We reported previously that <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-<em>10</em> mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Coordinated regulation of the activities of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) and its inhibitors is essential for skeletal development since loss-of-function experiments show that both BMPs and BMP inhibitory signals, such as noggin, are required to establish proper formation of skeletal tissues. In this paper, we asked how and when noggin would be functional to interact with BMPs during skeletogenesis in mammals. For this purpose, we first analyzed the spatial and temporal patterns of noggin, BMP-2, BMP-4, and BMP-7 expression during early skeletogenesis in mouse embryos. In situ hybridization study revealed that noggin expression was detected at a low level in limb mesenchyme, whereas BMP-7 was expressed at a high level throughout limb mesenchyme <em>10</em>.5 days postcoitum (dpc) in mouse embryos. One day later, noggin mRNA was expressed at a high level in the prechondrogenic condensations in appendicular and axial skeletal primordia, where sox9 transcripts were also expressed. At this stage, noggin-expressing cells were surrounded by those expressing BMP-7. The chondrogenic cell condensation continued to express noggin transcripts in 12.5 dpc and 13.5 dpc embryos, and again the noggin-expressing cells within the cartilaginous tissue were surrounded by those expressing BMP-7. We further examined interaction of noggin and BMPs by using organ cultures of 11.5 dpc mouse forelimbs and found that implantation of carriers containing BMP-7 <em>protein</em> into the forelimb explants induced noggin expression in the limb mesenchyme. BMP-7 also induced type II collagen and sox9 mRNAs in the same cell population, indicating that noggin induction occurred in the chondrogenic precursor cells. BMP-7 effects on noggin expression were observed in a dose-dependent manner within a dose range of <em>10</em>-<em>10</em>0 ng/microliter. These results suggest that BMP-7 induced expression of noggin transcripts within skeletal cell condensation and that this noggin expression in turn could act antagonistically to attenuate BMP action in the early skeletogenesis.

Muscle-based gene therapy and tissue engineering hold great promise for improving <em>bone</em> healing. However, the relative advantage of muscle-derived stem cells (MDSCs) or primary muscle-derived cells (MDCs) remains to be defined. We compared the ability of MDSCs and different subpopulations of MDCs (PP1 and PP3) to induce <em>bone</em> formation via ex vivo gene therapy. We were able to efficiently transduce the MDSCs and all the other evaluated populations of MDCs (efficiency of transduction = approximately 80%) by using a retroviral vector expressing human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4). All the transduced cell populations secreted high levels of BMP4 (140-300 ng/<em>10</em>(6) cells/24 h), but the MDSCs differentiated toward the osteogenic lineage more effectively than did the other muscle cell populations, as indicated by the expression of alkaline phosphatase, an early osteogenic marker. von Kossa staining indicated that mineralized <em>bone</em> formed as early as 7 days after implantation of any of the BMP4-expressing cell populations into immunocompetent syngeneic mice; however, MDSCs expressing BMP4 produced significantly more <em>bone</em> than did the other MDC populations, as evidenced by both histomorphometry and biochemical analysis. Further investigation revealed that MDSCs expressing BMP4 persisted for a significantly longer period of time at the <em>bone</em> forming sites than did the other BMP4-expressing MDC populations. Additionally, MDSCs expressing BMP4 triggered a smaller infiltration of CD4 lymphocytes within the <em>bone</em> forming areas than did the other MDC populations expressing BMP4. Finally, we demonstrated that MDSCs expressing BMP4 can heal a critical-sized skull <em>bone</em> defect in immunocompetent mice. In summary, this study shows that MDSCs are better than primary MDCs for use as cellular vehicles in BMP4-based ex vivo gene therapy to improve <em>bone</em> healing. The advantage of MDSCs may be attributable, at least in part, to their lower immunogenicity and higher capacity for in vivo survival.

