The UVB component of the solar spectrum induces DNA lesions that, in the absence of error-free DNA repair, may give rise during DNA replication to mutations in caretaker and gatekeeper genes. The DNA repair genes are the best candidates for caretaker genes as exemplified by the human hereditary xeroderma pigmentosum (XP) syndrome. Cultured XP cells are hypermutable after UVB irradiation. This increased mutation frequency is also found in gatekeeper genes, which govern signalling pathways implicated in the control of cellular proliferation, differentiation and survival of human epidermal keratinocytes. We describe and discuss the role of mutated gatekeeper genes in five specific signalling pathways which have been implicated in skin carcinogenesis. The pathways we focus on in this review are: (i) P16(INK4A)-CDK4/6-RB; (ii) P14(ARF)-HDM2-P53; (iii) Sonic hedgehog (SHH)/GLI; (iv) WNT/beta-catenin; and (v) <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em> (BMP)/SMAD. 70-80% of XP skin cancers exhibit one or several mutations in the P53, PTCH-1, SMO or CDKN2A genes, the type and frequency of mutated genes being different between squamous cell (SCCs) and basal cell carcinomas (BCCs). In XP cancers, the typically UVB-induced CC to TT tandem transitions represent approximately 60% of total mutations compared to <em>10</em>-15% in skin tumours from DNA repair-proficient patients. Acquired activation of the pathways described herein can alter proliferation and differentiation of keratinocytes, allowing a damaged cell to replicate and give rise to mutated daughter cells, then eventually to the development of the carcinogenic process following clonal selection.

METHODS

In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors.

OBJECTIVE

To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II.

BACKGROUND

Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before.

METHODS

Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-β1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed.

RESULTS

Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-β1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-β1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-β1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05).

CONCLUSIONS

NP cells in atelocollagens under the stimulation of TGF-β1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.

BACKGROUND

Hydroxyapatite (HA) is considered to be useful because of its high affinity for recombinant human bone morphogenetic protein (rhBMP), mechanical resistance to compressive force, and possible reduction of rhBMP dose.

OBJECTIVE

To evaluate the osteoinductivity of Escherichia coli-derived rhBMP-2 and the suitability of porous HA as an rhBMP-2 carrier.

METHODS

In vivo study using microcomputerized tomography (micro-CT) scanning.

METHODS

Seventy-six New Zealand white male rabbits were randomized into a single control group (n=14) without rhBMP-2 and four experimental groups (10 μg, 50 μg, 200 μg, and 500 μg of rhBMP-2; n=14 in each group). The subjects were divided into 3- and 6-week groups.

METHODS

Outcome was evaluated by radiography, bending test, three-dimensional micro-CT, and histologic examinations.

METHODS

Bilateral posterolateral fusion was carried out, and rhBMP-2 (0, 10, 50, 200, 500, 1,000, and 2,000 μg) was implanted into the bilateral transverse processes using HA as a carrier.

RESULTS

The fusion rates of the 3-week group were 83.3% for 50 and 200 μg of rhBMP-2 and 100% for 500 μg. The improved fusion rates of the 50 μg or higher groups compared with those of control were statistically significant. The fusion rates of the 6-week group were 75% for 10 μg of rhBMP-2 and 100% for 50 μg or higher. Similarly, the improved fusion rates of the 10 μg or higher groups compared with those of control were statistically significant. Significantly higher percent volumes were observed in the 3-week 200 μg of rhBMP-2 group and 6-week 200 μg of rhBMP-2 group than the 3-week HA group and 6-week HA group, respectively. Trabecular thickness was significantly higher in the 3-week 200 μg of rhBMP-2 group than the 3-week HA group. Histologic analysis of the 10 μg group showed bone tissues within the pores from 3 weeks, and this was observed more vividly in the 50, 200, and 500 μg groups. The 6-week 10 μg and 50 μg of rhBMP-2 groups had lower amounts of new tissue but higher portions of complete bone tissue within the HA specimen, along with higher formation of completely reconstituted bone tissues outside HA.

