Osteomyelitis has been one of the most common causes of post-operative problems and complications despite the advances in surgical techniques and the availability of newly developed antibiotics. Local antibiotic and growth factor delivery devices for treatment of various surgical infections have been studied recently, especially in the case of orthopedic infections. The report was to develop novel solvent-free biodegradable capsules for antibiotics and growth factors delivery. To fabricate a biodegradable capsule, polylactide-polyglycolide copolymers were pre-mixed with vancomycin. The mixture was then compression molded and sintered to form a cylinder with a cover of 8 mm in diameter. After the addition of 1 and <em>10</em> microg recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> (rhBMP-2) into the core, an ultrasonic welder was used to seal the capsules. An elution method was employed to characterize the in vitro release characteristics of the antibiotics and the rhBMP-2 over a 30-day period. The HPLC analysis and the bacterial inhibition test showed that biodegradable capsules released high concentrations and activity of vancomycin (well above the minimum inhibition concentration) in vitro for the period of time needed to treat <em>bone</em> infection; i.e. 4-6 weeks. In addition, the results of ELISA and ALP tests also suggested that the capsules released active rhBMP-2 for up to 30 days. By adopting this novel technique, we will be able to fabricate biodegradable capsules of various medicines for long-term drug deliveries.

BACKGROUND

Intraoperative reduction of low-grade lumbar spondylolisthesis (LGLS) remains disputed. There is currently no published data comparing midterm outcomes of reduction versus in situ fusion.

OBJECTIVE

This study aimed to compare mid-term clinical, radiological, and perioperative outcomes for reduction versus in situ fusion in LGLS with neurogenic symptoms.

METHODS

A retrospective review of prospectively collected spine registry data in a single institution was carried out.

METHODS

All patients who underwent minimally invasive transforaminal lumbar interbody fusion (MIS TLIF) for LGLS with neurogenic symptoms with a minimum 5-year follow-up comprised the patient sample.

METHODS

Self-reported measures were Oswestry Disability Index, North American Spine Society Neurogenic Symptom Score, Health Outcomes Survey Short Form-36 score, and Numerical Pain Rating Scale (back and leg pain). Radiological outcomes were fusion grading, adjacent segment degeneration (ASD), and implant failure or loosening. Perioperative outcomes were fluoroscopic time, operative time, intraoperative blood loss, opioid analgesia usage, time to ambulation, duration of hospitalization, and complication rate. Functional outcomes were patient satisfaction rate and rate of return to full function.

METHODS

A retrospective review was performed on prospectively collected registry data of patients undergoing MIS TLIF for LGLS with neurogenic symptoms, from 2004 to 2009. The operative technique and postoperative protocol were standardized. Two groups were formed based on complete reduction of the spondylolisthesis (reduction group [RG]) or the lack thereof (non-reduction group [NRG]) in the immediate postoperative radiograph. Outcomes at baseline, 6 months, 2 years, and 5 years postsurgery were compared.

RESULTS

There were 56 patients included (RG=30, NRG=26). The two groups had comparable baseline characteristics: demographics, body mass index, spondylolisthesis etiology, spinal level involved, <em>bone</em> graft and <em>bone</em> <em>morphogenetic</em> <em>protein</em> used, and all self-reported outcome measures. Perioperative outcomes were not significantly different. The early complication rate (RG=3.3%, NRG=19.2%, p=.086) and late complication rate (RG=<em>10</em>%, NRG=23.1%, p=.184) were similar. All patients achieved Bridwell grade 1 fusion from 2 years onward. Adjacent segment degeneration rate at 5 years was similar (RG=<em>10</em>%, NRG=0%, p=NS). Both groups showed significant postoperative improvement in all self-reported measures with no significant differences between the two groups at all follow-up points. Functional outcomes were equivalent.

CONCLUSIONS

Intraoperative reduction does not improve outcomes in LGLS with neurogenic symptoms after MIS TLIF. Adequate decompression and solid fusion are likely the keys to good mid-term outcomes.