Tooth eruption in the rat requires <em>bone</em> resorption resulting from a major burst of osteoclastogenesis on postnatal day 3 and a minor burst of osteoclastogenesis on postnatal day <em>10</em> in the alveolar <em>bone</em> of the first mandibular molar. The dental follicle regulates the major burst on postnatal day 3 by down-regulating its osteoprotegerin (OPG) gene expression to enable osteoclastogenesis to occur. To determine the role of receptor activator of nuclear factor-kappa B ligand (RANKL) in tooth eruption, its gene expression was measured on postnatal days 1-11 in the dental follicle. The results show that RANKL expression was significantly elevated on postnatal days 9-11 in comparison to low expression levels at earlier time-points. As OPG expression is high at this latter time-point, this increase in RANKL expression would be needed for stimulating the minor burst of osteoclastogenesis. Tumor necrosis factor-alpha enhances RANKL gene expression in vitro and it may be responsible for up-regulating RANKL in vivo. Transforming growth factor-beta1 and interleukin-1alpha also enhance RANKL gene expression in vitro but probably have no effect in vivo because they are maximally expressed early. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-2 acts to down-regulate RANKL expression in vitro and, in vivo, may promote alveolar <em>bone</em> growth in the basal region of the tooth.

Etanercept (ETN), which is a recombinant human soluble tumor necrosis factor (TNF) receptor that inhibits TNF activity, is effective in the treatment of rheumatoid arthritis. We investigated the effect of ETN on recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2)-induced ectopic <em>bone</em> formation in vivo. A block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG polymer) was used as the delivery system. Polymer discs (6 mm, 30 mg) containing 5 microg rhBMP-2 were implanted into the left dorsal muscle pouch of mice (n = 50). In the systemic administration groups (n = 5 per group), ETN was subcutaneously injected (25 mg/human = 12.5 microg/mouse) twice per week in a dose-dependent manner (placebo, 12.5 x <em>10</em>(-3), 12.5 x <em>10</em>(-1), 12.5, 125 microg), whereas a single dose of ETN (placebo, 12.5 x <em>10</em>(-3), 12.5 x <em>10</em>(-1), 12.5, 125 microg) was embedded in each rhBMP-2 polymer disc in the local administration groups (n = 5 per group). Three weeks after implantation, the mice were killed and the implants were analyzed. Implants in the optimally dosed groups had increased radiodensity, which was consistent with a significant increase in <em>bone</em> mineral content of the ossicles. <em>Bone</em> histomorphology revealed a significant increase in <em>bone</em> volume/total volume, number of osteoblasts, osteoblast surface/<em>bone</em> surface, and a significant decrease in the number of osteoclasts, osteoclast surface/<em>bone</em> surface in the optimal dosed systemic and locally administered groups. These data suggest that the optimal dose of ETN, administered either systemically or locally, enhanced the <em>bone</em>-inducing capacity of BMP with no apparent adverse systemic effects.

Fibrodysplasia ossificans progressiva (FOP) causes extensive heterotopic <em>bone</em> formation due to heterozygous mutations in the glycine-serine activation domain of ACVR1 (ALK2), a <em>bone</em> <em>morphogenetic</em> <em>protein</em> type I receptor. Anecdotal observations of facial similarity have been made by clinicians and parents, but no objective quantitative analysis of the faces of FOP patients has ever been undertaken. We delineated the common facial characteristics of 55 individuals with molecularly confirmed FOP by analyzing their face signature (face shape difference normalized against age and sex matched controls) and associated face signature graphs (with face signatures as vertices and adjacency corresponding to greatest similarity). Our analysis identified <em>10</em> affected individuals whose face signature is more homogeneous than others with FOP. This distinct subgroup showed the previously identified reduced mandible as well as newly identified features: underdevelopment of the upper orbit/supra-orbital ridge; infra-orbital prominence; and, low-set ears. These findings strongly suggest that the canonical FOP mutation variably affects the postnatal morphogenesis of the normotopic cranial skeleton in the upper midface and mandible and may have important diagnostic and functional implications.

OBJECTIVE

Bone morphogenetic proteins (BMPs) represent a distinct subset of the transforming growth factor-beta family best known for their role in joint formation and bone growth, and several recent clinical trials have begun to look at their efficacy in the augmentation of fracture healing. The goal of this research is to examine the effect of BMP-14, also known as growth differentiation factor-5 and cartilage- derived morphogenetic protein-1 (GDF-5, CDMP-1) on fracture healing by studying the long bone repair process in mice with a deficiency in this signaling peptide.

METHODS

The animal model used for these studies was the BMP-14 (-/-) brachypodism (bp) mouse. Phenotypically normal heterozygous (+/-) littermates were used as controls. Closed mid shaft femur fractures were created and stabilized with intramedullary fixation in 8-week-old female mice. Forty-eight mice per genotype group were examined. On postoperative days 4, 7, 10, 14, 21, 28, 35, and 42, the mice were killed and the femurs and repair tissue were harvested for analysis. At each time point, the fracture sites were analyzed radiographically, histologically, and biochemically. For all quantitative analyses, the data were normalized and analyzed statistically using a 2-factor ANOVA test.

