Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca(2+) complex)] on the osteoblast-like SaOS-2 cells and the osteoclast-like RAW 264.7 cells was determined. Both polymers are non-toxic for these cells up to a concentration of <em>10</em>0 µm. In contrast to polyP, polyP (Ca(2+) complex) significantly induced hydroxyapatite formation at a concentration>> <em>10</em> µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca(2+) complex) triggered in SaOS-2 cells transcription of BMP2 (<em>bone</em> <em>morphogenetic</em> <em>protein</em> 2), a cytokine involved in maturation of hydroxyapatite-forming cells. An additional activity of polyP (Ca(2+) complex) is described by showing that this polymer impairs osteoclastogenesis. At concentrations>> <em>10</em> µm polyP (Ca(2+) complex) slows down the progression of RAW 264.7 cells to functional osteoclasts, as measured by the expression of TRAP (tartrate-resistant acid phosphatase). Finally, it is shown that <em>10</em>-<em>10</em>0 µm polyP (Ca(2+) complex) inhibited phosphorylation of IκBα by the respective kinase in RAW 264.7 cells. We concluded that polyP (Ca(2+) complex) displays a dual effect on <em>bone</em> metabolizing cells. It promotes hydroxyapatite formation in SaOS-2 cells (osteoblasts) and impairs maturation of the osteoclast-related RAW 264.7 cells.

The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), cell proliferation, and gene expression, in particular <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) expression, of human <em>bone</em> marrow osteoprogenitor cells. Human <em>bone</em> marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (<em>10</em> nM) and ascorbate (<em>10</em>0 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human osteoprogenitor cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local <em>bone</em> turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation.

Uraria crinita is an edible herb used as a natural food for childhood skeletal dysplasia. Ethyl acetate, n-butanol, and aqueous fractions of a 95% ethanol crude extract of U. crinita were obtained and the active ingredients isolated and purified using a bioguided method. In this manner, we isolated and identified a new active flavone glycoside, apigenin 6-C-β-d-apiofuranosyl(1→2)-α-d-xylopyranoside (3) and <em>10</em> known components with stimulatory activity on human osteoblast cells. The new compound 3 at <em>10</em>0 μM significantly increased alkaline phosphatase activity (114.<em>10</em> ± 4.41%), mineralization (150.<em>10</em> ± 0.80%), as well as osteopontin (1.39 ± 0.01-fold), <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2, 1.30 ± 0.04-fold), and runt-related transcription factor 2 (Runx2, 1.43 ± 0.<em>10</em>-fold) mRNA expression through the activation of the BMP-2/Runx2 pathway. Two other components, dalbergioidin (1) and byzantionoside B (9), displayed similar effects. These results show that U. crinita and its active compounds may have the potential to stimulate <em>bone</em> formation and regeneration.

This study was designed to examine the effect of aging on <em>bone</em> formation induced by recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) combined with a fibrous collagen membrane (FCM). Implantation was done subperiosteally in bilateral palatal grooves in 34 male Wistar rats divided into three age groups: a <em>10</em>-week-old group (<em>10</em>w group), a 30-week-old group (30w group) and a 70-week-old group (70w group). RhBMP-2-combined FCMs were implanted on the left palatal grooves as BMP-implanted sites (BMP site), while rhBMP-2 was not implanted on the right palatal grooves as control sites. The rats were sacrificed 6 weeks after implantation, and histometric evaluations were performed. New <em>bone</em> formation was observed in every site of each age group and the new <em>bone</em> was almost completely continuous with the original <em>bone</em>. The new <em>bone</em> volume (NBV) of the BMP site was significantly higher than that of the control site in each age group. The NBV of both the control and BMP sites were highest in the <em>10</em>w group and lowest in the 70w group. The disparity of NBV between the control and BMP sites, which indicated the response to implanted BMP excluding the effect of skeletal growth and surgical stimulation, did not significantly differ among the age groups. These results indicate that rhBMP-2-combined FCM has the ability to induce new <em>bone</em> formation continuous with original <em>bone</em> even in senescent rats. Furthermore, it appeared that, in the case of palatal subperiosteal implantation, the responsiveness to implanted BMP was independent of age, although the total volume of newly formed <em>bone</em> declined with aging.

BACKGROUND

Farnesyltransferase (FT), an essential enzyme at the downstream of mevalonate pathway, was reported to be upregulated in hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHRs) compared with myocardium of Wistar-Kyoto rats (WKYs). This upregulation was accompanied with cardiac remodeling. This study was designed to determine whether FT inhibition can alter cardiac remodeling in SHRs.

