Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-<em>protein</em> coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first <em>10</em>-20 s), whereas changes in chromatin occurred at a later time point (∼<em>10</em> min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/<em>bone</em> <em>morphogenetic</em> <em>proteins</em> signaling pathway contributes to the establishment of baseline contractility in MSCs.

Vascular calcification is highly correlated with atherosclerosis and cardiovascular disease and is a significant predictor of cardiovascular morbidity and mortality. Studies in mice indicate a genetic contribution to this dystrophic extra osseous calcification. We sought to elaborate a method to induce dystrophic arterial calcification in mice and further examine the pathogenetical mechanisms involved in the phenotype. We established a method of freeze-thaw injury of the infrarenal aorta producing a limited tissue necrosis and histologically investigated the occurrence of dystrophic calcification within the aortic wall 1, 3 and 7 days after injury in C57BL/6 (a mouse strain shown to be resistant to dystrophic cardiac calcification after injury) and C3H/He (susceptible to dystrophic cardiac calcification). C57BL/6 mice exhibited no dystrophic calcification at all within the vessel wall upon injury of the infrarenal aorta (0/5 mice 1 day after injury and 0/<em>10</em> animals 7 days after injury). By contrast C3H/He mice displayed a remarkable extent of calcification mainly present within the media of the infrarenal aorta which was evident as early as 24 h (three out of five animals 1 day after injury) and reached its maximum extent 7 days after injury (<em>10</em> out of <em>10</em> animals at the seventh postoperative day, p<0.001 compared to C57BL/6 mice). Upon immuno-histological analysis calcification was accompanied by the occurrence of certain <em>bone</em>-matrix associated <em>proteins</em>. Osteopontin and <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em> 2/4 expression was detected co-localized with the calcified lesions. Our results demonstrate that freeze-thaw injury of the infrarenal aorta is a sufficient method to induce dystrophic arterial calcification in mice. We present evidence that the occurrence of arterial calcification in C3H/He mice seems to be actively regulated by certain <em>bone</em>-matrix associated <em>proteins</em>.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) promotes <em>bone</em> growth but must be retained at the delivery site for optimal efficacy in vivo. rhBMP-2 release from a collagen-based matrix has shown favorable pharmacokinetics. The present study assessed binding affinity and binding saturation of rhBMP-2 to a collagen matrix as a function of solution and rhBMP-2 isoform variables. Results indicate that rhBMP-2 binds to the collagen matrix with affinities on the order of <em>10</em>(3) to <em>10</em>(4) M(-1). Maximum binding, nu, was primarily a function of pH for heterogeneous rhBMP-2 and the extended (T(266)/T(266)) isoform. However, binding saturation of the <Q(283)/<Q(283) isoform was unaffected by pH. Overall, binding saturation was higher than the calculated saturation of a rhBMP-2 monolayer, suggesting both hydrophobic and ionic interactions in a multilayer formation. The contributions of pH and ionic strength to the linkage free energy of interaction was on the order of 1.3 kcal mol(-1) and approximately 0.3 kcal mol(-1), respectively. This thermodynamic approach can serve to optimize interactions between therapeutic <em>proteins</em> and delivery systems.

BACKGROUND

Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7).

METHODS

MDMSCs were cultured as a monolayer at a density of 1 × 105 cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression.

RESULTS

The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1.

CONCLUSIONS

The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage.

OBJECTIVE

Irradiation of the chest or chest wall has been shown to cause calcific aortic stenosis. However, the mechanisms are unknown. Aortic valve interstitial cells have been implicated in the pathogenesis of aortic stenosis; they have been shown to change from the phenotype of a myofibroblast to an osteoblastlike cell. We therefore hypothesized that irradiation of human aortic valve interstitial cells induces an osteogenic phenotype. In isolated human aortic valve interstitial cells, our purpose was to determine the effect of irradiation on the production of osteogenic factors: (1) bone morphogenetic protein 2, (2) osteopontin, (3) alkaline phosphatase, and (4) the transcription factor Runx2.

METHODS

Human aortic valve interstitial cells were isolated from normal aortic valves obtained from explanted hearts of patients undergoing cardiac transplantation (n = 4) and were grown in culture. The cells were grown to confluence, irradiated with 10 Gy using a cesium-137 irradiator, and then lysed 24 hours after irradiation. Cell lysates were analyzed via immunoblot and densitometry for bone morphogenetic protein 2, osteopontin, alkaline phosphatase, and Runx2. Statistical analysis was performed using analysis of variance, with P < .05 indicating significance.

