In vivo implants of demineralized dentin matrix into muscle induce the formation of <em>bone</em> within the muscle. As with <em>bone</em> matrix implants, the <em>bone</em> induction appears to follow a chondrogenic pathway. Outgrowth cells from explants of neonatal rat muscle respond to <em>bone</em> matrix, in vitro, by expressing a heightened synthesis of sulfated proteoglycans and type II collagen, phenotypic of cartilage. The in vitro cell culture system has been used as an assay to monitor the isolation of the factor responsible for expression of this phenotypic transformation. Soluble <em>proteins</em> extracted from rat incisor dentin matrix during demineralization with EDTA, and not precipitable with 1.0 M CaCl2, were active in the in vitro system. The active extract was fractionated by Sephacryl S-<em>10</em>0 chromatography in 6 M guanidine HCl, isoelectric focusing in Immobilines, and by reverse phase high performance liquid chromatography. All fractions were assayed for activity at every stage. The final active fraction from the reverse phase chromatography on a Zorbax Poly-F column was purified to homogeneity, and yielded a single spot on two-dimensional gel electrophoresis. The component, RP-4, had pI 5.4-5.5, and an apparent Mr 6,000-<em>10</em>,000, based on globular <em>protein</em> standards. Maximal activity with respect to both sulfate incorporation into proteoglycan and production of type II collagen was in the 1.0-<em>10</em> ng/ml range. The RP-4 had a unique amino-terminal amino sequence and was rich in Gly, Pro, Glx, and Ala residues. It was different from transforming growth factor-beta and the <em>bone</em> <em>morphogenetic</em> <em>protein</em> family of <em>proteins</em> in these essential features.

OBJECTIVE

The number of low-back fusion procedures for the treatment of spine disorders has increased steadily over the past <em>10</em> years. Lumbar pseudarthrosis is a potential complication of lumbar arthrodesis and can be associated with significant pain and disability. The aim of this study was to assess, using validated patient-reported outcomes measures, the long-term effectiveness of revision arthrodesis in the treatment of symptomatic pseudarthrosis.

METHODS

This is a retrospective study of 47 patients who underwent revision lumbar arthrodesis for pseudarthrosis-associated back pain. Baseline 2-year outcomes were assessed using the following: visual analog scale (VAS) for back pain, Oswestry Disability Index (ODI), Zung Self-Rating Depression Scale, time to narcotic independence, time to return to work, EuroQol health-state utility, and physical and mental quality of life (Short Form [SF]-12 Physical and Mental Component Summary scores).

RESULTS

The mean duration of time between prior fusion and development of symptomatic pseudarthrosis was 2.69 years. Bone morphogenetic protein was used in 4 cases (8.5%) of revision arthrodesis. A significant improvement in VAS back pain (7.31 ± 0.81 vs 5.06 ± 2.64, p = 0.001), ODI (29.74 ± 5.35 vs 25.42 ± 6.0, p = 0.001), and physical health SF-12 (23.83 ± 6.89 vs 27.85 ± 8.90, p = 0.001) scores was observed when comparing baseline and 2-year post-revision arthrodesis scores, respectively, with a mean cumulative 2-year gain of 0.35 quality-adjusted life years. The median time to narcotics independence was 12.16 (interquartile range 1.5-24.0) months and the median time to return to work was 4 months (interquartile range 3-5 months). By 2 years after revision surgery, no patients had experienced pseudarthrosis. The SF-12 Mental Component Summary (44.72 ± 7.90 vs 43.46 ± 7.51, p = 0.43) and Zung Self-Rating Depression Scale scores (39.36 ± 7.48 vs 41.39 ± <em>10</em>.72, p = 0.37) were not significantly improved by 2 years.

CONCLUSIONS

The authors' study suggests that revision lumbar arthrodesis for symptomatic pseudarthrosis provides improvement in low-back pain, disability, and quality of life. Revision lumbar arthrodesis should be considered a viable treatment option for patients with pseudarthrosis-related back pain. Mental health symptoms from pseudarthrosis-associated back pain may be more refractory to revision surgery.