METHODS

Twelve-week-old SHRs were randomized to receive infusion of either NS or FTI-276 (307 μg/kg/d i.v. each n=<em>10</em>). WKY rats served as normal controls (n=6). Echocardiography was performed before and after intervention. SHR hearts were perfused ex vivo for the evaluation of cardiac performance, collagen deposition and biochemical changes (activation of Ras, extracellular-signal regulated kinases/ERK1/2, procollagen type І/Ш, TGF-β1, connective tissue growth factor/CTGF, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7/BMP-7 expression).

RESULTS

FTI-276 intervention decreased interventricular septum wall thickness at end- diastole (IVSd) and relative wall thickness (RWT) of SHRs (P<0.05). Three week intervention with FTI-276 attenuated hydroxyproline content (P<0.05), collagen deposition (P<0.01), Ras activation, ERK1/2 phosphorylation (P<0.01) and mRNA expression of procollagen type I, TGF-β1 and CTGF and elevated mRNA expression of BMP-7 (P<0.05) in left ventricle of SHRs.

CONCLUSIONS

The present study indicated that FT inhibition could attenuate myocardial fibrosis and partly improve cardiac remodeling in SHRs. The beneficial effects might be mediated through suppression of the activation of Ras and ERK1/2 phosphorylation pathway. The enhanced mRNA expression of BMP-7 with inhibition of TGF-β1 and CTGF mRNA expression might be an important mechanism.

<em>Bone</em> formation is linked with osteogenic differentiation of mesenchymal stem cells (MSCs) in the <em>bone</em> marrow. Microgravity in spaceflight is known to reduce <em>bone</em> formation. In this study, we used a real microgravity environment of the SJ-<em>10</em> Recoverable Scientific Satellite to examine the effects of space microgravity on the osteogenic differentiation of human <em>bone</em> marrow-derived mesenchymal stem cells (hMSCs). hMSCs were induced toward osteogenic differentiation for 2 and 7 d in a cell culture device mounted on the SJ-<em>10</em> satellite. The satellite returned to Earth after going through space experiments in orbit for 12 d, and cell samples were harvested and analyzed for differentiation potentials. The results showed that space microgravity inhibited osteogenic differentiation and resulted in adipogenic differentiation, even under osteogenic induction conditions. Under space microgravity, the expression of <em>10</em> genes specific for osteogenesis decreased, including collagen family members, alkaline phosphatase ( ALP), and runt-related transcription factor 2 ( RUNX2), whereas the expression of 4 genes specific for adipogenesis increased, including adipsin ( CFD), leptin ( LEP), CCAAT/enhancer binding <em>protein</em> β ( CEBPB), and peroxisome proliferator-activated receptor-γ ( PPARG). In the analysis of signaling pathways specific for osteogenesis, we found that the expression and activity of RUNX2 was inhibited, expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 ( BMP2) and activity of SMAD1/5/9 were decreased, and activity of focal adhesion kinase (FAK) and ERK-1/2 declined significantly under space microgravity. These data indicate that space microgravity plays a dual role by decreasing RUNX2 expression and activity through the BMP2/SMAD and integrin/FAK/ERK pathways. In addition, we found that space microgravity increased p38 MAPK and <em>protein</em> kinase B (AKT) activities, which are important for the promotion of adipogenic differentiation of hMSCs. Space microgravity significantly decreased the expression of Tribbles homolog 3 ( TRIB3), a repressor of adipogenic differentiation. Y15, a specific inhibitor of FAK activity, was used to inhibit the activity of FAK under normal gravity; Y15 decreased <em>protein</em> expression of TRIB3. Therefore, it appears that space microgravity decreased FAK activity and thereby reduced TRIB3 expression and derepressed AKT activity. Under space microgravity, the increase in p38 MAPK activity and the derepression of AKT activity seem to synchronously lead to the activation of the signaling pathway specifically promoting adipogenesis.-Zhang, C., Li, L., Jiang, Y., Wang, C., Geng, B., Wang, Y., Chen, J., Liu, F., Qiu, P., Zhai, G., Chen, P., Quan, R., Wang, J. Space microgravity drives transdifferentiation of human <em>bone</em> marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

