There were few studies investigating the effects of the mechanical stimulation provided by daily low-intensity pulsed ultrasound (LIPUS) treatment. LIPUS is known to accelerate <em>bone</em> mineralization and regeneration; however, the precise cellular mechanism is unclear.Our purpose was to determine how daily LIPUS treatment affected cell viability, alkaline phosphatase activity, osteogenesis-related gene expression, and mineralized nodule formation in osteoblasts. The typical osteoblastic cell line ROS 17/2.8 cells were cultured in the absence or presence of LIPUS stimulation. Daily LIPUS treatments (1.5 MHz; 20 min) were administered at an intensity of 30 mW/cm(2) for 14 days. Expression of osteogenesis-related genes was examined at mRNA levels using real-time polymerase chain reaction and at <em>protein</em> levels using western blotting analysis. LIPUS stimulation did not affect the rate of cell viability. Alkaline phosphatase activity was increased after <em>10</em> days of culture with daily LIPUS stimulation. LIPUS significantly increased the expression of mRNAs encoding Runx2, Msx2, Dlx5, osterix, <em>bone</em> sialo<em>protein</em>, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, whereas it significantly reduced the expression of mRNA encoding the transcription factor AJ18. Mineralized nodule formation was markedly increased on Day 14 of LIPUS stimulation. LIPUS stimulation directly affected osteogenic cells, leading to mineralized nodule formation. LIPUS is likely to have a fundamental influence on key functional activities of osteoblasts in alveolar <em>bone</em>.

METHODS

A new mineralized collagen matrix combined with or without growth factor was used for the posterolateral spinal fusion in the rabbit lumbar spine.

OBJECTIVE

The availability of a new osteoconductive matrix with or without recombinant osteoinductive growth factors offers a possible alternative to the use of autogenous bone for grafting indications. This study evaluated the bone-forming activity of the biomimetic matrix: nano-hydroxyapatite/collagen/polylactic acid (nHAC/PLA) combined without or with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rabbit posterolateral spinal fusion.

BACKGROUND

Many bone grafting materials such as titanium alloy, ceramics, and polymers were used to repair bony defects. However, each has specific disadvantaged. The permanent implantation still has possibility to be eroded in vivo, which is caused by late breakdown and abscess formation. The acidic outcome of polymer biodegradation was also negatively affected in the later-stage results of bone repair. It needed a promising material for an alternative to the use of autogenous bone for grafting indications.

METHODS

Sixty-four rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC/PLA composite group (nHAC/PLA), autologous iliac crest bone mixed with nHAC/PLA composite group (ACB + nHAC/PLA), nHAC/PLA composite with recombinant human BMP-2 group (nHAC/PLA + rhBMP-2). The lumbar intertransverse process fusions were assessed by manual palpation, radiographic, histologic, and mechanical strength, and scanning electronic microscopy (SEM) in a 10-week observation.

RESULTS

Optimal formulations of the ACB + nHAC/PLA and nHAC/PLA + rhBMP-2 groups were shown to perform similar to ACB in both the fusion ratio and mechanical strength in the 6 and 10 weeks after surgery. From the microstructure analysis of the samples, there was no negative effect when the compound implanted this composite with autogenous iliac crest, and there was also new bone-like tissue formation implanted this composite without combined rhBMP-2 early at the second week after surgery.