OBJECTIVE

To report the results of the treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 (nglBMP-2) delivered from a designed fibrin matrix.

METHODS

Experimental trial in rodents and prospective clinical study in dogs and cats with nonunion fractures.

METHODS

Twenty adult female, albino, Sprague-Dawley rats; 8 client-owned cats and dogs.

METHODS

After development of a fibrin matrix and evaluation of nglBMP-2 in a rodent femoral defect model, 8 consecutive long bone nonunion fractures (no progression in healing in>> or = 3 months), were treated using 300 microg nglBMP-2 in a liquid fibrin precursor, injected into the defect gap after fracture revision and stabilization, or through a stab incision into the fracture site. The fibrin matrix was designed to clot in the wound after 60 seconds and to release the nglBMP-2 continuously over several days.

RESULTS

Using only fibrin gel, 7% of the rat femoral defect was filled with new formed bone compared with 79% defect filling using 2 microg nglBMP-2 (P=.006). Five and 10 microg nglBMP in fibrin resulted in union of all femoral defects with complete filling of the gap with new bone. Bony bridging and clinical healing was achieved in 7 patients within 24 weeks of administration of nglBMP-2.

CONCLUSIONS

Application of nglBMP-2 in a functional matrix can induce bone healing. Controlled release of nglBMP-2 from a fibrin matrix mimics the natural fracture hematoma.

CONCLUSIONS

nglBMP-2/fibrin can successfully replace a cancellous bone autograft in fracture treatment with an associated reduction in graft donor site morbidity and surgical time.

Multiple assessment methods are available to evaluate the performance of engineered scaffolds in accepted <em>bone</em> healing animal models. Evaluation and comparison of these methods can aid in the planning of future animal studies, as well as, inform clinical assessments as the engineered scaffolds translate into clinical studies and applications. To evaluate multiple <em>bone</em> assessment techniques, <em>bone</em> regrowth potential of tyrosine-derived polycarbonate (TyrPC) scaffolds loaded with various dosages of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) (0, <em>10</em>, 25, and 50 μg) was assessed after 16 weeks in vivo in a rabbit calvarial model. Traditional X-ray radiography and micro-computed tomography (micro-CT) analyses were used to quantify the volume and density of regenerated <em>bone</em>. Histomorphometric analysis was performed as the traditional gold standard of evaluation. While these techniques are fairly standard in <em>bone</em> tissue engineering, we also investigated 64-slice CT, a tool more commonly used clinically, for comparison and to guide translational efforts. The 64-slice CT scans were carried out at 4 and 16 weeks to monitor temporal <em>bone</em> healing patterns. Study results indicated a clear dose-dependent response of increasing regenerated <em>bone</em> volume with rhBMP-2 loaded on the TyrPC scaffolds after 16 weeks of implantation. Significantly more <em>bone</em> formation was observed at the highest dose of rhBMP-2 (50 μg), which is 25-50% of the previously recommended dose (<em>10</em>0-200 μg) for this defect. A significant difference was observed between the lowest and highest doses using radiographs (p<0.001), micro-CT (p=0.002), and CT (p<0.001) and a high correlation was found between techniques (R(2) values between 0.446 and 0.911). It was found that the number of animals required per group to detect significant dose effects ranged between 6 and 8 for the imaging methods while histomorphometric analysis would require 25 animals per group to detect similar differences (desired power=0.9, α=0.05). Radiographic analysis provided quantifiable % defect coverage and radio-opacity, micro-CT provided spatial volumetric and <em>bone</em> density measures, histomorphometry provided biological confirmation, and 64-slice CT allowed for establishing of clinically relevant translational guidelines. These methodologies allow for a standardized and comprehensive description of <em>bone</em> regeneration and provide guidelines for the planning of future preclinical and clinical studies.