CONCLUSIONS

Injection of 50 μg or more of E. coli-derived rhBMP-2 into a HA carrier induced earlier bone fusion in the intertransverse process of rabbits, which confirms the excellent bone forming ability of E. coli-derived rhBMP-2 and the suitability of HA as a carrier of rhBMP-2.

Modulating immune response to biomaterials through changing macrophage polarization has been proven to be a promising strategy to elicit beneficial outcomes in tissue repair. The objective of this study was to evaluate the response of macrophage polarization to titanium doped with magnesium (0.1~0.35%), which was prepared through the magnesium plasma immersion ion implantation (Mg PIII) technique. The M1/M2 polarization profile of macrophages was investigated using a murine cell line RAW 264.7 in vitro and a murine air pouch model in vivo. Our results demonstrated that the Mg PIII-treated titanium induced a higher percentage of M2 macrophages and higher concentrations of the anti-inflammatory cytokines interleukin (IL)-4 and IL-<em>10</em>. Genes encoding two growth factors, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) and vascular endothelial growth factor (VEGF) were up-regulated, thus indicating the ability of the M2 phenotype to promote wound healing. The nuclear factor κB (NF-κB) signalling pathway was down-regulated. In vivo the Mg PIII -treated titanium elicited a similar effect on macrophage polarization and induced thinner fibrous capsule formation and a decrease in infiltrated cells. These results indicate that Mg PIII treatment has the immunomodulatory potential to elicit the pro-healing M2-polarized macrophage phenotype, thus providing new insight into the development of immunomodulatory biomaterials.

Tissue formation and repair are dependent upon cascades of biological events, but the signals involved and the possible gene coexpression patterns during intramembranous <em>bone</em> repair are only poorly understood. We sought to place this mode of regeneration in context by profiling quantitative gene expression for a panel of 39 genes between days 1 and 14 following rat femoral marrow ablation. In situ hybridization was employed to localize a subset of genes. Additionally, principal components analysis was conducted to identify underlying factors suggestive of coexpression patterns. During inflammation (days 1-5), several genes, including cyclooxygenase-1 and -2, showed downregulation. Other proinflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, exhibited increasing levels around day 5. During repair (days 3-<em>10</em>), growth factors, receptors, and inhibitor genes for transforming growth factor- beta; basic fibroblast growth factor; <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 2, 4, and 7; vascular endothelial growth factor; and insulin-like growth factor-I were upregulated. In addition, the gene for core binding factor-alpha1 and markers of osteoblast function such as alkaline phosphatase, collagen type I, osteonectin, osteopontin, and osteocalcin had peak expression at day 5 or 7. The remodeling phase (days <em>10</em>-14) was characterized by peaks for cytokines associated with osteoclastic activity including receptor activator of nuclear factor-kappaB, receptor activator of nuclear factor-kappaB ligand (RANKL), cathepsin K, tumor necrosis factor-alpha, interleukin-6, and cyclooxygenase-2. In situ hybridization showed that the most common sites of increased signal were within osteoblastic cells on trabecular and endosteal surfaces. Principal components analysis identified eight underlying factors that together explained over 80% of the variance in the data.

Osteocalcin (OC) is an important constituent of <em>bone</em> that is synthesized by osteoblasts. Serum levels of OC have been used as a biochemical marker of <em>bone</em> turnover. To identify the genes influencing variation in serum OC levels, we conducted a genome-wide scan in 429 individuals comprising <em>10</em> large multigenerational families. OC levels were measured by immunoassay, and genetic markers were typed at approximately <em>10</em>-cM intervals across the genome. Quantitative trait linkage was tested using a multipoint analysis based on variance component methodology, adjusting for the effects of age, sex, and oral contraceptive use. Significance levels for linkage were obtained empirically, by Monte Carlo simulation. The heritability of OC levels in this population was 62 +/- 8%. We detected significant evidence for linkage between a quantitative trait locus influencing serum OC levels and markers on chromosome 16q, and suggestive evidence for linkage of OC levels with markers on chromosome 20q. The multipoint lod scores peaked at 3.35 on chromosome 16 and 2.78 on chromosome 20, corresponding to P values of 0.00004 and 0.00017, respectively. A potential candidate gene for <em>bone</em> formation in the linked region on chromosome 20 is CDMP1, which encodes cartilage-derived <em>morphogenetic</em> <em>protein</em> 1. Future studies should evaluate whether variation in CDMP1 or in other genes in the linked regions on chromosomes 16 and 20 influence the rate of <em>bone</em> turnover.

