METHODS

This was a prospective study evaluating the incidence of retrograde ejaculation (RE) after anterior lumbar interbody fusion (ALIF) based on laboratory analysis of semen and urine.

OBJECTIVE

The purpose of this study was to investigate the incidence of RE based on quantitative semen analysis and to compare the rate with that identified using a standardized qualitative questionnaire administered by telephone.

BACKGROUND

RE is a specific sexual dysfunction in which semen passes into the bladder during ejaculation. It has long been a known possible complication of ALIF associated with injury to the superior hypogastric plexus occurring during access to the anterior lumbar spine. Although reported in multiple studies, there is no standardized assessment for RE and many studies do not describe the methods by which it was assessed. Recently, there has been increased interest in ALIF-related RE with respect to the use of recombinant human bone morphogenetic protein (rhBMP-2). However, how RE was diagnosed remains loosely defined. One possible assessment of RE is to analyze semen and urine after ejaculation.

METHODS

Forty-one male patients undergoing ALIF with posterior pedicle screw instrumentation and fusion at L4-L5 and/or L5-L1 consented to participate in the study. The subjects went to a cryobank for preoperative semen and postejaculatory urine analysis. They returned to the cryobank 3 to 6 months after surgery for repeat testing. The semen analysis consisted of ejaculate volume, total sperm count, concentration, and motility, whereas the urine test reported postejaculatory voided urine for spermatozoa. Postoperatively, patients were called by the principle investigator who conducted an interview based on a standardized questionnaire (completed for 36 patients). Patients were considered to have RE based on the questionnaire if they reported less or no ejaculate fluid. These results were compared with those from the laboratory testing to evaluate the value of the questionnaire in assessing RE. rhBMP-2 was used in 21 of the cases. The incidence of RE was compared in these patients versus those in whom rhBMP-2 was not used.

RESULTS

Four patients (9.8%) were diagnosed with RE based on the laboratory analysis, 2 of which resolved spontaneously. Based on the questionnaire, 15 (41.7%) patients reported having RE, including the 4 that were confirmed by laboratory testing. Of the 21 patients treated with rhBMP-2, 2 (9.5%) were diagnosed with RE, 1 of which resolved. Of the 20 patients treated without rhBMP-2, 2 (10.0%) had RE, 1 of which resolved.

CONCLUSIONS

This study suggests that use of a questionnaire tends to overestimate the incidence of RE as compared with the quantitative semen and urine analysis. Contrary to recent studies using retrospective qualitative review, in this small group with quantitative analysis, use of rhBMP-2 was not related to an increased incidence of RE, with a rate of approximately 10% in both patients receiving, and those not receiving, rhBMP-2. Further study of a larger group using preoperative semen and urine analysis, postoperative standard questionnaire and postoperative semen analysis should be pursued to further investigate the occurrence of RE and to possibly assist in developing and validating a questionnaire for RE assessment.

OBJECTIVE

To examine the effectiveness of administering recombinant human bone morphogenetic protein-2 (rhBMP-2) in distraction osteogenesis.

METHODS

Twenty-one mature male Japanese white rabbits underwent unilateral mandibular osteotomy. After 5 days, the osteotomized mandibles were distracted 1mm/day for 10 days. On the first day (groups A-1 [n=4] and A-2 [n=4]) or on the last day (group B [n=5]) of distraction, rhBMP-2 mixed with collagen gel was injected into the distraction zone. In control groups (groups C-1 [n=4] and C-2 [n=4]), the mandibles were distracted without rhBMP-2 injection. At the end of the distraction period (groups A-1 and C-1) and after 2 weeks of consolidation (groups A-2, B, and C-2), the distracted mandibles were harvested and examined with soft radiographs, peripheral quantitative computed tomography (pQCT), and microscopy.

RESULTS

Radioopacity was more marked in the distraction zone of the groups with rhBMP-2 than in control groups. The mineral density of the cortical bone (BMD) was higher in groups B and A-2 than in group C-2. Histologically, bone formation was more advanced in groups A-2 and B than in group C-2. The cortical BMD was higher in group A-1 than in group C-1. Histologically, bone formation was more advanced in groups A-2 and B than in group C-2.

CONCLUSIONS

These results suggest that rhBMP-2 promotes bone formation in distraction osteogenesis.

OBJECTIVE

To investigate the relationship between endothelial-to-mesenchymal transition (EndMT) and myocardial fibrosis in acute viral myocarditis (VMC).

METHODS

Twenty-eight Balb/c mice were randomized into 3 groups: control group (n=8), VMC group(n=<em>10</em>) and intervention group(n=<em>10</em>). Mice in VMC and intervention groups were injected intraperitoneally(i.p) with single dose of coxsackievirus B3, mice in control group were injected with equal amount of viral-free vehicle. In the following day, mice in control and VMC groups were injected i.p with 0.1 ml of saline and intervention group with 0.1 ml of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7(rh-BMP7) at a concentration of 300 μg/kg. The mice hearts were harvested after 7 d, cardiac collagen volume fraction (CVF) was calculated on picrosirius red-stained sections. mRNA and <em>protein</em> expression of TGF-β1, CD31, VE-cadherin, fibroblast special <em>protein</em> 1 (FSP-1) and α-smooth muscle actin (α-SMA) and collagen 1α1 in myocardiac tissues were detected by real-time RT-PCR and Western blot analysis, respectively.