RESULTS

Irradiation induced an osteogenic phenotype in human aortic valve interstitial cells. Irradiation induced a 2-fold increase in bone morphogenetic protein 2, a 7-fold increase in osteopontin, a 3-fold increase in alkaline phosphatase, and a 2-fold increase in Runx2.

CONCLUSIONS

Radiation induces an osteogenic phenotype in human aortic valve interstitial cells. The irradiated cells had a significantly increased expression of the osteogenic factors bone morphogenetic protein 2, osteopontin, alkaline phosphatase, and Runx2. These data offer mechanistic insight into the pathogenesis of radiation-induced valvular heart disease.

OBJECTIVE

Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal, tendon, and ligament morphogenesis. The mRNA encoding GDF-5 is also expressed during odontogenesis, especially in dental follicle tissue. While this suggests that GDF-5 participates in the formation of alveolar bone and the periodontal ligament, cementum, and dental root, the physiologic role of GDF-5 in these tissues in adulthood remains unclear. We therefore investigated GDF-5 effects upon cultures of human periodontal ligament (HPDL) cells.

METHODS

HPDL cells were obtained from healthy periodontal ligaments of individuals. Tetrazolium reduction assay was carried out for cell proliferation assay. Alkaline phosphatase (ALP) activity was estimated by measuring light absorbance at 405 nm. Reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis were performed for gene expression in cultured HPDL cells. Sulfated glycosaminoglycan (sGAG) synthesis was evaluated by histochemical staining and a quantitative dye-binding method.

RESULTS

Expression of GDF-5 and its receptor was demonstrated in HPDL cells by RT-PCR. ALP activity in HPDL cells was significantly decreased by addition of rhGDF-5 at 10-1000 ng/ml (p < 0.05). Although northern analysis showed little change in gene expression for collagen alpha2(I) in rhGDF-5-stimulated HPDL cells, rhGDF-5 dose-dependently enhanced cell proliferation. This proliferative effect persisted for 16 d. Alcian blue staining and dye-binding assays indicated that sGAG synthesis was enhanced by rhGDF-5.

CONCLUSIONS

rhGDF-5 may provide an environment fostering periodontal healing or regeneration by affecting extracellular matrix metabolism.

Reconstruction of a segmental fracture with massive bone loss is still a challenge for orthopaedic surgeons. The aim of our study was to develop a suitable biodegradable thermosensitive hydrogel system as a carrier for bone morphogenetic protein (BMP)-2 delivery in the treatment of critical-sized femoral defects.

A block copolymer composed of monomethoxypoly(ethylene glycol) (mPEG), poly(lactic-co-glycolic acid) (PLGA) and 2, 2'-Bis (2-oxazolin) (Box) was synthesized by ring opening polymerization. The synthesized block copolymer was characterized by (1)H-NMR spectroscopy and gel permeation chromatography (GPC). Different biophysical and biochemical properties of the synthesized copolymer, including temperature-induced structure changes, degradation rate, pH changes during hydrolytic degradation, cell toxicity, and the release profile of BMP-2, were also evaluated and/or were compared with those of a well-characterized mPEG-PLGA copolymer. In animal testing, rabbits (n = 36) that received critically sized (10 mm) femoral defects were divided into 6 groups. These experimental groups included an untreated group, autograft, and groups treated with the synthesized copolymer carrying different concentrations of BMP-2 (0, 5, 10, and 20 μg/ml). Bone repair was evaluated using X-ray radiography, histological staining, micro-computed tomography (μCT), biomarker examination and biomechanical testing in a 12-week treatment period.

A new thermosensitive mPEG-PLGA/Box/mPEG-PLGA block copolymer, or named as BOX copolymer, was successfully prepared. Compared to the reported mPEG-PLGA in vitro, the prepared BOX copolymer at the same weight percent concentrations exhibited wider temperature ranges of gelation, slower degradation rates, higher the pH values, as well as less cytotoxicity. Furthermore, the BMP-2 release from BOX hydrogel exhibited a near-linear release profile in vitro. In animal experiments, treatment of critical-sized bony defects with 25 wt% BOX hydrogel carrying BMP-2 effectively promoted fracture healing during the 12-week trial period and higher concentrations of BMP-2 treatment correlated with better bone quality. Most importantly, clinical outcome and bone healing in the BOX-hydrogel group with 20 μg/ml BMP-2 were nearly equivalent to those in the autograft group in a 12-week treatment course.