We evaluated the combination effect of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) and cultured rat <em>bone</em> marrow mesenchymal stem cells (MSCs) in atelopeptide type I collagen (AC) solution on osteogenesis in a diffusion chamber (DC) to develop a <em>bone</em> substitute having consistent osteogenic capability for clinical applications. The cultured MSCs were obtained by <em>10</em>-day primary culture of fresh <em>bone</em> marrow cells of Fischer rats. We prepared three groups of DCs: AC solution with rhBMP-2, AC solution with cultured MSCs, and AC solution with rhBMP-2 and cultured MSCs. The prepared combined solutions were injected into DCs, which were subcutaneously implanted into the backs of syngeneic rats. DCs were harvested after 2, 4, or 8 weeks and analyzed for <em>bone</em>-forming capability by determining histological and osteoblastic biochemical markers. De novo <em>bone</em> formation was observed both inside and outside of the membrane filter of DCs in the group of AC solution with rhBMP-2 and cultured MSCs. The alkaline phosphatase activity and osteocalcin content in the group of AC solution with rhBMP-2 and cultured MSCs were significantly higher than those in the group of AC solution with cultured MSCs at any time. These findings indicate that AC aqueous solution is a useful material not only as a carrier of rhBMP-2 but also as a cell-anchorage for differentiation and proliferation of MSCs. Therefore, this study suggests that clinical repairs of <em>bone</em> defects are feasible using injectable AC solution with rhBMP-2 and cultured MSCs as a <em>bone</em> substitute.

The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the <em>bone</em> <em>morphogenetic</em> <em>proteins</em>, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and <em>bone</em> repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and <em>10</em> days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

The effects of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP-2) on osteochondrogenesis were examined in high-density cultures of periosteum-derived cells, which have the potential to differentiate into <em>bone</em> and hypertrophic cartilage in vitro. Proliferation of these cells was inhibited by treatment with rhBMP-2. The time course for alkaline phosphatase (ALP) expression was shortened and the mineralization of the culture was increased by supplementation with rhBMP-2. These stimulatory effects of rhBMP-2 were observed at doses of <em>10</em>-<em>10</em>0 ng/m. <em>Bone</em> Gla <em>protein</em> (BGP) was immunocytochemically detectable earlier in the culture treated with rhBMP-2, and the BGP-positive layer of the rhBMP-2-treated cultures was thicker than that of the control cultures. On the other hand, there was no difference in uronic acid content or the time course of alpha 1(II) collagen mRNA expression between the rhBMP-2-treated and the control cultures. These results indicate that rhBMP-2 shortens the time course of osteogenesis and increases the amount of <em>bone</em> formation, whereas chondrogenesis remains unaffected.

BACKGROUND

Injectable three-dimensional (3D) scaffolds have the advantages of fluidity and moldability to fill irregular-shaped defects, simple incorporation of bioactive factors, and limited surgical invasiveness. Adipose-derived stem cells (ADSCs) are multipotent and can be differentiated toward nucleus pulposus (NP)-like cells. A hypoxic environment may be important for differentiation to NP-like cells because the intervertebral disc is an avascular tissue. Hence, we investigated the induction effects of hypoxia and an injectable 3D chitosan-alginate (C/A) gel scaffold on ADSCs.

METHODS

The C/A gel scaffold consisted of medical-grade chitosan and alginate. Gel porosity was calculated by liquid displacement method. Pore microstructure was analyzed by light and scanning electron microscopy. ADSCs were isolated and cultured by conventional methods. Passage 2 BrdU-labeled ADSCs were co-cultured with the C/A gel. ADSCs were divided into three groups (control, normoxia-induced, and hypoxia-induced groups). In the control group, cells were cultured in <em>10</em>% FBS/DMEM. Hypoxia-induced and normoxia-induced groups were induced by adding transforming growth factor-β1, dexamethasone, vitamin C, sodium pyruvate, proline, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7, and 1% ITS-plus to the culture medium and maintaining in 2% and 20% O2, respectively. Histological and morphological changes were observed by light and electron microscopy. ADSCs were characterized by flow cytometry. Cell viability was investigated by BrdU incorporation. Proteoglycan and type II collagen were measured by safranin O staining and the Sircol method, respectively. mRNA expression of hypoxia-inducing factor-1α (HIF-1α), aggrecan, and Type II collagen was determined by reverse transcription-polymerase chain reaction.