RESULTS

Compared to controls, overt fibrosis was presented in necrotic area of myocardium in VMC group. Meanwhile, marked increase of TGF-β1 expression accompanied with EndMT characterized by loss of endothelial phenotype (decreased expression of CD31 and VE-cadherin), gain of mesenchymal proteins (overexpression of FSP-1 and α-SMA) and increased synthesis of collagen was also demonstrated. Both EndMT and cardiac fibrosis were simultaneously reversed by TGF-β1 inhibition.

CONCLUSIONS

EndMT is involved in cardiac fibrosis in acute viral myocarditis, TGF-β1 might be a main mediator.

Severe hematopoietic loss is one of the major therapeutic targets after radiation-combined injury (CI), a kind of injury resulting from radiation exposure combined with other traumas. In this study, we tested the use of ciprofloxacin (CIP) as a treatment, because of recently reported immunomodulatory effects against CI that may improve hematopoiesis. The CIP regimen was a daily, oral dose for 3 weeks, with the first dose 2 h after CI. CIP treatment improved 30-day survival in mice at 80% compared to 35% for untreated controls. Study of early changes in hematological parameters identified CI-induced progressive anemia by <em>10</em> days that CIP significantly ameliorated. CI induced erythropoietin (EPO) mRNA in kidney and <em>protein</em> in kidney and serum; CIP stimulated EPO mRNA expression. In spleens of CI mice, CIP induced <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) in macrophages with EPO receptors. Splenocytes from CIP-treated CI mice formed CD71⁺ colony-forming unit-erythroid significantly better than those from controls. Thus, CIP-mediated BMP4-dependent stress erythropoiesis may play a role in improving survival after CI.

Porousbiodegradable polymer scaffolds are widely utilized for <em>bone</em> tissue engineering, but are not osteoconductive like calcium phosphate scaffolds. We combine indirect solid freeform fabrication (SFF), ex vivo gene therapy, with biomineral coating to compare the effect of biomineral coating on <em>bone</em> regeneration for Poly (L-lactic acid) (PLLA) and Poly (ε-caprolactone) (PCL) scaffolds with the same porous architecture. Scanning electron microscope (SEM) and micro-computed tomography (μ-CT) demonstrate PLLA and PCL scaffolds have the same porous architecture and are completely coated. All scaffolds are seeded with human gingival fibroblasts (HGF) transduced with adenovirus encoded with either <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP-7) or green fluorescent <em>protein</em> (GFP), and implanted into mice subcutaneously for 3 and <em>10</em> weeks. Only scaffolds with BMP-7 transduced HGFs show mineralized tissue formation. At 3 weeks some blood vessel-like structures are observed in coated PLLA and PCL scaffolds, but there is no significant difference in <em>bone</em> ingrowth between the coated and uncoated scaffolds for either PLLA or PCL. At <em>10</em> weeks, however, coated scaffolds (both PLLA and PCL) have significantly more <em>bone</em> ingrowth than uncoated scaffolds, which have more fibrous tissue. Coated PLLA scaffolds have improved mechanical properties compared with uncoated PLLA scaffolds due to increased <em>bone</em> ingrowth.

This study tested the hypothesis that preactivated and disaggregated shape-changed platelet (PreD-SCP) therapy attenuates lung injury from acute respiratory distress syndrome (ARDS) induced by <em>10</em>0% oxygen inhalation and complicated by sepsis through peritoneal administration of 1.5 mg/kg lipopolysaccharide (LPS).

Adult male Sprague-Dawley rats, weighing 325 to 350 g, were randomized into group 1 (normal controls [NC]), group 2 (NC + PreD-SCP [3.0 × <em>10</em>, intravenous administration]), group 3 (ARDS-LPS), and group 4 (ARDS-LPS + PreD-SCP), and sacrificed by 72 h after ARDS induction.

The lung injury score was significantly higher in group 3 than that in other groups, and significantly higher in group 4 than that in groups 1 and 2, whereas the numbers of alveolar sacs and oxygen saturation (%) showed a reversed pattern compared with that of lung injury score among the four groups (all P < 0.0001) without significant difference between groups 1 and 2. The expressions of proinflammatory cells (CD11+, CD14+, CD68+) and proteins (tumor necrosis factor [TNF]-α, nuclear factor [NF]-κB, interleukin [IL]-1ββ, matrix metalloproteinase [MMP]-9, inducible nitric oxide synthase, intercellular adhesion molecule-1) exhibited a pattern identical to the lung injury score. Circulating levels of white blood cell, IL-6, TNF-α, myeloperoxidase and CCL5, and pulmonary protein expressions of oxidative stress (NOX-1/NOX-2, oxidized protein), apoptotic (Bax, cleaved caspase 3/poly (ADP-ribose) polymerase), fibrotic (Smad3, transforming growth factor [TGF]-β), and DNA damage (γ-H2AX) biomarkers showed an identical pattern, whereas protein expressions of antifibrotic (Smad1/5, bone morphogenetic protein [BMP]-2) and anti-inflammatory (Bcl-2) biomarkers demonstrated an opposite pattern compared with the proinflammatory indices among the four groups (all P < 0.001).

