OBJECTIVE

To compare mandibular bone regeneration with bone morphogenetic proteins-2 and -4 (BMP-2 and -4) at varying doses.

METHODS

Defects were created in the left hemi-mandibles of 82 Sprague-Dawley rats. The defects were filled with a hyaluronic acid polymer loaded with 0.01, 0.1, 1, or 10 microg of BMP-2 or -4. Control groups consisted of animals with unfilled defects, or with defects filled with the hyaluronic acid sponges loaded with growth factor dilution buffer. Animals were killed after 8 weeks, and the hemi-mandibles were analyzed histologically using stereologic techniques.

RESULTS

Mandibles implanted with carriers containing 10 microg of BMP-2 or -4 differed significantly from controls in terms of new bone area (p = 0.01 and p = 0.0001, respectively). Marrow space development occurred in a dose-dependent fashion (p < 0.0001 for both growth factors), and this effect was more pronounced for BMP-2 at larger doses (p < 0.0001 at 1 and 10 microg doses). New bone areas and volumes did not differ significantly between the growth factors. While defects implanted with BMP-4 tended to have thicker cortical bone and more trabecular bone, at least partial defect bridging was achieved in a greater number of defects implanted with BMP-2 (47%) than with BMP-4 (35%).

CONCLUSIONS

Although similar areas and volumes of new bone were induced with BMP-2 and -4, differences were noted in the quality of bone generated with each growth factor. The results indicate a threshold dose for acute administration between 1 and 10 mug BMP-2 for bony union in this model, and>> or =10 microg for BMP-4.

CONCLUSIONS

These findings suggest that differences in bone growth factor osteogenic potential deserve further study and may have an impact on the translation of osteoinductive protein therapy into clinical practice.

Osteoarthritis (OA) is a chronic degenerative joint disease showing altered <em>bone</em> metabolism. Osteoblasts contribute to the regulation of cartilage metabolism and <em>bone</em> remodeling. We have shown previously that induction of heme oxygenase-1 (HO-1) protects OA cartilage against inflammatory and degradative responses. In this study, we investigated the effects of HO-1 induction on OA osteoblast metabolism. HO-1 was induced with cobalt protoporphyrin IX (CoPP) and by transduction with LV-HO-1. In osteoblasts stimulated with interleukin (IL)-1β, CoPP enhanced mineralization, the expression of a number of markers of osteoblast differentiation such as Runx2, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, osteocalcin, and collagen 1A1 and 1A2, as well as the ratio osteoprotegerin/receptor activator of nuclear factor-κB ligand. HO-1 induction significantly reduced the expression of matrix metallo<em>protein</em>ase (MMP)-1, MMP-2 and MMP-3, and the production of pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6 whereas IL-<em>10</em> levels increased. HO-1 also exerted inhibitory effects on prostaglandin (PG)E(2) production which could be dependent on cyclooxygenase-2 and microsomal PGE synthase-1 down-regulation. The activity of senescence-associated β-galactosidase and the expression of the senescence marker caveolin-1 were significantly decreased after HO-1 induction. The inhibition of nuclear factor-κB activation induced by IL-1β in OA osteoblasts may contribute to some HO-1 effects. Our results have shown that HO-1 decreases the production of relevant inflammatory and catabolic mediators that participate in OA pathophysiology thus eliciting protective effects in OA osteoblasts.

WFIKKN1 and WFIKKN2 are large extracellular multidomain <em>proteins</em> consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both <em>proteins</em> have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN <em>proteins</em> with several additional representatives of the transforming growth factor (TGF)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN <em>proteins</em> bind TGFβ1, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)2 and BMP4 with relatively high affinity (K(d) ∼ <em>10</em>(-6) m). To assess the biological significance of these interactions we studied the effect of WFIKKN <em>proteins</em> on the activity of GDF8, GDF11, TGFβ1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFβ1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN <em>proteins</em> are antagonists of GDF8 and GDF11, but in the case of TGFβ1, BMP2 and BMP4 they function as growth factor binding <em>proteins</em>. It is suggested that the physical association of WFIKKN <em>proteins</em> with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space.