CONCLUSIONS

This study shows the effective results of nHAC/PLA in rabbit posterolateral spinal fusion combined with rhBMP-2. It is an alternative method to autograft by compounding this osteoconductive matrix with growth factors.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> 9 (BMP9) promotes the acquisition of the cholinergic phenotype in basal forebrain cholinergic neurons (BFCN) during development and protects these neurons from cholinergic dedifferentiation following axotomy when administered in vivo. A decline in BFCN function occurs in patients with Alzheimer's disease (AD) and contributes to the AD-associated memory deficits. We infused BMP9 intracerebroventricularly for 7 d in transgenic AD model mice expressing green fluorescent <em>protein</em> specifically in cholinergic neurons (APP.PS1/CHGFP) and in wild-type littermate controls (WT/CHGFP). We used 5-mo-old mice, an age when the AD transgenics display early amyloid deposition and few cholinergic defects, and <em>10</em>-mo-old mice, by which time these mice exhibit established disease. BMP9 infusion reduced the number of Aβ42-positive amyloid plaques in the hippocampus and cerebral cortex of 5- and <em>10</em>-mo-old APP.PS1/CHGFP mice and reversed the reductions in choline acetyltransferase <em>protein</em> levels in the hippocampus of <em>10</em>-mo-old APP.PS1/CHGFP mice. The treatment increased cholinergic fiber density in the hippocampus of both WT/CHGFP and APP.PS1/CHGFP mice at both ages. BMP9 infusion also increased hippocampal levels of neurotrophin 3, insulin-like growth factor 1, and nerve growth factor and of the nerve growth factor receptors, tyrosine kinase receptor A and p75/NGFR, irrespective of the genotype of the mice. These data show that BMP9 administration is effective in reducing the Aβ42 amyloid plaque burden, reversing cholinergic neuron abnormalities, and generating a neurotrophic milieu for BFCN in a mouse model of AD and provide evidence that the BMP9-signaling pathway may constitute a therapeutic target for AD.

A continuous source of osteoblasts for normal <em>bone</em> maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the <em>bone</em> marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-beta (TGF-beta1 and TGF-beta2) and TGF-beta-related <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-beta1-F2, a fusion <em>protein</em> engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-beta1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the <em>10</em>-day selection period, the surviving cells in the rhTGF-beta1-F2 test groups proliferated rapidly in response to serum factors (<em>10</em>% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative <em>bone</em> cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and beta-glycerophosphate (beta-GP). Although rhTGF-beta1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-beta1-F2 fusion <em>protein</em>, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized <em>bone</em> matrix (iDBM) and/or diffusion chambers into older rats they are able to produce <em>bone</em> and cartilage. The population of progenitor cells captured by rhTGF-beta1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> <em>10</em> (BMP<em>10</em>) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP<em>10</em> is restricted to the right atrium. The inactive pro-BMP<em>10</em> (∼60 kDa) is processed into active BMP<em>10</em> (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of pro<em>protein</em> convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP<em>10</em> by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP<em>10</em> in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP<em>10</em> mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP<em>10</em> is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP<em>10</em>, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP<em>10</em>.

BACKGROUND

The current management of large mandibular resection defects involves harvesting of autogenous bone grafts and repeated bending of generic reconstruction plates. However, the major disadvantage of harvesting large autogenous bone grafts is donor site morbidity and the major drawback of repeated reconstruction plate bending is plate fracture and difficulty in reproducing complex facial contours. The aim of this study was to describe reconstruction of three mandibular ameloblastoma resection defects using tissue engineered constructs of beta-tricalcium phosphate (β-TCP) granules, recombinant human bone morphogenetic protein-2 (rhBMP-2), and Good Manufacturing Practice (GMP) level autologous adipose stem cells (ASCs) with progressively increasing usage of computer-aided manufacturing (CAM) technology.

METHODS

Patients' three-dimensional (3D) images were used in three consecutive patients to plan and reverse-engineer patient-specific saw guides and reconstruction plates using computer-aided additive manufacturing. Adipose tissue was harvested from the anterior abdominal walls of three patients before resection. ASCs were expanded ex vivo over 3 weeks and seeded onto a β-TCP scaffold with rhBMP-2. Constructs were implanted into patient resection defects together with rapid prototyped reconstruction plates.

RESULTS

All three cases used one step in situ bone formation without the need for an ectopic bone formation step or vascularized flaps. In two of the three patients, dental implants were placed 10 and 14 months following reconstruction, allowing harvesting of bone cores from the regenerated mandibular defects. Histological examination and in vitro analysis of cell viability and cell surface markers were performed and prosthodontic rehabilitation was completed.

CONCLUSIONS

Constructs with ASCs, β-TCP scaffolds, and rhBMP-2 can be used to reconstruct a variety of large mandibular defects, together with rapid prototyped reconstruction hardware which supports placement of dental implants.

Neural precursors have been derived from human embryonic stem cells (hESC) using the <em>bone</em> <em>morphogenetic</em> <em>protein</em> antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naive hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About <em>10</em>% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.

