OBJECTIVE

Large mandibular resection defects historically have been treated using autogenous bone grafts and reconstruction plates. However, a major drawback of large autogenous bone grafts is donor-site morbidity.

METHODS

This report describes the replacement of a 10-cm anterior mandibular ameloblastoma resection defect, reproducing the original anatomy of the chin, using a tissue-engineered construct consisting of β-tricalcium phosphate (β-TCP) granules, recombinant human bone morphogenetic protein-2 (BMP-2), and Good Manufacturing Practice-level autologous adipose stem cells (ASCs). Unlike prior reports, 1-step in situ bone formation was used without the need for an ectopic bone-formation step. The reconstructed defect was rehabilitated with a dental implant-supported overdenture. An additive manufactured medical skull model was used preoperatively to guide the prebending of patient-specific hardware, including a reconstruction plate and titanium mesh. A subcutaneous adipose tissue sample was harvested from the anterior abdominal wall of the patient before resection and simultaneous reconstruction of the parasymphysis. ASCs were isolated and expanded ex vivo over the next 3 weeks. The cell surface marker expression profile of ASCs was similar to previously reported results and ASCs were analyzed for osteogenic differentiation potential in vitro. The expanded cells were seeded onto a scaffold consisting of β-TCP and BMP-2 and the cell viability was evaluated. The construct was implanted into the parasymphyseal defect.

RESULTS

Ten months after reconstruction, dental implants were inserted into the grafted site, allowing harvesting of bone cores. Histologic examination and in vitro analysis of cell viability and cell surface markers were performed and prosthodontic rehabilitation was completed.

CONCLUSIONS

ASCs in combination with β-TCP and BMP-2 offer a promising construct for the treatment of large, challenging mandibular defects without the need for ectopic bone formation and allowing rehabilitation with dental implants.

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess <em>bone</em> healing, the genetically modified ("gene activated") tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as <em>10</em> days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed <em>bone</em> were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal <em>bone</em> and cartilage more expeditiously, and at lower cost, than is presently possible.

We have purified and characterized recombinant Xenopus <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (xBMPs): homodimers of xBMP-4, 7 and heterodimers (xBMP-4/7) produced by a baculovirus expression system. Highly purified xBMPs had homogeneous NH2-termini predicted from a consensus motif, Arg-X-X-Arg, while they possessed diverse sugar chains. Implantation of xBMPs together with pure collagen carrier in rats induced new <em>bone</em> formation in a dose-dependent manner. The xBMP-4/7 heterodimer showed the strongest activity, with an effective dose of 1-30 micrograms, while more than <em>10</em> micrograms of xBMP-4 or 7 homodimer was required for a significant effect. Histological examination revealed that xBMP-4/7 implants showed intramembranous ossification without chondrogenesis. In primary cultures of rat <em>bone</em> marrow stromal cells, xBMP-4/7 induced alkaline phosphatase 3-fold more strongly than xBMP-7 and 20-fold more than xBMP-4. These results suggest that the heterodimeric form of BMP would generate the strongest signal triggering osteogenic differentiation of osteoprogenitor cells in adult tissues.

Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, <em>10</em> ng/mL TGF-β1, <em>10</em>0 ng/mL GDF-5, or <em>10</em>0 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs.

Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. Herein, we investigated differentiation of human embryonic stem cells (hESCs) down the trophoblast lineage by culture with <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) over a <em>10</em>-day period. Within 2 days, the stemness markers POU5F1 and NANOG were markedly down-regulated, followed temporally by up-regulation of the CDX2, KRT7, HLA-G, ID2, CGA, and CGB trophoblast markers. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. Through whole-genome analysis, more than 3800 genes displayed statistically significant and 2-fold or greater changes in expression during the time course. Of those genes that showed the largest increases, many were involved in processes associated with trophoblast biology; however, novel genes were also identified. Some of them are hypothesized to be associated mainly with extracellular matrix remodeling (e.g., NID2) and cell migration and invasion (e.g., RAB25). Using Ingenuity pathways analysis software to identify signaling pathways involved in trophoblast differentiation or function, we discovered that many genes are involved in WNT/beta-catenin, ERK/MAPK, NFKB, and calcium signaling pathways, suggesting potential roles for these families in trophoblast development. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development.