Cartilage derived <em>morphogenetic</em> <em>proteins</em> (CDMPs, also known as growth and differentiation factors, GDFs) are a subgroup of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) gene family. As most BMPs, they are known to induce cartilage or <em>bone</em> formation when implanted subcutaneously or intramuscularly on an appropriate carrier. However, similar implantation experiments with CDMPs have also reported the formation of a tendon-like tissue, without any cartilage or <em>bone</em>. A solution to this apparent contradiction might be offered by the mechanical tissue differentiation theory, suggesting that tissue differentiation depends on the mechanical environment. This study analyzes the response to CDMP-2 implants at different sites and under different loading conditions in the rat. Collagen sponges carrying CDMP-2 were implanted subcutaneously, intramuscularly or inside a freshly created defect in the achilles tendon. Large amounts of <em>bone</em> were induced subcutaneously, smaller amounts intramuscularly, and in the tendons, only small amounts of <em>bone</em> or cartilage were seen in few animals. Thus, the amount of <em>bone</em> appeared inversely related to the degree of mechanical stimulus. To confirm this, CDMP was also injected into tendon defects that were either loaded or partially unloaded. All the unloaded tendons showed <em>bone</em> induction after one CDMP-2 injection, whereas only 4 of <em>10</em> loaded ones showed any cartilage or <em>bone</em> (p = 0.0005). Single injections of a similar dose of CDMP-2 have previously been shown to augment tendon repair by increasing the size of the tendon callus. This study suggests that the response to CDMP-2 is dependent on the mechanical situation at the site where it is applied.

Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH.

Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured.

Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival.

Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation.

A composite inductive allograft consisting of an allogeneic, autolysed, antigen-free cortical <em>bone</em> carrier lyophilized with partially purified human <em>bone</em> <em>morphogenetic</em> <em>protein</em> was implanted in 30 consecutive femoral reconstructions that resulted from failure of fracture healing. There were 24 atrophic shortened femoral nonunions, four equal length femoral nonunions, and two femoral malunions. There were <em>10</em> men and 20 women with an average age of 47 years (range, 28-75 years). Allogeneic, autolysed antigen-free cortical <em>bone</em> was used as a structural alloimplant and as a delivery system for partially purified human <em>bone</em> <em>morphogenetic</em> <em>protein</em>. The composite implant of human <em>bone</em> <em>morphogenetic</em> <em>protein</em>/allogeneic, autolysed antigen-free cortical <em>bone</em> was used in conjunction with one-stage lengthening of the extremity, restoration of mechanical axis and rotational alignment. In 26 of 30 femurs, the human <em>bone</em> <em>morphogenetic</em> <em>protein</em>/allogeneic autolysed antigen-free cortical <em>bone</em> consisted of an allogeneic cortical <em>bone</em> implant incorporated into a one-stage lengthening of atrophic femoral nonunion. In four patients with equal length femoral nonunions, the human <em>bone</em> <em>morphogenetic</em> <em>protein</em>/allogeneic, autolysed antigen-free implant was placed as an medical femoral shaft onlay graft. Internal remodeling of the implant occurred within 8 to 12 weeks after implantation. Lengthening defects greater than 2 cm were supplemented with intercalary autogeneic <em>bone</em> graft. Twenty-four femurs healed at an average of 6 months at an average followup of 55 months. Four of six plate fatigue failures were salvaged with repeat plating. Two patients were lost to followup. The human <em>bone</em> <em>morphogenetic</em> <em>protein</em>/allogeneic, autolysed antigen-free <em>bone</em> allograft is an excellent structural and delivery system that induces host <em>bone</em> formation and implant remodeling allowing salvage of difficult femoral nonunions.