Non-viral delivery of genes involved in stimulation of <em>bone</em> formation has been pursued for clinical <em>bone</em> repair, but no effort has been made to assess <em>protein</em> expression levels after in vivo delivery. This is critical to better understand gene delivery efficiencies and to compare different modes of non-viral delivery. This study investigated expression levels of basic fibroblast growth factor (bFGF) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) after delivering expression vectors (plasmid DNA) with polymeric carriers in a rat subcutaneous implant model. The polymers used were a 2 kDa molecular weight polyethylenimine modified with linoleic acid (PEI-LA) and the 25 kDa PEI (PEI25) used for non-viral gene delivery in animal models. The PEI-LA mediated delivery of the plasmid DNAs in 293T cells led to ∼3.5 and ∼13 ng/<em>10</em>(6) cells/day secretion of bFGF and BMP-2 in vitro, respectively. Using the reporter <em>protein</em>, Green Fluorescence <em>Protein</em> (GFP), transfection in implants was readily detected by the presence of GFP-positive cells and a polymeric carrier was needed for this GFP expression. No bFGF and BMP-2 were detected in the scaffolds due to high background in detection assays and/or rapid diffusion of the secreted <em>proteins</em> from the implant site. However, using an ex vivo culture system, significant levels of BMP-2, but not bFGF, secretion were observed from the scaffolds. The BMP-2 secretion from PEI-LA delivered expression vector was equivalent and/or superior to PEI25 depending on the plasmid DNA implant dose. Gelatin scaffolds were able to sustain ∼0.3 ng/sponge/day BMP-2 secretion as compared to collagen scaffolds (∼0.1 ng/sponge/day). These values were equivalent to secretion rates reported with some viral delivery systems from independent studies.

It has been generally accepted that <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of <em>10</em>0 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and <em>10</em> days after treatment. At 7 days, no sign of cartilage and/or <em>bone</em> formation was detected. However, at <em>10</em> days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous <em>bones</em> of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing <em>bone</em> formation at <em>10</em> days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.

Porous beta-tricalcium phosphate (betaTCP) has osteoconductive properties. The adsorption of human recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) onto TCP could realize an osteoinductive <em>bone</em> substitute. We evaluated it on an animal model using dual-energy X-ray absorptiometry (DEXA) and solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. BetaTCP cylinders loaded with rhBMP-2 were implanted into rabbits' femoral condyle <em>bone</em> defects, and betaTCP alone as control into the contralateral femur. We studied two different doses of rhBMP-2 (<em>10</em> and 40 microg) on two groups of four animals. Evaluation consisted in radiography, histology, and histomorphometry, DEXA, and NMR spectroscopy using an original method of quantification. With both doses of rhBMP-2, we observed on radiographs an increase of trabecular <em>bone</em> around implants. Histology showed resorption of the ceramic, trabecular <em>bone</em> with osteoblasts and osteoid substance around the implants, and colonization inside the porous betaTCP by new <em>bone</em> formed. Histomorphometry showed that the osteoid surface (OS/BS) was greatest with the high dose of rhBMP-2. The difference was slight between the low dose of rhBMP-2 and control. DEXA showed a dose-dependent increase of <em>bone</em> mineral density of rhBMP-2-loaded betaTCP vs. control. NMR spectroscopy confirmed that the amount of new <em>bone</em> formed in betaTCP was greater when betaTCP carried rhBMP-2, and increased with the dose of rhBMP-2 used. We showed that betaTCP was a good matrix for rhBMP-2, which gave it osteoinductive properties in an orthotopic site, in a dose-dependent manner. Thus, such composite biomaterial seems to be of great interest in reconstructive <em>bone</em> surgery. Further studies are needed in clinical practice to determine optimal doses.

The vascular endothelial growth factor (VEGF) pathway is critical for tumor angiogenesis. However, VEGF pathway inhibition has been limited by intrinsic and acquired resistance. Simultaneously targeting multiple steps involved in tumor angiogenesis is a potential means of overcoming this resistance. Activin like kinase 1 (ALK1) and endoglin (ENG) have effects on angiogenesis that are distinct from those of VEGF. Whereas VEGF is important for vessel initiation, ALK1 and endoglin are involved in vessel network formation. Thus, ALK1 and endoglin pathway inhibitors are attractive partners for VEGF-based combination antiangiogenic therapy. Genetic evidence supports a role for this receptor family and its ligands, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP) 9 and <em>10</em>, in vascular development. Patients with genetic alterations in ALK1 or endoglin develop hereditary hemorrhagic telangiectasia, a disorder characterized by abnormal vessel development. There are several inhibitors of the ALK1 pathway advancing in clinical development for treatment of various tumor types, including renal cell and ovarian carcinomas. Targeting of alternate angiogenic pathways, particularly in combination with VEGF pathway blockade, holds the promise of optimally inhibiting angiogenically driven tumor progression. Clin Cancer Res; 20(11); 2838-45. ©2014 AACR.