Wide variation in glucocorticoid (Gc) sensitivity exists between individuals which may influence susceptibility to, and treatment response of, inflammatory diseases. To determine a genetic fingerprint of Gc sensitivity <em>10</em>0 healthy human volunteers were polarized into the <em>10</em>% most Gc-sensitive and <em>10</em>% most Gc-resistant following a low dose dexamethasone (0.25 mg) suppression test. Gene expression profiling of primary lymphocytes identified the 98 most significantly Gc regulated genes. These genes were used to build a subnetwork of Gc signaling, with 54 genes mapping as nodes, and 6 non-Gc regulated genes inferred as signaling nodes. Twenty four of the 98 genes showed a difference in Gc response in vitro dependent on the Gc sensitivity of their donor individuals in vivo. A predictive model was built using both partial least squares discriminate analysis and support vector machines that predicted donor glucocorticoid sensitivity with 87% accuracy. Discriminating genes included <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor, type II (BMPRII). Transfection studies showed that BMPRII modulated Gc action. These studies reveal a broad base of gene expression that predicts Gc sensitivity and determine a Gc signaling network in human primary T lymphocytes. Furthermore, this combined gene profiling, and functional analysis approach has identified BMPRII as a modulator of Gc signaling.

In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29,000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (<em>10</em>0- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-<em>10</em> in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage.

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P(4)VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4-<em>10</em>%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. <em>Bone</em> marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic genetic disorder of progressive heterotopic ossification associated with dysregulated production of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4), a potent osteogenic morphogen. Postnatal heterotopic ossification in FOP is often heralded by hectic episodes of severe post-traumatic connective tissue swelling and intramuscular edema, followed by an intense and highly angiogenic fibroproliferative mass. The abrupt appearance, intense size, and rapid intrafascial spread of the edematous preosseous fibroproliferative lesions implicate a dysregulated wound response mechanism and suggest that cells and mediators involved in inflammation and tissue repair may be conscripted in the growth and progression of FOP lesions. The central and coordinate role of inflammatory mast cells and their mediators in tissue edema, wound repair, fibrogenesis, angiogenesis, and tumor invasion prompted us to investigate the potential involvement of mast cells in the pathology of FOP lesions. We show that inflammatory mast cells are present at every stage of the development of FOP lesions and are most pronounced at the highly vascular fibroproliferative stage. Mast cell density at the periphery of FOP lesional tissue is 40- to 150-fold greater than in normal control skeletal muscle or in uninvolved skeletal muscle from FOP patients and <em>10</em>- to 40-fold greater than in any other inflammatory myopathy examined. These findings document mobilization and activation of inflammatory mast cells in the pathology of FOP lesions and provide a novel and previously unrecognized target for pharmacologic intervention in this extremely disabling disease.

METHODS

In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells.

OBJECTIVE

To demonstrate the effect of BMP-2 on mRNAs expression (collagen type I, collagen type II, aggrecan, and osteocalcin), proteoglycan synthesis, expression of alkaline phosphatase, bone nodule formation in human IVD cells.

BACKGROUND

BMP-2 was widely known as a powerful agent for osteoinduction and a crucial growth factor for early chondrogenesis and maintenance of cartilaginous phenotype. BMP-2 proved to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells. Nevertheless, the effect of BMP-2 on IVD cells, whether chondrogenic or osteogenic, was not thoroughly elucidated in transcriptional level and histochemical stains.

METHODS

Human IVDs were harvested and enzymatically digested. Then IVD cells were cultured three-dimensionally in alginate beads. Osteoblasts were cultured from cancellous bone of ilium for histochemical stains. Recombinant human BMP-2 (rhBMP-2) was produced by Chinese hamster ovary cells after transduction of BMP-2 cDNA, then concentrated and purified. Then IVD cell cultures were exposed to various concentrations of rhBMP-2. Reverse transcription-polymerase chain reaction for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin was performed. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. As a histochemical examination, alkaline phosphatase and Alizarin red-S stains were used to detect osteogenic marker and bone nodule formation, respectively.