Injuries of the Achilles tendon are relatively common with potentially devastating outcomes. Healing Achilles tendons form a fibrovascular scar resulting in a tendon which may be mechanically weaker than the native tendon. The resulting strength deficit causes a high risk for reinjury and other complications. Treatments using biologics aim to restore the normal properties of the native tendon and reduce the risk of rerupture and maximize tendon function. The purpose of this review was to summarize the current findings of various therapies using biologics in an attempt to improve the prognosis of Achilles tendon ruptures and tendinopathies. A PubMed search was performed using specific search terms. The search was open for original manuscripts and review papers limited to publication within the last <em>10</em> years. From these searches, papers were included in the review if they investigated the effects of biological augmentation on Achilles tendon repair or healing. Platelet-rich plasma may assist in the healing process of Achilles tendon ruptures, while the evidence to support its use in the treatment of chronic Achilles tendinopathies remains insufficient. The use of growth factors such as hepatocyte growth factor, recombinant human platelet-derived growth factor-BB, interleukin-6, and transforming growth factor beta as well as several <em>bone</em> <em>morphogenetic</em> <em>proteins</em> have shown promising results for Achilles tendon repair. In vitro and preclinical studies have indicated the potential effectiveness of <em>bone</em> marrow aspirate as well. Stem cells also have positive effects on Achilles tendon healing, particularly during the early phases. Polyhydroxyalkanoates (PHA), decellularized tendon tissue, and porcine small intestinal submucosa (SIS) are biomaterials which have shown promising results as scaffolds used in Achilles tendon repair. The application of biological augmentation techniques in Achilles tendon repair appears promising; however, several techniques require further investigation to evaluate their clinical application.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP‑1R) are incretin receptors that play important roles in regulating insulin secretion from pancreatic β cells. Incretin receptors are also thought to play a potential role in <em>bone</em> metabolism. Osteoblasts in animals and humans express GIPR; however, the presence of GLP-1R in these cells has not been reported to date. Thus, the aim of this study was to determine whether GLP-1R and GIPR are expressed in osteoblastic cells, and whether their expression levels are regulated by the extracellular glucose concentration. Mouse osteoblastic MC3T3-E1 cells were cultured in medium containing normal (5.6 mM) or high (<em>10</em>, 20 or 30 mM) glucose concentrations, with or without <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). RT-PCR, western blot analysis and immunofluorescence were carried out to determine GIPR and GLP-1R mRNA and <em>protein</em> expression levels. Cell proliferation was also assessed. The GLP-1R and GIPR mRNA expression levels were higher in the MC3T3-E1 cells cultured in medium containing high glucose concentrations with BMP-2 compared with the cells cultured in medium containing normal glucose concentrations with or without BMP-2. GLP-1R <em>protein</em> expression increased following culture in high-glucose medium with BMP-2 compared with culture under normal glucose conditions. However, the cellular localization of GLP-1R was not affected by either glucose or BMP-2. In conclusion, our data demonstrate that the expression of GLP-1R and GIPR is regulated by glucose concentrations in MC3T3-E1 cells undergoing differentiation induced by BMP-2. Our results reveal the potential role of incretins in <em>bone</em> metabolism.

The use of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) in spine fusion has led to concerns regarding a potential accompanying inflammatory response. This study evaluates a combination therapy (TrioMatrix®; Pioneer Surgical, Inc., Marquette, MI) comprised of a demineralized <em>bone</em> matrix (DBM), hydroxyapatite, and a nanofiber-based collagen scaffold in a rodent spine fusion model. Thirty-six athymic rats that underwent a posterolateral intertransverse spinal fusion were randomly assigned to 1 of 5 treatment groups: absorbable collagen sponge alone (ACS, negative control), <em>10</em> µg rhBMP-2 on ACS (positive control), TrioMatrix®, Grafton® (Osteotech, Inc., Eatontown, NJ), and DBX® (Synthes, Inc., West Chester, PA). Both TrioMatrix® and rhBMP-2-treated animals demonstrated <em>10</em>0% fusion rates as graded by manual palpation scores 8 weeks after implantation. This rate was significantly greater than those of the ACS, Grafton®, and DBX® groups. Notably, the use of TrioMatrix® as evaluated by microCT quantification led to a greater fusion mass volume when compared to all other groups, including the rhBMP-2 group. T2-weighted axial MRI images of the fusion bed demonstrated a significant host response associated with a large fluid collection with the use of rhBMP-2; this response was significantly reduced with the use of TrioMatrix®. Our results therefore demonstrate that a nanocomposite therapy represents a promising, cost-effective <em>bone</em> graft substitute that could be useful in spine fusions where BMP-2 is contraindicated.