PreD-SCP therapy effectively improved lung injury in ARDS complicated by sepsis.

The delivery of osteogenic factors is a proven therapeutic strategy to promote <em>bone</em> regeneration. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) constitute a family of cytokines with well-known osteogenic and <em>bone</em> regenerative abilities. However, clinical uses of BMPs require high doses that have been associated with complications such as osteolysis, ectopic <em>bone</em> formation, or hematoma formation. In the present work, we sought to improve <em>bone</em> tissue engineering through an approach that combines the use of <em>bone</em> marrow-derived mesenchymal stem cells (BMMSCs), composite scaffolds, and osteoinductive agents. We employed a composite gelatin/CaSO4 scaffold that allows for an early expansion of seeded BMMSCs, which is followed by an increased level of osteogenic differentiation after <em>10</em> days in culture. Furthermore, this scaffold enhanced <em>bone</em> formation by BMMSCs in a mouse model of critical-sized calvarial defect. More importantly, our results demonstrate that ex vivo pretreatment of BMMSCs with low amounts of BMP-2 (2 nM) and Wnt3a (50 ng/mL) for 24 h cooperatively increases the expression of osteogenic markers in vitro and <em>bone</em> regeneration in the critical-sized calvarial defect mouse model. These data provide a strong rationale for the development of an ex vivo cooperative use of BMP-2 and Wnt3a. Osteogenic factor cooperation might be applied to reduce the required amount of growth factors while obtaining higher therapeutic effects.

METHODS

Prospective animal and human clinical pilot trial.

OBJECTIVE

The purpose of this study was to determine and test the dose of Ne-Osteo growth factor extract and carrier required for consistent radiographic bone induction in humans.

BACKGROUND

Preclinical studies have demonstrated that Ne-Osteo, an extract-containing bone morphogenetic proteins, was successful at generating spine fusion in rabbits and rhesus monkeys. Consistent fusions have yet to be achieved in nonhuman primates and humans.

METHODS

Adult rhesus monkeys underwent single-level posterolateral intertransverse lumbar arthrodesis with either 3.0 mg (N = 4), 5.0 mg (N = 4), 12.5 mg (N = 4), or 25 mg (N = 4) of Ne-Osteo per side. Animals were killed after 24 weeks. In the human clinical trial, 22 patients (18 females, 4 males) had lumbar spinal stenosis and/or spondylolisthesis requiring spine arthrodesis. To minimize patient risk of nonunion, patients received autogenous bone graft from the posterior iliac crest on one side and Ne-Osteo growth factor on the other. The dose was 12.5 mg, 25 or 50 mg, or 25 mg Ne-Osteo per side performed in the three phases, respectively.

RESULTS

Three of four monkeys that received 12.5 mg Ne-Osteo per side and four of four that received 25 mg per side achieved solid fusions. In phase I of the human clinical trial, two of six patients showed radiographic bone induction (plain radiograph, CT scans-blindly evaluated) on the Ne-Osteo side (12.5-mg dose). In phase II, both sides were graded as fused in five of six patients. Although graded as fused, the 6-month scans demonstrated a ring of new bone with the center filling in slower (12-24 mo) than was predicted by nonhuman primate studies. As a result, phase III carrier was designed to have a more porous/open early fusion mass than with the dense DBM paste (used in phase I and II) by mixing in local bone or cancellous allograft chips. Results using the 25- and 50-mg doses were the same, so 25 mg was used in phase III. In phase III, 9 of 10 autograft were fused by 12 months. Five of five patients with Ne-Osteo plus local bone and four of five with allograft chips were fused by 6 months. The one patient in this group that did not heal on either the autograft or the Ne-Osteo side was a smoker.

CONCLUSIONS

A graft composite of Ne-Osteo bone growth factor with human DBM with or without cancellous allograft or local bone autograft was capable of achieving a contiguous spine fusion mass in 15 of 16 patients at a dose of at least 25 mg per side. This result was comparable with the results using iliac crest autograft (94%) in this side-by-side model. These results warrant confirmation in a definitive trial using Ne-Osteo on both sides of the spine and thus avoiding the need for iliac crest bone graft harvest.