BACKGROUND

Bone morphogenetic proteins (BMPs) have the potential to improve incorporation of allograft bone in revision surgery. This could result in improved fixation and graft incorporation.

METHODS

We evaluated the effect of mixing OP-1 (BMP-7) with morselized allograft in hip revisions. In a case-control study, 20 acetabular revisions (10 in the study group, 10 controls) and 41 femoral revisions (11 in study group, 30 controls) were done using impaction allografting. The migration of the cups and stems was studied with radiostereometric analysis (RSA) for up to 5 years. Changes of bone mineral density around the femoral component were measured with dual energy X-ray adsorptiometry for 2 years. Bone remodeling and the extent of radiolucent lines were evaluated on conventional radiographs after 5 years. The clinical results were documented using Harris hip score.

RESULTS

In the cup study, there was no significant difference in implant migration between the study and control groups. 4 sockets in the study group were classified as radiographically loose after 5 years and 2 of them were revised after 5 years. After 2 years, the stems in the study group showed slightly increased posterior tilt (0.3 degrees , p = 0.03). 1 stem in the study group loosened and was revised during the third year of observation.

CONCLUSIONS

The sample size and study design in our evaluation did not allow any firm conclusions. Absence of any trend to improved fixation and early revisions in the study group prompted us to stop recruitment to this study.

Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of <em>bone</em> growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2). Nanoindentation studies of <em>bone</em> tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine <em>bone</em>, eight weeks post-treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius <em>bones</em>, following the implantation of MSCs. High-resolution micro-computed tomography (micro-CT) was performed <em>10</em> and 35 weeks post MSC implantation, followed by micro-finite element (micro-FE) analysis. The results have shown that the regenerated <em>bone</em> tissue remodels over time, as indicated by a significant decrease in <em>bone</em> volume, total volume, and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2-1.5 times higher compared to the contralateral intact limbs, at <em>10</em> and 35 weeks post-treatment. These results could be attributed to the fusion that occurred in between the ulna and radius <em>bones</em>. In conclusion, although MSCs induce <em>bone</em> formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair.

OBJECTIVE

Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) is becoming a common clinical approach to enhance bone repair. There is little or no information in the literature on the dose of rhBMP-2 required for effective healing of critical-sized defects such as those associated with trauma. In this study, we used a segmental defect model to assess the dose response of rhBMP-2 using quantitative and qualitative endpoints.

METHODS

Femoral defects in rats were replaced with absorbable collagen sponges carrying rhBMP-2 (0, 1, 5, 10 or 20 μg; N=5). At 4-weeks new bone formation was assessed using quantitative (radiography and microcomputed tomography) and qualitative (histology and backscattered-SEM) endpoints statistically compared.

RESULTS

rhBMP-2 showed increased bridging in the gap. Quantitative evaluation presented a bi-phasic dose response curve. Histological assessment revealed that with rhBMP-2 the defect showed the presence of spongy bone with the trabeculae layered with active osteoblasts and osteoclasts. The density and compactness of the bone varied with the dose of rhBMP-2.

CONCLUSIONS

Our findings revealed that all doses of rhBMP-2 result in new bone formation. However, there is an optimum dose of 12 μg of rhBMP-2 for bone repair in this model, above which and below which less stimulation of bone occurs.

OBJECTIVE

To compare mRNA gene expression during fracture healing in young and adult rats.

METHODS

Gene expression was measured at zero, 1, 2, 4, 6, 8 and <em>10</em> weeks after fracture (6 rats/age/time point) in rats at 6 and 26 weeks of age at surgery.

METHODS

AAALAC-accredited vivarium of an independent academic medical center.

METHODS

Female Sprague-Dawley rats at 6 and 26 weeks of age.

METHODS

An intramedullary rod was placed retrograde in the left femur, and a simple transverse closed middiaphyseal fracture was induced.

