure exposure zone, whereas titanium particles recruit and activate osteoclasts in areas in direct contact to particles. Fluid pressure induced weaker expression of the selected inflammatory related genes, although the eventual degree of osteoclast induction was similar in both models. Using TNFαRa (4 mg/kg) (Enbrel) and dexamethasone (2 mg/kg) as specific and more general suppressors of inflammation we showed that the TNFαRa failed to generate statistically impaired osteoclast generation while dexamethasone was much more potent. These results demonstrate that fluid pressure induces osteoclasts at a different localization than titanium particles by a molecular pathway less associated with TNFα and the innate system, which open up for other pathways controlling pressure induced osteoclastogenesis. J. Cell. Biochem. 113: 1224–1234, 2012.

Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression

Angulated femurs are present prenatally both in CYP26B1 deficient humans with a reduced capacity to degrade retinoic acid (RA, the active metabolite of vitamin A), and mice overexpressing vascular endothelial growth factor a (Vegfa). Since excessive ingestion of vitamin A is known to induce spontaneous fractures and as the Vegfa-induced femur angulation in mice appears to be caused by intrauterine fractures, we analyzed bones from a CYP26B1 deficient human and rats with hypervitaminosis A to further explore Vegfa as a mechanistic link for the effect of vitamin A on bone. We show that bone from a human with CYP26B1 mutations displayed periosteal osteoclasts in piles within deep resorption pits, a pathognomonic sign of hypervitaminosis A. Analysis of the human angulated fetal femur revealed excessive bone formation in the marrow cavity and abundant blood vessels. Normal human endothelial cells showed disturbed cell-cell junctions and increased CYP26B1 and VEGFA expression upon RA exposure. Studies in rats showed increased plasma and tissue Vegfa concentrations and signs of bone marrow microhemorrhage on the first day of excess dietary vitamin A intake. Subsequently hypervitaminosis A rats displayed excess bone formation, fibrosis and an increased number of megakaryocytes in the bone marrow, which are known characteristics of Vegfa overexpression. This study supports the notion that the skeletal phenotype in CYP26B1 deficient human bone is caused by excess RA. Our findings suggest that an initial part of the vitamin A mechanism causing bone alterations is mediated by excess Vegfa and disturbed bone marrow microvessel integrity.