Recombinant human growth/differentiation factor 5 (rhGDF-5) and recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP-2), as members of the transforming growth factor Β family, influence <em>bone</em> formation and differentiation. This in vitro osteoblast cell culture study investigated the molecular biologic effect of these growth factors on regulator gene expression of the homebox <em>proteins</em> MSX1 and MSX2 as well as distal-less homebox 5 (Dlx5) and runt-related transcription factor 2 (Runx2/Cbfa1). Concerning effector genes, the messenger ribonucleic acids of osteocalcin (OCN) were quantified using a reverse transcriptase real-time polymerase chain reaction. Over a period of 15 days, osteoblasts were stimulated and analyzed at days 1, 2, 5, <em>10</em>, and 15. rhGDF-5 and rhBMP-2 were applied in concentrations of <em>10</em>0, 500, and 1,000 ng/mL. The results showed enhanced gene expression of MSX1 and MSX2 by the lower rhGDF-5 concentration (<em>10</em>0 ng/mL) during the first 48 hours and a marginally enhanced gene expression of Runx2 and OCN in a dose-dependent manner. The rhBMP-2 stimulation showed enhanced MSX1 and MSX2 gene expression with peaks at 24 and 240 hours; Runx2 and OCN were more highly expressed than the unstimulated control with the <em>10</em>0-ng/mL concentration. rhGDF-5 seems to stimulate early osteoblast differentiation and extracellular matrix production, while rhBMP-2 seems to boost early and late osteoblast differentiation. Low growth factor concentrations appeared to be more effective in terms of gene expression.

BACKGROUND

Repair of complex cranial defects is hindered by a paucity of appropriate donor tissue. Bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 1 (TGFβ1) have been shown separately to induce bone formation through physiologically distinct mechanisms and potentially improve surgical outcome for cranial defect repair by obviating the need for donor tissue. We hypothesize that a combination of BMP2 and TGFβ1 would improve calvarial defect healing by augmenting physiologic osteogenic mechanisms.

RESULTS

Coronal suturectomies (3×15 mm) were performed in 10-day-old New Zealand White rabbits. DermaMatrix™ (3×15mm) patterned with four treatments (vehicle, 350 ng BMP2, 200 ng TGFβ1, or 350 ng BMP2+200 ng TGFβ1) was placed in suturectomy sites and rabbits were euthanized at 6 weeks of age. Two-dimensional (2D) defect healing, bone volume, and bone density were quantified by computed tomography. Regenerated bone was qualitatively assessed histologically. One-way analysis of variance revealed significant group main effects for all bone quantity measures. Analysis revealed significant differences in 2D defect healing, bone volume, and bone density between the control group and all treatment groups, but no significant differences were detected among the three growth factor treatment groups. Qualitatively, TGFβ1 treatment produced bone with morphology most similar to native bone. TGFβ1-regenerated bone contained a suture-like tissue, growing from the lateral edge of the defect margin toward the midline. Unique to the BMP2 treatment group, regenerated bone contained lacunae with chondrocytes, demonstrating the presence of endochondral ossification.

CONCLUSIONS

Total healing in BMP2 and TGFβ1 treatment groups is not significantly different. The combination of BMP2+TGFβ1 did not significantly increase bone healing compared with treatment with BMP2 or TGFβ1 alone postoperatively at 4 weeks. We highlight the potential use of TGFβ1 to regenerate calvarial bone and cranial sutures. TGFβ1 therapy significantly augmented bony defect healing at an earlier time point when compared with control, regenerated bone along the native intramembranous ossification pathway, and (unlike BMP2 alone or in combination with TGFβ1) permitted normal suture reformation. We propose a novel method of craniofacial bone regeneration using low-dose, spatially controlled growth factor therapies to minimize potentially harmful effects while maximizing local bioavailability and regenerating native tissues.

The <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor IB (BMPR-IB) gene was studied as a candidate gene for the prolificacy of goats. According to mRNA sequence of ovine BMPR-IB gene, ten pairs of primers were designed to detect single nucleotide polymorphisms (SNPs) of exon 1, exon 2, exon 6 to exon <em>10</em> and 3' untranslated region (UTR) of the BMPR-IB gene in both high prolificacy breed (Jining Grey goat) and low prolificacy breeds (Wendeng Dairy and Inner Mongolia Cashmere goats) by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method. Only the products amplified by primers P8 and P<em>10</em> of the 3'UTR displayed polymorphisms. For primer P8, three genotypes (AA, AB and BB) were detected in Jining Grey and Wendeng Dairy goats, two genotypes (AA and AB) were in Inner Mongolia Cashmere goats. Sequencing revealed one mutation (71C→T) of the BMPR-IB gene in genotype BB compared with AA. The differences of least squares mean (LSM) for litter size between genotypes AA, AB and BB were non-significant (P>> 0.05) in Jining Grey goats. For primer P<em>10</em>, three genotypes (CC, CD and DD) were detected in Jining Grey and Wendeng Dairy goats and one genotype (CC) in Inner Mongolia Cashmere goats. Sequencing revealed one mutation (130T→C) of the BMPR-IB gene in genotype DD compared with CC. The differences of LSM for litter size between genotypes CC, CD and DD were non-significant (P>> 0.05) in Jining Grey goats. These results preliminarily showed that the detected loci of the BMPR-IB gene had no significant effect on prolificacy of Jining Grey goats.