RESULTS

C/A gels had porous exterior surfaces with 80.57% porosity and 50-200 üm pore size. Flow cytometric analysis of passage 2 rabbit ADSCs showed high CD90 expression, while CD45 expression was very low. The morphology of induced ADSCs resembled that of NP cells. BrdU immunofluorescence showed that most ADSCs survived and proliferated in the C/A gel scaffold. Scanning electron microscopy showed that ADSCs grew well in the C/A gel scaffold. ADSCs in the C/A gel scaffold were positive for safranin O staining. Hypoxia-induced and normoxia-induced groups produced more proteoglycan and Type II collagen than the control group (P < 0.05). Proteoglycan and Type II collagen levels in the hypoxia-induced group were higher than those in the normoxia-induced group (P < 0.05). Compared with the control group, higher mRNA expression of HIF-1α, aggrecan, and Type II collagen was detected in hypoxia-induced and normoxiainduced groups (P < 0.05). Expression of these genes in the hypoxia-induced group was significantly higher than that in the normoxia-induced group (P < 0.05).

CONCLUSIONS

ADSCs grow well in C/A gel scaffolds and differentiate toward NP-like cells that produce the same extracellular matrix as that of NP cells under certain induction conditions, which is promoted in a hypoxic state.

Heterotopic ossification is a pathological condition in which <em>bone</em> forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and <em>bone</em> formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing <em>10</em> nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5 weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.

<em>Bone</em> metabolism is influenced by systemic and local acting hormons. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) as representatives of the latter substances are known to have the ability for ectopic <em>bone</em> formation. Within this study, we investigated the influence of different growth factors on the proliferation- and differentiation rate of osteoblast-like cells. For that purpose, human osteoblast-like cells (HPOC) were incubated in the presence of either recombinant BMP-4 of the genome of xenopus laevis (rxBMP-4), recombinant human BMP 2 (rhBMP-2), transforming growth factor-beta (TGF-beta) or basic fibroblast growth factor (rh-bFGF) in two different concentrations each (<em>10</em> ng/ml and 50 ng/ml). Cell proliferation was measured within a MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromid] assay, the amount of cell differentiation by the activity of alcaline phosphatase. Rx-BMP-4 induced a differentiation of HPOC to almost the same extent as rhBMP-2, whereas the addition of rh-bFGF, applied at the same concentration, failed to have any influence on cell differentiation. However, rh-bFGF provoked an increase in cell proliferation when compared with unstimulated HPOC, while rhBMP-2 and rxBMP-4 showed no effect on proliferation. TGF-beta influenced <em>bone</em> proliferation as well as differentiation significantly. The equipotent effect of recombinant human BMP-2 and recombinant BMP-4 obtained from Xenopus laevis with regard to differentiation and proliferation of human primary osteoblast-like cells originates either in the fact that target cells have receptors for BMP 2 as well as BMP 4, or that both BMP's link to the same receptor with almost the same affinity.

The aims of this study were to modulate inflammation in synovial explants with the compounds: dexamethasone, rapamycin, bone morphogenetic protein 7 (BMP-7) and pravastatin, and to investigate the modulatory capacity of the compounds on specific macrophage phenotypes.

Synovial explants from osteoarthritis (OA) patients were treated with 10(-6) M dexamethasone, 100 ng/mL rapamycin, 500 ng/mL BMP-7 or 50 μM pravastatin. Half of the explants were pre-stimulated with IFNγ + TNFα to simulate acute inflammation. Inflammatory state of the synovium was assessed with gene expression analysis. Primary human monocytes were isolated and stimulated towards macrophage phenotypes M(IFNγ + TNFα), M(IL-4) and M(IL-10) with the respective cytokines, followed by treatment with the compounds.