These data support that the use of BOX hydrogel (25 wt%) as a drug delivery system is a promising method in the treatment of large bone defects.

<em>Bone</em> loss around tooth extraction sites can occur, thus making future placement of dental implants difficult. Alveolar <em>bone</em> regeneration can be guided by the application of a nanofibrous <em>bone</em> graft coupled with osteoinductive <em>proteins</em>/peptides, following tooth loss or tooth extraction. In the present study, we demonstrate the potential of mineralized nanofiber segments coupled with calcium-binding <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) mimicking peptides for periodontal <em>bone</em> regeneration. Thin electrospun nanofiber membranes of PLGA-collagen-gelatin (2:1:1 wt ratios) were mineralized in <em>10</em>× modified simulated body fluid (<em>10</em>× mSBF) and cryocut to segments of 20 µm. For predetermined weights of the mineralized nanofiber segments, it was possible to load various amounts of heptaglutamate E7-domain-conjugated BMP-2 peptide. Mineralized short fiber grafts (2 mg), with and without E7-BMP-2 peptides, were implanted into 2 mm × 2 mm (diameter × depth) critical-sized socket defects created in rat maxillae, following extraction of the first molar teeth. A sustained release profile of E7-BMP-2 from the mineralized nanofiber segments was recorded over 4 weeks. X-ray microcomputed tomography (µ-CT) analysis of peptide-loaded nanofiber graft filled defects revealed ∼3 times greater new <em>bone</em> volume and <em>bone</em> mineral density over 4 weeks in comparison to unfilled control defects. Further, histopathology data confirmed the formation of greater new osseous tissue in the BMP2 peptide-loaded, mineralized nanofiber segment group than that of fibrous connective tissue in the unfilled defect group. Altogether, the mineralized nanofiber segments coupled with E7-BMP-2 peptides may be an effective treatment option for alveolar <em>bone</em> loss and defects. STATEMENT OF SIGNIFICANCE: With the high incidence of dental implants/fixtures for missing teeth, the success of the surgical procedures in restorative dentistry is dictated by the quality and quantity of the supporting alveolar <em>bone</em>. To address the problem of alveolar <em>bone</em> loss and defects due to tumor, periodontitis, or even postextraction remodeling, the present study is the first report on the application of mineralized nanofiber fragments coupled with calcium-binding osteoinductive BMP-2 peptides as a synthetic graft material for oral <em>bone</em> regeneration. The ease of fabrication and application of cryocut mineralized nanofiber fragments as maxillofacial <em>bone</em> defect fillers present a promising alternative to the current dental <em>bone</em> graft formulations. Furthermore, the nanofiber segments may also be utilized for several biomedical applications including hemostasis, soft tissue engineering, and wound healing.

During development, growth factors (GFs) such as <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo, the extracellular matrix (ECM) not only provides support for adherent cells, but also acts as reservoir of GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell trans-membrane receptors, such as integrins. In conveying adhesion-mediated signaling to the intracellular compartment, integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors. Here, we present a strategy for the immobilization of BMP-2 onto cellular fibronectin (cFN), a key <em>protein</em> of the ECM, to investigate GF-mediated signaling and migration. Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin as cross-linker. Characterization with quartz crystal microbalance with dissipation monitoring and enzyme-linked immunosorbent assay confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h. To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2), we investigated short- and long-term responses of C2C12 myoblasts, which are an established in vitro model for BMP-2 signaling, in comparison to soluble BMP-2 (sBMP-2) or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation of the complex to the nucleus, corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after 6 days in sBMP-2 and iBMP-2. We next implemented this approach in the fabrication of cFN micropatterned stripes by soft lithography. These stripes allowed cell-surface interaction only on the patterned cFN, since the surface in between was passivated, thus serving as platform for studies on directed cell migration. During a <em>10</em>-h observation time, the migratory behavior, especially the cells' net displacement, was increased in presence of BMP-2. As such, this versatile tool retains the bioactivity of GFs and allows the presentation of ECM adhesive cues.