During endochondral ossification (EO), cartilage is replaced by <em>bone</em>. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 x <em>10</em>(6) cells per 35-mm plate and grown for 17 days in BGJ(b) medium supplemented with <em>10</em>% fetal bovine serum, 50 microg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent <em>bone</em> matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of <em>bone</em> like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive (40)Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> <em>10</em> (BMP<em>10</em>) belongs to the TGFβ-superfamily. Previously, we had demonstrated that BMP<em>10</em> is a key regulator for ventricular chamber formation, growth, and maturation. Ablation of BMP<em>10</em> leads to hypoplastic ventricular wall formation, and elevated levels of BMP<em>10</em> are associated with abnormal ventricular trabeculation/compaction and wall maturation. However, the molecular mechanism(s) by which BMP<em>10</em> regulates ventricle wall growth and maturation is still largely unknown. In this study, we sought to identify the specific transcriptional network that is potentially mediated by BMP<em>10</em>. We analyzed and compared the gene expression profiles between α-myosin heavy chain (αMHC)-BMP<em>10</em> transgenic hearts and nontransgenic littermate controls using Affymetrix mouse exon arrays. T-box 20 (Tbx20), a cardiac transcription factor, was significantly up-regulated in αMHC-BMP<em>10</em> transgenic hearts, which was validated by quantitative RT-PCR and in situ hybridization. Ablation of BMP<em>10</em> reduced Tbx20 expression specifically in the BMP<em>10</em>-expressing region of the developing ventricle. In vitro promoter analysis demonstrated that BMP<em>10</em> was able to induce Tbx20 promoter activity through a conserved Smad binding site in the Tbx20 promoter proximal region. Furthermore, overexpression of Tbx20 in myocardium led to dilated cardiomyopathy that exhibited ventricular hypertrabeculation and an abnormal muscular septum, which phenocopied genetically modified mice with elevated BMP<em>10</em> levels. Taken together, our findings demonstrate that the BMP<em>10</em>-Tbx20 signaling cascade is important for ventricular wall development and maturation.

BACKGROUND

Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions.

METHODS

Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates.

RESULTS

Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.

CONCLUSIONS

Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

METHODS

Intertransverse process spinal fusion using recombinant human bone morphogenetic protein-7 (rhBMP-7) was performed in intact and ovariectomized female rats.

OBJECTIVE

To examine fusion rates in intact and ovariectomized female rats using rhBMP-7 to determine if spine fusion is dependent on estrogen status.

BACKGROUND

Rat spinal fusion has been established as a consistent, efficient model for posterolateral intertransverse process fusion. Previous experiments have confirmed the efficacy of pellets containing the carrier, insoluble collagen bone matrix (ICBM), and rhBMP-7 to augment intertransverse process single level fusion in a rat model. Studying these implications in an osteoporosis model is of clinical value because there are many patients undergoing spinal fusion surgery that have osteoporotic bone disease, and there is a steady increase in this group of patients.

METHODS

A total of 15 ovariectomized and 15 intact Sprague-Dawley female rats were randomly assigned to groups receiving 25 mg ICBM alone, 25 mg ICBM + 10 microg rhBMP-7, and 25 mg ICBM + 30 microg rhBMP-7. Spinal fusion was evaluated by manual motion testing at each lumbar segment, radiographic evaluation using the Lenke grading system, and histology.

RESULTS

Ovariectomized and intact rats receiving 25 mg carrier ICBM alone did not show spinal fusion. With 25 mg ICBM + 10 microg rhBMP-7, there was not a significant difference in fusion rates between intact and ovariectomized rats (P = 0.63). Ovariectomized rats receiving 25 mg ICBM + 30 microg rhBMP-7 showed significantly lower fusion rates than intact rats (P = 0.013).

CONCLUSIONS

These data suggest that spinal fusion using rhBMP-7 is estrogen-dependent in rats. At the dosages used, rhBMP-7 was unable to overcome the inhibitory effects of estrogen deficiency on spinal fusion.

BACKGROUND

The goal of periodontal regenerative therapies is to reconstruct periodontal tissues such as bone, cementum, and periodontal ligament cells (PDL). The need to establish predictable treatment modalities is important for reconstruction of these tissues. The aim of this study was to determine the effects of a low molecular extract of bovine bone protein (BP) containing bone morphogenetic proteins (BMPs) 2, 3, 4, 6, 7, 12, and 13, alone or in combination with platelet-derived growth factor (PDGF) and/or insulin-like growth factor (IGF) on osteoblast differentiation in vitro.

METHODS

BP, mixed with a collagen matrix, was added to a poly (DL-lactide-co-glycolide) polymer (PLG) and placed at orthotopic sites in the skullcaps of Sprague-Dawleys rats. At day 28, rats were sacrificed for histological analysis. All sites treated with the polymer/BP produced bone while control sites (without BP) showed no bone formation. Having established the biological activity of BP, in vitro studies were initiated using MC3T3-E1 cells, a mouse osteoprogenitor cell line. The ability of BP and other growth factors to alter cell proliferation was determined by Coulter counter, and differentiation was determined by Northern analysis for specific genes.