In this study, the effects of incubating two clonal rat osteoblastic cell lines at different stages of differentiation, ROB-C26 (C26) and ROB-C20 (C20), with transforming growth factor-beta1 (TGF-beta1) on the gene expression of decorin, biglycan, and alkaline phosphatase were examined. C26 cells are a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes and is differentiated into osteoblasts after treatment with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2. C20 cells are a more differentiated osteoblastic cell line. Our Northern blot studies demonstrated that after treatment with TGF-beta1 (0, 0.1, 1.0, 5.0, and <em>10</em> ng/ml), a dose- and time-dependent decrease in decorin mRNA expression was found in C26 cells. In contrast, the effect of decorin mRNA with TGF-beta1 was not determined in C20 cells, since decorin mRNA expression was extremely low in this cell line even in the absence or presence of TGF-beta1. Although TGF-beta1 treatment resulted in no appreciable effect on biglycan mRNA expression in both cell lines in a dose- and time-dependent manner, it decreased significantly the expression of alkaline phosphatase in both cell lines at the gene and <em>protein</em> level. Reverse transcriptase-polymerase chain reaction analysis revealed the gene expression of decorin, and TGF-beta type I and type II receptors in both cell lines. These results indicate that osteoblasts progenitor cells express both decorin and biglycan mRNAs. In contrast, more differentiated and mature osteoblastic cells express preferentially biglycan mRNA. TGF-beta1 exerts different effects on the expression of decorin and biglycan mRNAs, and is a potent inhibitor of the gene expression of alkaline phosphatase during osteoblast differentiation.

BACKGROUND

Discography is an important diagnostic approach to identify the painful discs. However, the benefit of discography, a procedure involving needle puncture and injection of the diagnostic agent into the intervertebral disc, is controversial and has been reported to be associated with accelerated degeneration.

OBJECTIVE

To investigate the effect of lovastatin on the prevention of degeneration caused by a discography simulation procedure in rat caudal discs.

METHODS

In vivo study using rat caudal discs.

METHODS

A single flexible 27-gauge needle puncture into rat caudal discs was performed under fluoroscopic monitoring. Different concentrations (0.1, 1, 5, and <em>10</em> μM) of lovastatin were prepared and injected into randomly chosen caudal discs. RNA expression of selected genes, histologic, and immunohistochemical staining were performed to evaluate the phenotypic effects of lovastatin on rat caudal discs.

RESULTS

Simulation of the discography procedure by puncturing the rat caudal discs with a 27-gauge needle and injection of saline solution induced degenerative changes in the nucleus pulposus with minimal damage to the annulus fibrosus. Aggrecan, Type II collagen, and SOX9 expressions were upregulated, whereas Type I collagen expression was significantly suppressed in discs treated with 5 and <em>10</em> μM lovastatin. Discs treated with 5 and <em>10</em> μM lovastatin were subjected to alcian blue staining and immunohistochemistry that revealed higher levels of glycosaminoglycans and an increase in the number of cells producing S-<em>10</em>0 proteins, Type II collagen, and bone morphogenetic protein-2 (BMP-2), respectively. The most effective phenotypic repair was observed in discs treated with <em>10</em> μM lovastatin.

CONCLUSIONS

Intradiscal administration of lovastatin solution upregulated the expressions of BMP-2 and SOX9 and promoted chondrogenesis of rat caudal discs after needle puncture and substance injection. Therefore, we suggest that lovastatin promotes disc repair and can be used as a potential therapeutic agent for biological repair of disc degeneration after the diagnostic discography procedure.

BACKGROUND

A major challenge for dental implantology is to consistently obtain appropriate bone augmentation before implant placement. The aim of this study is to evaluate the effect of recombinant human bone morphogenetic protein 2 (rhBMP-2) associated with bone substitute materials beta-tricalcium phosphate (β-TCP), biphasic calcium phosphate (BCP), and bovine bone mineral on vertical guided bone regeneration (GBR) in rabbit calvarium.

METHODS

Four titanium cylinders were fixed to the calvarium of 22 rabbits. In group 1 (n = 10), three cylinders were randomly filled with one of the test materials, and one cylinder was filled with a blood clot (CL). In group 2 (n = 12), the cylinders were randomly assigned to the same materials and CL but with the addition of rhBMP-2. Bone labels were injected over the course of 13 weeks, and euthanasia was performed 14 weeks after surgery in both groups.

