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To determine whether male and female skeletons are equally responsive to mechanical load, the left ulnae in a group of juvenile male (n = 7), and age-matched female (n = 9) rats received a short daily period of controlled dynamic loading in vivo (1200 cycles at 2 Hz each day for 10 days) in addition to their normal exercise. Axial loads for each group were adjusted to engender a peak dynamic strain of -4000 microstrain at the medial face of the ulna midshaft, applied and released at a rate of +/-30,000 microstrain/sec. Fluorescent labels were administered at the start and finish of the loading period. Over the course of daily loading, the body mass of the male rats increased 2.5 times faster than that of the females (6.3 g/day vs. 2.5 g/day). The increase in periosteal interlabel bone area due to growth and normal exercise was also 2.5 times greater in the males than in the females. Both genders showed statistically significant (p < 0.05) increases in periosteal new bone deposition in the ulna of their loaded compared with their control limb. The pattern of osteogenic response was similar in males and females and featured increased mineral apposition rate on the lateral surface of the ulna, and arrest of modeling-drift-related resorption with its reversal to bone formation on the medial surface. In males, the absolute loading-related increase in bone area was six times greater than that in females. However, when the absolute size of the loading-related change in periosteal interlabel new bone deposition was expressed relative to that due to growth, there was no difference between males and females (Mean +/- SEM: 37 +/- 12% for males, 34 +/- 12% for females). These data confirm that the ulna of young actively growing rats of both genders responds to a short daily period of loading with an altered modeling response that involves increased bone formation and decreased resorption. Although the absolute amount of new bone formation stimulated by loading is greater in males than in females there is no difference between genders following correction for the higher rate of bone deposition seen in the males in association with their faster rate of growth.
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PMID:Growth rate rather than gender determines the size of the adaptive response of the growing skeleton to mechanical strain. 1179 3

Due to their osteogenic germination potential, periosteum-derived osteoprogenitor cells are a potential source for tissue engineering a bone graft that could be used to regenerate skeletal defects. In this study we evaluated if ectopic bone formation could be induced by a construct made of human periosteal cells and a novel scaffold architecture whose mechanical properties are in the range of cancellous bone. Biopsies from human calvarial periosteum were harvested and cells were isolated from the inner cambial layer. Fifty thousand periosteal cells were seeded into the scaffolds measuring 6 x 6 x 2 mm. The cell-scaffold constructs were cultured for a period of 3 weeks prior to implantation into balb C nude mice. Mice were sacrificed and implants were analyzed 6 and 17 weeks postoperatively. Immunohistochemical analysis confirmed the osteoblastic phenotype of the seeded cells. Formation of focal adhesions and stress fibers could be observed in both scaffold architectures. Three-dimensional cell proliferation was observed after 2 weeks of culturing with centripetal growth pattern inside the pore network. The deposition of calcified extracellular matrix was observed after 3 weeks of culturing. In vivo, endochondral bone formation with osteoid production was detectable via von Kossa and Osteocalcin staining after 6 and 17 weeks. Histology and SEM revealed that the entire scaffold/bone grafts were penetrated by a vascular network. This study showed the potential of bone tissue engineering by using human periosteal cells in combination with a novel scaffold technology.
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PMID:Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology. 1209 36

Blocks of two porous synthetic hydroxyapatites (HA) with porosity fraction of 30-40 and 50-60 vol%, respectively and a coralline derived porous HA were evaluated in vitro in presence of the osteogenic line MC3T3-E1 and of L929 fibroblasts. The two tested biomaterials did not affect cellular proliferation (MTT test), but the contact inhibited alkaline phosphatase activity. Porous aggregates resulted perfectly biocompatible in the tests performed, since observations performed by light microscopy did not show any cell morphological change, osteoblast presented a stellar shape and typical pseudopodes. SEM observations showed intercellular matrix containing fibers on HA-based porous aggregates.
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PMID:Growth of osteoblast-like cells on porous hydroxyapatite ceramics: an in vitro study. 1220 71