The purpose of this study was three-fold: (a) to develop a new small animal model to evaluate dental implant systems that recapitulates aspects of the challenging intraoral environment, (b) screen several scaffolds for in vivo <em>bone</em> forming efficacy when used to deliver non-glycosylated <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) together with a miniaturized titanium (Ti) dental implant, and (c) identify correlations between in vitro BMP-2 release rates and in vivo results. The scaffolds tested were: (1) collagen-hydroxyapatite composite (Col/HA), (2) polyethylene glycol hydrogel (PEG-hydrogel), and (3) Col/HA infused with PEG-hydrogel (Col/HA/PEG-hydrogel). BMP-2 delivery directly from the Ti implants rather than from the scaffolds was also tested. MicroCT analyses at 4 weeks showed that the maximum volume and height of new <em>bone</em> occurred when BMP-2 (<em>10</em> μg) was delivered from the Col/HA/PEG-hydrogel scaffolds. BMP-2 delivery from the Ti implant was not as effective as from the scaffolds. While in vitro BMP-2 release was highest for the PEG-hydrogel, the scaffold most successful in vivo was the Col/HA/PEG-hydrogel scaffold because it had the necessary mechanical strength to perform well in the mandibular <em>bone</em> environment. The in vitro release studies suggested a threshold dose of 5 μg which was borne out by the in vivo dose response studies.

To determine if recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) can be adsorbed onto porous ceramic hydroxyapatite (HA) and promote the integration of HA to host <em>bone</em>, 54 subperiosteal pockets were created on the skulls of 19 adult Pasteurella-free white rabbits. Fourteen HA implants were saturated with saline and placed in subperiosteal pockets (control), 22 HA implants were saturated with saline and placed into subperiosteal pockets after burring 1-2 mm of calvarium to expose bleeding cancellous <em>bone</em>, and 18 HA implants were saturated with rhBMP-2 and placed into subperiosteal pockets. The animals were sacrificed at 1 month with examination to determine implant mobility. Histology was used to determine the amount of <em>bone</em> growth into the implant. Of the 14 control sites, <em>10</em> implants were found to be freely mobile, five demonstrated host <em>bone</em> resorption, and only one exhibited <em>bone</em> growth into the implant. Of the 22 burred sites, eight were freely mobile and <em>10</em> demonstrated <em>bone</em> growth into the implant (p = 0.04). Of the 18 rhBMP-2 sites, only two were freely mobile, none demonstrated host <em>bone</em> resorption, and 16 exhibited <em>bone</em> growth into the implant (p = 0.00002). This study supports the use of porous ceramic HA as a biocompatible, osteoconductive implant material for use in craniomaxillofacial augmentation and reconstruction. It also provides evidence that rhBMP-2 enhances osseointegration, thereby fixing the implant in position against the host-<em>bone</em> interface. In the clinical setting, osseous fixation of the implant should aid in preventing displacement, minimizing host <em>bone</em> resorption, and decreasing the incidence of extrusion.

This study investigates the influence of the controlled release of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP-7) from cross-linked chitosan microparticles on pre-osteoblasts (OB-6) in vitro. BMP-7 was incorporated into microparticles by encapsulation during the particle preparation and coating after particle preparation. Chitosan microparticles had an average diameter of 700 µm containing ∼<em>10</em>-15 ng of BMP-7. The release study profile indicates that nearly 98% of the BMP-7 coated on the microparticles was released in a period of 18 days while only 36% of the BMP-7 encapsulated in the microparticles was released in the same time period. Cell attachment study indicated that the BMP-7 coated microparticles have many cells adhered on the microparticles in comparison with microparticles without growth factors on day <em>10</em>. DNA assay indicated a statistical significant increase (p < 0.05) in the amount of DNA obtained from BMP-7 encapsulated and coated microparticles in comparison with microparticles without any growth factors. A real-time RT-PCR experiment was performed to determine the expression of a few osteoblast specific genes-Dlx5, runx2, osterix, osteopontin, osteocalcin, and <em>bone</em> sialo<em>protein</em>. The results thus suggest that chitosan microparticles obtained by coacervation method are biocompatible and helps in improving the encapsulation efficiency of BMP-7. Also BMP-7 incorporated in the microparticles is being released in a controlled fashion to support attachment, proliferation and differentiation of pre-osteoblasts, thus acting as a good scaffold for <em>bone</em> tissue regeneration.

BACKGROUND

Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on an absorbable collagen sponge is a U.S. Food and Drug Administration-approved therapy effective at generating bone formation. In pediatric patients for whom other therapeutic options have been exhausted, rhBMP-2 is used off-label to address problematic bony defects. In the skeletally immature patient, the safety of rhBMP-2 therapy remains uncertain. Experiments are needed that investigate the effect of rhBMP-2 on growth and development in clinically relevant models.

METHODS

Ten juvenile rabbits underwent creation of a parietal skull defect that was treated with either 0.2 mg/cc rhBMP-2/absorbable collagen sponge or a neutral buffer solution/absorbable collagen sponge. Amalgam markers were placed at suture confluences to track suture separation and skull growth. Cranial growth was assessed radiographically at 10, 25, 42, and 84 days of age. Means and standard deviations for the various craniofacial growth variables were calculated and compared. Mean differences were considered significant for values of p < 0.05. At 84 days, sutures were analyzed by means of micro-computed tomographic scanning and histologic staining.

RESULTS

Treatment with rhBMP-2 resulted in fusion of the coronal sutures bilaterally, with variable fusion of the sagittal suture by cephalometric, radiographic, and histologic analysis. There were statistically significant changes to coronal suture growth, sagittal suture growth, skull height, craniofacial length, and intracranial volume (p < 0.05).