RESULTS

In the rhBMP-2 treated cultures, there was increased newly synthesized proteoglycan (67% in 300 ng/mL and 200% in 1,500 ng/mL of rhBMP-2) and up-regulated expression of aggrecan, collagen type I, and collagen type II mRNA over untreated control. However, rhBMP-2 did not up-regulate expression of osteocalcin mRNA in the given dose and culture period. IVD cell cultures with rhBMP-2 showed no evidence of bone formation in histochemical stains, i.e., alkaline phosphatase and Alizarin red-S, while osteoblast culture exhibited strong positive stains.

CONCLUSIONS

The rhBMP-2 clearly up-regulated mRNA expression of chondrogenic components and also stimulated proteoglycan synthesis without expression of osteogenic phenotype. Taken together, this study raise the possibility of rhBMP-2 can be anabolic agent for regenerating matrix of intervertebral disc.

This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) for <em>bone</em> regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods' surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods' surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with <em>10</em> and 20 microg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with <em>10</em> microg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 microg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days <em>10</em> and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points.

The spermatogenesis and oogenesis-specific transcription factor Sohlh2 is normally expressed only in premeiotic germ cells. In this study, Sohlh2 and several other germ cell transcripts were found to be induced in mouse embryonic stem cells when cultured on a feeder cell line that overexpresses <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4. To study the function of Sohlh2 in germ cells, we generated mice harboring null alleles of Sohlh2. Male Sohlh2-deficient mice were infertile because of a block in spermatogenesis. Although normal prior to birth, Sohlh2-null mice had reduced numbers of intermediate and type B spermatogonia by postnatal day 7. By day <em>10</em>, development to the preleptotene spermatocyte stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia, and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type A2 spermatogonia and accumulated in M-phase prior to death. A similar phenotype was observed in Sohlh2-null mice on postnatal days 14, 21, 35, 49, 68, and 151. In adult Sohlh2-mutant mice, the ratio of undifferentiated type A spermatogonia (DAZL+/PLZF+) to differentiating type A spermatogonia (DAZL+/PLZF-) was twice normal levels. In culture, undifferentiated type A spermatogonia isolated from Sohlh2-null mice proliferated normally but linked the mutant phenotype to aberrant cell surface expression of the receptor-tyrosine kinase cKit. Thus, Sohlh2 is required for progression of differentiating type A spermatogonia into type B spermatogonia. One conclusion originating from these studies would be that testicular factors normally regulate the viability of differentiating spermatogonia by signaling through Sohlh2. This regulation would provide a crucial checkpoint to optimize the numbers of spermatocytes entering meiosis during each cycle of spermatogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (Bmp) signaling is critical for the development and patterning of the mouse pituitary from the initial induction of Rathke's pouch to cell specification in the anterior lobe. We examined the regulation of Bmp signaling during pituitary development by analyzing null embryos for noggin, a Bmp 2 and 4 antagonist. Noggin is expressed in the ventral diencephalon during Rathke's pouch induction, in the underlying cartilage plate during cell specification and in the adult anterior pituitary gland. Noggin null embryos have a variable pituitary phenotype, which ranges from a rostrally displaced Rathke's pouch to induction of secondary pituitary tissue. While cell specification in the anterior pituitary appears normal, patterning in the ventral diencephalon is disrupted; Bmp4 activity is expanded resulting in Fibroblast growth factor <em>10</em> repression and in a rostral shift in the boundary between the Bmp4 and Sonic hedgehog expression domains. The expanded domain of Bmp4 activity also results in additional invaginations of oral ectoderm and can shift the position of Rathke's pouch or create secondary pituitary tissue. This work demonstrates the importance of attenuating the activity of Bmp signaling during pituitary induction in order to maintain the proper balance of signaling factors necessary for pituitary organogenesis.