Dexamethasone had an anti-inflammatory effect on IFNγ + TNFα stimulated and osteoarthritic synovium, likely due to suppression of pro-inflammatory M(IFNγ + TNFα) macrophages while enhancing anti-inflammatory M(IL4) and M(IL10) macrophages. Rapamycin and BMP-7 further enhanced inflammation in stimulated synovium, but rapamycin did not have a clear effect on non-stimulated synovium. Rapamycin suppressed M(IL-4) and M(IL-10) macrophages without affecting M(IFNγ + TNFα). BMP-7 suppressed M(IFNγ + TNFα) and enhanced M(IL-10) in the macrophage cultures. Pravastatin did not affect synovium, but enhanced M(IL-10).

These data indicate that macrophage phenotype modulation can be used to guide joint inflammation and thereby contribute to the development of new therapies to delay the progression of OA. The varying effects of the compounds on synovium of different degrees of inflammation, indicate that the modulatory capacity of the compounds depends on OA stage and underlines the importance of identifying this stadium for adequate treatment.

The Xenopus laevis DNA fragment encoding a <em>protein</em> homologous with human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-1 (BMP-1) was amplified by polymerase chain reaction (PCR) from cDNA derived from stage 26 (st.26) embryonic RNA. Subsequently this fragment was used as a probe to isolate cDNA clones by screening of a X. laevis st.24 embryonic cDNA library. Two partial clones (22 and 63) were obtained and the missing 5'-end of the clone 22 was extended by the anchored PCR technique. The nucleotide sequence of the resulting clone (22AN) contained an open reading frame coding for a <em>protein</em> with 707 deduced amino acids. Three sizes of mRNA (2.9, 5.2 and 6.6 kb) were detected in blastula (st.9) and early gastrula (st.<em>10</em>) embryos, and in hatched tadpole (st.40), but little or no expression was observed in morula (st.7) and late gastrula (st.12) embryos, suggesting a physiological role(s) of X.laevis BMP-1 in normal embryonic development.

Differential screening-selected gene aberrative in neuroblastoma (DAN) is a member of a cystine knot <em>protein</em> family that includes Cerberus and Gremlin. First isolated in a screen to identify genes down-regulated in transformed rat fibroblasts, DAN has subsequently been cloned in Xenopus, mouse, and human. Overexpression of DAN suppresses the transformed phenotype and retards the cell's entry into S phase. Biochemical analyses have demonstrated DAN's ability to bind <em>bone</em> <em>morphogenetic</em> <em>proteins</em> and antagonize their signaling activity. In this study, chick DAN was cloned and sequenced, revealing a conserved cystine knot region as well as an N-glycosylation site. A riboprobe was designed from the 3' chick DAN coding sequence and used for analysis of DAN in the developing chick embryo by in situ hybridization. Chick DAN was expressed beginning at stage <em>10</em> in the developing somites and the medial otic epithelium. Expression in the neural layer of the eye became apparent at stage 14. By stage 17, expression had expanded to the base of the hindbrain. Limb bud labeling began at stage 20, whereas expression in the branchial arches appeared at stage 25. Chick DAN expression generally corresponded to that of mouse DAN expression as shown by comparative in situ hybridization. However, chick DAN was found in the otic epithelium and notochord, whereas mouse DAN was restricted to the overlying otic ectomesenchyme and was absent from the notochord. This observation suggests that DAN may play different roles in chick and mouse otic and notochord development.

BACKGROUND

We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair.

RESULTS

H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-<em>10</em> and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of <em>bone</em> <em>morphogenetic</em> <em>proteins</em>.

CONCLUSIONS

Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remodeling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists.

<em>Protein</em> kinase A (PKA), a serine/threonine kinase, regulates <em>bone</em> formation, and enhances <em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-induced osteoblast differentiation. However, the mechanisms of how PKA controls the cellular response to BMP are not well known. We investigated the effects of modulating PKA activity during BMP2-induced osteoblast differentiation, and found that PKA regulates the function of Dlx3. Dlx3 plays crucial roles in osteoblast differentiation and it is expressed in most skeletal elements during development. We found that PKA activation increases BMP2-induced expression of Dlx3 <em>protein</em>, and enhances the <em>protein</em> stability, DNA binding, and transcriptional activity of Dlx3. In addition, PKA activation induces the phosphorylation of Dlx3 at consensus PKA phosphorylation target site(s). Lastly, substitution of serine <em>10</em> in Dlx3 to alanine significantly reduces, if not completely abolishes, the phosphorylation of Dlx3 and the regulation of Dlx3 function by PKA. These results suggest that Dlx3 is a novel target of PKA, and that PKA mediates BMP signaling during osteoblast differentiation, at least in part, by phosphorylating Dlx3 and modulating the <em>protein</em> stability and function of Dlx3.