CONCLUSIONS

The use of rhBMP-2 in this juvenile animal model resulted in skeletal changes that may be undesirable in a clinical setting. The appearance of these fused sutures suggested a direct effect of rhBMP-2. Further work is required to limit the effect of rhBMP-2 to the target defect when used in the immature skeleton.

A one-step concept for <em>bone</em> regeneration has been postulated in which human adipose stem cells (hASCs) are harvested, triggered to differentiate, seeded on carriers, and implanted in the same operative procedure. Toward this goal it was investigated whether short (minutes) incubation with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) suffices to trigger osteogenic differentiation of hASCs seeded on calcium phosphate carriers. hASCs were treated with or without BMP-2 (<em>10</em> ng/mL) for 15 min, and seeded on β-tricalcium phosphate granules (β-TCP; sized <0.7 mm or >0.7 mm) or biphasic calcium phosphate (BCP; 60%/40% or 20%/80% hydroxyapatite/β-TCP). Attachment was determined after <em>10</em>-30 min. Proliferation (DNA content) and osteogenic differentiation (alkaline phosphatase activity, gene expression) were analyzed up to 3 weeks of culture. hASC attachment to the different scaffolds was similar, and unaffected by BMP-2. It stimulated gene expression of the osteogenic markers core binding factor alpha 1, collagen-1, osteonectin, and osteocalcin in hASCs seeded on BCP and β-TCP. Downregulation of osteopontin expression by BMP-2 was seen in BCP-seeded cells only. BMP-2 treatment inhibited expression of the adipogenic marker peroxisome proliferator-activated receptor gamma. In conclusion, 15 min BMP-2 preincubation of hASCs seeded on BCP/β-TCP scaffolds had a long-lasting stimulating effect on osteogenic differentiation in vitro. These results strongly support a one-step clinical concept for <em>bone</em> regeneration.

The differentiation and maturation of osteoprogenitor cells into osteoblasts are processes which are thought to be modulated by transforming growth factors-beta (TGF-beta) as well as by <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). Osteogenic <em>protein</em>-1 (OP-1, also known as BMP-7) is a member of the BMP family, and it is considered to have important regulatory roles in skeletal embryogenesis and <em>bone</em> healing. Rat <em>bone</em> marrow cells were cultured in vitro in a collagen-gel medium containing 0.5% fetal bovine serum (FBS) for <em>10</em> days in the presence of 40 ng/ml recombinant human OP-1 (rhOP-1). Under these conditions, survival of the <em>bone</em> marrow cell population was dependent on the presence of rhOP-1. Subsequently, the selected cells were cultured-for 6 days in medium containing 40 ng rhOP-1 and <em>10</em>% FBS. During the last 2 days, dexamethasone (<em>10</em>(-8) M) and beta-glycerophosphate (2 mM) were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels, colony number and size were determined. Chondro-osteogenic differentiation in vitro was evaluated in terms of the expression of alkaline phosphatase, the production of osteocalcin and the formation of mineralized matrix. After culturing in vitro, cells were placed inside diffusion chambers or inactivated demineralized <em>bone</em> matrix (DBM) cylinders and implanted subdermically into the backs of old rats for 28 days. Biochemical, histological and immunocytochemical analyses provided evidence of cartilage and osteoid tissue inside the diffusion chambers, whereas <em>bone</em> was also observed inside the DBM implants. In conclusion, this experimental procedure is capable of selecting a cell population from <em>bone</em> marrow which, in the presence of rhOP-1, achieves skeletogenic potential under in vitro as well as in vivo environments.

Between 5% to <em>10</em>% of tibial fractures progress to nonunion, causing substantial disability. <em>Bone</em> autografts, along with internal fixation, are the usual treatment for these failures, but the morbidity associated with autogenous tissues remains problematic. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> are currently available for clinical use and preclinical models, as well as an increasing number of patients treated with these molecules demonstrate their safety and efficacy. Osteogenic <em>Protein</em>-1, OP-1, has been evaluated in a randomized, prospective, multi-institution study of tibial nonunions. Sixty-one patients with 63 nonunions received OP-1 and intramedullary rod fixation, and were compared with 61 patients with 61 nonunions treated with fresh autogenous <em>bone</em> graft and the same fixation. Clinical outcomes (success in 81% of OP-1 and 85% of autograft-treated patients) and radiographic evaluation (healing in 75% of OP-1 and 84% of autograft-treated patients) were statistically indistinguishable at 9 months following treatment. No OP-1 or graft-related adverse events occurred. More than 20% of the autograft group had significant donor-site pain 6 months following surgery. OP-1 is a safe and effective alternative to autogenous <em>bone</em> in treatment of tibial nonunions.