BACKGROUND

The efficacy of recombinant growth factors in vivo is highly dependent on the delivery vehicle. The authors investigated the osteoinductive effects of recombinant human bone morphogenetic proteins (BMP)-2 implanted together with a complex of heparin and chitosan.

METHODS

Sixty rats were used. Three different carriers in gel formulation (type I collagen, heparin/type I collagen, and heparin/chitosan) were mixed with either 0, 10, or 50 microg of BMP-2, making the number of groups nine. The gels were injected into the quadriceps muscles of both legs in 45 rats (n = 10 per group). Freeze-dried formulations of the carriers were also tested with the same amounts of BMP-2 using 15 rats (n = 5 per group). Four weeks after implantation, the quality and amount of newly formed bone were assessed.

RESULTS

Chitosan was shown to protect the heparinase-mediated degradation of heparin in vitro. The osteoinductive effects of BMP-2 in combination with heparin/chitosan were superior as compared with BMP-2 implanted together with type I collagen. Interestingly, the heparin/chitosan complex induced a small amount of bone also without BMP-2 added. The heparin/chitosan was completely absorbed after 4 weeks as determined by histologic evaluation, and a normal active bone formation was present. The freeze-dried formulations of the carriers demonstrated similar osteoinductive effects as the gels.

CONCLUSIONS

An osteoinductive formula for clinical use is needed for general bone reconstruction. Heparin in complex with chitosan has the ability to stabilize or activate the growth factor in vivo and induce the generation of new bone in good yields.

BACKGROUND

Recombinant human bone morphogenetic protein-2 (rh-BMP2) has osteoinductive properties. The sequence of osteogenesis induced by rh-BMP2 in a rat calvarial bone defect model was evaluated histologically.

METHODS

Atelopeptide type I collagen was selected as the carrier matrix for rh-BMP2. This collagen was mixed with 50 microg of rh-BMP2 and treated by lyophilization to make a suitable form for filling bone defects. Thirty Wistar rats (male; 10 weeks old) underwent craniotomy resulting in a round bone defect with a diameter of 4 mm. Three weeks after the operation, the defect site was exposed to implant collagen with rh-BMP2 in the experimental group and collagen only in the control group. Three, 6 and 9 weeks after implantation, calvarial specimens were harvested and stained with haematoxylin/eosin. In addition, bromodeoxyuridine (BrdU) immunohistochemical staining was performed.

RESULTS

In the experimental group, marked ossification was found. In contrast, there was no tendency towards osteogenesis in the control group, 9 weeks after collagen implantation. In BrdU immunohistochemical staining, the number of BrdU-positive cells was significantly higher in the experimental group than in the control group, and was significantly higher at the periosteal site than at other sites at 3 and 6 weeks after implantation.

CONCLUSIONS

rh-BMP2 exhibited osteoinductive properties, and markedly stimulated proliferation of periosteal cells.

A robust method for inducing <em>bone</em>-formation without an autograft has not been established. Currently, both platelet-rich plasma (PRP) and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) have been widely investigated for their clinical use in such cases. However, their synergistic effect is still controversial and previously shown diversity of this effect depends on various factors such as the <em>bone</em> substitutes involved. In this study, we investigated the synergistic effect of PRP and BMP2 on an alloplastic substitute as potentiators to induce in vivo <em>bone</em>-formation. A <em>10</em> mm diameter bony defect in rabbit calvarium was reconstructed using biphasic calcium phosphate (BCP) ceramics with or without PRP, recombinant human (rh) BMP2, and their combination. At 6 and 12 weeks after implantation, rabbits were euthanized and the radiographic and histomorphometric features of <em>bone</em> reconstruction were analyzed. The results showed that defects filled by rhBMP2/BCP with or without PRP had high <em>bone</em> density at 6 and 12 weeks in radiological evaluation. However, in histomorphometric analysis, the defects filled by rhBMP2/BCP with PRP showed significant new <em>bone</em> formation compared with that by rhBMP2/BCP without PRP, especially at 6 weeks. We propose that the synergistic effect of PRP and rhBMP2 gives highly osteoinductive properties to alloplastic substitutes in vivo.