BACKGROUND

The nonunion rate after posterolateral spinal fusion can be as high as 35%. This has stimulated interest in the development of techniques for enhancing new bone formation, including the addition of bioactive peptides or the use of cell-based therapies, including genetically modified cells. In previous studies we have demonstrated that exposing autologous, marrow-derived osteoprogenitor cells to a recombinant human bone morphogenetic protein-6 (rhBMP-6) containing extracellular matrix induces osteoblastic differentiation, and that these cells are capable of increasing new bone formation. Growth of autologous cells on a synthetic rhBMP-6 containing matrix yields a population of stimulated osteoprogenitor cells, without the expense of adding large amounts of rhBMP-6 directly, or the risks inherent in the use of genetically altered cells.

OBJECTIVE

This study was performed to evaluate the potential of rhBMP-6 stimulated osteoprogenitor cells (stOPC) to enhance the rate and strength of posterolateral spinal fusion.

METHODS

Prospective in vivo animal study

METHODS

Radiographic evidence of spinal fusion, biomechanical testing of explanted spines, histological analysis of new bone formation

METHODS

Single-level posterolateral spinal arthrodeses were performed in 69 New Zealand white rabbits. Autologous marrow stem cells were concentrated and then plated on an rhBMP-6-rich extracellular matrix synthesized by genetically engineered mouse C3H10T1/2 cells. Animals in Groups I (n=18) and II (n=18) received autografts of 30M and 60M rhBMP-6 stOPCs in guanidine extracted demineralized bone matrix (gDBM), respectively, whereas those in Group III (n=13) received iliac crest bone graft (ICBG). Those in Group IV (n=10) received gDBM, and those in Group V (n=10) underwent decortication only. Assessment of fusion was made with serial radiographs, manual palpation of the explanted spines, and biomechanical testing. The fusion masses from two animals each in Groups I, II, and IV were evaluated histologically.

RESULTS

Fifty-three animals were available for analysis at the conclusion of the study. In these animals, the arthrodesis rate was significantly higher after treatment with rhBMP-6 stOPCs (77% for both Groups I and II by palpation) than ICBG, gDBM, or decortication alone (Group III=55%, IV=20% and V=0%, respectively). Similarly, the peak loads to failure of the fusion masses in Groups I and II (212.5+/-37.8 N and 234.6+/-45.7 N) were significantly greater than the corresponding values in the other groups (Group III=155.9+/-36.4N, Group IV=132.7+/-59.9N, and Group V=92.8+/-18.4N), though when only the fused specimens in Groups I, II, and III were compared, only Group II was significantly different than Group III (234.6+/-45.7N and 155.9+/-36.4N, respectively). The fusion masses in the rhBMP-6 stOPC-treated animals were typified by a thin, fusiform cortical shell, newly formed trabecular bone emanating from the decorticated transverse processes, and residual unremodeled gDBM carrier particles. The fusion masses in the gDBM treated bones were morphologically similar, though they contained less newly formed bone.

CONCLUSIONS

The use of rhBMP-6 stOPCs in a carrier of gDBM significantly enhanced the rate and strength of single-level posterolateral spinal arthrodeses in the New Zealand white rabbit, compared with ICBG, gDBM, and decortication alone. Our results confirm that the stimulation of marrow-derived osteoprogenitor cells by growing them on a rhBMP-6 containing extracellular matrix is feasible. Further investigation is warranted to determine the relative contribution of rhBMP-6 stimulation and the number of cells implanted, as well as strategies for optimizing the technique for clinical application.