RESULTS

The mean volume and area of tissue growth was greater in group 2 (with rhBMP-2) than in group 1 (without rhBMP-2), irrespective of the material used (P <0.001). The mean volume of tissue growth in the CL cylinder was smaller than that observed with all other materials (P <0.001) in both groups. The mean area of regenerated bone in the CL cylinder was smaller than that observed in the β-TCP cylinder (P = 0.028). The histologic study revealed more lamellar bone in the rhBMP-2 group, with a greater level of biodegradation of all the bone substitute materials tested.

CONCLUSIONS

The use of rhBMP-2/absorbable collagen sponge (ACS) combined with all of the bone substitute materials tested resulted in a greater amount of bone formation than that produced with the bone substitute materials alone or rhBMP-2/(ACS) and CL using the rabbit calvarium GBR model.

OBJECTIVE

Cell-matrix interactions promote cartilage homeostasis. We previously found that Smad1, the transcriptional modulator of the canonical bone morphogenetic protein 7 (BMP-7) pathway, interacted with the cytoplasmic domain of CD44, the principal hyaluronan receptor on chondrocytes. To elucidate the physiologic function of CD44-Smad1 interactions, as well as the role of hyaluronan, we studied the response of chondrocytes isolated from CD44(-/-) and BALB/c (wild-type [WT]) mice to stimulation with BMP-7.

METHODS

In primary murine chondrocytes, CD44 expression was decreased by small interfering RNA (siRNA) transfection or was enhanced by plasmid transfection. Pericellular hyaluronan was removed by hyaluronidase treatment, or its endogenous synthesis was inhibited. Changes in response to BMP-7 stimulation were evaluated by Western blotting of Smad1 phosphorylation and aggrecan messenger RNA (mRNA) expression.

RESULTS

Chondrocytes from CD44(-/-) mice and WT mice transfected with CD44 siRNA were less responsive than untransfected chondrocytes from WT mice to BMP-7. CD44(-/-) mouse chondrocytes transfected with pCD44 showed increased sensitivity to BMP-7. Significant increases in aggrecan mRNA were observed in WT mouse chondrocytes in response to 10 ng/ml of BMP-7, whereas at least 100 ng/ml of BMP-7 was required for CD44(-/-) mouse chondrocytes. However, in chondrocytes from CD44(-/-) and WT mice, hyaluronidase treatment decreased cellular responses to BMP-7. Treatment of both bovine and murine chondrocytes with 4-methylumbelliferone to reduce the synthesis of endogenous hyaluronan confirmed that hyaluronan promoted BMP-7 signaling.

CONCLUSIONS

Taken together, these investigations into the mechanisms underlying BMP-7 signaling in chondrocytes revealed that while hyaluronan-dependent pericellular matrix is critical for BMP-7 signaling, the expression of CD44 promotes the cellular response to lower concentrations of BMP-7.

This study aimed to determine whether treadmill walking (TW) prevents the progression of post-traumatic osteoarthritic changes in cartilage-subchondral bone unit, and whether the exercise timing changes the exercise efficacy in destabilized medial meniscus (DMM) rat knees.

Twelve-week-old male Wistar rats underwent DMM surgery on their right knees and sham surgery on their left knees and were assigned to either the sedentary (n = 10) or walking (n = 24) groups. The rats in the walking group were subjected to TW from day 2 through 4 weeks, from 4 through 8 weeks, or from day 2 through 8 weeks (n = 8 per group). Osteoarthritic changes of cartilage and subchondral bone were assessed with micro-computed tomography, histology, and immunohistochemistry 8 weeks after surgery.

TW prevented the progression of cartilage and subchondral bone lesions induced by the DMM, and increased bone morphogenetic protein (BMP)-2 and -6 expressions in superficial zone chondrocytes and bone-lining cells including osteoblasts. Furthermore, the TW-induced increase in BMPs varied with the exercise timing. Beginning TW 4 weeks after DMM surgery was the best option for increasing BMPs, coinciding with the most robust prevention of osteoarthritic changes.

TW increased the expression of BMPs and prevented the progression of cartilage-subchondral bone lesions in rat knees with a DMM. Selective exercise timing may be a key factor in the development of an exercise regimen for preventing the progression of post-traumatic osteoarthritis (PTOA). Furthermore, exercise may have favorable effects even after the PTOA has been developed.

OBJECTIVE

The osteogenic potential of a graft is based on the parallel lines ability of cells to survive transplantation and to respond to local factors that stimulate new bone formation. Here we investigated the potential of cells that had grown out of porcine cortical bone grafts obtained by 3 preparation techniques to respond to mitogenic and osteogenic stimuli.