Combining autologous <em>bone</em> graft and recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) to treat long-<em>bone</em> fracture aseptic atrophic nonunions theoretically could promote <em>bone</em> healing at higher rates than each of these grafting agents separately. We retrospectively reviewed prospectively collected data on patient general characteristics, clinical outcomes, and complications over 3 years to determine the healing rates and the incidence of complications and adverse events of this "graft expansion rationale." There were 45 patients (32 male) with a median age of 43 years (range, 19-76 years). Minimum followup was 12 months (mean, 24.5 months; range, 12-65 months). There were seven humeral, 19 femoral, and 19 tibial nonunions. The median number of prior operations was two (range, 1-7). All fractures united. Clinical and radiographic union occurred within a median of 5 months (range, 3-14 months) and 6 months (range, 4-16 months), respectively. Thirty-nine (87%) patients returned to their preinjury occupation at a mean of 4.2 months (range, 3-6 months). The median visual analog scale pain score was 0.9 (range, 0-2.8; maximum <em>10</em>), and the median functional score was 86 (range, 67-95; maximum <em>10</em>0) at the final followup. BMP-7 as a <em>bone</em>-stimulating agent combined with conventional autograft resulted in a nonunion healing rate of <em>10</em>0% in these 45 patients.

METHODS

Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, <em>10</em>% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo <em>bone</em> formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze-thaw is substantively muted, both in vitro and in vivo. The use of recombinant <em>proteins</em>, however, may be used to mitigate the deleterious effects of the freeze-thaw process.

METHODS

Retrospective case series.

OBJECTIVE

To present results of recombinant human bone morphogenetic protein-2 (rhBMP-2) use in medically nonresponsive pyogenic vertebral osteomyelitis (PVO), treated by anterior/posterior debridement and instrumented fusion in the cervical, thoracic, and lumbosacral spine.

BACKGROUND

Surgical options for PVO vary, as do their outcomes, and can be complicated by recurrence, pseudarthrosis, and death. Although rhBMP-2 use in spinal fusion is increasing, its utility in PVO is unknown. Additionally, use in areas of infection is listed as a contraindication, although this is not supported by laboratory (animal) studies or clinical studies in long bones.

METHODS

Between 2003 and 2005, 14 patients who underwent circumferential fusion for PVO were included in this study. Average patient age was 54 years (range, 27-77 years). Eight (57%) patients had 3 or more vertebral bodies involved. Diagnostic studies included radiographs, CT, MRI, and markers of infection [(C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), white blood count (WBC)]. All patients underwent anterior fusion with rhBMP-2 inserted in structural allograft (11 patients) or titanium cylindrical cages (3 patients), followed by posterior instrumented fusion with autogenous iliac crest graft (8 occurring on the same day). Follow-up averaged 22 months (range, 11-30 months). All were studied with plain radiographs, including flexion-extension lateral films and fine cut CT scans with reconstruction. Pain ratings were recorded by visual analog scores (VAS).

RESULTS

Clinical resolution of infections, normalization of lab values, and bony fusion, based on dynamic radiographs and CT scans, were seen in all patients at latest follow-up. Staphylococcus aureus was the most frequently identified organism (8 patients). Four (29%) patients had positive blood cultures (all MRSA). Predisposing comorbidities were present in 12 patients. Six patients had epidural abscesses. Eight (57%) patients presented with neurologic deficits, ranging from paraparesis to quadriplegia. Complete recovery was seen in 7 (quadriplegia unchanged). At 1 year, mean VAS pain scores improved significantly (P < 0.05) from 7.9 (range, 3-10) to 2.8 (range, 0-6). Perioperative complications (non-BMP related) occurred in 2 patients. There were no surgically-related deaths.

CONCLUSIONS

rhBMP-2 use, in combination with antibiotics and circumferential instrumented fusion, provides a safe and successful surgical treatment of medically nonresponsive PVO, with solid fusions obtained, good clinical results, and no adverse side effects from the BMP.