Osteoblasts are the primary cells responsible for <em>bone</em> formation and are thought to originate from mesenchymal osteoprogenitor cells within skeletal tissues. To elucidate the osteoblastic differentiation process, fetal rat calvariae (FRC) were enzymatically digested and fractionated to provide an osteoprogenitor-enriched cell population. The third fraction of cells from the five sequential digestions tested showed a significant osteogenic response to dexamethasone (Dex), a well-known differentiation hormone, which was demonstrated by high alkaline phosphatase activity early in culture and enhanced calcium deposition and <em>bone</em> nodule formation in late stage cultures. These data indicate that fraction three contains a large number of osteoprogenitor cells. During the osteoblastic differentiation of the third fraction of FRC cells, the formation of collagen cross-links (pyridinoline and deoxypyridinoline) was time-dependently accelerated with the accumulation of collagens, which coincided with an onset of mineralization of the cultures, i.e., calcium deposition and <em>bone</em> nodule formation. Moreover, noncollagenous matrix <em>proteins</em>, <em>bone</em> sialo<em>protein</em> and osteocalcin, were also increased at both mRNA and <em>protein</em> level in Dex-treated cultures with advancing culture periods. Further examination for mRNA expression of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) and TGF-beta1 revealed a notable elevation in BMP-6 mRNA expression on days 3 and <em>10</em>, and no significant change in TGF-beta1 expression. These observations suggested that the progressive formation of collagen cross-links, production of noncollagenous <em>proteins</em>, and up regulation of BMP-6 mRNA play an important role in the osteoblastic differentiation process of osteoprogenitor cells isolated from FRC. This culture system provides us a suitable model for in vitro <em>bone</em> formation.

Recombinant human (rh) <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the <em>bone</em> metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (<em>10</em>(-<em>10</em>)-<em>10</em>(-8) M) of PGE2, whereas a high concentration (<em>10</em>(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of <em>10</em>(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately <em>10</em> times higher affinity for BMP-2 than DS, yet it equally stabilized the <em>protein</em>, as determined by alkaline phosphatase activity. Ectopic <em>bone</em> formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of <em>bone</em> compared with BMP-2 delivered uncomplexed to GAG.

We evaluated the effects of mechanical stimulation on the osteogenic differentiation of human intraoral mesenchymal stem and progenitor cells (MSPCs) using the Flexcell FX5K Tension System that mediated cyclic tensile stretch on the cells. MSPCs were isolated from human mandibular retromolar <em>bones</em> and characterized using flow cytometry. The positive expression of CD73, CD90, and CD<em>10</em>5 and negativity for CD14, CD19, CD34, CD45, and HLA-DR confirmed the MSPC phenotype. Mean MSPC doubling time was 30.4 ± 2.1 hrs. The percentage of lactate dehydrogenase (LDH) release showed no significant difference between the mechanically stimulated groups and the unstimulated controls. Reverse transcription quantitative real-time PCR revealed that <em>10</em>% continuous cyclic strain (0.5 Hz) for 7 and 14 days induced a significant increase in the mRNA expression of the osteogenesis-specific markers type-I collagen (Col1A1), osteonectin (SPARC), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2), osteopontin (SPP1), and osteocalcin (BGLAP) in osteogenic differentiated MSPCs. Furthermore, mechanically stimulated groups produced significantly higher amounts of calcium deposited into the cultures and alkaline phosphatase (ALP). These results will contribute to a better understanding of strain-induced <em>bone</em> remodelling and will form the basis for the correct choice of applied force in oral and maxillofacial surgery.

We investigated the effects of transplantation of osteoblastic cells with a <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)/carrier complex on <em>bone</em> repair by in vitro and in vivo experiments. Poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS) was used as a carrier for cell transplantation. In the in vitro experiments, three cell types, C3H<em>10</em>T1/2 cells, MC3T3-E1 cells, and primary osteoblastic cells, isolated from newborn rat calvariae (ROB cells), were cultured for 2 weeks on PGS alone or PGS containing BMP-2 (PGS/BMP). C3H<em>10</em>T1/2 cells cultured on PGS/BMP expressed several markers related to differentiation of both osteoblasts and chondrocytes, such as alkaline phosphatase (ALP) activity and mRNAs for osteocalcin and aggrecan, whereas the cells cultured on PGS alone expressed no such markers. MC3T3-E1 cells cultured on PGS/BMP exhibited a more ALP-positive cells than those cultured on PGS alone. PGS/BMP promoted ROB cell differentiation into both osteoblasts and chondrocytes. In the in vivo experiments, we transplanted ROB cells, which had been cultured on PGS alone or PGS/BMP in vitro for 2 weeks, into <em>bone</em> defects created in rat calvariae. Transplantation of ROB cells cultured on PGS alone generated little new <em>bone</em>. Transplantation of ROB cells cultured on PGS, which absorbed a low dose (<em>10</em> ng) of rhBMP-2,; induced significantly higher <em>bone</em> mineral content than PGS/BMP alone, although application of a high dose (1 microg) of rhBMP-2 induced no difference in <em>bone</em> mineral content between transplantation of PGS/BMP with or without ROB cells. These results show that transplantation of osteoblastic cells after induction of osteoblast maturation in vitro by cultivation on PGS/BMP is a potent technique for cell therapy of <em>bone</em> repair.