We carried out a prospective study to determine whether the addition of a recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> (rhBMP-2) to a machined allograft spacer would improve the rate of intervertebral body fusion in the spine. We studied 77 patients who were to undergo an interbody fusion with allograft and instrumentation. The first 36 patients received allograft with adjuvant rhBMP-2 (allograft/rhBMP-2 group), and the next 41, allograft and demineralised <em>bone</em> matrix (allograft/demineralised <em>bone</em> matrix group). Each patient was assessed clinically and radiologically both pre-operatively and at each follow-up visit using standard methods. Follow-up continued for two years. Every patient in the allograft/rhBMP-2 group had fused by six months. However, early graft lucency and significant >> <em>10</em>%) subsidence were seen radiologically in 27 of 55 levels in this group. The mean graft height subsidence was 27% (13% to 42%) for anterior lumbar interbody fusion, 24% (13% to 40%) for transforaminal lumbar interbody fusion, and 53% (40% to 58%) for anterior cervical discectomy and fusion. Those who had undergone fusion using allograft and demineralised <em>bone</em> matrix lost only a mean of 4.6% (0% to 15%) of their graft height. Although a high rate of fusion (<em>10</em>0%) was achieved with rhBMP-2, significant subsidence occurred in more than half of the levels (23 of 37) in the lumbar spine and 33% (6 of 18) in the cervical spine. A 98% fusion rate (62 of 63 levels) was achieved without rhBMP-2 and without the associated graft subsidence. Consequently, we no longer use rhBMP-2 with allograft in our practice if the allograft has to provide significant structural support.

Permanent alcohol abuse may lead to chronic liver injury with deleterious sequelae such as liver cirrhosis and hepatocellular carcinoma. Mechanisms of fibrogenesis encompass recruitment of inflammatory cells at the site of injury and cytokine mediated activation of hepatic stellate cells (HSC) with accumulation of interstitial collagens. HSC transdifferentiation and accompanying apoptosis result in destruction of liver architecture and are therefore key steps of disease progression. TGF-beta represents the main profibrogenic cytokine in liver fibrosis and other fibroproliferative disorders by inducing extracellular matrix deposition as part of the wound healing response. In parallel, TGF-beta triggers hepatocytes that are strongly responsive for this cytokine, to undergo apoptosis, thereby providing space for HSC proliferation and generation of a collagenous matrix. Anti TGF-beta approaches were established and successfully utilized for the treatment of experimental fibrogenesis. Dominant negative TGF-beta receptors (TbetaR), generated by fusing the Fc domain of human IgG and the N-terminal (extracellular) fragment of TbetaRII (Fc:TbetaRII) were applied to suppress fibrosis. Similarly TGF-beta binding <em>proteins</em> like decorin, antagonistic cytokines such as <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7, hepatocyte growth factor, IL-<em>10</em>, or IFN-gamma were as efficient as camostat mesilate, a protease inhibitor that possibly abrogated proteolytic activation of TGF-beta. Further, our group recently overexpressed Smad7 in bile duct ligation induced liver fibrosis and achieved efficient inhibition of intracellular TGF-beta signaling, thereby counteracting profibrogenic effects in cultured HSC and in vivo. A direct link between the effect of alcohol and TGF-beta exists through reactive oxygen species that are generated in liver cells by alcohol metabolism and represent activators of TGF-beta signaling. Thus, soluble TbetaRII expression reduced experimental fibrogenesis in vitro and in vivo partially by decreasing intracellular ROS and inhibiting NADH oxidase. Approaches that specifically target profibrogenic TGF-beta signaling are promising to treat alcoholic liver disease in the future. However, to ensure safety for the patients to be treated, approaches with strong specificity need to be established. Therefore, it is essential to delineate the profibrogenic actions of TGF-beta and the influence of alcohol abuse in molecular detail.