Between 2 and <em>10</em> years of age, the developing craniofacial skeleton poses a significant reconstructive challenge. Local autogenous <em>bone</em> is largely unavailable, distant <em>bone</em> grafts are fraught with significant morbidity and limited yield, and alloplastic materials are incompatible with the growing calvarium and facial skeleton. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 2, a member of a class of <em>proteins</em> first noticed in the 1960s to promote <em>bone</em> deposition in soft tissues, offers a potential solution to the <em>bone</em> shortage historically faced by the pediatric craniofacial surgeon. A review of English language literature was conducted from the 1960s to the present.Attention was focused on BMP-2's osteoinductive mechanism, basic science and translational laboratory findings, and multidisciplinary clinical experiences. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2 has been embraced by spine surgeons, is gaining in popularity for long-<em>bone</em> repair, and is making its way into the plastic surgery literature. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2 may provide a basis for an off-the-shelf tissue-engineered <em>bone</em> construct that is compatible with the growing craniofacial skeleton while free from the morbidities of distant graft harvest. Questions remain, however, regarding the safety and efficacy of this compound in the context of pediatric craniofacial surgery. In an effort to facilitate the clinician's risk-benefit analysis of this emerging technology, we present a primer on the basic science of BMP-2, a discussion of possible morbidities associated with its use, a review of laboratory and clinical trials with this substance to date, and an analysis of strategies to maximize its efficacy in craniofacial surgery.

Osteogenic <em>protein</em>-1 (OP-1, BMP-7) is a member of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> subfamily of the TGF-ss superfamily that selectively stimulates dendritic neuronal outgrowth. In previous studies, we found that the intracisternal injection of OP-1, starting at one day after stroke, enhanced sensorimotor recovery of the contralateral limbs following unilateral cerebral infarction in rats. In the current study, we further explored the time window during which intracisternal OP-1 enhances sensorimotor recovery, as assessed by limb placing tests. We found that intracisternal OP-1 (<em>10</em> microg) given 1 and 3 days, or 3 and 5 days, but not 7 and 9 days after stroke, significantly enhanced recovery of forelimb and hindlimb placing. There was no difference in infarct volume between vehicle- and OP-1-treated animals. The mechanism of OP-1 action might be stimulation of new dendritic sprouting in the remaining uninjured brain.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) and basic fibroblast growth factor (bFGF) are the focus of research pertaining to the stimulation of <em>bone</em> formation. We ascertained the effects of different concentrations rhBMP-2 on proliferation and differentiation of <em>bone</em> marrow stromal cells (BMSCs) in vitro and on ectopic <em>bone</em> formation in rats. BMSCs were obtained from beagle dogs and cultured in medium containing different concentrations rhBMP-2 and bFGF (0, 25, 50, <em>10</em>0, or 200 ng/mL). In a separate experiment, BMSCs were treated with different ratios (1:1, 2:1, 4:1, or 8:1) of rhBMP to bFGF (in each case the concentration of rhBMP was <em>10</em>0 ng/mL and the bFGF concentrations <em>10</em>0, 50, 25, or 12.5 ng/mL). Proliferation and differentiation of BMSCs were quantified by assessing methyl thiazole tetrazolium (MTT) and alkaline phosphatase (ALP) over 6 consecutive days. Von Kossa staining was performed on day 6. For the in vivo tests, porous calcium phosphate cement (CPC) was seeded with BMSCs (5 x <em>10</em>(4)) in medium containing <em>10</em>0 ng/mL rhBMP-2, 50 ng/mL bFGF or combined <em>10</em>0 ng/mL rhBMP-2 and 50 ng/mL bFGF. These cells were then subcutaneously implanted in four sites in nude rats. <em>Bone</em> formation was detected by histology at weeks 4 and 12 and quantified using a KS400 computer based image analysis system. It was determined that combined rhBMP-2 and bFGF at a ratio of 2:1 (<em>10</em>0:50 ng/mL) promoted significantly increased BMSC proliferation and differentiation of BMSCs compared to rhBMP-2 or bFGF alone (p < 0.05). CPC with combined <em>10</em>0 ng/mL rhBMP-2 and 50 ng/mL bFGF stimulated more <em>bone</em> formation than either <em>10</em>0 ng/mL rhBMP-2 or <em>10</em>0 ng/mL bFGF (p < 0.05). These results show that a combination of rhBMP-2 and bFGF effectively induces early BMSC proliferation and differentiation in vitro. When combined, rhBMP-2 and bFGF synergistically promote new <em>bone</em> formation.