BACKGROUND

Demineralized bone matrix and recombinant human bone morphogenetic protein-2 or 7 (BMP-2 or BMP-7)-containing collagenous matrix have been shown to induce new bone formation in orthotopic and heterotopic sites. We examined the ability of subcutaneous implants of collagen combined with adenoviral vector containing the BMP-2 gene (AdBMP-2) to induce bone formation in rats. We also evaluated whether targeting the AdBMP-2 vector through an alternative receptor pathway, fibroblast growth factor (FGF), would increase the vector's potency.

METHODS

In a time-course study, rat subcutaneous sites were implanted with (1) AdBMP-2 in rat-bone-derived collagen or (2) rat-bone-derived collagen alone. Samples were collected three, seven, fourteen, or thirty-five days after treatment. In a dose-response study, bone induction by AdBMP-2 in collagen (AdBMP-2/collagen) or by AdBMP-2 and FGF2 Fab' anti-adenovirus knob protein antibody in collagen (FGF2-AdBMP-2/collagen) was tested at fourteen days. Viral vector doses of 1 x 10(9) PN (viral particle number), 3 x 10(9) PN, 1 x10(10) PN, 3 x 10(10) PN, or 1 x 10(11) PN per implant were used. Equal amounts of collagen (25 mg) were used to formulate all implants. Explanted tissues were evaluated histologically to determine bone formation, specific activity of alkaline phosphatase, and calcium content.

RESULTS

AdBMP-2/collagen implants induced robust bone formation. New bone was formed by the fourteenth day after implantation. In contrast, little or no bone was induced by the implant containing collagen alone. FGF2-AdBMP-2/collagen implants stimulated significantly more bone formation (p < 0.05) than did AdBMP-2/collagen implants, regardless of the dose of viral particles.

CONCLUSIONS

Local delivery of AdBMP-2 in a collagen matrix rapidly induces bone formation, and targeting the virus through FGF receptors enhances the osteogenic potential of AdBMP-2.

In order to explore the methods for commercialized <em>bone</em> tissue engineering, engineered <em>bones</em> should be cultivated in bioreactors to realize three-dimensional culture under well-defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1-month-old Zelanian rabbits were inoculated on to the BDBS (bio-derived <em>bone</em> scaffolds) to investigate the three-dimensional fabrication of engineered <em>bone</em> in an RWVB (rotating-wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent <em>protein</em>, were respectively seeded at 2 x <em>10</em>(6) and 1 x <em>10</em>(6) cells x ml(-1) on to the BDBS and then cultured in a T-flask and an RWVB for 1 week. The morphologies and structure of the fabricated <em>bone</em> were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von-Kossa staining on mineralized nodules, type I collagen and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell-seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T-flask. <em>Bone</em> tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three-dimensional fabrication of engineered <em>bone</em> could be realized.

Cultures of embryonic rat septum were exposed for 24-48 h to 2-5 nm okadaic acid (OA), an inhibitor of pp1A and pp2A phosphatases. This stress killed approximately 75% of neurons. A neurotrophin (NT) combination (nerve growth factor and brain-derived neurotrophic factor, each <em>10</em>0 ng/mL) plus a <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP6 or BMP7, 5 nm) reduced the death of both cholinergic and non-cholinergic neurons, and preserved choline acetyltransferase (ChAT) activity assayed 2-6 days post-stress. This NT + BMP combination preserved ChAT activity better than either NTs or BMPs alone, and was effective even if trophic factor addition was delayed until 12 h after stress onset. A general caspase inhibitor (qVD-OPH, <em>10</em> micro g/mL) also increased survival of stressed cholinergic neurons, but its protection of ChAT activity was shorter lived than that produced by the NT + BMP combination. Neither the NT + BMP combination nor the caspase inhibitor reduced the OA-induced increase in tau phosphorylation. These findings indicate that NTs and BMPs have synergistic protective effects against an OA stress, and suggest that at least some of these protective effects occur upstream of caspase activation.