Alendronate (ALN) is an antiresorptive agent widely used for the treatment of osteoporosis. Its suppressive effect on osteoclasts has been extensively studied. However, the effect of ALN on <em>bone</em> formation is not as clear as its effect on resorption. The objective was to determine the effect of short-term ALN on <em>bone</em> formation and tooth extraction wound healing. Molar tooth extractions were performed in mice. ALN, parathyroid hormone (PTH), or saline (vehicle control) was administered. PTH was used as the <em>bone</em> anabolic control. Mice were euthanized at 3, 5, 7, <em>10</em>, and 21 d after extractions. Hard tissue healing was determined histomorphometrically. Neutrophils and lymphatic and blood vessels were quantified to evaluate soft tissue healing. Gene expression in the wounds was assessed at the RNA level. Furthermore, the vossicle <em>bone</em> transplant system was used to verify findings from extraction wound analysis. Alkaline phosphatase (ALP) was visualized in the vossicles to assess osteoblast activity. ALN exhibited no negative effect on <em>bone</em> formation. In intact tibiae, ALN increased <em>bone</em> mass significantly more than PTH did. Consistently, significantly elevated osteoblast numbers were noted. In the extraction sockets, <em>bone</em> fill in the ALN-treated mice was equivalent to the control. Genes associated with <em>bone</em> <em>morphogenetic</em> <em>protein</em> signaling, such as bmp2, nog, and dlx5, were activated in the extraction wounds of the ALN-treated animals. <em>Bone</em> formation in vossicles was significantly enhanced in the ALN versus PTH group. In agreement with this, ALN upregulated ALP activity considerably in vossicles. Neutrophil aggregation and suppressed lymphangiogenesis were evident in the soft tissue at 21 d after extraction, although gross healing of extraction wounds was uneventful. <em>Bone</em> formation was not impeded by short-term ALN treatment. Rather, short-term ALN treatment enhanced <em>bone</em> formation. ALN did not alter <em>bone</em> fill in extraction sockets.

BACKGROUND

Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear.

METHODS

We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways.

RESULTS

FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages.

CONCLUSIONS

Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

OBJECTIVE

This study compared the histologic parameters and outcomes of two types of grafts in large vertical maxillary defects: a composite graft of recombinant human bone morphogenetic protein-2/acellular collagen sponge (rhBMP-2/ACS), crushed cancellous freeze-dried allogeneic bone (CCFDAB), and platelet-rich plasma (PRP); and size-matched 100% autogenous grafts.

METHODS

Twenty patients each were treated with a composite graft, which contained 1.05 mg rhBMP-2/ACS per two-tooth segment together with CCFDAB and PRP, or a 100% autogenous graft prior to implant placement. Grafting material was contained within a titanium mesh crib.

RESULTS

Two grafts in each group were lost as a result of early mesh exposure and infection. Three grafts in each group developed a late exposure of the mesh that did not affect bone regeneration. The remaining 18 autogenous grafts all regenerated sufficient bone for implant restoration (100%), and 17 of 18 (97.4%) of the composite grafts regenerated sufficient bone for implant restoration. The autogenous grafts included 54% ± 10% of new viable bone but also included residual nonviable graft particles. The composite grafts contained 59% ± 12% viable new bone and no remaining nonviable bone particles. The composite grafting technique resulted in less blood loss and shorter surgical time but greater and longer-lasting edema. The costs of both grafts were nearly equal.

CONCLUSIONS

A composite graft of rhBMP-2/ACS-CCFDAB-PRP regenerates bone in large vertical ridge augmentations as predictably as 100% autogenous graft with less morbidity, equal cost, and more viable new bone formation without residual nonviable bone particles, but with more edema. This composite graft represents an in situ tissue engineering concept that is able to achieve results equivalent to autogenous grafts in large vertical ridge augmentations without donor bone harvesting.

Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH) is a rare disease characterized by proliferation and occlusion of small pulmonary arterioles, leading to progressive elevation of pulmonary artery pressure and pulmonary vascular resistance, and right ventricular failure. Historically, it has been associated with a high mortality rate, although, over the last decade, treatment has improved survival. PAH includes idiopathic PAH (IPAH), heritable PAH (HPAH), and PAH associated with certain medical conditions. The aetiology of PAH is heterogeneous, and genetics play an important role in some cases. Mutations in BMPR2, encoding <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2, a member of the transforming growth factor-β superfamily of receptors, have been identified in 70% of cases of HPAH, and in <em>10</em>-40% of cases of IPAH. Other genetic causes of PAH include mutations in the genes encoding activin receptor-like type 1, endoglin, SMAD9, caveolin 1, and potassium two-pore-domain channel subfamily K member 3. Mutations in the gene encoding T-box 4 have been identified in <em>10</em>-30% of paediatric PAH patients, but rarely in adults with PAH. PAH in children is much more heterogeneous than in adults, and can be associated with several genetic syndromes, congenital heart disease, pulmonary disease, and vascular disease. In addition to rare mutations as a monogenic cause of HPAH, common variants in the gene encoding cerebellin 2 increase the risk of PAH by approximately two-fold. A PAH panel of genes is available for clinical testing, and should be considered for use in clinical management, especially for patients with a family history of PAH. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic <em>bone</em> formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H<em>10</em>T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, <em>10</em>% stretch). In the presence of mechanical stress, the expression of BMP2 <em>protein</em> in C3H<em>10</em>T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to <em>bone</em> transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.

METHODS

Prospective, randomized, controlled preclinical trial.

OBJECTIVE

This study seeks to characterize the localized and systemic host response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a well established rodent spine arthrodesis model utilizing cytokine analysis and magnetic resonance imaging (MRI).

BACKGROUND

Although high fusion rates are achieved with rhBMP-2 in the spine, several complications have also been reported, including a localized response leading to radiculitis and seroma formation. The mechanism in which this occurs clinically is yet unknown.

METHODS

One hundred female Fischer rats underwent a posterolateral intertransverse lumbar spinal fusion, with paraspinal muscle tissue resection, using iliac crest autograft, type I absorbable collagen sponge (ACS), 10- or 100-μg rhBMP-2/ACS. The animals underwent magnetic resonance imaging evaluation, serum cytokine analysis, manual palpation, and gross tissue inspection at 2, 4, 7, 10, and 21 days, postoperatively.

RESULTS

Qualitative evaluation of MR images demonstrated a transient fluid collection at the surgery site in the rhBMP-2 animals as early as 4 and 7 days that was greater than the autograft or ACS groups. Quantitative analysis on T2-weighted axial images demonstrated greater signal intensity in the rhBMP-2 animals compared with the ACS and autograft groups in a time-dependent fashion. Higher concentrations of several cytokines were also detected at 2, 4, and 7 days, including interleukin 1β, interleukin 18, tumor necrosis factor α, macrophage inflammatory protein 1α, and monocyte chemotactic protein 1 in animals treated with rhBMP-2/ACS relative to ACS alone.

CONCLUSIONS

Our data suggest that the in vivo host response to rhBMP-2 in an animal model may be associated with circulating proinflammatory and osteoclastic cytokines.

The <em>10</em>-12 nm diameter microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main <em>protein</em> component is the calcium-dependent glyco<em>protein</em> fibrillin, which assembles into microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin microfibrils are further engaged in a number of cell matrix interactions such as with integrins, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 (FBN1) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of <em>10</em>-12 nm diameter microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the <em>10</em>-12 nm diameter microfibril and perform such diverse roles.

Zoledronic acid (ZA), which is one of the most potent and efficacious bisphosphonates, has been commonly used in clinical practice for the treatment of various <em>bone</em> disorders. The extensive use of ZA has been associated with increasing occurrence of jaw complications, now known as bisphosphonate‑associated osteonecrosis of the jaw (BRONJ). However, the mechanism underlying BRONJ remains to be fully elucidated. The aim of the present study was to investigate the effects of different concentrations of ZA on the MC3T3‑E1 murine preosteoblast cell line cells and examine the possible pathogenesis of BRONJ. In the present study, the effect of ZA on the viability, apoptosis, differentiation and maturation of MC3T3‑E1 cells, as well as its relevant molecular mechanism, were examined The results of a Cell Counting Kit 8 assay, a flow cytometric Annexin‑V/propidium iodide assay and western blot analysis demonstrated that ZA exhibited a significant inhibition of cell viability and induction of apoptosis at concentrations>><em>10</em> µM. Subsequently, the effect of ZA on cell differentiation at concentrations <1 µM were investigated. In this condition, ZA inhibited <em>bone</em> nodule formation and decreased the activity of alkaline phosphatase. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses indicated that ZA downregulated the expression levels of the marker genes and <em>proteins</em> associated with osteogenic differentiation. Further investigation revealed that the suppression of differentiation by ZA was associated with decreased expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>‑2 (BMP‑2) and downregulation of the phosphorylation levels in the downstream extracellular signal‑regulated kinase 1/2 and p38 pathways. These adverse effects of ZA were observed to be concentration‑dependent. The results from the present study suggested that ZA at higher concentrations induces cytotoxicity towards osteoblasts, and ZA at lower concentrations suppresses osteoblast differentiation by downregulation of BMP-2. These results assist in further understanding the mechanisms of BRONJ.