Signals from the transforming growth factor beta family members are transmitted in the cell through specific receptor-activated Smads and a common partner Smad4. Two Smad4 genes (alpha and beta/<em>10</em>, or smad4 and smad4.2) have been isolated from Xenopus, and conflicting data are reported for Smad4beta/<em>10</em> actions in mesodermal and neural induction. To further understand the functions of the Smad4s in early frog development, we analyzed their activities in detail. We report that Smad<em>10</em> is a mutant form of Smad4beta that harbors a missense mutation of a conserved arginine to histidine in the MH1 domain. The mutation results in enhanced association of Smad<em>10</em> with the nuclear transcription corepressor Ski and leads to its neural inducing activity through inhibition of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling. In contrast to Smad<em>10</em>, both Smad4alpha and Smad4beta enhanced BMP signals in ectodermal explants. Using antisense morpholino oligonucleotides (MOs) to knockdown endogenous Smad4 <em>protein</em> levels, we discovered that Smad4beta was required for both activin- and BMP-mediated mesodermal induction in animal caps, whereas Smad4alpha affected only the BMP signals. Neither Smad4 was involved directly in neural induction. Expression of Smad4beta-MO in early frog embryos resulted in reduction of mesodermal markers and defects in axial structures, which were rescued by either Smad4alpha or Smad4beta. Smad4alpha-MO induced only minor deficiency at late stages. As Smad4beta, but not Smad4alpha, is expressed at high levels maternally and during early gastrulation, our data suggest that although Smad4alpha and Smad4beta may have similar activities, they are differentially utilized during frog embryogenesis, with only Smad4beta being essential for mesoderm induction.

OBJECTIVE

Povidone-iodine [polyvinylpyrrolidone-iodine complex (PVP-I)] is a broad-spectrum antimicrobial agent, frequently used in dentistry. In this study we investigated the short- and longterm effects on osteoblast number, viability, and function after short exposure to PVP-I with and without additional bone-morphogenetic protein-2 (BMP-2).

METHODS

Confluent osteoblast-like cell line (MC3T3-E1, subclone 24) cultures were exposed to pure PVP-I solution (7.7 mg/ml) and dilutions of 1:10, 1:100 and 1:1000 for 10 seconds and washed with phosphate buffer solution. Cell proliferation and viability was determined by MTT and differentiation status by alkaline phosphatase (ALP) activity 6 days after initial plating. In a separate experiment, long-term cell proliferation, viability and function were assessed 4 weeks after PVP-I treatment by MTT and deposited calcium using an Alizarin-red staining test.

RESULTS

PVP-I decreased ALP activity substantially. Stimulation by BMP-2 recovered ALP activity to near control levels at 1:100 and 1:1000 dilutions of PVP-I. The MTT assay showed reduced proliferation of the preosteoblastic cells for all treatments, irrespective whether BMP-2 was used or not. Only at PVP-I dilutions of 1:1000 proliferation rate was back to normal levels (95.6±2.4 %). No adverse long-term effect of PVP-I on mineralization of the extracellular matrix (Alizarinred) for dilutions higher than 1:100 was observed. Interestingly, undiluted and 1:10 diluted PVP-I even showed a significant increase in mineral deposition, especially in the presence of BMP-2.

CONCLUSIONS

Short-time application of PVP-I in concentrations of 1:10 and higher lead to decreased viability and impaired differentiation. However, surviving cells showed good recovery and mineralization potential.

BACKGROUND

The stability of titanium implants is determined by the rigid load-bearing connections that are formed by the bone, a process that involves a complex network of cells, pro- and anti-inflammatory mediators, and growth factors. The osseointegration processes at the interfaces of machined and porous implants were studied using molecular and histological techniques.

METHODS

Two machined and two porous titanium implants were inserted into the tibiae of four minipigs. The animals were sacrificed at 15, 30, 60, and 90 days post-implantation. The levels of bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-beta1, and tumor necrosis factor (TNF)-alpha were quantified in the peri-implant osseous samples. The levels of interleukin (IL)-1beta, IL-6, IL-10, and TNF-alpha in the serum were also assessed.

RESULTS

Histomorphological analysis showed evidence of bone ossification around the porous implant at 60 days. Surrounding the machined implants, highly sclerotic fibrous pads started the healing response at 90 days, and the levels of TGF-beta1 and BMP-4 began to increase at 60 days, at which time bone ossification around the porous implants was already evident. TNF-alpha was not present in the bone next to the implants. The serum levels of cytokines IL-1beta, IL-6, and IL-10 were not increased. The serum level of TNF-alpha increased during the healing process.

CONCLUSIONS

We observed that the levels of BMP-4 and TGF-beta1, which play essential roles in the osteogenesis process, increased earlier around the porous implants than around the machined implants. Similarly, the ossification process was initiated earlier at the surfaces of the porous implants than at the surfaces of the machined implants.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) in combination with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) or transforming growth factor-beta1 (TGF-beta1) are under evaluation for <em>bone</em> tissue engineering. Posttranslational modification of type I collagen is essential for functional <em>bone</em> tissue with adequate physical and mechanical properties. We investigated whether BMP-2 (<em>10</em>-<em>10</em>0 ng/mL) and/or TGF-beta1 (1-<em>10</em> ng/mL) affect gene expression of alpha2(I) procollagen and collagen-modifying enzymes, that is, lysyl oxidase and lysyl hydroxylases 1, 2, and 3 (encoded by PLOD1, 2, and 3), by human AT-MSCs. BMP-2, but not TGF-beta1, increased alkaline phosphatase activity after 28 days, indicating osteogenic differentiation of AT-MSCs. At day 4, both BMP-2 and TGF-beta1 upregulated alpha2(I) procollagen and PLOD1, which was downregulated at day 28. TGF-beta1, but not BMP-2, downregulated PLOD3 at day 28. Lysyl oxidase was upregulated by TGF-beta1 at day 4 and by BMP-2 at day 7. Neither BMP-2 nor TGF-beta1 affected PLOD2. In conclusion, these results suggest that AT-MSCs differentially respond to BMP-2 and TGF-beta1 with changes in gene expression of collagen-modifying enzymes. AT-MSCs may thus be able to appropriately modify type I collagen to form a functional <em>bone</em> extracellular matrix for tissue engineering, dependent on the growth factor added.