METHODS

mRNA gene expression was measured for 34 genes for extracellular matrix, osteoblasts, bone morphogenic protein, inflammation, cytokine, and receptor genes.

RESULTS

The young rats reached radiographic union by 4 weeks after fracture, whereas the adult rats took 8 to <em>10</em> weeks to unite. All genes studied increased in mRNA expression with a peak at 1 to 2 weeks after fracture. All genes in the young rats then subsided to baseline by 4 weeks after fracture. However, during the longer period needed for radiographic union in the adult rats, only genes related to bone matrix, osteoblastic markers, angiogenesis, and the fibroblast growth factors remained significantly up-regulated at 4 and 6 weeks after fracture. Genes related to cartilage, Indian hedgehog, the bone morphogenetic proteins, and transforming growth factor-beta came to undetectable baseline values in the adult rats prior to radiographic union.

CONCLUSIONS

Most stimulators of bone healing are not expressed during the later stages of fracture repair in adult rats. Other genes must control bone growth to bridge the fracture gap.

In the current investigation, we report osseous regeneration in critical-size rat calvarial defects using recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) and novel delivery systems based on biomaterials. The novel systems combine rhBMP-2 with dry powder microparticles of poly(D,L-lactide-co-glycolide) (PLGA). The mixture of rhBMP-2 with PLGA microparticles is added to an aqueous solution of biopolymer to yield a semisolid paste. The biopolymers tested include autologous blood clot, hydroxypropyl methylcellulose, and sodium alginate cross-linked with calcium ion. Insoluble collageneous <em>bone</em> matrix was also studied as a control. Test articles were made at 0-, <em>10</em>-, and 30-micrograms doses of rhBMP-2 and imiplanted in 8-mm-diameter rat calvarial defects (which will not heal if left untreated). The animals were examined 21 days after implantation by radiography, radiomorphometry, histology, and histomorphometry. All tested materials containing rhBMP-2 restored radiopacity and normal contouring to the calvarial defects. Samples without added rhBMP-2 yielded only soft tissue within the defects. Histology showed restoration of inner and outer <em>bone</em> tables plus marrow constituents. The PLGA microparticles were significantly resorbed at the 21-day time point. Although small differences between delivery systems were evident at 0- and <em>10</em>-micrograms rhBMP-2 doses, all test articles performed essentially equivalently at the 30-micrograms dose. Thus, novel delivery systems for rhBMP-2 offer the promise of combining the intrinsic bioactivity of the osteoinductive <em>protein</em> with pharmaceutically acceptable biomaterials.

The myelin <em>protein</em> P(0) has a major structural role in Schwann cell myelin, and the expression of P(0) <em>protein</em> and mRNA in the Schwann cell lineage has been extensively documented. We show here, using in situ hybridization, that the P(0) gene is also activated in a number of other tissues during embryonic development. P(0) mRNA is first detectable in <em>10</em>-day-old embryos (E<em>10</em>) and is at this time seen only in cells in the cephalic neural crest and in the otic placode/pit. P(0) expression continues in the otic vesicle and at E12 P(0) expression in this structure largely overlaps with expression of another myelin gene, proteolipid <em>protein</em>. In the developing ear at E14, P(0) expression is complementary to expression of serrate and c-ret mRNAs, which later are expressed in sensory areas of the inner ear, while expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-4 and P(0), though largely complementary, shows small areas of overlap. P(0) mRNA and <em>protein</em> are detectable in the notochord from E<em>10</em> to at least E13. In addition to P(0) expression in a subpopulation of trunk crest cells at E11/E12 and in Schwann cell precursors thereafter, P(0) mRNA is also present transiently in a subpopulation of cells migrating in the enteric neural crest pathway, but is down-regulated in these cells at E14 and thereafter. P(0) is also detected in the placode-derived olfactory ensheathing cells from E13 and is maintained in the adult. No signal is seen in cells in the melanocyte migration pathway or in TUJ1 positive neuronal cells in tissue sections. The activation of the P(0) gene in specific tissues outside the nervous system was unexpected. It remains to be determined whether this is functionally significant, or whether it is an evolutionary relic, perhaps reflecting ancestral use of P(0) as an adhesion molecule.

