Microstructured zirconia surfaces modulate osteogenic marker genes in human primary osteoblasts.

In dentistry, zirconia has been used since the early 1990s for endodontic posts, more recently for implant abutments and frameworks for fixed dental prostheses. Zirconia is biocompatible and mechanically strong enough to serve as implant material for oral implants. Although several zirconia implant systems are available, currently the scientific and clinical data for zirconia implants are not sufficient to recommend them for routine clinical use. Here the influence of microstructured yttria-stabilized zirconia (YZ) on human primary osteoblast (HOB) behavior was determined. YZ surfaces were treated by sandblasting (YZ-S), acid etching (YZ-SE) and additionally heat treatment (YZ-SEH). Morphological changes of HOB were determined by scanning electron microscopy. Actin cytoskeleton was investigated by laser scanning microscopy and analyzed by novel actin quantification software. Differentiation of HOB was determined by real time RT-PCR. Improved mechanical interlocking of primary HOB into the porous microstructure of the acid etched and additionally heat treated YZ-surfaces correlates with drastically increased osteocalcin (OCN) gene expression. In particular, OCN was considerably elevated in primary HOB after 3 days on YZ-SE (13-fold) as well as YZ-SEH (12-fold) surfaces. Shorter actin filaments without any favored orientation on YZ-SE and YZ-SEH surfaces are associated with higher roughness (Ra) values. Topographically modified yttria-stabilized zirconia is a likely material for dental implants with cell stimulating properties achieving or actually exceeding those of titanium.

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds.

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down-regulated in co-cultures on polystyrene, they were up-regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co-culture, direct cell-cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells.

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors.

The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to ß1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors.

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells.

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

Influence of manganese ions on cellular behavior of human osteoblasts in vitro.

Divalent cations like Mn(2+) are known to strongly influence the integrin affinity to ligands and - in consequence - cell adhesion to extracellular matrix proteins. Therefore, divalent cation supplementation of biomaterials could be a promising approach to improve the ingrowth and the integration of implants. We were interested, whether manganese ions affect cellular functions like spreading, proliferation as well as gene expression in human osteoblasts. MG-63 osteoblastic cells were cultured in DMEM with 10% FCS. MnCl(2) was added at a concentration range of 0.01-0.5mM for 24h and 48 h. Spreading (cell area in microm(2)) of PKH26-stained cells (cell membrane dye) was analyzed using confocal microscopy. Cell proliferation was measured by flow cytometry. Quantification of the phosphorylation status of signaling proteins was estimated using the Bio-Plex 200 system. Gene expression of osteogenic markers at the mRNA and protein level was analyzed by quantitative real time RT-PCR and Western blot, respectively. The results demonstrated that at higher concentrations of Mn(2+) cells revealed a spindle shaped morphology. Further analyses indicated a reduced spreading, proliferation as well as phosphorylation of signaling proteins due to the influence of Mn(2+) in a concentration-dependent manner. Although expression of bone sialo protein (BSP) at the mRNA level increased both after 24h and 48 h in the presence of manganese, no increased expression of BSP was detected at the protein level. The expression of alkaline phosphatase (ALP) and collagen 1 (Col 1) mRNA decreased at >0.1mM MnCl(2). We speculate that the effect of manganese cations on cell functions is strongly concentration-dependent and the release of manganese when incorporated in a biomaterial surface has to be thoroughly adjusted.

Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials.

The survival and functioning of a bone biomaterial requires a rapid and stable vascularization after implantation. However, the mechanisms involved in the context of the complex healing microenvironment are poorly understood. To evaluate the vascularization potential of bone biomaterials, angiogenic stimuli were added to human dermal microvascular endothelial cells (HDMEC) growing on three-dimensional (3-D) bone biomaterials consisting of porous hydroxyapatite, porous calcium phosphate, porous nickel-titanium, successfully being used in humans, and also silk fibroin nets. HDMEC did not migrate to form microcapillary-like structures as they did on cell culture plastic. In cocultures of HDMEC and primary human osteoblast cells (HOS) or the human osteoblast-like cell line MG-63 on these biomaterials, a tissue-like self-assembly of cells occurred with time, with endothelial cells forming microcapillary-like structures containing a lumen and giving a strong PECAM-1 expression at cell interfaces. These microcapillary-like structures were intertwined between cell layers of osteoblasts and did not form when exogenous angiogenic stimuli were added to these cocultures. The life span of HDMEC was also significantly enhanced by coculture; with HDMEC being present for up to at least 42 days, compared to the monoculture where cells began to die rapidly after 1 week without passage. This coculture system may be applicable to a prevascularization strategy for biomaterials prior to implantation. Irrespective of this, the coculture model holds promise for studies to deepen our understanding of bone regeneration on 3-D substrates. Most importantly, these data raise important questions concerning the exact nature of pro-angiogenic drug- or gene-delivery systems to be incorporated into scaffolds. Our results underline the necessity to take into account the in situ production of growth factors by invading mesenchymal cells in the regenerative niche.