The objective of this study was to examine the osteoinductive capacity of different concentrations of BMP-2 on bone marrow stromal cells in vitro. Further, we intended to determine whether titanium provided with an increased surface roughness is more efficient in osteoblast differentiation than machined titanium. Therefore, 20,000 cells/ml were seeded and cultured on machined and grit-blasted titanium discs for 4, 8 and 16 days. Different concentrations of rhBMP-2 (0, 10, 100, 1000 ng/ml) were supplemented to the medium for 8 days of culturing. To evaluate cellular proliferation and differentiation, specimens were examined for DNA, alkaline phosphatase activity, and calcium content. Morphological appearance of the specimens at 8 and 16 days of incubation was evaluated using scanning electron microscopy. Two separate experimental runs were performed. Evaluation of the DNA and alkaline phosphatase data revealed that a significant difference existed for these data between both experimental runs. Further analysis of the DNA figures learned that roughening of the titanium surface and addition of BMP-2 had no effect on cell proliferation. The alkaline phosphatase analysis and calcium measurements revealed that BMP-2 stimulated the early differentiation of osteogenic cells on machined titanium substrates in a dose-dependent manner. After 16 days of culture, no significant differences in calcium content could be observed anymore between machined and roughened titanium surfaces. Further, the data revealed that the machined surfaces showed a significant increase in calcium deposition when 100 and 1000 ng/ml BMP-2 were supplemented to the medium. However, the roughened surfaces showed this significant enhancement in calcium content only with 1000 ng/ml BMP-2. In addition, SEM evaluation revealed a dose-dependent response to BMP-2. Increasing BMP-2 concentrations resulted in more calcified globular accretions on bone surfaces than when no BMP-2 was added. On the basis of our results, we conclude that (1) due to the heterogeneous nature of bone marrow, experimental results with primary rat bone marrow cells are difficult to reproduce from one experiment to the other, and (2) addition of rhBMP-2 in the medium stimulates the early differentiation and matrix mineralization of osteogenic cells on machined titanium surfaces in a dose-responsive manner. Further, we concluded that our roughened titanium surfaces had no effect on proliferation and differentiation of primary derived rate bone marrow cells.
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PMID:Observations on the effect of BMP-2 on rat bone marrow cells cultured on titanium substrates of different roughness. 1261 75

Electrostatic spray deposition (ESD) is a recently developed technique to deposit a calcium phosphate (CaP) coating upon substrates. With this technique, an organic solvent containing calcium and phosphate is pumped through a nozzle. Between the nozzle and substrate a high voltage is applied. As a consequence, droplets coming out the nozzle disperse into a spray, and this spray is deposited upon the substrate. When the solvent has evaporated, a coating is formed on the substrate. ESD allows for a variation in coating composition and morphology. Titanium alloy (TiAl6V4) substrates were coated with a CaP layer using two different methods; radio frequency magnetron sputtering, and ESD. These surfaces were characterized with X-ray diffraction, Fourier transform infrared spectroscopy, an universal surface tester, scanning electron microscopy, and energy dispersive spectrometry. Subsequently, bone marrow cells were isolated from rat femora and cultured 1, 4, 8, 14 and 16 days. Cell proliferation, alkaline phosphatase activity, and osteocalcin concentration were assayed. RT-PCR was done for collagen type I and osteocalcin. SEM was also performed to observe cellular behaviour during culture. Two separate runs of the experiment were performed. In the first run, osteoblast-like cells on both CaP coatings showed similar results in all assays. In the second run, proliferation and osteogenic expression had increased on ESD coatings. On basis of these results, we conclude that the novel ESD coating behaved similar to, or even better than the known RF magnetron sputter coating. Thus, ESD could be a valid addition to already existing CaP coating processes.
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PMID:Electrostatic spray deposition (ESD) of calcium phosphate coatings, an in vitro study with osteoblast-like cells. 1474 16

Alkaline-heat-treated titanium self-forms an apatite surface layer in vivo. The aim of the present study was to materialistically characterize the surface of alkaline-heat-treated titanium immersed in simulated body fluid (AHS-TI) and to examine the differentiation behavior of osteoblasts on AHS-TI. SEM, thin-film XRD, FTIR, and XPS analyses revealed that AHS-TI contained a 1.0- micro m-thick, low-crystalline, and [002] direction-oriented carbonate apatite surface. Human osteoblast-like SaOS-2 cells were cultured on polystyrene, titanium, and AHS-TI, and RT-PCR analyses of osteogenic differentiation-related mRNAs were conducted. On AHS-TI, the expression of bone sialoprotein mRNA was up-regulated as compared with that on polystyrene and titanium (p < 0.05). On AHS-TI, the expression of osteopontin and osteocalcin mRNAs was up-regulated as compared with that on polystyrene (p<0.05). The results indicate that the apatite was bone-like and accelerated the osteogenic differentiation of SaOS-2, suggesting that alkaline-heat treatment might facilitate better integration of titanium implants with bone.
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PMID:Characterization of apatite formed on alkaline-heat-treated Ti. 1515 53

Sheep mesenchymal stem cells (MSCs) were isolated and expanded using the principle of plastic adherence. Their identity as progenitor cells was confirmed by induction along the osteoblastic lineage using osteogenic supplements and observation of calcific deposits by von Kossa staining. MSCs were seeded onto two types of hyaluronan-based cylindrical scaffolds in high concentrations and cultured for varying time points up to three weeks. Culture medium was supplied using the following conditions: statically, on a shaker, by stirring with a magnetic stirrer or by perfusion in a tubular flow circuit. Total cell metabolism was assessed by MTT assay and the quality of cell coverage and matrix formation observed by SEM and histological analysis of thin sections of the constructs. Perfusion culture was established as the most appropriate culturing conditions, with cell metabolism increasing by approximately 300% over three weeks. The coverage of the scaffold surface was very good and the deposition of collagenous matrix was superior in these conditions compared to the, static and other dynamic culture conditions.
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PMID:Metabolic and histological analysis of mesenchymal stem cells grown in 3-D hyaluronan-based scaffolds. 1533 5