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis, which can be done in juvenile rabbits. Passagefour bromodeoxyuridine (BrdU)-labeled ADSCs were cultured, assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability. Two-month-old, healthy male rabbits (1.2 to 1.4 kg, n=45) underwent ischemic induction and were randomly divided into five groups (group A: animal model control; group B: drilling; group C: drilling & ADSCs; group D: drilling & BMP-2; and group E: drilling & ADSCs & BMP-2). Magnetic resonance imaging (MRI), X-ray imaging, hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4, 6 and <em>10</em> weeks after treatment. Approximately 90% of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability. Similar results were observed in the rabbits in groups C and E at weeks 6 and <em>10</em>. The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P<0.01). Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P<0.05). In summary, drilling through the growth plate combined with ADSC and BMP-2 treatments induced new <em>bone</em> formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

Background: A wealth of research has reported on the anti-obesity effects of green tea extract (GTE). Although browning of white adipose tissue (WAT) has been reported to attenuate obesity, no study has disclosed the effects of GTE on browning in Sprague Dawley rats. Objectives: The aims of the study were to investigate the effects of GTE on anti-obesity and browning, and their underlying mechanisms. Methods: Four groups of rats (n=<em>10</em>/group) were used including a normal diet with vehicle treatment, and a high-energy diet (HED) with vehicle or GTE by oral gavage at 77.5 or 155 mg/kg/day for 8 weeks. Body weight, fat accumulation, and serum biochemical parameters were used to evaluate obesity. The gene expressions were analyzed using RT-qPCR and western blotting. Results: GTE modulated HED-induced body weight, fat accumulation, and serum levels of triacylglycerol, total cholesterol, low-density lipo<em>protein</em>, free fatty acids, aspartate aminotransferase, and alanine aminotransferase. Moreover, GTE enhanced the serum high-density lipo<em>protein</em>. Most importantly, the biomarkers of beige adipose tissue were up-regulated in WAT in GTE-given groups. GTE induced genes involved in different pathways of browning, and reduced transducin-like enhancer <em>protein</em>-3 in WAT. Conclusion: Our results suggest that GTE may improve obesity through inducing browning in HED-fed rats. Abbreviations: ALT: Alanine transaminase; AST: Aspartate transaminase; BAT: Brown adipose tissue; BMP-7: <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-7; BW: Body weight; CIDEA: Cell death activator; CPT-1: Carnitine palmitoyltransferase-1; EFP: Epididymal fat pad; FFA: Free fatty acid; FGF-21: Fibroblast growth factor-21; GTE: Green tea extract; HDL: High-density lipo<em>protein</em>; HED: high-energy diet; LDL: Low-density lipo<em>protein</em>; MFP: Mesenteric fat pad; PGC-1α: Activates PPAR-γ coactivator-1; PPAR-γ: Peroxisome proliferator-activated receptor-γ; PRDM-16: PR domain containing 16; RFP: Renal fat pad; SD: Sprague Dawley; TC: Total cholesterol; TG: Triacylglycerol; TLE-3: Transducin-like enhancer <em>protein</em>-3: UCP-1: Uncoupling <em>protein</em>-1; WAT: White adipose tissue.

BACKGROUND

Statins have been reported to promote bone formation. However, taken orally, their bioavailability is low to the bones. Implant therapies require a local repair response, topical application of osteoinductive agents, or biomaterials that promote implant fixation.

METHODS

The present study evaluated the effect of a single local injection of simvastatin on screw fixation in an ovariectomized rat model of osteoporosis.

RESULTS

Dual-energy X-ray absorptiometry, micro-computed tomography, histology, and biomechanical tests revealed that 5 and 10 mg simvastatin significantly improved bone mineral density by 18.2% and 22.4%, respectively (P<0.05); increased bone volume fraction by 51.0% and 57.9%, trabecular thickness by 16.4% and 18.9%, trabeculae number by 112.0% and 107.1%, and percentage of osseointegration by 115.7% and 126.3%; and decreased trabeculae separation by 34.1% and 36.6%, respectively (all P<0.01). Bone mineral apposition rate was significantly increased (P<0.01). Furthermore, implant fixation was significantly increased (P<0.05), and bone morphogenetic protein 2 (BMP2) expression was markedly increased. Local injection of a single dose of simvastatin also promoted angiogenesis. Vessel number, volume, thickness, surface area, and vascular volume per tissue volume were significantly increased (all P<0.01). Vascular endothelial growth factor (VEGF), VEGF receptor-2, von Willebrand factor, and platelet endothelial cell adhesion molecule-1 expression were enhanced.