RESULTS

When compared with cells treated with 2% serum alone, PDGF enhanced cell numbers at 10 and 20 ng/ml; IGF produced no significant effect at these doses; and BP at 10 and 20 microg/ml decreased cell proliferation. Northern analysis revealed that PDGF blocked gene expression of osteopontin (OPN) and osteocalcin (OCN), while BP and IGF promoted gene expression of bone sialoprotein (BSP) and OPN. The combination of BP and IGF enhanced expression of OPN beyond that of either BP or IGF alone. PDGF was able to block the effects of IGF on gene expression, but not those of BP.

CONCLUSIONS

These results indicate that BP, PDGF, and IGF influence cell activity differently, and thus raise the possibility that combining factors may enhance the biological activity of cells.

The clinical significance of hydroxyapatite (HAP) as a <em>bone</em> substitute has become apparent in recent years and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) a substance which induces <em>bone</em> has attracted much attention. In this study, a 1.2 cm diameter <em>bone</em> defects created on rabbit cranium were treated with the BMP-2 gene (cDNA plasmid) introduced with porous HAP after completion of hemostasis and the resultant <em>bone</em> formation was analyzed histopathologically. The amounts of <em>bone</em> formation was compared BMP-2 cDNA plasmids were not combined with cationic liposomes as a vector. Four groups of rabbits were compared. In the HAP group the cranial <em>bone</em> defect was treated with HAP containing 40 microg of liposomes and a dummy gene (PU). The BMP gene HAP group was treated with HAP soaked in liposomes and <em>10</em> microg of the BMP-2 gene. In addition, a group was treated with the gene without implanting HAP. <em>Bone</em> formation on the cranial defects was evaluated 3, 6 and 9 weeks after the operation, by X-ray and histopathological examinations. Three weeks after the operation there was vigorous <em>bone</em> formation in the cranial defect in the group which received the BMP-2 gene without HAP, and complete ossification was observed at 9 weeks. In the group which received HAP containing the BMP-2 gene, although new <em>bone</em> formation was evident surrounding the scaffold 3 weeks post-operation, the induced <em>bone</em> tissue did not fill all the pores of the scaffold even at 9 weeks post-operation. These results confirm the clinical usefulness of gene therapy for <em>bone</em> formation, using the BMP-2 gene combined with cationic liposomes as a vector. It is possible that the effects of administering the BMP-2 gene will be improved by specializing the microstructure of scaffold for gene therapy.

BACKGROUND

Prosthetic-driven implant dentistry requires predictable procedures for alveolar ridge augmentation. The objective of this pilot study was to evaluate bone regeneration in mandibular, full-thickness, alveolar ridge, saddle-type defects following surgical implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a novel hyaluronan (HY) sponge carrier. This sponge was fabricated from auto-crosslinked HY.

METHODS

Alveolar ridge defects (approximately 15 x 10 x 10 mm), 2 per jaw quadrant, were surgically prepared in each of 3 young adult American fox hounds. Four defects were immediately implanted with rhBMP-2/HY. Three defects were implanted with rhBMP-2 in an absorbable collagen sponge (ACS) carrier (positive control). The rhBMP-2 solution (1.5 ml at 0.2 mg/ml) was soak-loaded onto the HY and ACS sponges. Three defects were implanted with HY sponges soak-loaded with buffer without rhBMP-2 (negative control), while 2 defects served as surgical controls. The animals were euthanized at 12 weeks postsurgery for histometric analysis.

RESULTS

Clinically, alveolar ridge defects receiving rhBMP-2/ACS exhibited a slight supracrestal expansion, while defects receiving rhBMP-2/HY were filled to contour. In contrast, the HY and surgical controls exhibited ridge collapse. rhBMP-2/HY-treated defects exhibited a dense bone quality without radiolucent regions observed in defects treated with rhBMP-2/ACS. The histometric analysis showed 100% bone fill for the rhBMP-2/ACS defects and 94%, 58%, and 65% bone fill for the rhBMP-2/HY, HY, and surgical control defects, respectively.