Conditioning protocols involving mechanical stress independently or with chemical cues such as growth factors (GFs) possess significant potential to enhance <em>bone</em> regeneration. However, utilization of thermal stress conditioning alone or with GFs for <em>bone</em> therapy has been under-investigated. In this study, a preosteoblast cell line (MC3T3-E1) was exposed to treatment with water bath heating (44°C, 4 and 8 min) and osteoinductive GFs (<em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and transforming growth factor-β1) individually or in combination to investigate whether these stimuli could promote induction of <em>bone</em>-related markers, an angiogenic factor, and heat shock <em>proteins</em> (HSPs). Cells remained viable when heating durations were less than 20 min at 40ºC, 16 min at 42ºC, and <em>10</em> min at 44ºC. Increasing heating duration at 44°C, promoted gene expression of HSPs, osteocalcin (OCN), and osteopontin (OPN) at 8 h post-heating (PH). Heating in combination with GFs caused the greatest gene induction of osteoprotegerin (OPG; 6.9- and 1.6-fold induction compared to sham-treated and GF only treated groups, respectively) and vascular endothelial growth factor (VEGF; 16.0- and 1.6-fold compared to sham and GF-only treated groups, respectively) at 8 h PH. Both heating and GFs independently suppressed the matrix metallo<em>protein</em>ase-9 (MMP-9) gene. GF treatment caused a more significant decrease in MMP-9 <em>protein</em> secretion to non-detectable levels compared to heating alone at 72 h PH. Secretion of OCN, OPN, and OPG increased with the addition of GFs but diminished with heating as measured by ELISA at 72 h PH. These results suggest that conditioning protocols utilizing heating and GFs individually or in combination can induce HSPs, <em>bone</em>-related <em>proteins</em>, and VEGF while also causing downregulation of osteoclastic activity, potentially providing a promising <em>bone</em> therapeutic strategy.

OBJECTIVE

Although situational risk factors for incisional hernia formation are known, the methods used to determine who would be most susceptible to develop one are unreliable. We hypothesized that patients with recurrent incisional hernias may possess unique gene expression profiles.

METHODS

Skin and intact fascia were collected from 15 normal control (NC) patients with no hernia history and 18 patients presenting for recurrent incisional hernia (RH) repair. Microarray analysis was performed using whole genome microarray chips on NC (n = 8) and RH (n = 9). These samples were further investigated using a pathway-specific PCR array containing fibrosis-related genes.

RESULTS

Microarray data revealed distinct differences in the gene expression profiles between RH and NC patients. One hundred and sixty-seven genes in the skin and 7 genes in the fascia were differentially expressed, including 8 directly involved in collagen synthesis. In particular, GREMLIN1, or <em>bone</em> <em>morphogenetic</em> <em>protein</em> antagonist 1, was under expressed in skin (fold = 0.49, p < <em>10</em>(-7), q = 0.0009) and fascia (fold = 0.23, p < <em>10</em>(-4), q = 0.095) of RH patients compared with NC. The PCR array data supported previous reports of decreased collagen I/III ratios in skin of RH versus NC (mean = 1.51 ± 0.73 vs. mean = 2.26 ± 0.99; one-sided t test, p = 0.058).

CONCLUSIONS

To our knowledge, this is the first microarray-based analysis to show distinct gene expression profiles between the skin and fascia of RH and NC patients and the first report of an association between GREMLIN1 and incisional hernia formation. Our results suggest that gene expression profiles may act as surrogate markers that stratify patients into different groups at risk for hernia development prior to their initial surgery.

BACKGROUND

Mesenchymal Stem Cells (MSCs) are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord mesenchymal Stem Cells (hUCMSCs) can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell (PGC) was performed in vitro under specific condition.

METHODS

Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing <em>10</em>% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em> 4 (BMP4) and it was followed by retinoic acid (RA). Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and <em>proteins</em>, respectively.

RESULTS

Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to transdifferentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture.

CONCLUSIONS

Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications.

OBJECTIVE

To compare follistatin (FST) and activin (Act) expression in normal and glaucomatous trabecular meshwork (TM) cells and tissues and determine if exogenous TGF-β2 regulates the expression of FST and Act in TM cells.