Dynamic hydrogels have emerged as an important class of biomaterials for temporal control over growth factor delivery. In this study we formed dynamic hydrogel microspheres from <em>protein</em>-polymer conjugates using an aqueous two-phase suspension polymerization process. This polymerization process enabled rapid microsphere formation without the use of an organic phase, surfactants, mechanical strain or toxic radical initiators. The microspheres' size distribution was modulated by varying the <em>protein</em>-polymer conformation in the pre-polymer solution. Notably, the <em>protein</em>'s ligand-induced, nanometer-scale conformational change translated to maximum hydrogel volume changes of 76±<em>10</em>%. The magnitude of the microspheres' volume change was tuned by varying the crosslinking time and ligand identity. After characterizing the microspheres' dynamic properties, we encapsulated two important therapeutic <em>proteins</em>, vascular endothelial growth factor (VEGF) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), in the hydrogel microspheres and characterized how the microspheres' dynamic properties controlled their release. Significantly, the aqueous two-phase suspension polymerization process enabled high encapsulation efficiencies (65.8±4.8% and 79.5±3.0% for VEGF and BMP-2, respectively). Also, the microspheres' ligand-induced volume change triggered VEGF and BMP-2 release at specific, predetermined times. There are hundreds of <em>proteins</em> that undergo well-characterized conformational changes that could be processed into hydrogel microspheres via aqueous two-phase suspension polymerizations. Therefore, this approach could be used to form dynamic, growth-factor-releasing hydrogel microspheres that respond to a broad range of specific biochemical ligands.

Inhibition of bone formation after surgery to correct craniosynostosis would alleviate the need for secondary surgeries and decrease morbidity and mortality. This study used a single dose of Noggin protein to prevent resynostosis and improve postoperative outcomes in a rabbit model of craniosynostosis.

BACKGROUND

Craniosynostosis is defined as the premature fusion of one or more of the cranial sutures, which causes secondary deformations of the cranial vault, cranial base, and brain. Current surgical intervention involves extirpation of the fused suture to allow unrestricted brain growth. However, resynostosis of the extirpated regions often occurs. Several bone morphogenetic proteins (BMPs), well-described inducers of ossification, are involved in bone healing. This study tested the hypothesis that a postoperative treatment with Noggin, an extracellular BMP inhibitor, can inhibit resynostosis in a rabbit model of human familial nonsyndromic craniosynostosis.

METHODS

Thirty-one New Zealand white rabbits with bilateral coronal suture synostosis were divided into three groups: (1) suturectomy controls (n = 13); (2) suturectomy with BSA in a slow-resorbing collagen vehicle, (n = 8); and (3) suturectomy with Noggin in a slow-resorbing collagen vehicle (n = 10). At 10 days of age, a 3 x 15-mm coronal suturectomy was performed. The sites in groups 2 and 3 were immediately filled with BSA-loaded gel or Noggin-loaded gel, respectively. Serial 3D-CT scan reconstructions of the defects and standard radiographs were obtained at 10, 25, 42, and 84 days of age, and the sutures were harvested for histological analysis.

RESULTS

Radiographic analysis revealed that Noggin-treated animals had significantly greater coronal suture marker separation by 25 days and significantly greater craniofacial length at 84 days of age compared with controls. 3D-CT analysis revealed that Noggin treatment led to significantly greater defect areas through 84 days and to increased intracranial volumes at 84 days of age compared with other groups. Histological analysis supported CT data, showing that the untreated and BSA-treated groups had significant healing of the suturectomy site, whereas the Noggin-treated group had incomplete wound healing.

CONCLUSIONS

These data support our hypothesis that inhibition of BMP activity using Noggin may prevent postoperative resynostosis in this rabbit model. These findings also suggest that Noggin therapy may have potential clinical use to prevent postoperative resynostosis in infants with craniosynostosis.