OBJECTIVE

To study the effects of maxillary sinus floor elevation by a tissue engineered bone complex with bone morphogenetic protein-2 (BMP-2) gene modified bone marrow stromal cells (bMSCs) and a novel porous ceramic scaffold (OsteoBone) in rabbits.

METHODS

bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with adenovirus AdBMP-2 and with AdEGFP gene (without BMP-2 gene sequence) as a control, respectively, in vitro. These bMSCs were then combined with OsteoBone scaffold at a concentration of 2 x 10(7)cells/ml and used to elevate the maxillary sinus floor in rabbits. Eight rabbits were randomly allocated into groups and sacrificed at weeks 2 and 4. For each time point, 8 maxillary sinus floor elevation surgeries were made bilaterally in 4 rabbits for the two groups (n=4 per group): group A (AdBMP-2-bMSCs/material) and group B (AdEGFP-bMSCs/material). All samples were evaluated by histologic and histomorphometric analysis.

RESULTS

The augmented maxillary sinus height was maintained for both groups over the entire experimental period, while new bone area increased over time for group A. At week 4 after operation, bone area in group A was significantly more than that in group B (P<0.05), and was more obviously detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed in the EGFP group, which suggests that those implanted bMSCs had contributed to the new bone formation.

CONCLUSIONS

bMSCs modified with AdBMP-2 gene can promote new bone formation in elevating the rabbit maxillary sinus. OsteoBone scaffold could be an ideal carrier for gene enhanced bone tissue engineering.

Alteration in the control of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-regulated genes and increased expression of endothelin (ET)-1 are both believed to play important roles in the still incompletely understood pathobiology of pulmonary vascular remodeling and fibrosis. Recent studies have drawn attention to the contribution of adventitial fibroblast activation in these phenomena. Because chloride channels are involved in the control of physiological function of fibroblasts, we hypothesized that these channels are differentially regulated by BMPs and ET. We measured chloride ion currents by whole-cell path-clamping in cultured primary human pulmonary fibroblasts. The application of BMP2 prevented activation of these currents by hypotonic challenge in a time- and dose-dependent manner, partially via <em>protein</em> kinase C signaling. Maximal inhibition was observed after 45-minute incubation of cells in the presence of <em>10</em> ng/ml of BMP2. ET-1 did not activate chloride channels acutely; however, prolonged treatment of cells with ET-1 (<em>10</em>0 nM, 2 h) induced the appearance of lysophosphatidic acid-activated chloride currents (a marker of differentiated myofibroblasts), and this induction could be effectively blocked by BMP2 pretreatment (<em>10</em> ng/ml). BMP2 also prevented stimulation of α-smooth muscle actin gene expression and cell migration of fibroblasts induced by ET-1. We conclude that ET-1 and BMP2 have opposing effects on chloride channel activity in human fibroblasts. This is a potentially relevant mechanism involved in pulmonary vascular remodeling and fibrosis.

BACKGROUND

Traumatic muscle loss (i.e., volumetric muscle loss [VML] injury) impairs adjacent fracture healing but is often left untreated. A promising therapy for this application is a decellularized extracellular matrix (ECM) because of their capacity to regenerate a vascularized tissue bed. This study tested the hypothesis that repair of VML concomitant to fracture with a small intestine submucosa (SIS)-ECM improves musculoskeletal healing.

METHODS

In male Lewis rats (~375 g), a 3-mm segmental <em>bone</em> defect (SBD) was created in concomitance with a 6-mm, full-thickness VML injury to the adjacent tibialis anterior (TA) muscle. For all rats (n = <em>10</em>), the SBD was treated with internal plate fixation and delivery of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (1 μg) on a collagen sponge. The VML either had no repair or SIS-ECM repair (n = 5/group). <em>Bone</em> regeneration within the SBD (BV/TV [<em>bone</em> volume as a fraction of total volume]) was assessed via in vivo micro-computed tomography at 2, 4, and 6 weeks and histology at 6 weeks after injury. Tibialis anterior muscle in vivo strength and histologic assessments were performed at 6 weeks after injury.

