Platelet released growth factors boost expansion of bone marrow derived CD34(+) and CD133(+) endothelial progenitor cells for autologous grafting.

Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM + 5%, FCS + 5% PRGF and IMDM + 10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell transplantation.

A review of the cost effectiveness of bisphosphonates in the treatment of post-menopausal osteoporosis in Switzerland.

The economic burden associated with osteoporosis is considerable. As such, cost-effectiveness analyses are important contributors to the diagnostic and therapeutic decision-making process. The aim of this study was to review the cost effectiveness of treating post-menopausal osteoporosis with bisphosphonates and identify the key factors that influence the cost effectiveness of such treatment in the Swiss setting. A systematic search of databases (MEDLINE, EMBASE and the Cochrane Library) was conducted to identify published literature on the cost effectiveness of bisphosphonates in post-menopausal osteoporosis in the Swiss setting. Outcomes were compared with similar studies in Western European countries. Three cost-effectiveness studies of bisphosphonates in this patient population were identified; all were from a healthcare payer perspective. Outcomes showed that, relative to no treatment, treatment with oral bisphosphonates was predicted to be cost saving for most women aged ≥70 years with osteoporosis or at least one risk factor for fracture, and cost effective for women aged ≥75 years without prior fracture when used as a component of a population-based screen-and-treat programme. Results were most sensitive to changes in fracture risk, cost of fractures, cost of treatment, nursing home admissions and adherence with treatment. Swiss results were generally comparable to those in other European settings. Assuming similar clinical efficacy, lowering treatment cost (through the use of price-reduced brand-name or generic drugs) and/or improving adherence should both contribute to further improving the cost effectiveness of bisphosphonates in women with post-menopausal osteoporosis. Published evidence indicates that bisphosphonates are estimated to be similarly cost effective or cost saving in most treatment scenarios of post-menopausal osteoporosis in Switzerland and in neighbouring European countries.

Nuclear alpha NAC influences bone matrix mineralization and osteoblast maturation in vivo.

Nascent-polypeptide-associated complex and coactivator alpha (alpha NAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, alpha NAC is translocated to the nuclei of osteoblasts, where it acts as an AP-1 coactivator to increase osteocalcin gene transcription. To determine the physiological role of nuclear alpha NAC, we engineered a knock-in mouse model with a serine-to-alanine mutation at position 43 (S43A). The S43A mutation resulted in a decrease in the amount of nuclear alpha NAC with reduced osteocalcin gene promoter occupancy, leading to a significant decrease in osteocalcin gene transcription. The S43A mutant bones also expressed decreased levels of alpha(1)(I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the alpha(1)(I) collagen gene as a novel alpha NAC target. The reduced quantity of bone matrix in S43A mutant bones was mineralized faster, as demonstrated by the significantly reduced mineralization lag time, producing a lower volume of immature, woven-type bone characterized by poor lamellation and an increase in the number of osteocytes. Accordingly, the expression of the osteocyte differentiation marker genes DMP-1 (dentin matrix protein 1), E11, and SOST (sclerostin) was increased. The accelerated mineralization phenotype was cell autonomous, as osteoblasts isolated from the calvaria of S43A mutant mice mineralized their matrix faster than did wild-type cells. Thus, inhibition of alpha NAC nuclear translocation results in an osteopenic phenotype caused by reduced expression of osteocalcin and type I collagen, accelerated mineralization, and immature woven-bone formation.

Osteopromotion with a plasmatransglutaminase on a beta-TCP ceramic.

We investigated the osteopromotive properties of plasmatransglutaminase (F XIII), bone marrow and venous blood on a resorbable beta-tricalcium phosphate (beta-TCP) scaffold. A baseline binding and release study of F XIII from the scaffold showed a continuous release of 18% of the total dose after 48 h. The main study consisted of 18 adult sheep with cylindrical defects in both tibiae. The defects were filled with a beta-TCP cylinder impregnated either with bone marrow, venous blood, F XIII or sheep were treated with 1250 IU F XIII intravenously over 14 days (n = 4 in each group). The defects were left open in two sheep. QCT and histology was performed after 6 and 12 weeks. The best bone ingrowth was seen after 6 weeks in the bone marrow group and after 12 weeks in the local F XIII group. The highest ingrowth on the inside of the cylinder proving the osteopromoting potential of F XIII was found in the local F XIII group. In our opinion F XIII is a good and readily available osteopromoting agent which can be used with beta-TCP in cases of bone deficit to promote bone regeneration.

Human endothelial cells inhibit BMSC differentiation into mature osteoblasts in vitro by interfering with osterix expression.

It is well accepted, that there is communication between osteoblasts and endothelial cells. However, the influence of endothelial cells on the differentiation of bone precursors into mature osteoblasts is not yet well understood. We therefore studied the effect of human umbilical vein endothelial cells (HUVEC) on human bone marrow stromal cell (BMSC) differentiation towards an osteoblastic phenotype by culturing them in two different types of HUVEC-BMSC cultures (indirect contact, HUVEC-conditioned medium). Typical bone markers (45Ca incorporation, ALP activity, and gene expression of collagen 1 (COL1), osteonectin (ON), matrix metalloproteinase 13 (MMP-13), bone morphogenetic protein 2 (BMP-2)) and transcription factors (Runx2, osterix (OSX)) were evaluated at different time points. The effect of stimulating HUVEC with vascular endothelial growth factor (VEGF) before co-cultures with BMSC was also evaluated. As expected, BMSC in osteogenic medium (OM) (with dexamethasone) differentiated towards the osteoblastic phenotype, as measured by increased matrix mineralization, high ALP activity, and elevated expression of specific osteoblastic marker genes, when compared to BMSC in non-OM. HUVEC reversibly inhibited osteoblastic differentiation of BMSC in OM, independent of the co-culture type. When HUVEC were removed from the cultures, BMSC differentiated into osteoblasts, albeit at a faster rate than BMSC that were never co-cultured with HUVEC. Stimulation of HUVEC with VEGF before co-culture enhanced the inhibitory effect of HUVEC on BMSC differentiation. This inhibitory effect was connected to a reversible suppression of OSX gene expression and was specific to endothelial cells. We conclude that HUVEC can inhibit dexamethasone-induced BMSC differentiation into osteoblasts in vitro, by interfering with OSX expression, thereby arresting BMSC differentiation at a preosteoblastic stage.