CONCLUSIONS

The conclusions are based on data from 2 of 3 animals in the study. In one animal, no response to rhBMP-2 was observed with either carrier, and the animal may have been a non-responder of unknown nature. With this limitation, the observations herein suggest that: 1) HY supports significant bone induction by rhBMP-2; 2) the rhBMP-2-induced bone assumes qualities of the immediate resident bone; 3) HY alone exhibits no apparent osteoconductive potential; and 4) HY appears to resorb within a 12-week healing interval in the absence or presence of rhBMP-2. Thus, HY appears to be a suitable candidate carrier for rhBMP-2.

The Forkhead Box (Fox) family of transcription factors plays important roles in regulating expression of genes involved in cellular proliferation and differentiation. In a previous study, we showed that newborn foxf1(+/-) mice with diminished Foxf1 levels exhibited abnormal formation of pulmonary alveoli and capillaries and died postnatally. Interestingly, surviving newborn foxf1(+/-) mice exhibited increased pulmonary Foxf1 levels and normal adult lung morphology, suggesting that wild-type Foxf1 levels are required for lung development and function. The present study was conducted to determine whether adult foxf1(+/-) mice were able to undergo lung repair similar to that observed in wild-type mice. We demonstrated that adult foxf1(+/-) mice died from severe lung hemorrhage after butylated hydroxytoluene (BHT) lung injury and that this phenotype was associated with a <em>10</em>-fold decrease in pulmonary Foxf1 expression and increased alveolar endothelial cell apoptosis that disrupted capillary integrity. Furthermore, BHT-induced lung hemorrhage of adult foxf1(+/-) mice was associated with a drastic reduction in expression of the Flk-1, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4, surfactant <em>protein</em> B, platelet endothelial cell adhesion molecule, and vascular endothelial cadherin genes, whereas the expression of these genes was either transiently diminished or increased in wild-type lungs after BHT injury. Because these <em>proteins</em> are critical for lung morphogenesis and endothelial homeostasis, their decreased mRNA levels are likely contributing to BHT-induced lung hemorrhage in foxf1(+/-) mice. Collectively, our data suggest that sustained expression of Foxf1 is essential for normal lung repair and endothelial cell survival in response to pulmonary cell injury.

Fracture repair is a complex process involving timed cellular recruitment, gene expression, and synthesis of compounds that regenerate native tissue to restore the mechanical integrity, and thus function of injured <em>bone</em>. While the majority of fractures heal without complication, this takes time and a subset of patients ( approximately <em>10</em>%) experience healing delays, extending their morbidity and treatment costs. Consequently, there is a need for efficacious therapeutics for the intervention of fracture healing. Recent studies into the molecular control of fracture repair and advances in the understanding of the skeleton as a whole have resulted in the identification of numerous novel targets and compounds for such intervention. These include traditional agents such <em>bone</em> <em>morphogenetic</em> <em>proteins</em> and other growth factors, but also relatively newer compounds such as parathyroid hormone and modulators of the Wnt signaling pathway. These agents, along with others, are discussed in the current article in terms of their investigative status and potential for clinical implementation. Hopefully, these agents, as well as others yet to be discovered, will demonstrate sufficient clinical utility for successful intervention of fracture healing. This may have significant implications for the duration of morbidity and costs associated with traumatic <em>bone</em> fractures.