Our recent study showed that a novel member of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) family, BMP-<em>10</em>, was decreased in prostate cancer. In the present study, we investigated the implication of BMP-<em>10</em> in breast cancer, particularly the relation of its expression with clinical aspects. The expression of BMP-<em>10</em> was examined in a cohort of human breast cancer specimens (normal, n = 23; cancer, n = 97), using both quantitative real-time PCR and immunohistochemical staining. The full-length human BMP-<em>10</em> was cloned into a mammalian expression plasmid vector and then transfected into breast cancer cells. The effect on growth, cell matrix adhesion, motility, and invasion of MDA-MB-231 cells by BMP-<em>10</em> was then investigated using in vitro growth assays. Immunohistochemical staining and quantitative real-time PCR revealed a decreased expression of BMP-<em>10</em> in breast cancer. Further analysis of BMP-<em>10</em> transcript level against the clinical aspect demonstrated that the decreased BMP-<em>10</em> expression correlated with disease progression, <em>bone</em> metastasis, and poor prognosis. The disease-free survival of the patients with a higher level of BMP-<em>10</em> was 132.8 (95% CI, 122.0-143.5) months, significantly longer compared to 93.7 (95% CI, 60.3-127.2) months for patients with a lower level of BMP-<em>10</em> expression (P = 0.043). The overexpression of BMP-<em>10</em> has broad inhibitory effects on the in vitro growth, invasion, and motility of breast cancer cells. Taken together, BMP-<em>10</em> can inhibit the cell growth of breast cancer cells, and decreased BMP-<em>10</em> expression correlates to poor prognosis and disease progression, particularly the lymphatic and <em>bone</em> metastasis. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-<em>10</em> (BMP-<em>10</em>) may function as a tumor suppressor in breast cancer.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) can be used to enhance the repair of congenital or acquired <em>bone</em> pathologies when formulated in the appropriate carrier. Poly [D,L-(lactide-co-glycolide)] (PLGA) has been shown to be an effective carrier of rhBMP-2. We investigated several particle sizes PLGA and several doses of rhBMP-2 in a rat orthotopic model. We also investigated the effects of a fibrinolytic inhibitory agent, epsilon aminocaproic acid (EACA), on the healing response. Our data indicate that higher doses of rhBMP-2 resulted in increased failure torque (408 +/- 70 N-mm or 60% of the intact value) and higher incidence of union (<em>10</em>0%). The induced <em>bone</em> in femurs treated with the smaller particle size PLGA achieved the greatest torsional stiffness and strength. The presence of rhBMP-2 was necessary for new <em>bone</em> to form, but the presence of EACA did not change these results; the use of the PLGA carrier appeared to increase <em>bone</em> strength and stiffness. In fact, with higher doses of rhBMP-2 in PLGA, the stiffness of the new <em>bone</em> was equal to that of intact controls (64 +/- 20 N-mm/deg [intact femurs] versus 45 +/- <em>10</em> N-mm/degree [medium dose in small PLGA], 61 +/- 17 N-mm/degree [high dose in small PLGA], and 36 +/- 11 N-mm/degree [medium dose in large PLGA]; P>> .05). In conclusion, PLGA implanted with rhBMP-2 effectively aided in healing large segmental defects in rat femurs.

Human adipose-derived mesenchymal stem cells (AD-MSCs) attracted much interest as a promising alternative to autologous chondrocytes and <em>bone</em> marrow-derived mesenchymal stem cells for cartilage regeneration. Developing a suitable culture technique to direct AD-MSCs into the chondrogenic lineage could be a crucial prerequisite for the cartilage defect repair application of AD-MSCs. Herein, we prepared the PEGDG-crosslinked porous three-dimensional (3D) hyaluronic acid (HA) scaffold and evaluated for its feasibility to induce proliferation and chondrogenic differentiation of the AD-MSCs. In addition, the effect of <em>bone</em>-<em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and platelet-derived growth factor (PDGF) on chondrogenic differentiation was further investigated. Proliferation and chondrogenic differentiation were evaluated by cell morphology, DNA contents, s-GAG contents, and level of mRNA expression of relevant marker genes. When cultured with reference chondrogenic medium (RCM; serum-free DMEM-HG supplemented with <em>10</em> ng/mL of transforming growth factor-β1 (TGF-β1), 50 nM ascorbate, <em>10</em>0 nM dexamethasone, and 5 μg/mL of ITS), better proliferation and chondrogenic differentiation of AD-MSCs were obtained in the 3D HA scaffold culture as compared to the micromass culture, a standard 3D culture system. Moreover, the level of chondrogenic differentiation of AD-MSCs in the HA scaffold-RCM culture system was further increased by BMP-2, and decreased by PDGF. These results suggested that the HA scaffold with RCM was a promising chondrogenic culture system of AD-MSCs, and that BMP-2 could potentially serve as a chondrogenic supplement for AD-MSCs. However, PDGF was determined to be an inappropriate supplement based on its inhibition of the chondrogenic differentiation of AD-MSCs.