RESULTS

Biochemically, peak values of normalized proteoglycan content were approximately 3 times less in the mutant fractures early in the time course of healing compared with the controls (P < 0.05). Histologically, BMP-14-deficient fractures exhibited a delay in peak area cell density, callus organization, and bone formation compared with controls. Radiographic analysis demonstrated that the peak callus was 2 weeks delayed and approximately 2 times less in the mutants compared with controls (P < 0.05). Radiographic grading of callus also demonstrated a significant difference after day 14.

CONCLUSIONS

Based on histologic, radiographic, and biochemical analysis, BMP-14-deficient mice display a short-term delay in healing of approximately 1 to 2 weeks. The observed abnormalities seem to be the result of a delay in cellular recruitment and chondrocyte differentiation in the early stages fracture repair in the absence of BMP-14. These results support the hypothesis that BMP-14 deficiency leads to a delay in fracture healing. Further studies are warranted to more closely examine the role of BMP-14 in normal fracture healing and the mechanism by which it works.

Nonsteroidal antiinflammatory drugs (NSAIDs) are known to potentially impair the fracture healing process. The aim of the present study was to determine if the impairment of <em>bone</em> healing by systemic NSAID application is, at least in part, due to an interaction of NSAIDs with the <em>bone</em> anabolic BMP-7 pathway. Therefore, we first analyzed fracture healing in control and diclofenac-treated mice, where we not only found a significant impairment of fracture healing due to diclofenac treatment as assessed by biomechanical testing and microCT imaging, but also found high coexpression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) and cyclooxygenase-2 (COX-2) within the fracture callus of both groups. To experimentally address the possible interaction between BMP-7 and COX-2, we then induced ectopic <em>bone</em> formation in control (n = <em>10</em>) and diclofenac-treated mice (n = <em>10</em>) by application of BMP-7 (recombinant human OP-1, rhOP-1) into the hamstring muscles. After 20 days of treatment, each ectopic <em>bone</em> nodule was analyzed by contact-radiography, microCT, histology, and histomorphometry. Diclofenac application decreased the trabecular number and <em>bone</em> mass in the ectopic <em>bone</em> nodules significantly due to reduced osteoblast number and activity. These data demonstrate that the <em>bone</em> anabolic effect of BMP-7 and fracture healing is impaired by diclofenac application, and suggest that the potential negative impact of NSAIDs on fracture healing is, at least in part, due to interference with BMP-7 signaling.

BACKGROUND

Secondary alveolar cleft reconstruction using autologous iliac crest bone graft is currently the standard treatment for alveolar clefts. Although effective, harvesting autologous bone may result in considerable donor-site morbidity, most commonly pain and the potential for long-term sensory disturbances. In an effort to decrease patient morbidity, a novel technique using recombinant human bone morphogenetic protein (rhBMP)-2 encased in a demineralized bone matrix scaffold was developed as an alternative to autografting for secondary alveolar cleft reconstruction.

METHODS

A chart review was conducted for the 55 patients who underwent secondary alveolar cleft reconstruction over a 2-year period with a mean follow-up of 21 months. Of these, 36 patients received rhBMP-2/demineralized bone matrix scaffold (including 10 patients with previously failed repairs using iliac crest bone grafting) and 19 patients underwent iliac crest bone grafting. Postoperatively, bone stock was evaluated using occlusal radiographs rated according to the Bergland and Chelsea scales.

RESULTS

Alveolar clefts repaired using rhBMP-2/demineralized bone matrix scaffold were 97.2 percent successful compared with 84.2 percent with iliac crest bone grafting. Radiographically, initial repairs with rhBMP-2/demineralized bone matrix scaffold were superior to iliac crest bone grafting according to both Bergland and Chelsea scales, and significantly more patients in the rhBMP-2/demineralized bone matrix scaffold group had coronal bridging. The postoperative intraoral infection rate following iliac crest bone grafting was significantly greater than for rhBMP-2/demineralized bone matrix scaffold. The cost of rhBMP-2/demineralized bone matrix scaffold products was offset by cost savings associated with a reduction in operative time averaging 102 minutes.