Hepcidin is a hepatocellular hormone that inhibits the release of iron from certain cell populations, including enterocytes and reticuloendothelial cells. The regulation of hepcidin (HAMP) gene expression by iron status is mediated in part by the signaling molecule <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6 (BMP6). We took advantage of the low iron status of juvenile mice to characterize the regulation of Bmp6 and Hamp1 expression by iron administered in three forms: 1) ferri-transferrin (Fe-Tf), 2) ferric ammonium citrate (FAC), and 3) liver ferritin. Each of these forms of iron enters cells by distinct mechanisms and chemical forms. Iron was parenterally administered to <em>10</em>-day-old mice, and hepatic expression of Bmp6 and Hamp1 mRNAs was measured 6 h later. We observed that hepatic Bmp6 expression increased in response to ferritin but was unchanged by Fe-Tf or FAC. Hepatic Hamp1 expression likewise increased in response to ferritin and Fe-Tf but was decreased by FAC. Exogenous ferritin increased Bmp6 and Hamp1 expression in older mice as well. Removing iron from ferritin markedly decreased its effect on Bmp6 expression. Exogenously administered ferritin and the derived iron localized in the liver primarily to sinusoidal lining cells. Moreover, expression of Bmp6 mRNA in isolated adult rodent liver cells was much higher in sinusoidal lining cells than hepatocytes (endothelial>>) stellate>> Kupffer). We conclude that exogenous iron-containing ferritin upregulates hepatic Bmp6 expression, and we speculate that liver ferritin contributes to regulation of Bmp6 and, thus, Hamp1 genes.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) is known for its osteoinductive potential in <em>bone</em> tissue engineering. Calcium phosphate (Ca-P) cements are injectable, osteoconductive ceramic materials in which a macroporous structure can be induced during the setting reaction. In this study, the osteoinductive capability of rhBMP-2 loaded porous Ca-P cement was evaluated. Porous Ca-P cement discs were made and loaded with rhBMP-2 in vitro and implanted subcutaneously in the back of New Zealand white rabbits. The implantation period was either 2 or <em>10</em> weeks. Histological analysis of retrieved specimens revealed evident <em>bone</em> formation in the rhBMP-2 loaded Ca-P cement discs (pore fill: 18+/-6%) after <em>10</em> weeks of implantation. <em>Bone</em> formation occurred only in rhBMP-2 loaded porous Ca-P cement discs. Degradation of the Ca-P cement could not be confirmed after <em>10</em> weeks of implantation. The scaffold maintained its shape and stability during this time period. We conclude that porous Ca-P cement is a suitable carrier material for ectopic <em>bone</em> engineering.

BACKGROUND

Bone morphogenetic proteins (BMPs) are actively involved in ossification, and BMP-2 participates throughout the entire process. Gene therapy for bone regeneration using adenovirus-expressing BMPs has been successful in small mammals, but it has not been satisfactory in large mammals.

METHODS

We generated a 3-component implant (3C graft) comprising autologous mesenchymal stem cells (MSCs), ex vivo transduced with an adenovirus vector-expressing BMP-2 and embedded in a demineralized human bone matrix (DBM).

RESULTS

In vitro studies demonstrated vector-induced osteogenesis; osteoblast population and mineralization of the extracellular matrix were greater in the vector-transduced cultures than in the controls (nontransduced MSCs stimulated with osteogenic media were used as positive controls, and nontransduced MSCs served as a negative control). The 3-component grafts were used to fill osteotomies created by bone distraction surgery in mongrel dogs. Control groups comprised dogs with bone distraction alone and dogs with nontransduced MSC grafts. The radiography follow-up, performed 10 weeks after distraction, demonstrated a remarkable reduction in the consolidation period compared with controls. Postmortem mandibles submitted for anatomic and histologic analyses showed improved remodeling and bone maturation in the 3C-grafted dogs. Inflammatory infiltrates were not observed in any of the treated areas, and no liver toxicity was detected.

CONCLUSIONS

We demonstrated acceleration of osteogenesis in a dog model for bone distraction by using an implant of BMP-2 modified MSCs. These results are helpful for future clinical trials of mandible bone distraction.

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a <em>10</em>00-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-I, insulin, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, parathyroid hormone, growth hormone, dexamethasone, 1alpha25-dihydroxy vitamin D3, L-3,3',5'-triodothyronine, interleukine-1 receptor antagonist, 17beta-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with <em>10</em> ng/ml FGF-2, while the cell number synergistically gained a <em>10</em>-12-fold increase with 5 microg/ml insulin or <em>10</em>0 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of <em>10</em>0 ng/ml for FGF-2, and especially for the combination of <em>10</em>0 ng/ml FGF-2 and 5 microg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than <em>10</em>,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.

METHODS

An in vitro biologic study comparing the effects of a series of bone morphogenetic proteins (BMPs) and Sox9 on the extracellular matrix accumulation by bovine anulus fibrosus (AF) cells.

OBJECTIVE

To compare the effects of adenoviral-mediated overexpression of various BMPs and Sox9 on extracellular matrix accumulation by AF cells in vitro.

BACKGROUND

Repair of the disrupted AF, which is perceived to be a potential therapy to diminish nucleus pulposus (NP) herniation, may also offer a treatment strategy for severe symptomatic degenerative disc disease. To date, no systematic comparison of a large group of growth factors in the AF has been published. In this study, we compared the effects of the adenoviral-mediated overexpression of 12 BMPs and Sox9 on extracellular matrix production by AF cells.

