Controlling fibroblast adhesion and proliferation by 1D Al2O3 nanostructures.

The fibrotic encapsulation, which is mainly accompanied by an excessive proliferation of fibroblasts, is an undesired phenomenon after the implantation of various medical devices. Beside the surface chemistry, the topography plays also a major role in the fibroblast-surface interaction. In the present study, one-dimensional aluminium oxide (1D Al2O3) nanostructures with different distribution densities were prepared to reveal the response of human fibroblasts to the surface topography. The cell size, the cell number and the ability to form well-defined actin fibres and focal adhesions were significantly impaired with increasing distribution density of the 1D Al2O3 nanostructures on the substratum.

VEGF-loaded mineral-coated microparticles improve bone repair and are associated with increased expression of epo and RUNX-2 in murine non-unions.

A poor vascular supply of the fracture gap is a key factor for the development of atrophic non-unions. Mineral-coated microparticles (MCM) represent a sophisticated carrier system for the delivery of vascular endothelial growth factor (VEGF). Hence, we investigated whether VEGF-loaded MCM improve bone repair in non-unions. For this purpose, we analyzed binding and release kinetics of MCM for VEGF in vitro. Moreover, we applied VEGF-loaded or -unloaded MCM in a murine non-union model in vivo and studied the process of bone healing by means of biomechanical, radiological, histomorphometric, and Western blot techniques. MCM-free non-unions served as controls. The binding efficiency of MCM for VEGF was 46 ± 3% and the release profile revealed an initial minor burst release followed by a sustained release over a 50-day study period, thus, mimicking the physiological expression profile of VEGF during bone healing. In vivo, bone defects treated with VEGF-loaded MCM exhibited a higher bending stiffness, a higher fraction of bone volume/tissue volume and a larger callus area on days 14 and 70 when compared to the other groups. Western blot analyses on day 14 revealed a higher expression of VEGF, erythropoietin (EPO), and runt-related transcription factor 2, but not of EPO-receptor in bone defects treated with VEGF-loaded MCM. These findings demonstrate that the use of MCM for VEGF delivery shows great potential due to the ability to maintain protein stability and functionality in vivo. Moreover, the application of VEGF-loaded MCM represent a promising strategy for the treatment of non-unions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Induction of osteogenic differentiation by nanostructured alumina surfaces.

Permanent orthopedic implants are becoming increasingly important due to the demographic development. Their optimal osseointegration is key in obtaining good secondary stability. For anchorage dependent cells, topographic features of a surface play an essential role for cell adhesion, proliferation, differentiation and biomineralization. We studied the topographical effect of nanostructured alumina surfaces prepared by chemical vapor deposition on osteogenic differentiation and growth of human osteoblasts. Chemical vapor deposition of the single source precursor (tBuOAIH2)2 led to synthesis of one dimensional alumina nanostructures of high purity with a controlled stoichiometry. We fabricated different topographic features by altering the distribution density of deposited one dimensional nanostructures. Although the topography differed, all surfaces exhibited identical surface chemistry, which is the key requirement for systematically studying the effect of the topography on cells. Forty-eight hours after seeding, cell density and cell area were not affected by the nanotopography, whereas metabolic activity was reduced and formation of actin-fibres and focal adhesions was impaired compared to the uncoated control. Induction of osteogenic differentiation was demonstrated via up-regulation of alkaline phosphatase, bone sialoprotein, osteopontin and Runx2 at the mRNA level, demonstrating the potential of nanostructured surfaces to improve the osseointegration of permanent implants.

Bromelain down-regulates myofibroblast differentiation in an in vitro wound healing assay.

Bromelain, a pineapple-derived enzyme mixture, is a widely used drug to improve tissue regeneration. Clinical and experimental data indicate a better outcome of soft tissue healing under the influence of bromelain. Proteolytic, anti-bacterial, anti-inflammatory, and anti-oedematogenic effects account for this improvement on the systemic level. It remains unknown, whether involved tissue cells are directly influenced by bromelain. In order to gain more insight into those mechanisms by which bromelain modulates tissue regeneration at the cellular level, we applied a well-established in vitro wound healing assay. Two main players of soft tissue healing--fibroblasts and microvascular endothelial cells--were used as mono- and co-cultures. Cell migration, proliferation, apoptosis, and the differentiation of fibroblasts to myofibroblasts as well as interleukin-6 were quantified in response to bromelain (36 × 10(-3) IU/ml) under normoxia and hypoxia. Bromelain attenuated endothelial cell and fibroblast proliferation in a moderate way. This proliferation decrease was not caused by apoptosis, rather, by driving cells into the resting state G0 of the cell cycle. Endothelial cell migration was not influenced by bromelain, whereas fibroblast migration was clearly slowed down, especially under hypoxia. Bromelain led to a significant decrease of myofibroblasts under both normoxic (from 19 to 12 %) and hypoxic conditions (from 22 to 15 %), coincident with higher levels of interleukin-6. Myofibroblast differentiation, a clear sign of fibrotic development, can be attenuated by the application of bromelain in vitro. Usage of bromelain as a therapeutic drug for chronic human wounds thus remains a very promising concept for the future.

Excess dietary methionine does not affect fracture healing in mice.

BACKGROUND: An elevated serum concentration of homocysteine (hyperhomocysteinemia) has been shown to disturb fracture healing. As the essential amino acid, methionine, is a precursor of homocysteine, we aimed to investigate whether excess methionine intake affects bone repair. MATERIAL/METHODS: We analyzed bone repair in 2 groups of mice. One group was fed a methionine-rich diet (n=13), and the second group received an equicaloric control diet without methionine supplementation (n=12). Using a closed femoral fracture model, bone repair was analyzed by histomorphometry and biomechanical testing at 4 weeks after fracture. Blood was sampled to measure serum concentrations of homocysteine, the bone formation marker osteocalcin, and the bone resorption marker collagen I C-terminal crosslaps RESULTS: Serum concentrations of homocysteine were significantly higher in the methionine group than in the control group, while serum markers of bone turnover did not differ significantly between the 2 groups. Histomorphometry revealed no significant differences in size and tissue composition of the callus between animals fed the methionine-enriched diet and those receiving the control diet. Accordingly, animals of the 2 groups showed a comparable bending stiffness of the healing bones. CONCLUSIONS: We conclude that excess methionine intake causes hyperhomocysteinemia, but does not affect fracture healing in mice.

Sildenafil accelerates fracture healing in mice.

Sildenafil, a cyclic guanosine monophosphate (cGMP)-dependent phospodiesterase-5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro-angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro-angiogenic and osteogenic cysteine-rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61-associated pathway.