Vascular calcification is a highly regulated process sharing features of <em>bone</em> mineralization. Since endothelium regulates many of the processes during atherogenesis, we monitored the expression of genes involved in calcification upon exposure of human coronary artery endothelial cells (HCAECs) to atherogenic stimuli. Genes studied were: core binding factor alpha-1 (Cbfa1/Runx2), a pivotal transcriptional regulator of osteogenesis; <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2), an inducer of cartilage and <em>bone</em>; and matrix gla-<em>protein</em> (MGP), a potent inhibitor of calcification, which exerts its action by blocking BMP2. HCAECs were treated with oxidized-low density lipo<em>protein</em> (ox-LDL, 80 microg/mL) or tumor necrosis factor-alpha (TNFalpha, <em>10</em> ng/mL), and the expression of Cbfa1, BMP2, and MGP was quantified by real-time PCR. Cbfa1 was expressed at low levels in untreated HCAECs, and its expression did not change with ox-LDL or TNFalpha treatment. The expression of BMP2 and MGP increased early after exposure to ox-LDL or TNFalpha (at 2-8 h), and the increase was not evident at 24 h. Ox-LDL exerted a stronger effect on MGP than on BMP2 expression. The effects of ox-LDL, but not TNFalpha, on MGP and BMP2 expression were inhibited by pretreatment of cells with an antibody directed at LOX-1, a lectin-like receptor for ox-LDL (<em>10</em> microg/mL). Thus, the endothelium, when exposed to atherogenic stimuli, ox-LDL in particular, regulates the process of calcification by enhancing the expression of the <em>bone</em> inhibitory MGP, while the expression of Cbfa1 remains unchanged. Upregulation of BMP2 may represent a feedback upregulation in response to increase in MGP. The effect of ox-LDL appears to be mediated by LOX-1 activation.

Biochemical studies have shown that Smad7 blocks signal transduction of transforming growth factor beta (TGFbeta); however, its in vivo functions are largely unknown. To determine the functions of Smad7, we have expressed Smad7 in transgenic mice, utilizing a keratin K5 promoter (K5.Smad7). K5.Smad7 mice exhibited pathological changes in multiple tissues and died within <em>10</em> days after birth. These mice were born with open eyelids and corneal defects, significantly delayed and aberrant hair follicle morphogenesis, and hyperproliferation in the epidermis and other stratified epithelia. Furthermore, K5.Smad7 mice developed severe thymic atrophy and massive thymocyte death, suggesting that Smad signaling in thymic epithelia is essential for thymocyte survival. Interestingly, in addition to a reduction in Smad phosphorylation, the <em>protein</em> levels of the receptors for TGFbeta, activin and <em>bone</em> <em>morphogenetic</em> <em>protein</em> were significantly decreased in the affected tissues of K5.Smad7 mice. Our study provides evidence that Smad7 is a potent in vivo inhibitor for signal transduction of the TGFbeta superfamily during development and maintenance of homeostasis of multiple epithelial tissues.

A challenge in the tissue engineering of alveolar <em>bone</em> surrounding oral or dental implants is achieving the targeted and sustained delivery of growth-promoting molecules at the osteotomy site. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) has demonstrated the ability to stimulate <em>bone</em> regeneration in multiple skeletal sites, including the craniofacial complex. This study evaluates in vivo gene delivery of BMP-7 for <em>bone</em> tissue engineering around titanium dental implants. The maxillary first molar teeth of 44 Sprague-Dawley rats were extracted and allowed to heal for a period of 1 month. Large osteotomy defects were created in the edentulous ridge areas followed by the placement of dental implant fixtures. Recombinant adenoviral vectors encoding either the BMP-7 or the luciferase gene were delivered to the osseous defects using a collagen matrix. The kinetics of the gene expression was measured using in vivo bioluminescence optical imaging, while <em>bone</em> regeneration was evaluated under light and scanning electron microscopy. The results revealed sustained, targeted transgene expression for up to <em>10</em> days at the osteotomy sites with nearly undetectable levels by 35 days. Treatment of dental implant fixtures with Ad/BMP-7 resulted in enhancement of alveolar <em>bone</em> defect fill, coronal new <em>bone</em> formation, and new <em>bone</em>-to-implant contact. In vivo gene therapy of BMP-7 offers potential for alveolar <em>bone</em> engineering applications.