METHODS

Adult monolayer-cultured bovine AF cells were transduced with adenoviral vectors containing human BMP and green fluorescence protein (GFP) genes (AdBMPs), or Sox9 and GFP genes (AdSox9), or GFP gene alone (AdGFP, as negative control). Proteoglycan and collagen accumulation, and cell proliferation were measured for each of the treatment groups 6 days after viral transduction.

RESULTS

AF cells transduced with BMP-2, -3, -5, -7, -8, -12, -13, -14, and -15, and Sox9 accumulated significantly more collagen than AF cells transduced with AdGFP (control). AF cells transduced with AdBMP-2, -4, -7, -10, -12, and -13, and AdSox9 accumulated significantly more proteoglycans than AF cells transduced with AdGFP.

CONCLUSIONS

We have demonstrated the relative effectiveness of 12 different BMPs and Sox9 on the stimulation of proteoglycan and/or collagen accumulation by AF cells. This study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by AF. This information should prove useful to those seeking to develop a strategy for repair of the AF in humans.

OBJECTIVE

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide which has antifibrogenic effects at physiological concentrations in various tissues. AcSDKP is produced locally in the liver, however, little is known about its biological effect in this organ. We hypothesize that basal levels of endogenous AcSDKP decrease during the development of liver fibrosis and preservation of basal AcSDKP attenuates liver fibrosis.

METHODS

Endogenous levels of AcSDKP in the liver were measured by enzyme immunoassay after 2, 6, and <em>10</em> weeks of carbon tetrachloride (CCl(4))-induced liver fibrosis in rats. Subcutaneous osmotic pump infusion of vehicle or AcSDKP (800 microg/kg/day) was administered to CCl(4)-treated rats for 8 weeks to study the effect of exogenous AcSDKP on liver fibrosis. The effect of AcSDKP on profibrogenic properties of hepatic stellate cells was studied in vitro.

RESULTS

Endogenous AcSDKP was significantly decreased in the liver of CCl(4)-treated rats. Chronic AcSDKP infusion preserved basal levels of AcSDKP and reduced liver injury, inflammation, fibrosis, and profibrogenic transforming growth factor-beta signaling. This was demonstrated by decreased aminotransferase serum levels, CD45 positive cells, collagen accumulation, alpha-smooth muscle actin positivity, transforming growth factor-beta1, phosphorylated Smad2/3 protein, increased bone morphogenetic protein-7, and phosphorylated Smad1/5/8. Further, AcSDKP exerts antifibrogenic effects on hepatic stellate cells (HSCs) by downregulation of HSC activation in vitro.

CONCLUSIONS

Maintaining physiological levels of AcSDKP is critical in negatively regulating the development of fibrosis in chronic liver injury. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

We have previously shown that the animal pole tissue from a st.<em>10</em>+ early gastrula Xenopus embryo stimulates the primary differentiation of erythrocytes in the ventral mesoderm in combination culture. To characterize the nature of this stimulation, various sizes and different portions of animal pole tissue were combined with the ventral mesoderm explants. The erythrocyte differentiation in terms of globin expression depended on the size of the animal pole tissue that was combined with the ventral mesoderm. No difference was observed in the potency of stimulation between the ventral and dorsal halves of animal pole tissue. We also found that animal pole tissue from as late as st.7 failed to stimulate both mRNA and <em>protein</em> levels of globin in the explant. Histological studies of the combination explant with st.7 animal pole tissue showed that epidermis, vesicle structure, and blood-cell-like cells developed in the explant, but very few blood cells expressed globin molecules. However, the stimulation of erythroid differentiation was restored if total (20 ng) or poly(A)+ (0.2 ng) RNA from st.<em>10</em>+ animal pole tissue was previously injected at the 2-cell stage and the resulting animal pole tissue at st.7 was combined with st.<em>10</em>+ ventral mesoderm. Erythroid differentiation was also restored by injection with 1 ng of Xenopus <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4 (XBMP-4) RNA. The effect of an extremely small dose of poly(A)+ RNA on erythroid differentiation suggests that in addition to XBMP-4 there exist substances, expressed later than st.7 in the animal pole region, which can stimulate erythrocyte differentiation in the ventral mesoderm.