CONCLUSIONS

rhBMP-2 encased in a demineralized bone matrix scaffold appears to be a viable alternative for secondary alveolar cleft repair. Patients are spared donor-site morbidity and achieve excellent results, decreasing operative time, and increasing operating room use.

METHODS

Therapeutic, III.

BACKGROUND

Icariine is a flavonoid isolated from a traditional Chinese medicine Epimedium pubescens and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis. But the mechanism is unclear. The aim of the study was to research the effect of Icariine on the proliferation and differentiation of human osteoblasts.

METHODS

Human osteoblasts were obtained by inducing human marrow mesenchymal stem cells (hMSCs) directionally and were cultured in the presence of various concentrations of Icariine. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of Icariine on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of calcified nodules were assayed to observe the effect on cell differentiation. The expression of bone morphogenetic protein 2 (BMP-2) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

Icariine (20 microg/ml) increased significantly the proliferation of human osteoblasts. And, Icariine (10 microg/ml and 20 microg/ml) increased the activity of ALP and the amount of calcified nodules of human osteoblasts significantly (P < 0.05). BMP-2 mRNA synthesis was elevated significantly in response to Icariine (20 microg/ml).

CONCLUSIONS

Icariine has a direct stimulatory effect on the proliferation and differentiation of cultured human osteoblast cells in vitro, which may be mediated by increasing production of BMP-2 in osteoblasts.

To clarify the mechanism of the stimulatory effect of statins on <em>bone</em> formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of <em>bone</em> from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (<em>10</em>(-9) M, <em>10</em>(-8) M, <em>10</em>(-7) M, <em>10</em>(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the <em>bone</em>-forming effect of statins, probably through the BMP2 pathway.

Chorioamnionitis and antenatal corticosteroids mature the fetal lung functionally but disrupt late-gestation lung development. Because Sonic Hedgehog (Shh) signaling is a major pathway directing lung development, we hypothesized that chorioamnionitis and antenatal corticosteroids modulated Shh signaling, resulting in an altered fetal lung structure. Time-mated ewes with singleton ovine fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) and/or maternal intramuscular betamethasone 7 and/or 14 days before delivery at 120 days gestational age (GA) (term = 150 days GA). Intra-amniotic LPS exposure decreased Shh mRNA levels and Gli1 <em>protein</em> expression, which was counteracted by both betamethasone pre- or posttreatment. mRNA and <em>protein</em> levels of fibroblast growth factor <em>10</em> and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4, which are important mediators of lung development, increased 2-fold and 3.5-fold, respectively, 14 days after LPS exposure. Both 7-day and 14-day exposure to LPS changed the mRNA levels of elastin (ELN) and collagen type I alpha 1 (Col1A1) and 2 (Col1A2), which resulted in fewer elastin foci and increased collagen type I deposition in the alveolar septa. Corticosteroid posttreatment prevented the decrease in ELN mRNA and increased elastin foci and decreased collagen type I deposition in the fetal lung. In conclusion, fetal lung exposure to LPS was accompanied by changes in key modulators of lung development resulting in abnormal lung structure. Betamethasone treatment partially prevented the changes in developmental processes and lung structure. This study provides new insights into clinically relevant prenatal exposures and fetal lung development.

Basic fibroblast growth factor (bFGF) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) are actively pursued for stimulation of <em>bone</em> formation. To assess their promise for systemic therapy of osteoporosis, we ascertained the effects of bFGF and BMP-2 on <em>bone</em> marrow cells in vitro. <em>Bone</em> marrow cells were obtained from young (8 weeks) and adult (32 weeks) rats by femoral aspiration and were exposed to osteogenic medium (ie, basal medium with <em>10</em> mM beta-glycerolphosphate and <em>10</em>0 nM dexamethasone) containing the growth factors. The cell viability in osteogenic medium was reduced after 3 weeks but not if the concentration of beta-glycerolphosphate/dexamethasone was reduced to 3 mM/30 nM. Unlike BMP-2, bFGF at 2-50 ng/mL was capable of enhancing long-term cell viability. Continuous treatment of <em>bone</em> marrow cells for 3 weeks resulted in dose-dependent stimulation of mineralization by BMP-2, but not by bFGF, whose activity was optimal at 2-<em>10</em> ng/mL. To explore the effect of short-term exposure, <em>bone</em> marrow cells were treated with growth factors for 1 week and subsequent mineralization was investigated. BMP-2 exposure increased the extent of mineralization, but bFGF was not effective after the short exposure. We concluded bFGF was more potent (ie, required lower concentration) for stimulating osteogenic parameters, but BMP-2 effects were lasting on the <em>bone</em> marrow cells.