In craniofacial surgery, bone is needed to augment misshapen areas and to fill gaps during repair of congenital anomalies and injuries resulting into bone deficiencies. Examples of conditions requiring bone tissue include missing alveolar bone in cleft palates, bony nasal pyramid defects following removal of fistulous tracts or cysts and defects following removal of sinus and mandibular tumors. Moreover, maxillofacial neurosensory deficiencies may be caused by various surgical procedures, such as tooth extraction, osteotomies, pre-prosthetic procedures, excision of tumors or cysts, surgery of TMJ, and surgical treatment of fractures and cleft lip/palate. Therefore, a tissue engineering approach to craniofacial surgery has a crucial importance: the use of various composites with osteoconductive ceramics, polymers, bioactive factors, cells, or a combination of them, offers the possibility of rapid tissue regeneration and integration with the host tissue. In this study, a composite consisting of two well-known biomaterials, collagen/hydroxyapatite (Col/HAp), was used as a drug delivery device for neurotrophin - nerve growth factor beta (NGF beta). This delivery device, enriched with neurogenic-osteogenic factor, was analyzed in vitro and in vivo. It was implanted into calvaria defects of 20 Wistar rats, weighing 200-250 g. Implants were left in place for different periods of time. Controls were as follows: (a) contralateral defect without any implant; and (b) contralateral defect implanted with composite without NGF factor. The rats were euthanized after 30 days, and the implant sites and explants were examined clinically, histologically, SEM and histomorphometrically. Our results evidenced stimulation of periosteal and endocortical woven and lamellar bone formation, with increases in bone mass and decreases in bone marrow. We found that NGF enhanced the remodeling activity in the intracortical region, and induced an increase in the intracortical cavity number and area by the end of the study. In vitro results were in line with in vivo ones. We believe that the composite proposed in this study has considerable advantages in tissue engineering and is very suitable as a biomaterial for the filling of irregular defects in maxillo-facial surgery. Two areas of clinical research will be impacted by this system. The first is pharmaceutical research on drug delivery and high-throughput screening of neurotrophic-osteogenic compounds. Transplantation research is the second area that will benefit from the system.
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PMID:Nerve growth factor beta(NGF beta) delivery via a collagen/hydroxyapatite (Col/HAp) composite and its effects on new bone ingrowth. 1534 79

Tricalcium phosphate/hydroxyapatite (TCP/HA), hydroxyapatite (HA), chitosan and calcium sulphate (CaSO4) were studied and evaluated for possible bone tissue engineered construct acting as good support for osteogenic cells to proliferate, differentiate, and eventually spread and integrate into the scaffold. Surface morphology visualized by SEM showed that scaffold materials with additional fibrin had more cell densities attached than those without, depicting that the presence of fibrin and collagen fibers were truly a favourite choice of cells to attach. In comparison of various biomaterials used incorporated with fibrin, TCP/HA had the most cluster of cells attached.
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PMID:Evaluation of suitable biodegradable scaffolds for engineered bone tissue. 1546 86

It was recently claimed that titanium metal and its alloys can bond to the living bone, without being coated by apatite (VPS coatings), but by being chemically and heat-treated. The bioactivity of treated titanium is of interest because of the opportunity to obtain orthopaedic or dental implants presenting, at the same time, high toughness, strength and fatigue resistance as well as bone-bonding ability. The bioactive behaviour of the treated implants is due to the presence of a modified surface, which, during soaking in body fluid, promotes the precipitation of apatite. The apatite formed is strongly bonded to the substrate and promotes living bone bonding. In this work were characterised samples of Ti-6Al-7Nb alloy with surfaces presenting a different chemical and mechanical state. The aim of the research was twofold. The first objective was to characterise chemically and heat-treated samples with different surface topography, in order to define the best conditions for osteogenic integration. The second aim was to assess the corrosion behaviour of the bioactive implants, because they expose a microporous and quite thin modified surface layer. No-treated and passivated samples, with a surface state closed to that nowadays used on implants, were used as reference. The surface structure, morphology, electrochemical behaviour and bioactivity of the different samples were assessed by means of XRD, SEM-EDS, anodic polarizations, open circuit measurements and in-vitro tests. Results evidence that it is possible to modify the surface of the Ti-6Al-7Nb alloy in order to obtain the formation of a bioactive layer and that the substrate roughness influences the characteristics of the surface layer formed. It was also evidenced that the as treated surfaces present inadequate corrosion behaviour, so a new two-step chemical treatment has been developed in order to obtain a bioactive material with good corrosion resistance.
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PMID:New chemical treatment for bioactive titanium alloy with high corrosion resistance. 1574 11


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