BACKGROUND

Simvastatin is one of the cholesterol lowering drugs. Recent studies demonstrated that it has a bone stimulatory effect. Periodontal ligament (PDL) cells are believed to play an important role in periodontal regeneration; that is, they may differentiate into specific cells which make cementum, bone, and attachment apparatus. It would be of interest whether simvastatin has a positive effect on PDL cells. Therefore, effects of simvastatin on cell proliferation and osteoblastic differentiation in PDL cells were analyzed.

METHODS

Human PDL cells were cultured in monolayer with simvastatin for 24 and 72 hours and cell metabolism and proliferation were determined. To analyze osteoblastic differentiation, human PDL cells were cultured in organoid culture for 7, 14, and 21 days and alkaline phosphatase (ALP) activity, osteopontin (OPN), bone morphogenetic protein (BMP) -2, osteocalcin (OCN), and calcium contents were measured. They were co-treated by simvastatin and mevalonate.

RESULTS

Simvastatin enhanced cell proliferation and metabolism dose-dependently after 24 hours. Simvastatin also stimulated ALP activity of human PDL cells dose-dependently, and maximum effect was obtained at the concentration of 10(8) M. In time dependent analysis, 10(8) M simvastatin stimulated ALP activity and osteopontin content after 7 days and calcium contents after 21 days. BMP-2 and OCN contents were not detected. Moreover this statin-enhanced ALP activity was abolished by mevalonate.

CONCLUSIONS

These results suggest that at low concentration, simvastatin exhibits positive effect on proliferation and osteoblastic differentiation of human PDL cells, and these effects may be caused by the inhibition of the mevalonate pathway.

Primary pulmonary hypertension (PPH) is a rare but severe and progressive disease characterised by obstructive lesions of small pulmonary arteries. Patients with PPH often have mutations in the <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type II (BMPR2) gene, whereas some carry mutations in the activin receptor-like kinase 1 (ALK-1) gene, generally associated with hereditary haemorrhagic telangiectasia (HHT) type 2, a vascular dysplasia affecting multiple organs. The aim of this study was to determine whether members of families with PPH and confirmed or probable HHT had ALK-1 mutations. ALK-1 and BMPR2 mutation analysis was performed on deoxyribonucleic acid from affected members of four families with PPH and confirmed or suspected HHT. ALK-1 mutations were identified in all four families and three novel mutations found in exon <em>10</em>, leading to truncated <em>proteins</em>. In the fourth family, a missense mutation, previously reported in four independent HHT families, was detected in exon 8. Analysis of the BMPR2 gene revealed no exonic mutations in the probands with both PPH and HHT. The present data bring to <em>10</em> the number of reported families with primary pulmonary hypertension and hereditary haemorrhagic telangiectasia type 2, representing 16% of the 61 families with known activin receptor-like kinase 1 mutations. Such mutations might predispose to primary pulmonary hypertension, and specialists should be aware of the potential link between these two disorders.

BACKGROUND

Previous studies have shown a limited potential for bone augmentation following guided bone regeneration (GBR) in horizontal alveolar defects. Surgical implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge carrier (ACS) significantly enhances bone regeneration in such defects; however, sufficient quantities of bone for implant dentistry are not routinely obtained. The objective of this study was to evaluate the potential of rhBMP-2/ACS to enhance GBR using a space-providing, macro-porous expanded polytetrafluoroethylene (ePTFE) device.