Mesenchymal stem cells (MSCs) were treated with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently and time-dependently. Together they caused a strong synergistic effect on the osteogenic differentiation of MSCs, with lower concentrations of each factor being enough to show the synergistic promotion (50 ng BMP-2/ml, 1 ng VEGF/ml and <em>10</em> ng bFGF/ml). When both VEGF and bFGF were added in the early proliferating stage (the first 7 days) and BMP-2 was added in the late differentiation stage (the last 7 days), osteogenic differentiation of MSCs could be enhanced more effectively.

OBJECTIVE

The goal of this study was to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would induce new bone formation in an internally stabilized segmental defect with a chronic bacterial infection in the rat femur and whether treatment with systemic antibiotic would enhance this effect.

METHODS

A 6-mm unilateral femoral segmental defect was surgically created in 120 Sprague-Dawley rats, internally stabilized with a polyacetyl plate and 6 Kirschner wires, and contaminated with 10(4) colony-forming units of Staphylococcus aureus. After 2 weeks, all defects were surgically debrided and implanted with 0, 20, or 200 microg of rhBMP-2 in a type 1 bovine collagen sponge. Half of the animals in each treatment group received 4 weeks of systemic antibiotic, and half did not. Animals were euthanized at 4 or 12 weeks after debridement. Bone formation within and adjacent to the defect was assessed using microcomputed tomography, torsional failure testing and undecalcified histology.

RESULTS

No substantial callus formed in the infected defects without rhBMP-2. Significantly more mineralized callus was induced with the higher dose of rhBMP-2 than with the lower dose (P = 0.001), with systemic antibiotic therapy than without (P < 0.001), and at 12 weeks after debridement compared with 4 weeks (P < 0.001).

CONCLUSIONS

Recombinant human bone morphogenetic protein-2 maintained its osteoinductive capability in the presence of a chronic infection, and this property was enhanced by systemic antibiotic. This study presents an intervention that may potentially accelerate fracture healing in the presence of infection and colonized hardware, thereby permitting earlier removal of the hardware, and more timely and effective treatment of infection.

Current methods for engineering immobilized, 'solid-phase' growth factor patterns have not addressed the need for presentation of the growth factors in a biologically-relevant context. We developed an inkjet printing methodology for creating solid-phase patterns of unmodified growth factors on native biological material substrates. We demonstrate this approach by printing gradients of fluorescently labeled <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and insulin-like growth factor-II (IGF-II) bio-inks on fibrin-coated surfaces. Concentration gradients were created by overprinting individual substrate locations using a dilute bio-ink to modulate the surface concentration of deposited growth factor. Persistence studies using fluorescently-labeled BMP-2 verified that the gradients retained their shape for up to 7 days. Desorption experiments performed with (125)I-BMP-2 and (125)I-IGF-II were used to quantify the surface concentration of growth factor retained on the substrate for up to <em>10</em> days in serum containing media after rinsing of the unbound growth factor. The inkjet method is programmable so the gradient shape can be easily modified as demonstrated by printed linear gradients with varying slopes and exponential gradients. In addition, the versatility of this method enabled combinatorial arrays of multiple growth factors to be created by printing overlapping patterns. The overlapping printing method was used to create a combinatorial square pattern array consisting of various surface concentrations of BMP-2 and fibroblast growth factor-2 (FGF-2). C2C12 myogenic precursor cells were seeded on the arrays and alkaline phosphatase staining was performed to determine the effect of FGF-2 and BMP-2 surface concentration on guiding C2C12 cells towards an osteogenic lineage. These results demonstrate the utility of inkjet printing for creating orthogonal growth factor gradients to investigate how combinations of immobilized growth factors influence cell fate.

BACKGROUND

Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated.

RESULTS

Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume.

CONCLUSIONS

TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.