RESULTS

Compared with no repair, SIS-ECM presented -21% (p = 0.09) and -27% (p = 0.004) BV/TV at 4 and 6 weeks after injury, respectively. At 6 weeks, the SBD gap length was shorter for the no repair than that for the SIS-ECM (2.64 ± 0.30 and 3.67 ± 0.41 mm, respectively; p = 0.09), whereas the distances from the end of each cortical segment to the center of the first stabilization screw were longer (1.86 ± 0.25 and 0.85 ± 0.30 mm, respectively; p = 0.035), indicating enhanced resorption in the SIS-ECM group. Both groups presented similar magnitude TA muscle strength deficits compared with their contralateral limbs (<em>10</em>-150 Hz: no repair, -58% to 67%; SIS-ECM, -51% to 74%). The TA muscle of the SIS-ECM group was remarkable for its presentation of fibrosis, edema, and immune cell presence.

CONCLUSIONS

Small intestine submucosa-ECM VML repair impaired open fracture healing and failed to improve skeletal muscle strength.

Osteogenic <em>Protein</em>-1 (OP-1/BMP-7) is a <em>bone</em> <em>morphogenetic</em> <em>protein</em> in the transforming growth factor-beta superfamily and has been shown to be expressed temporally and spatially during epithelial-mesenchymal interactions mediating tissue morphogenesis in early embryogenesis. In order to identify the primary role(s) for OP-1 in development, we carried out whole rat embryo cultures, over a 72-h period from primitive streak stages to early limb bud stages, in rat sera containing either OP-1 blocking antibodies (<em>10</em> micrograms/ml) or nonreactive IgG. Rat embryos cultured with control antibodies developed normally, while those cultured with anti-OP-1 antibodies consistently exhibited over-all reduced size and absence of eyes. Histological sections revealed a greater reduction in neural retina development in the embryos treated with anti-OP-1 blocking antibodies. In situ hybridization and immunolocalization analyses indicate that OP-1 is expressed in the neuroepithelium of the optic vesicle at E11.5, is limited to the presumptive neural retina and developing lens placode, and is subsequently expressed in the neural retina, lens and developing cornea at E12.5-E13.5. Our results indicate that OP-1 mediates the inductive signals involved in mammalian eye development.

In this study, the capacity of hBMP-4 gene therapy combined with tissue-engineering techniques to improve the repair of mandibular osseous defects in rabbits was explored. A mammalian plasmid vector expressing enhanced green fluorescent <em>protein</em>-human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4 (pEGFP-hBMP-4) was initially constructed through subcloning techniques. <em>Bone</em>-marrow stromal cells (bMSCs) from New Zealand White rabbits were cultured and either transfected with pEGFP-hBMP-4 or pEGFP, or left untransfected in vitro. Once the transfer efficiency was determined through the expression of EGFP, cells from the three groups were combined with natural non-organic <em>bone</em> (NNB) at a concentration of 50 x <em>10</em>(6)cells/ml and placed in 15 mm x 6 mm bilateral, full-thickness, mandibular defects surgically made in 12 rabbits. Together with NNB control, there were six samples per group. Four weeks after surgery, the implants were harvested and evaluated histomorphologically. Under optimal experimental conditions, gene transfer efficiency reached a maximum of 38.2+/-9.4%. While the percentage of new <em>bone</em> area in the NNB control group was 8.8+/-3.1%, in the untransfected bMSC group 22.5+/-8.2%, and in the pEGFP group 18.1+/-9.0%, a significantly higher amount of 32.5+/-6.1% was observed in the pEGFP-hBMP-4 group. These results suggest that transfection of bMSCs with hBMP-4 enhances their inherent osteogenic capacity for maxillofacial <em>bone</em> tissue-engineering applications.