The role of follistatin as an activin-binding <em>protein</em> has dominated the study of this molecule for the last <em>10</em> years. However, there is emerging evidence that follistatin has a role in modulating the biology of other members of the transforming growth factor beta (TGF-beta) superfamily. This review summarizes the current concepts encompassing follistatin biochemistry as well as molecules with which it is functionally associated. Moreover, the importance of the two follistatin isoforms (follistatin-288 and follistatin-315) is discussed with particular emphasis on the regulation of the ovary. In addition to activin, this review discusses the functions of other members of the TGF-beta superfamily, for example growth differentiation factor 9 (GDF-9), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP-15), BMP-6, BMP-4 and BMP-7, in the ovary, and the potential interactions between follistatin and these growth factors. The complex network of TGF-beta superfamily growth factor members involved in the modulation of ovarian function and the interactions of follistatin with these <em>proteins</em> is highlighted.

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-<em>10</em> (FGF-<em>10</em>), which is expressed in the mesenchyme near the distal tips, and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-<em>10</em> bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant <em>protein</em> C at the tips nearest the diffusible <em>protein</em> source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-<em>10</em> bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-<em>10</em> bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.

METHODS

Enzyme-linked immunosorbent assay was used to detect bone morphogenetic proteins (BMPs) 2, 4, and 7 in 9 commercially available ("off the shelf") demineralized bone matrix (DBM) product formulations using 3 different manufacturer's production lots of each DBM formulation.

OBJECTIVE

To evaluate and compare the quantity of BMPs among several different DBM formulations (inter-product variability), as well as examine the variability of these proteins in different production lots within the same DBM formulation (intra-product variability).

BACKGROUND

DBMs are commonly used to augment available bone graft in spinal fusion procedures. Surgeons are presented with an ever-increasing variety of commercially available human DBMs from which to choose. Yet, there is limited information on a specific DBM product's osteoinductive efficacy, potency, and constancy.

METHODS

There were protein extracts from each DBM sample separately dialyzed 4 times against distilled water at 4 degrees C for 48 hours. The amount of BMP-2, BMP-4, and BMP-7 was determined using enzyme-linked immunosorbent assay. RESULTS.: The concentrations of detected BMP-2 and BMP-7 were low for all DBM formulations, only nanograms of BMP were extracted from each gram of DBM (20.2-120.6 ng BMP-2/g DBM product; 54.2-226.8 ng BMP-7/g DBM). The variability of BMP concentrations among different lots of the same DBM formulation, intra-product variability, was higher than the variability of concentrations among different DBM formulations, inter-product variability (coefficient of variation range BMP-2 [16.34% to 76.01%], P < 0.01; BMP-7 [3.71% to 82.08%], P < 0.001). BMP-4 was undetectable.

CONCLUSIONS

The relative quantities of BMPs in DBMs are low, in the order of 1 x 10(-9) g of BMP/g of DBM. There is higher variability in concentration of BMPs among 3 different lots of the same DBM formulation than among different DBM formulations. This variability questions DBM products' reliability and, possibly, efficacy in providing consistent osteoinduction.

METHODS

Observational study with prospective CT analysis.

OBJECTIVE

To assess the incidence and clinical sequelae of epidural bone formation following the adjunctive use of recombinant bone morphogenetic protein 2 (rhBMP2) with local autogenous bone graft use of (rhBMP2) in minimal access interbody (PLIF and TLIF) fusions.

BACKGROUND

The use of rhBMP2 for interbody fusion is associated with high fusion rates. However, for posterior approaches, concerns regarding heterotopic bone formation within the epidural space have been raised.