Nanoparticle (NP)-based delivery has gained importance for improving the potency of therapeutic agents. The bovine serum albumin (BSA) NPs, obtained by a coacervation process, was modified by electrostatic adsorption of cationic polyethylenimine (PEI) to NP surfaces for delivery of <em>bone</em>-inducing growth factor, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). Different concentrations of PEI were utilized for coating BSA NPs to stabilize the colloidal system and to control the release of BMP-2. The NPs were characterized by size and zeta potential measurements, as well as by Scanning Electron Microscopy and Atomic Force Microscopy. The encapsulation efficiency was typically >90% in all NP preparations. In vitro release kinetics showed that the PEI concentration used for coating the NPs efficiently controlled the release of BMP-2, demonstrating a gradual slowing, sustained release pattern during a <em>10</em>-day study period. The bioactivity of the encapsulated BMP-2 and the toxicity of the NPs were examined by the alkaline phosphatase (ALP) induction assay and the MTT assay, respectively, using C2C12 cells. The results indicated that PEI was the primary determinant of NP toxicities, and BSA NPs coated with 0.1 mg/mL PEI demonstrated tolerable toxicity, retained the bioactivity of BMP-2, and efficiently slowed the release rate of BMP-2. We conclude that BMP-2 encapsulated in BSA NPs might be an efficient way to deliver the <em>protein</em> for in vivo <em>bone</em> induction.

The epidermis is maintained by epidermal stem cells (ESCs) that reside in distinct niches and contribute to homeostasis and wound closure. Keratinocytes at the nonhealing edges of venous ulcers (VUs) are healing-incompetent, hyperproliferative, and nonmigratory, suggesting deregulation of ESCs. To date, genes which regulate ESC niches have been studied in mice only. Utilizing microarray analysis of VU nonhealing edges, we identified changes in expression of genes harboring regulation of ESCs and their fate. In a prospective clinical study of <em>10</em> VUs, we confirmed suppression of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor (BMPR) and GATA binding <em>protein</em> 3 (GATA3) as well as inhibitors of DNA-binding <em>proteins</em> 2 and 4 (ID2 and ID4). We also found decreased levels of phosphorylated glycogen synthase kinase 3 (GSK3), nuclear presence of β-catenin, and overexpression of its transcriptional target, c-myc, indicating activation of the Wnt pathway. Additionally, we found down-regulation of leucine-rich repeats and immunoglobulin-like domains <em>protein</em> 1 (LRIG1), a gene important for maintaining ESCs in a quiescent state, and absence of keratin 15 (K15), a marker of the basal stem cell compartment suggesting local depletion of ESCs. Our study shows that loss of genes important for regulation of ESCs and their fate along with activation of β-catenin and c-myc in the VU may contribute to ESC deprivation and a hyperproliferative, nonmigratory healing incapable wound edge.

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats. Osthol was supplemented into cultured medium at <em>10</em>⁻⁷, <em>10</em>⁻⁶, <em>10</em>⁻⁵ and <em>10</em>⁻⁴ mol/l, respectively. No stimulating effect was found on cell proliferation, but <em>10</em>⁻⁵ mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. Results of ALP histochemical staining and mineralized <em>bone</em> nodule assays both showed that the number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with <em>bone</em> development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the <em>protein</em> expression of p38 mitogen-activated <em>protein</em> kinase (MAPK) and type I collagen. p38MAPK <em>protein</em> and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control. The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts. The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs.

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with significant morbidity and mortality in patients with various lung and heart diseases. PAH is characterized by vascular obstruction which leads to a sustained increased pulmonary vascular resistance, vascular remodeling, and right ventricular hypertrophy and failure. Limited PAH therapies indicate that novel approaches are urgently needed for the treatment of PAH. Nuclear factor-κB (NF-κB) has been shown to play an important role in different cardiac pathologies; however, the role of NF-κB remains limited in the setting of PAH. Here, we investigated whether NF-κB inhibition in the lungs using Club (Clara) cell-<em>10</em> promoter driving IκBα mutant had any effect in monocrotaline (MCT)-induced PAH mouse model. Our data revealed that MCT-induced PAH and right ventricular hypertrophy were associated with NF-κB activation, inflammatory response, and altered expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2, inhibitor of differentiation, and Notch-3 signaling molecules in wild-type mice; and all these alterations were prevented in IκBα mutant mice treated with MCT. Moreover, endothelial cell apoptosis and endothelial-to-mesenchymal transition occurred in the lungs of MCT-treated wild-type mice and were restored in IκBα mutant+MCT mice, indicating an association with NF-κB signaling. In lung microvascular endothelial cells, IκBα (AA) mutant plasmid restored the decreased <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2 <em>protein</em> level and reversed the endothelial-to-mesenchymal transition process induced by transforming growth factor-β1. We conclude that NF-κB regulates <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2-inhibitor of differentiation-Notch-3 axis genes and the subsequent endothelial cell apoptosis and endothelial-to-mesenchymal transition events in the lungs, providing new mechanistic information about MCT-induced PAH and right ventricular hypertrophy.