METHODS

Total RNA was isolated from TM cell strains, and mRNA expression for FST 317/344 isoforms and Act was determined via RT-PCR and quantitative PCR (qPCR). Western immunoblotting and immunocytochemistry determined FST and Act A <em>protein</em> levels in normal TM (NTM) and glaucomatous TM (GTM) cells. Cells were treated with recombinant human TGF-β2 <em>protein</em> at 0 to <em>10</em> ng/mL for 0 to 72 hours. qPCR, Western immunoblotting, immunocytochemistry, and ELISA immunoassay were utilized to determine changes in FST and Act A mRNA and <em>protein</em> levels. In addition, NTM and GTM tissue samples were examined by immunohistochemistry for expression of FST, FST 315, FST 288, and Act A.

RESULTS

Both FST mRNA and protein levels were significantly elevated in GTM cells. FST mRNA transcripts FST 317/344 were also significantly elevated in GTM cells. Immunohistochemistry showed FST levels were significantly elevated in GTM tissues. Exogenous TGF-β2 significantly induced FST mRNA and protein expression. Immunohistochemistry demonstrated that Act A protein levels were significantly higher in NTM tissues compared to GTM tissues.

CONCLUSIONS

FST is elevated in GTM cells and tissues. FST is known to be an inhibitor of bone morphogenetic proteins (BMPs), which, coupled with the ability of TGF-β2 to upregulate FST levels, may indicate a possible role of FST in the pathogenesis of glaucoma. These results suggest that additional endogenous molecules in human TM may regulate TGF-β2 signaling via inhibition of BMP family members.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are known to play an important role in the regulation of cell proliferation, survival, differentiation and apoptosis in many vertebrates and invertebrates through the TGF-β signaling pathway. Although the TGF-β signaling pathway exists in schistosomes, BMP homologue, a ligand of TGF-β in Schistosoma japonicum, has not yet been identified. In this study, a BMP homologue of S. japonicum was cloned and characterized. The full length SjBMP cDNA is 3,020 bp and encodes 928 amino acids, which include a TGF-β superfamily conserved domain at the C-terminus. BLAST analysis showed that, SjBMP has 68%, 51% and 43% homology with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica at the amino acid level, respectively. According to data from real-time PCR, SjBMP was expressed in lung-stage schistosomula, 21-day liver-stage schistosomula, 50-day adult worms (the male and female), and eggs. The PCR data also indicated that, there was a ≈ 27- and ≈ 37-fold increase of SjBMP transcripts in the lung-stage schistosomula and eggs, respectively, and that there was relatively more SjBMP transcript in the adult male worm than in the adult female, in which the hepatic schistosomula was set as the calibrator for calculation. In situ hybridization based on FITC-labeled specific antisense oligonucleotide probes showed that SjBMP mRNA localized to the ovary of female worms and the integument and epithelium of female and male worms. After treatment with double-stranded RNA (dsRNA) at a concentration of 8 × <em>10</em>(-2) μg/ml, which was added to the culture medium every other day for a week, the level of SjBMP mRNA in the cultured adult mixed-sex S. japonicum decreased at a range of ≈ 25-98% within 7 days compared with the level of SjBMP mRNA in the blank control group. On the 2nd day, the number of eggs produced per pair of worms decreased 28.7%, and the percent of normal eggs also decreased (12.7% vs. 4.3%) in the SjBMP dsRNA-treated group when compared with the eggs laid by the blank control group. No difference was detected between the two groups on the 7th day of treatment, because the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group. In addition, no significant difference in the morphological structure of the adult worms was observed. Thus, the preliminary in vitro experiment indicated that SjBMP may be involved in the oviposition behavior of S. japonicum, and further studies based on the recombinant virus vector-induced steady knockdown of SjBMP or in vivo experiments are required for more in-depth investigation.

A variety of different growth factors, most notably <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for <em>10</em> days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: <em>10</em>0 ng/ml; days 7-<em>10</em>: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (<em>10</em> μmol/L) for periods up to 72 h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and α-smooth muscle actin (α-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and <em>protein</em> expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to <em>10</em> μmol/L AA increased the mRNA and <em>protein</em> expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 <em>protein</em> levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin plays a critical role in the modulation of reno-protective action of BMP, and that inhibition of gremlin will be a promising means of developmenting novel treatments for AAN.