METHODS

Bone grafts were harvested from 2 pigs. Cortical bone was taken in cylindrical form and ground in a bone mill or harvested via drilling and aspiration.

RESULTS

Cell outgrowth was observed in all cortical bone cylinders, bone cylinders homogenized by mill, and 5 of 10 explants of bone dust collected upon drilling. After a 2-week culture period, the number of outgrown cells did not significantly differ among the 3 preparations. Bone cells showed increased proliferation in response to platelet-released supernatants as determined by 3 [H]-thymidine incorporation assay. When cultured under conditions that favor the expression of an osteogenic phenotype, the outgrown cells expressed alkaline phosphatase activity and transcripts of the osteoblast-specific marker osteocalcin. Individual cell preparations showed accumulation of mineral salts in their extracellular matrix. Bone cells also increased alkaline phosphatase activity in response to bone morphogenetic protein (BMP)-2, BMP-6, and BMP-7. The mitogenic and osteogenic response was obtained with cells from the mandible as well as from the maxilla, irrespective of the preparation technique.

CONCLUSIONS

These data show that cortical bone grafts contain cells that have the ability to proliferate and differentiate into the osteogenic lineage, suggesting that these cells can contribute to bone regeneration following transplantation.

Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since <em>protein</em> adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for <em>10</em> days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory <em>protein</em>-1delta (MIP-1δ), platelet-derived growth factor-BB, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in <em>bone</em> homeostasis and wound healing, such as growth hormone, transforming growth factor-β3, and insulin-like growth factor-binding <em>proteins</em>, as well as several angiogenic mediators, including endocrine gland-derived vascular endothelial factor, fibroblast growth factor-7, and placental growth factor, were significantly promoted by Fg. This work provides a new perspective on the inflammatory response in the context of <em>bone</em> repair/regeneration mediated by a pro-inflammatory <em>protein</em> (Fg) adsorbed onto a biomaterial (Ch) that does not otherwise exhibit osteogenic properties.

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote <em>bone</em> formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (<em>10</em>.9-fold enrichment, P = 2.4 × <em>10</em>-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to <em>10</em>% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Synthetic graft materials are considered as possible substitutes for cancellous <em>bone</em>, but lack osteogenic and osteoinductive properties. In this study, we investigated how composite scaffolds of βTCP containing osteogenic human <em>bone</em> marrow mesenchymal stem cells (hBMSCs) and osteoinductive <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) influenced the process of fracture healing. hBMSCs were loaded into βTCP scaffolds 24 h before implantation in a rat critical-sized <em>bone</em> defect. hBMSCs were either stimulated with rhBMP-2 or transduced with BMP-2 by gene transfer. The effect of both <em>protein</em> stimulation and gene transfer was compared for osteogenic outcome. X-rays were conducted at weeks 0, 1, 3, 6, 9 and 12 post-operatively. In addition, <em>bone</em>-labelling fluorochromes were applied at 0, 3, 6 and 9 weeks. Histological analysis was performed for the amount of callus tissue and cartilage formation. At 6 weeks, the critical-sized defect in 33% of the rats treated with the Ad-BMP-2-transduced hBMSCs/βTCP scaffolds was radiographically bridged. In contrast, in only <em>10</em>% of the rats treated with rhBMP2/hBMSCs, 12 weeks post-treatment, the <em>bone</em> defect was closed in all treated rats of the Ad-BMP-2 group except for one. Histology showed significantly higher amounts of callus formation in both Ad-BMP-2- and rhBMP-2-treated rats. The amount of neocartilage was less pronounced in both BMP-2-related groups. In summary, scaffolds with BMP-2-transduced hBMSCs performed better than those with the rhBMP2/hBMSCs <em>protein</em>. These results suggest that combinations of osteoconductive biomaterials with genetically modified MSCs capable of secreting osteoinductive <em>proteins</em> may represent a promising alternative for <em>bone</em> regeneration. Copyright © 2015 John Wiley & Sons, Ltd.

BACKGROUND

In this study the induction of bone formation in an axially vascularized bone matrix using mesenchymal stem cells (MSCs) and application of bone morphogenetic protein 2 (BMP2) was analyzed in the arteriovenous loop (AVL) model.

METHODS

An AVL was created in the medial thigh of 42 rats and placed in a porous titanium chamber filled with a particulated porous hydroxyapatite and beta-tricalcium phosphate matrix and fibrin. In group A the fibrin was loaded with 5×10(6) DiI-stained fibrin gel-immobilized primary MSCs from syngenic Lewis rats, in group B the matrix was loaded with 60 μg/mL BMP2 and in group C both, BMP2 and MSCs were applied at implantation time point. After 6 and 12 weeks, specimens were investigated by means of histological, morphometrical, and micro-computed tomography analysis.