CONCLUSIONS

A single local injection of simvastatin significantly increased bone formation, promoted osseointegration, and enhanced implant fixation in ovariectomized rats. The underlying mechanism appears to involve enhanced BMP2 expression and angiogenesis in the target bone.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, <em>bone</em> metastases were more common (<em>10</em> vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in <em>bone</em>. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings.

This study evaluated the levels of <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (<em>10</em>, 50, or <em>10</em>0 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.

Uterine fibroids are benign uterine smooth muscle tumors that are present in up to 8 out of <em>10</em> women by the age of 50. Many of these women experience symptoms such as heavy and irregular menstrual bleeding, early pregnancy loss, and infertility. Traditionally believed to be inert masses, fibroids are now known to influence endometrial function at the molecular level. We present a comprehensive review of published studies on the effect of uterine fibroids on endometrial function. Our goal was to explore the current knowledge about how uterine fibroids interact with the endometrium and how these interactions influence clinical symptoms. Our review shows that submucosal fibroids produce a blunted decidualization response with decreased release of cytokines critical for implantation such as leukocyte inhibitory factor and cell adhesion molecules. Furthermore, fibroids alter the expression of genes relevant for implantation, such as <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type II, glycodelin, among others. With regard to heavy menstrual bleeding, fibroids significantly alter the production of vasoconstrictors in the endometrium, leading to increased menstrual blood loss. Fibroids also increase the production of angiogenic factors such as basic fibroblast growth factor and reduce the production of coagulation factors resulting in heavy menses. Understanding the crosstalk between uterine fibroids and the endometrium will provide key insights into implantation and menstrual biology and drive the development of new and innovative therapeutic options for the management of symptoms in women with uterine fibroids.

OBJECTIVE

To investigate the correlation of the functional disequilibrium of regulatory T cells (Treg)/T-helper (Th17) cells with calcification and to explore the significance of their influence on the outcome of cardiovascular disease (CVD) in uremic patients after hemodialysis (HD).

METHODS

Out of 66 uremia patients, 36 patients had CVD after HD (maintenance hemodialysis (MHD) group1) and 30 patients did not have CVD (MHD group2). Twenty healthy volunteers were selected as normal control group. Peripheral blood mononuclear cells were isolated and treated with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2). Treg and Th17 frequencies were measured by flow cytometry. Forkhead/winged helix transcription factor (Foxp3) and retinoic acid receptor-related orphan receptor-γt (ROR-γt) mRNA expressions were measured by real-time quantitative polymerase chain reaction. Levels of interleukin (IL)-<em>10</em> and IL-17 were detected by enzyme-linked immunosorbent assay.

RESULTS

When compared with controls, rhBMP-2 upregulates Treg/Th17 functional disequilibrium in uremia patients, displaying higher Treg and Th17 frequencies, Foxp3 and ROR-γt expressions, and levels of cytokines (p < 0.05). These differences were also significant between MHD group1 and group2 (p < 0.05). It was also observed that Treg/Th17 functional disequilibrium was not only correlated with a calcification state but also consistent with the CVD.

CONCLUSIONS

The Treg/Th17 cell function disequilibrium might act synergistically with calcification in the high incidence of CVD after HD.

The genomic stability and integrity of host strains are critical for the production of recombinant <em>proteins</em> in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant <em>proteins</em> in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant <em>proteins</em> such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~<em>10</em>(-8)/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (<em>10</em>(-8)/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant <em>protein</em> production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant <em>proteins</em>, emphasizing the importance of the development of IS-free host strains.