BACKGROUND

Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear.

RESULTS

In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-<em>10</em>0 microm in depth, which we termed primary; and (ii) smaller microcavities of <em>10</em>-20 microm in diameter and 3-<em>10</em> microm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct <em>bone</em>-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior <em>bone</em> formation was elicited compared to cells seeded on the smooth scaffold.

CONCLUSIONS

In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future.

OBJECTIVE

Bone morphogenetic protein 2 (BMP-2) is a member of the main subgroup of bone morphogenetic proteins within the transforming growth factor-beta superfamily. BMP-2 is involved in numerous cellular functions including development, cell proliferation, apoptosis, and extracellular matrix synthesis. We examined BMP-2 expression in human scleral fibroblasts (HSF) and assessed the effects of recombinant human BMP-2 (rhBMP-2) on HSF proliferation, matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2).

METHODS

We used confocal fluorescence microscopy (CFM) to study BMP-2 distribution in HSF cells and frozen human scleral sections. The influence of rhBMP-2 on cell proliferation at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml) was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effects of rhBMP-2 on the cell cycle were investigated with flow cytometric analysis. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine MMP-2 and TIMP-2 mRNAs and secreted proteins in HSF that were incubated with rhBMP-2.

RESULTS

BMP-2 protein expression from human sclera was confirmed by CFM. Cell proliferation was significantly increased with 100 ng/ml rhBMP-2 in a time-dependent manner (p<0.05). The HSF cell cycle moved to the S and S+G(2)M phases after rhBMP-2 stimulation at 100 ng/ml compared to normal cells (p<0.05). TIMP-2 mRNA levels were significantly increased in HSF incubated for 24 h with 100 ng/ml rhBMP-2 (p<0.01). A 48 h incubation with 10 ng/ml or 100 ng/ml rhBMP-2 resulted in significantly increased TIMP-2 mRNA and protein expression and significantly decreased MMP-2 mRNA expression (p<0.01) while MMP-2 protein expression significantly decreased at 100 ng/ml rhBMP-2 (p<0.01).

CONCLUSIONS

Human sclera fibroblasts expressed BMP-2, which promoted cell proliferation, and elicited changes in MMP-2 and TIMP-2, might influence extracellular matrix synthesis.

BACKGROUND

Bone-lengthening can be accomplished by means of distraction osteogenesis. In the present study, we examined the effect of a single dose of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the rate of new-bone formation during distraction osteogenesis in the rat.

METHODS

Fourteen Long-Evans rats were randomized into two groups of seven rats each. An external fixator was applied to the left femur, and a transverse osteotomy was performed. One group was treated with rhBMP-7 in an aqueous solvent, and the other group received the solvent alone and served as the control. rhBMP-7 was administered on the day of surgery by means of a single injection into the osteotomy gap. Distraction was started seven days after surgery at a rate of 0.25 mm every twelve hours. Distraction was continued for twenty days, resulting in a total of 10 mm of lengthening. The regenerate was monitored with use of radiographs and bone-mineral-density measurements at the conclusion of distraction and at two and four weeks after the cessation of distraction. The lengthened femora were harvested, and biomechanical studies were carried out to determine the stiffness and maximum torque to failure.

RESULTS

Radiographs showed accelerated regenerate ossification in the BMP-7 group, with a larger amount of new bone compared with the control group. The bone-mineral-density values were dramatically enhanced on Day 20 in the BMP-7 group (103.6 +/- 12.6 mg/cm (2) ) compared with the control group (26.2 +/- 15.1 mg/cm (2) ). These differences continued to be greater at two and four weeks after the cessation of distraction. Normalized values of stiffness (percent stiffness) reached 76.5% +/- 5.4% in the BMP-7 group, compared with 6.6% +/- 0.5% in the control group. The percent maximum torque at failure was 81.1% +/- 1.2% in the BMP-7 group, compared with 20.8% +/- 0.3% in the control group.

CONCLUSIONS

A single injection of rhBMP-7 at the time of osteotomy surgery stimulated the rate of regenerate ossification and increased bone-mineral density during distraction osteogenesis. The biomechanical properties of the newly formed bone were also increased by BMP-7 injection.