RESULTS

After implantation of an AVL a dense vascular network was visible in all groups. In group A, newly generated bone islands were detected in the periphery of the main vascular axis. Using BMP2 alone (group B), small islands of newly formed bone were visible evenly distributed in all parts of the constructs. In group C nearly the whole matrix was interspersed with bone formations. In all groups there was an increase of bone formation between the 6 and 12 weeks explantation time points.

CONCLUSIONS

This study demonstrates for the first time the successful generation of axially vascularized bone substitutes using MSCs and BMP2 in the AVL rat model using a one step procedure. Using the combination of BMP2 and MSCs there was a significant increase of bone formations detectable compared to the BMP2 or MSCs alone groups.

BACKGROUND

In response to increasing use of lumbar fusion for improving back pain, despite unclear efficacy, particularly among injured workers, some insurers have developed limited coverage policies. Washington State's workers' compensation (WC) program requires imaging confirmation of instability and limits initial fusions to a single level. In contrast, California requires coverage if a second opinion supports surgery, allows initial multilevel fusion, and provides additional reimbursement for surgical implants. There are no studies that compare population-level effects of these policy differences on utilization, costs, and safety of lumbar fusion.

OBJECTIVE

The purpose of this study was to compare population-level data on the use of complex fusion techniques, adverse outcomes within 3 months, and costs for two states with contrasting coverage policies.

METHODS

The study design was an analysis of WC patients in California and Washington using the Agency for Healthcare Research and Quality's State Inpatient Databases, 2008-2009.

METHODS

All patients undergoing an inpatient lumbar fusion for degenerative disease (n=4,628) were included the patient sample.

METHODS

Outcome measures included repeat lumbar spine surgery, all-cause readmission, life-threatening complications, wound problems, device complications, and costs.

METHODS

Log-binomial regressions compared 3-month complications and costs between states, adjusting for patient characteristics.

RESULTS

Overall rate of lumbar fusion operations through WC programs was 47% higher in California than in Washington. California WC patients were more likely than those in Washington to undergo fusion for controversial indications, such as nonspecific back pain (28% versus 21%) and disc herniation (37% versus 21%), as opposed to spinal stenosis (6% versus 15%), and spondylolisthesis (25% versus 41%). A higher percentage of patients in California received circumferential procedures (26% versus 5%), fusion of three or more levels (<em>10</em>% versus 5%), and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (50% versus 31%). California had higher adjusted risk for reoperation (relative risk [RR] 2.28; 95% confidence interval [CI], 2.27-2.29), wound problems (RR 2.64; 95% CI, 2.62-2.65), device complications (RR 2.49; 95% CI, 2.38-2.61), and life-threatening complications (RR 1.31; 95% CI, 1.31-1.31). Hospital costs for the index procedure were greater in California ($49,430) than in Washington ($40,114).

CONCLUSIONS

Broader lumbar fusion coverage policy was associated with greater use of lumbar fusion, use of more invasive operations, more reoperations, higher rates of complications, and greater inpatient costs.

BACKGROUND

Simvastatin increases the expression of bone morphogenetic protein 2 (BMP-2) in osteoblasts, therefore it is important to investigate the influence of statins on bone formation, fracture healing and implant integration. The aim of the present study was to investigate the effect of simvastatin, locally applied from intramedullary coated and bioactive implants, on bone integration using biomechanical and histomorphometrical analyses.

METHODS

Eighty rats received retrograde nailing of the femur with titanium implants: uncoated vs. polymer-only (poly(D,L-lactide)) vs. polymer plus drug coated (either simvastatin low- or high dosed; "SIM low/ high"). Femurs were harvested after 56 days for radiographic and histomorphometric or biomechanical analysis (push-out).

RESULTS

Radiographic analysis revealed no pathological findings for animals of the control and SIM low dose group. However, n=2/10 animals of the SIM high group showed osteolysis next to the implant without evidence of bacterial infection determined by microbiological analysis. Biomechanical results showed a significant decrease in fixation strength for SIM high coated implants vs. the control groups (uncoated and PDLLA). Histomorphometry revealed a significantly reduced total as well as direct bone/implant contact for SIM high- implants vs. controls (uncoated and PDLLA-groups). Total contact was reduced for SIM low vs. uncoated controls. Significantly reduced new bone formation was measured around SIM high coated implants vs. both control groups.