Osseointegration [direct <em>bone</em>-implant contact (BIC)] is a primary goal following installation of endosseous dental implants. Such <em>bone</em> contact provides stability for the dental implant over time. The objective of this study was to evaluate <em>bone</em> formation and BIC at long-term, functionally loaded, endosseous dental implants placed into <em>bone</em> induced by recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier. Mandibular, saddle-type, alveolar ridge defects (approximately 15 x <em>10</em> x <em>10</em> mm), two per jaw quadrant, were surgically induced in each of six young adult American fox hounds. The defects were immediately implanted with rhBMP-2/ACS. Two defects per animal additionally received a nonresorbable expanded polytetrafluoroethylene (ePTFE) membrane or a bioresorbable polyglycolide fiber membrane. Healing was allowed to progress for 3 months, when the ePTFE membrane was removed, and machined, threaded, titanium dental implants were installed into the rhBMP-2/ACS induced <em>bone</em> and into the adjacent resident <em>bone</em>. At 4 months of osseointegration, the implants were exposed to receive abutments and prosthetic treatment (two- or three-unit bridges). Some implants were removed for histologic analysis. The remainder of implants were exposed to functional loading for 12 months at which time the animals were killed for histometric analysis. One animal died prematurely due to kidney failure unrelated to the experimental protocol and was not included in the analysis. The 12-month block sections from a second animal were lost in the histological processing. Four sites receiving rhBMP-2/ACS and ePTFE or resorbable membranes experienced wound failure and membrane exposure, and subsequently exhibited limited <em>bone</em> formation. Defects without wound failure filled to contour with the adjacent alveolar <em>bone</em>. The newly formed <em>bone</em> exhibited features of the resident <em>bone</em> with a re-established cortex; however, it commonly included radiolucent areas that resolved over time. Dental implants block biopsied at 4 months exhibited limited, if any, crestal resorption, whereas those exposed to functional loading for 12 months exhibited some crestal resorption. Implants biopsied at 4 months exhibited a mean (+/- SD) BIC of 40.6 +/- 8.2% in rhBMP-2/ACS induced <em>bone</em> vs. 52.7 +/- 11.4% in resident <em>bone</em>. Dental implants exposed to 12 months of functional loading exhibited a mean BIC of 51.7 +/- 7.1% in rhBMP-2/ACS induced <em>bone</em> vs. 74.7 +/- 7.0% in resident <em>bone</em>. There were no significant differences between dental implants placed into rhBMP-2/ACS induced <em>bone</em> and resident <em>bone</em> for any parameter at any observation interval. In conclusion, rhBMP-2/ACS-induced <em>bone</em> allows installation, osseointegration, and long-term functional loading of machined, threaded, titanium dental implants in dogs.

Twisted gastrulation (TWSG1), an extracellular regulator of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling, is critical for embryonic brain development. Mice deficient in TWSG1 have abnormal forebrain development manifesting as holoprosencephaly. The expression and potential roles of TWSG1 in postnatal brain development are less well understood. We show that Twsg1 is expressed in the adult mouse brain in the choroid plexus (CP), hippocampus, and other regions, with the strongest expression observed in CP. TWSG1 was also detected in a human fetal brain at mid-gestation, with highest levels in the epithelium of CP. Bmp1, Bmp2, Bmp4-Bmp7 as well as BmprIA and BmprII, but not BmprIB, were expressed in CP. BMP antagonists Chordin (Chrd) and Noggin were not detected in CP, however Chrd-like 1 and brain-specific Chrd-like (Brorin) were expressed. Electrophysiological study of synaptic plasticity revealed normal paired-pulse facilitation and long-term potentiation in the CA1 region of hippocampus in Twsg1(-/-) mice. Among the homozygous mutants that survive beyond the first 2 weeks, the prevalence of hydrocephalus was 4.3%, compared to 1.5% in a wild type colony (P=0.0133) between 3 and <em>10</em> weeks of life. We detected a high level of BMP signaling in CP in wild type adult mice that was 17-fold higher than in the hippocampus (P=0.005). In contrast, transforming growth factor beta (TGFbeta) signaling was predominant in the hippocampus. Both BMP signaling and the expression of BMP downstream targets Msx1 and Msx2 were reduced in CP in Twsg1(-/-) mice. In summary, we show that Twsg1 is expressed in the adult mouse and human fetal CP. We also show that BMP is a branch of TGFbeta superfamily that is dominant in CP. This presents an interesting avenue for future research in light of the novel roles of CP in neural progenitor differentiation and neuronal repair, especially since TWSG1 appears to be the main regulator of BMP present in CP.