The aim of this study was to explore the effect of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and fibroblast growth factor-2 (FGF-2)- paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI)-on murine <em>bone</em> marrow stem cell (mBMSC) differentiation in an ex vivo cardiac microenvironment. For this purpose, green fluorescent <em>protein</em> (GFP) expressing hematopoietic lineage negative (lin-) c-kit ligand (c-kit) and stem cell antigen-1 (Sca-1) positive (GFP-lin-/c-kit+/sca+) mBMSC were co-cultured with neonatal rat ventricular cardiomyocytes (NVCMs). GFP+ mBMSC significantly induced the expression of BMP-2 and FGF-2 in NVCMs, and approximately 4% GFP+ mBMSCs could be recovered from the co-culture at day <em>10</em>. The addition of BMP-2 in concert with FGF-2 significantly enhanced the amount of integrated GFP+ mBMSCs by 5-fold ( approximately 20%), whereas the addition of anti-BMP-2 and/or anti-FGF-2 antibodies completely abolished this effect. An analysis of calcium cycling revealed robust calcium transients in GFP+ mBMSCs treated with BMP-2/FGF-2 compared to untreated co-cultures. BMP-2 and FGF-2 addition led to a significant induction of early (NK2 transcription factor related, locus 5; Nkx2.5, GATA binding <em>protein</em> 4; GATA-4) and late (myosin light chain kinase [MLC-2v], connexin 43 [Cx43]) cardiac marker mRNA expression in mBMSCs following co-culture. In addition, re-cultured fluorescence-activated cell sorting (FACS)-purified BMP-2/FGF-2-treated mBMSCs revealed robust calcium transients in response to electrical field stimulation which were inhibited by the L-type calcium channel (LTCC) inhibitor, nifedipine, and displayed caffeine-sensitive intracellular calcium stores. In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. This study provides the first evidence that mBMSCs with long-term self-renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross-talk within an appropriate cardiac microenvironment.

Abstract The natural history of familial pulmonary arterial hypertension (PAH) typically involves mutations in and/or haploinsuffciency of BMPR2 (gene for <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type 2) but with low penetrance (<em>10</em>%-15%), delayed onset (in the third or fourth decade), and a gender bias (two- to fourfold more prevalent in postpubertal women). Thus, investigators have sought an understanding of "second-hit" modalities that might affect BMPR2 anterograde trafficking and/or function. Indeed, vascular lung lesions in PAH have been reported to contain enlarged "vacuolated" endothelial and smooth muscle cells with dilated endoplasmic reticulum (ER) cisternae, increased ER structural <em>protein</em> reticulon 4 (also called Nogo-B), and enlarged and fragmented Golgi apparatus. We recently replicated this cellular phenotype in primary human pulmonary arterial endothelial cells and human pulmonary arterial smooth muscle cells in culture by acute knockdown of the estradiol 17β (E2)-responsive <em>proteins</em> signal transducer and activator of transcription 5a (STAT5a) and STAT5b using small interfering RNAs (siRNAs). We have now investigated whether functional haploinsufficiences of these molecules, alone or in combination with other modalities, might interfere with anterograde membrane trafficking using (a) the quantitative tsO45VSV-G-GFP trafficking assay and (b) assays for cell-surface localization of Flag-tagged BMPR2 molecules. The G glyco<em>protein</em> of the vesicular stomatitis virus (VSV-G) trafficking assay was validated in EA.hy926 endothelial cells by showing that cells exposed to monocrotaline pyrrole displayed reduced anterograde trafficking. Thereafter, the combinatorial knockdowns of STAT5a, STAT5b, BMPR2, and/or endothelial nitric oxide synthase as well as exposure to E2 or 2-methoxyestradiol were observed to significantly inhibit VSV-G trafficking. These combinations also led to intracellular trapping of wild-type Flag-tagged BMPR2. Overexpression of the PAH disease-derived F14 and KDF mutants of BMPR2, which were trapped in the ER/Golgi, also inhibited VSV-G trafficking in trans. Moreover, probenecid, a chemical chaperone in clinical use today, partially restored cell-surface localization of the KDF but not the F14 mutant. These data identify several combinatorial modalities that inhibit VSV-G anterograde trafficking and cause mislocalization of BMPR2. These modalities merit consideration in defining aspects of the late-developing and gender-biased natural history of human PAH.