CONCLUSIONS

This animal study suggests impaired implant integration with local application of simvastatin from intramedullary titanium implants after 8 weeks when compared to uncoated or carrier-only coated controls.

OBJECTIVE

The purpose of this study was to explore the mechanisms of postoperative intestinal motility disorders in intestinal atresia patients by investigating the expression profiles of proteins, including calretinin (CR), glial-derived neurotrophic factor (GDNF), bone morphogenetic protein 2 (BMP-2), c-kit, α-smooth muscle actin (α-SMA), and S-100 protein; to decipher the correlation between the area of the pathological segment and the alteration of the above 6 proteins; and thereby to provide a clinical specific reference values to determine the removal length for intestinal tract resection.

METHODS

Immunohistochemistry technique was applied to detect the CR, c-kit, GDNF, BMP-2, α-SMA, and S-100 protein in specimens of atretic, proximal, and distal intestine from 25 cases of intestinal atresia and samples of intestinal walls from 10 non-atresia control specimens. The alteration of the enteric nervous system, nerve growth and its regulatory factors, the interstitial cells of Cajal (ICCs), and the enteric muscle system were examined, with particular attention being paid to pathological changes and the lesion area.

RESULTS

The expression of all of the abovementioned 6 proteins in the proximal side of the atresia was significantly lower than in control group. The expression of the abovementioned proteins tended to be higher farther away from the atresia site. The expressions of both GDNF and BMP-2 had returned to normal level at 10 cm proximal to the atresia site, whereas the expressions of CR, c-kit, α-SMA, and S-100 protein only returned to normal at 15 cm proximal to the atresia site. On the distal side, the expression of all 6 markers at 3 cm distal to the atresia site was normal.

CONCLUSIONS

Pathological deterioration of the myenteric ganglia, nerve growth factor, and ICCs are the causes of intestinal motility disorders after the surgical repair of intestinal atresia. Our data support resecting an intestinal segment extending from 15 cm proximal to 3 cm distal to the atretic segment. In proximal jejunal atresia, when it is not possible to resect 15 cm, we suggest resecting as much of the hypertrophic proximal intestine as possible. Based on our data, we believe this surgical practice could improve postoperative dysmotility in these patients.

There is increasing evidence that inorganic calcium-polyphosphates (polyP) are involved in human bone hydroxyapatite (HA) formation. Here we investigated the morphology of the particles, containing calcium phosphate (CaP) with different concentrations of various Na-polyP concentrations, as well as their effects in cell culture. We used both SaOS-2 cells and human mesenchymal stem cells. The polymeric phosphate readily binds calcium ions under formation of insoluble precipitates. We found that addition of low concentrations of polyP (<10wt.%, referred to the CaP deposits) results in an increased size of the HA crystals. Surprisingly, at higher polyP concentrations (>10wt.%) the formation of crystalline HA is prevented and amorphous polyP/HA hybrid particles with a size of ≈50nm are formed, most likely consisting of polyP molecules linked via Ca(2+) bridges to the surface of the CaP deposits. Further studies revealed that the polyP-CaP particles cause a strong upregulation of the expression of the genes encoding for two marker proteins of bone formation, collagen type I and alkaline phosphatase. Based on their morphogenetic activity the amorphous polyP-CaP particles offer a promising material for the development of bone implants, formed from physiological inorganic precursors/polymers.

UNASSIGNED

Hydroxyapatite (HA) is a naturally occurring mineral of vertebrate bone. Natural HA, a bio-ceramic material which is crystalline to different scale, has been used as a biomaterial to fabricate scaffolds for in situ bone regeneration and other tissue engineering purposes. In contrast to natural HA, synthetic apatite is much less effective. In general, while HA is bioactive, its interaction and biocompatibility with existing bone tissue is low. These properties have been attributed to a minimal degradability in the physiological environment. In the present study we introduce a new Ca-phosphate (CaP) fabrication technology, starting from calcium chloride and dibasic ammonium phosphate with the HA characteristic Ca/P molar ratio of 10:6 and report that after addition >10% (by weight) of polyphosphate (polyP) amorphous CaP/HA samples were obtained. Those samples elicits strong morphogenetic activity let us to conclude that polyP/HA-based material might be beneficial for application as bone substitute implant.

We examined the effect of recombinant human osteogenic <em>protein</em>-1 (OP-1, or <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7), a member of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (<em>10</em>-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and <em>protein</em> synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-beta 1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.