BACKGROUND

The secondary hyperparathyroidism of chronic kidney disease (CKD) produces a high turnover osteodystrophy that is associated with peritrabecular fibrosis. The nature of the cells involved in the development of peritrabecular fibrosis may represent osteoprogenitors expressing a fibroblastic phenotype that are retarded from progressing through osteoblast differentiation.

METHODS

To test the hypothesis that osteoblast differentiation is retarded in secondary hyperparathyroidism due to CKD producing <em>bone</em> marrow fibrosis, we administered <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP-7), a physiologic regulator of osteoblast regulation, to C57BL6 mice that had CKD produced by electrocautery of one kidney followed by contralateral nephrectomy two weeks later. Following the second surgical procedure, a subgroup of mice received daily intraperitoneal injections of BMP-7 (<em>10</em> microg/kg). Three to six weeks later, the animals were sacrificed, blood was obtained for measurements of blood urea nitrogen (BUN) and parathyroid hormone (PTH) levels, and the femora and tibiae were processed for histomorphometric analysis.

RESULTS

The animals had significant renal insufficiency with BUN values of 77.79 +/- 22.68 mg/dL, and the level of renal impairment between the CKD untreated mice and the CKD mice treated with BMP-7 was the same in the two groups. PTH levels averaged 81.13 +/- 51.36 and 75.4 +/- 43.61 pg/mL in the CKD and BMP-7 treated groups, respectively. The animals with CKD developed significant peritrabecular fibrosis. In addition, there was an increase in osteoblast surface and osteoid accumulation as well as increased activation frequency and increased osteoclast surface consistent with high turnover renal osteodystrophy. Treatment with BMP-7 eliminated peritrabecular fibrosis, increased osteoblast number, osteoblast surface, mineralizing surface and single labeled surface. There was also a significant decrease in the eroded surface induced by treatment with BMP-7.

CONCLUSIONS

These findings indicate that BMP-7 treatment in the setting of high turnover renal osteodystrophy prevents the development of peritrabecular fibrosis, affects the osteoblast phenotype and mineralizing surfaces, and decreases bone resorption. This is compatible with a role of osteoblast differentiation in the pathophysiology of osteitis fibrosa.

OBJECTIVE

The goal of this study was to demonstrate the incidence of fusion and soft-tissue swelling in multilevel anterior cervical discectomies and fusions (ACDFs) using polyetheretherketone (PEEK) spacers with recombinant human bone morphogenetic protein-2 (rhBMP-2) impregnated in a Type I collagen sponge and titanium plates.

METHODS

A single surgeon performed 30 multilevel ACDFs using PEEK spacers with an rhBMP-2 impregnated collagen sponge (0.4 ml, or the equivalent of 0.6 mg rhBMP-2). Soft-tissue swelling was assessed using cervical spine radiographs on postoperative Day 1 and at 2, 6, and 10 weeks and 6 months after surgery. Incidence of dysphagia was assessed with the Cervical Spine Research Society Swallowing-Quality of Life tool. Clinical success was evaluated with the Neck Disability Index, neck pain scores, and arm pain scores. Final fusion was assessed with CT by an independent neuroradiologist.

RESULTS

Patients were followed for 6 months unless they had an incomplete fusion; those patients were reassessed at 9 months. Twenty-four patients underwent 2-level ACDFs and 6 underwent 3-level ACDFs were performed on patients with the following risk factors for pseudarthrosis: smoking (33%), diabetes (13%), and obesity (body mass index ≥ 30 [43%]). Seventeen percent of the patients had multiple risk factors. Soft-tissue swelling peaked at 2 weeks regardless of level of surgery or number of levels treated surgically and decreased to near preoperative levels by 6 months. At 2 weeks, Swallowing-Quality of Life evaluation showed 19% of patients frequently choking on food, 4.8% frequently choking when drinking, and 47.6% with frequent food sticking in the throat. Scores continued to improve, and at 6 months, 0% had frequent choking on food, 6.7% had frequent difficulty drinking, and 6.7% had frequent food sticking in the throat. The Neck Disability Index, neck pain, and arm pain scores all improved progressively over 6 months. Incidence of fusion was 95% at 6 months and 100% at 9 months. There were no rehospitalizations or reoperations for soft-tissue swelling or dysphagia.

CONCLUSIONS

Multilevel ACDF procedures using PEEK grafts and rhBMP-2 can be performed safely in patients with multiple risk factors for pseudarthrosis with excellent fusion outcomes.