No abstract

Pro-forms of growth factors have received intensive scientific attention recently because in some cases different biological activities have been ascribed compared with the mature growth factors. Examples are the pro-apoptotic role of the nerve growth factor (NGF) proform (proNGF) or the latency of the transforming growth factor (TGF)-␤ pro-form (proTGF-␤). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-␤ family, mature BMP-2, proBMP-2, and the isolated pro-peptide were recombinantly produced in Escherichia coli cells, and a biophysical comparison was performed. Protocols were developed that allowed efficient refolding and subsequent purification of the proteins. ProBMP-2 could be processed to an N-terminally truncated form of BMP-2, digit removed BMP-2 (drBMP-2), that possessed biological activity, i.e. it induced ectopic bone formation. Bone inducing activity was also displayed by proBMP-2. The three proteins were characterized both by fluorescence and CD spectroscopy. From these analyses, predominant ␤-sheet secondary structural elements in the pro-peptide were deduced. The thermodynamic stability of the pro-peptide was determined by chemical unfolding assays. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. However, in contrast to NGF/proNGF, the pro-peptide did not stimulate oxidative folding of the mature part in vitro.

At the time we prefer OCT instead of ACT given the correct indication.

The osteogenic potential of bone and bone matrix has been characterised only by its biological effects and the parameters influencing it. Recently, the osteoinductive ability of bone matrix had been defined chemically by the description of a bone morphogenetic protein (BMP), and the parameters of osteogenic factors from different species have now been recognised. The current state of isolation, purification and characterisation of these factors is summarised in this review. General aspects of the isolation and testing of BMP preparations, and the results of orthotopic application of BMP implants, including clinical cases, are reported.

No abstract

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM beta-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3-6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells.

No abstract

The histological and enzymatic effects of single-dose irradiation of 7 Gray (Gy) versus fractionated irradiation of 5 x 2 Gy on the suppression of heterotopic ossification were examined over a period of 60 days in adult male Wistar rats (n = 57). The standardized osteogenesis model system in rats 19, 10, 11, 16, 19] was used for this purpose. The course of developing ossifications was documented quantitatively and qualitatively by means of quantitative computed tomography/osteodensitometry and digital luminescence radiography. Assessment of the activities of the enzymes alkaline and acid phosphatase throughout the experiment as well as characterization of the isoenzyme of alkaline phosphatase (AP) in connection with histological observations displayed a metaplasia of the ingrowing connective tissue into bone-typical cells during osteoinduction. Thus, the increase of AP is the first sign of a functional transformation of mesenchymal stem cells into chondroid bone cells. The increase in the acid phosphatase level with a maximum of activity between the 15th and 30th day (according to the respective treatment group) is highly suggestive of a remodeling process paralleling incipient chondroclast and osteoclast activity. In the animal groups undergoing irradiation, the above-mentioned increase of enzymes occurred after a delay. Furthermore, the maximum values observed were lower than those in the group not undergoing irradiation. Both findings were more manifest in the animal group which underwent 5 x 2 Gy of radiation than in the group which underwent single-dose irradiation of 7 Gy. Radiation suppresses matrix-induced osteogenesis. The histological and enzymatic course of this process was unchanged in the animals which did not undergo irradiation. However, it was quantitatively reduced and accompanied by a retardation of osteogenesis. Both effects were again reduced with fractionated irradiation of 5 x 2 Gy, which is theoretically dose-equivalent to a 1 x 7 Gy application. Histological examinations revealed damage to the migratory, proliferating mesenchymal stem cell population by irradiation doses which had relatively small effects on preosteoblasts, osteoblasts, chondroblasts and other specialized cell forms. Therefore, it may be concluded that the smaller degree of heterotopic ossification in the irradiated groups was due to damage of and a decrease in the number of mesenchymal stem cells at the implant site. Our results stress the necessity of instituting postoperative irradiation therapy as early as possible to prevent heterotopic ossification. In view of experimentally proven better effects, fractionated irradiation has to be preferred to a dose-equivalent single-dose radiation, especially considering the fewer side-effects noted with fractionated irradiation.