METHODS

Bilateral, critical size, supra-alveolar, peri-implant defects were surgically created in four Hound Labrador mongrel dogs. Two turned and one surface-etched 10-mm titanium dental implant were placed 5 mm into the surgically reduced alveolar ridge creating 5-mm supra-alveolar defects. rhBMP-2/ACS (rhBMP-2 at 0.2 mg/ml) or buffer/ACS was randomly assigned to left and right jaw quadrants in subsequent animals. The space-providing, macro-porous ePTFE device was placed to cover rhBMP-2/ACS and control constructs and dental implants. Gingival flaps were advanced for primary wound closure. The animals were euthanized at 8 weeks postsurgery for histologic and histometric analysis.

RESULTS

Bone formation was significantly enhanced in defects receiving rhBMP-2/ACS compared to control. Vertical bone gain averaged (+/- SD) 4.7 +/- 0.3 and 4.8 +/- 0.1 mm, and new bone area 10.3 +/- 2.0 and 8.0 +/- 2.5 mm2 at turned and surface-etched dental implants, respectively. Corresponding values for the control were 1.8 +/- 2.0 and 1.3 +/- 1.3 mm, and 1.8 +/- 1.3 and 1.2 +/- 0.6 mm2. Bone-implant contact in rhBMP-2-induced bone averaged 6.4 +/- 1.4% and 9.6 +/- 7.5% for turned and surface-etched dental implants, respectively (P=0.399). Corresponding values for the control were 14.6 +/- 19.4% and 23.7 +/- 9.7% (P=0.473). Bone-implant contact in resident bone ranged between 43% and 58% without significant differences between dental implant surfaces.

CONCLUSIONS

rhBMP-2/ACS significantly enhances GBR at turned and surface-etched dental implants. The dental implant surface technology does not appear to substantially influence bone formation.

We studied the effect of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and vitamin D(3) on the osteogenic differentiation of adipose stem cells (ASCs). ASCs were treated with <em>10</em>, 50, and <em>10</em>0 ng/ml of BMP-2, and <em>10</em>(-8), <em>10</em>(-7), <em>10</em>(-6)M vitamin D(3). Then, to investigate the effects of combined treatment, ASCs were treated with BMP-2 and vitamin D(3) dose-dependently and time-dependently. The osteogenic differentiation was assessed by alkaline phosphatase (ALP) activities/staining and the mineralization was evaluated by Alizarin red S staining. ALP activity and mineralization dose-dependently increased in early stages (ALP on 7th day and mineralization on the 14th day) while all three doses of BMP-2 or vitamin D(3) showed comparable effects in late stages (ALP on the 14th day and mineralization on the 21st day) in ASCs. BMP-2 and vitamin D(3) had synergistic effect on the osteogenic differentiation of ASCs. While all three doses of BMP-2 acted similarly in reinforcing the effect of vitamin D(3), vitamin D(3) dose-dependently augmented the osteogenic effect of BMP-2. When BMP-2 was constantly treated, vitamin D(3) effect did not differ depending on the period of vitamin D(3) treatment. However, when vitamin D(3) was constantly treated, the BMP was more effective when treated for the last 7 days than when treated for the first 7 days. In conclusion, BMP-2 and vitamin D(3) promote osteogenic differentiation of ASCs, and can work synergistically. These results can be used to induce effective and economical osteogenic induction of ASCs for <em>bone</em> tissue engineering.

Several of the different <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are involved in development and progression of specific tumors. For hepatocellular carcinoma (HCC) only BMP4 and BMP6 are described to be important for carcinogenesis. However, up to now neither the influence of other BMPs on tumor progression, nor the responsible signaling pathways to mediate target gene expression in HCC are known. In order to characterize BMP expression pattern in HCC cell lines, we performed RT-PCR analysis and revealed enhanced expression levels of several BMPs (BMP4, 6, 7, 8, 9, <em>10</em>, 11, 13 and 15) in HCC. Thus, we treated HCC cells with the general BMP inhibitors chordin and noggin to determine the functional relevance of BMP overexpression and observed decreased migration and invasion of HCC cells. A cDNA microarray of noggin treated HCC cells was performed to analyze downstream targets of BMPs mediating these oncogenic functions. Subsequent analysis identified collagen XVI as 'Smad signaling specific' and nidogen-2 as 'MAPK/ERK signaling specific' BMP-target genes. To examine which signaling pathway is mainly responsible for the oncogenic role of BMPs in HCC, we treated HCC cells with dorsomorphin to determine the influence of BMP activated Smad signaling. Interestingly, also migratory and invasive behavior of dorsomorphin treated HCC cells was diminished. In summary, our findings demonstrate enhanced expression levels of several BMPs in HCC supporting enhanced migratory and invasive phenotype of HCC cells mainly via activation of Smad signaling.