METHODS

An independent CT analysis of 33 consecutive patients following minimal access lumbar fusion (PLIF [n = 10] or TLIF [n = 23]) with [n = 23] and without [n = 10] rhBMP2 was performed. Bone formation was graded in a centrifugal manner (intradiscal, anular/ALL/PLL, epidural [canal/foramen] and beyond the spine). In all BMP cases, a constant dose of 4.2 mg/disc level was administered (lowest commercially available dose). In all cases, local autograft was used. Review and assessment of prospectively collected outcomes data were performed.

RESULTS

Average clinical and CT (minimum 6 months) follow-up was 25.0 and 7.9 months, respectively. Bridging bone (fusion) was seen in 100% of the BMP group and 90% without BMP. Epidural bone formation occurred in 20.8% with the use of BMP (5 levels: n = 1 spinal canal and n = 4 within the foramen) compared with 8.3% (1 level: canal) without BMP. Foraminal bone formation was seen only in the TLIF group. All epidural bone formation was heterotopic, and no ectopic bone formation occurred. There were no clinical sequelae associated with heterotopic bone formation. The mean preoperative and postoperative Oswestry Disability Index was 50.2% (range, 25%-75%) and 11.3% (range, 0%-38%) respectively.

CONCLUSIONS

Although the adjunctive use of rhBMP2 is associated with a higher incidence of heterotopic bone, there does not seem to be any associated clinical sequelae.

While parathyroid hormone-related <em>protein</em> (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13-day embryos, and the role of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP-6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (<em>10</em>(-7) M) at the time of plating completely abolished BMP-6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP-6 and type X collagen mRNA, with BMP-6 again preceding type X collagen expression. The addition of exogenous BMP-6 (<em>10</em>0 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP-6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP-6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP-6, it indirectly regulates Ihh expression through BMP-6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP-6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP-6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP-6.

Demineralized freeze-dried <em>bone</em> allograft (DFDBA) has been used extensively in periodontal therapy. The rationale for use of DFDBA includes the fact that <em>proteins</em> capable of inducing new <em>bone</em>; i.e., <em>bone</em> <em>morphogenetic</em> <em>proteins</em>, can be isolated from <em>bone</em> grafts. Commercial <em>bone</em> banks have provided DFDBA to the dental practitioner for many years; however, these organizations have not verified the osteoinductive capacity of their DFDBA preparations. The aim of this study was to determine the ability of commercial DFDBA preparations to induce new <em>bone</em> formation. DFDBA with particle sizes ranging from 200 to 500 microns was received from six <em>bone</em> banks using various <em>bone</em> production methods. Different lots of DFDBA from the same tissue bank were sometimes available. A total of 14 lots were examined. The surface area of <em>bone</em> particles in each sample was measured morphometrically and the pH of a solution containing the particles after suspension in distilled water determined. Samples from each DFDBA lot were implanted intramuscularly (<em>10</em> mg) or subcutaneously (20 mg) into three different animals and tissue biopsies harvested after 4 weeks. One sample from each tissue bank was implanted and harvested after 8 weeks. At harvest, each area where DFDBA had been implanted was excised and examined by light microscopy. The ability of DFDBA to produce new <em>bone</em> was evaluated and the amount of residual <em>bone</em> particles measured. The results show that <em>bone</em> particles from all tissue banks had a variety of shapes and sizes, both before implantation and after 1 or 2 months of implantation. The pH of particle suspensions also varied between batches, as well as between tissue banks. None of the DFDBA induced new <em>bone</em> formation when implanted subcutaneously. Intramuscular implants from three banks induced new <em>bone</em> formation after 1 and 2 months. DFDBA from two banks caused new <em>bone</em> formation only after 2 months. However, DFDBA from one bank did not induce new <em>bone</em> at all. Particle size before implantation correlated with particle size after implantation. However, particle size did not correlate with ability to induce <em>bone</em>. The results show that commercial DFDBA differs in both size and ability to induce new <em>bone</em> formation, but that the two are not related. The study also indicates that wide variation in commercial <em>bone</em> bank preparations of DFDBA exist and that ability to induce new <em>bone</em> formation also varies widely. Furthermore, the results suggest that methods or assays for evaluating the ability of DFDBA to induce new <em>bone</em> should be developed and standardized.