We investigated pellet-shaped implants prepared from biphasic calcium phosphate (BCP) ceramics with five different ratios of hydroxyapatite (HAP) to beta-tricalcium phosphate (beta-TCP) to evaluate these ceramics as <em>bone</em> substitutes. BCP ceramics impregnated with different doses of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP-2) (1, 5, and <em>10</em> microg) were used for experimental purposes and ceramics without rhBMP-2 were used for control. The pellets were implanted under the pericranium in adult Wistar male rats and were harvested 8 weeks after implantation. The retrieved pellets were then examined radiologically, histologically, and histomorphometrically. The results revealed that the pellets treated with rhBMP-2 exhibited new <em>bone</em> and <em>bone</em> marrow, whereas control pellets produced fibrous connective tissues. The formation of new <em>bone</em> induced by rhBMP-2 was dose dependent. The extent of <em>bone</em> and <em>bone</em> marrow formation and the degree of resorption of the ceramic particles were significantly higher in the pellets composed of 25% HAP-75% TCP. In this study, bioresorption of the ceramic produced favorable conditions for rhBMP-2-induced <em>bone</em> formation.

Germ-line mutations in the <em>bone</em> <em>morphogenetic</em> <em>protein</em> type II receptor (BMPR2; BMPR-II) gene, a transforming growth factor-β (TGFβ) receptor superfamily member, cause the majority of cases of heritable pulmonary arterial hypertension (PAH). Pulmonary arterial hypertension is a subset of pulmonary hypertension (PH) disorders, which also encompass hypoxia-related lung diseases. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), via BMPR-II, activate the canonical Smad1/5/9 pathway, whereas TGFβs (TGFβ1-3) activate the Smad2/3 pathway via the ALK5 receptor. Dysregulated TGFβ1 signalling is pathogenic in fibrotic diseases. We compared two rat PH models, monocrotaline-induced PAH (MCT-PAH) and chronic normobaric hypoxia (fractional inspired O2 <em>10</em>%), to address whether BMPR-II loss is common to PH and permits pathogenic TGFβ1 signalling. Both models exhibited reduced lung BMPR-II expression, but increased TGFβ1 signalling and decreased BMP signalling were observed only in MCT-PAH. Furthermore, a pharmacological ALK5 inhibitor prevented disease progression in the MCT-PAH model, but not in hypoxia. In vitro studies using human pulmonary artery smooth muscle cells showed that TGFβ1 directly inhibits BMP-Smad signalling. In conclusion, BMPR-II loss is common to the hypoxic and MCT-PAH models, but systemic ALK5 inhibition is effective only in the MCT model, highlighting a specific role for TGFβ1 in vascular remodelling in MCT-PAH, potentially via direct inhibition of BMP signalling.

The biological effects of endogenous <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP-4), a member of the transforming growth factor beta family, on embryonic development of Xenopus laevis were investigated by using a functionally defective mutant of the BMP-4 receptor (delta mTFR11), which blocks the BMP signaling pathway. Injection of delta mTFR11 RNA into either the animal pole area or ventral marginal cells at the two-cell stage induced a dorsal phenotype in the explant of ventral mesoderm with animal pole tissue from stage <em>10</em>+ embryo, even though the normal fate of this explant is a "mesenchymal ball" containing blood cells. These explants with the dorsal phenotype contained muscle, neural tissue, eye capsule, and cement gland. Northern blot analysis showed an increase of cardiac alpha-actin mRNA and a decrease of T alpha-globin mRNA expression, providing further evidence of a conversion from ventral to dorsal phenotype. Although injection of delta mTFR11 RNA did not induce mesoderm in an animal cap culture, the same tissue injected with delta mTFR11 RNA can alter the differentiation fate of uninjected ventral mesodermal explant from ventral to dorsal type, suggesting specific interaction of animal pole tissue and prospective ventral mesoderm in vivo.