BACKGROUND

Human adipose tissue-derived stromal cells (hADSCs) can be induced to differentiate along an osteoblastic lineage under stimulation of dexamethasone (DEX). Recent studies, however, have questioned the efficacy of glucocorticoids such as DEX in mediating the osteogenesis process of skeletal progenitor cells and processed lipoaspirate cells. Is it possible to find a substitute for DEX? Therefore, this study was designed to investigate osteogenic capacity and regulating mechanisms for osteoblastic differentiation of hADSCs by comparing osteogenic media (OM) containing either 1, 25-dihydroxyvitamin D(3) (VD) or DEX and determine if VD was an ideal substitute for DEX as an induction agent for the osteogenesis of hADSCs.

METHODS

Osteogenic differentiation of hADSCs was induced by osteogenic medium (OM) containing either <em>10</em> nmol/L VD or <em>10</em>0 nmol/L DEX. Differentiation of hADSCs into osteoblastic lineage was identified by alkaline phosphatase (ALP) staining, von Kossa staining, and reverse transcription-polymerase chain reaction assays for mRNA expression of osteogenesis-related genes such as type I collagen (COL I), <em>bone</em> sialo<em>protein</em> (BSP), osteocalcin (OC), <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2, BMP-4, BMP-6, BMP-7, runt-related transcription factor 2/core binding factor alpha1 (Runx2/Cbfa1), osterix (Osx), and LIM mineralization <em>protein</em>-1 (LMP-1).

RESULTS

von Kossa staining revealed that the differentiated cells induced by both VD and DEX were mineralized in vitro. They also expressed osteoblast-related markers, such as ALP, COL I, BSP, and OC. Runx2/Cbfa1, Osx, BMP-6, and LMP-1 were upregulated during VD and DEX-induced hADSC osteoblastic differentiation, but BMP-4, BMP-7 were not. BMP-2 was only expressed in VD-induced differentiated cells.

CONCLUSIONS

VD or DEX-induced hADSCs differentiate toward the osteoblastic lineage in vitro. Runx2/Cbfa1, Osx, BMP-2, BMP-6, and LMP-1 are involved in regulating osteoblastic differentiation of hADSCs, but BMP-4, BMP-7 are not. VD, but not DEX, induces expression of BMP-2 during osteogenic induction of hADSCs. VD is an ideal substitute for DEX for osteogenic induction of hADSCs.

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/<em>10</em>T1/2 murine embryonic fibroblasts by recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied <em>10</em> min on/<em>10</em> min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/<em>10</em>T1/2 cell line in a duration-dependent manner.

Activin receptor-like kinase-2 (Alk2) has been shown to be a promiscuous type I receptor for the transforming growth factor beta (TGFbeta) family of growth and differentiation factors, such as activin, <em>bone</em> <em>morphogenetic</em> <em>proteins</em>, and Müllerian inhibiting substance (MIS). We have studied the putative role of Alk2 in activin signaling using MA-<em>10</em> cells, a mouse transformed Leydig cell line, in which endogenous expression of cytochrome P450 c17 hydroxylase/C17-20 lyase mRNA is inhibited by both MIS and activin A. Overexpression of Alk2 in MA-<em>10</em> cells inhibited the activation of the activin-responsive CAGA-luciferase reporter and, conversely, transfection of siRNA for Alk2 increased the response. In contrast, overexpression of the MIS type II receptor in MA-<em>10</em> cells increased the activin-mediated induction of CAGA-luciferase approximately fivefold, which we hypothesized occurs by MIS type II receptor sequestering endogenous Alk2. Binding experiments with (125)I-labeled activin show that the underlying mechanism of Alk2-mediated inhibition of activin signaling involves Alk2 blocking the access of activin to its type II receptor, which we show can bind Alk2 in the absence of ligand. These results show that the complement of other type I receptors in addition to the ligand-specific type I receptor can provide an important mechanism for modulating cell-specific responses to members of the TGFbeta family.

The present study was performed to determine the histological, ultrastructural, and radiographic changes that occur over time at intramuscular BMP-9 gene therapy treatment sites. Several members of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) family have the potential to induce osteochondrogenesis when the <em>protein</em> is delivered to rodents, canines, rabbits, and nonhuman primates. Previous studies have also demonstrated that BMP gene therapy utilizing adenoviral vectors can also stimulate orthotopic and heterotopic <em>bone</em> formation in rodents and rabbits. Athymic nude and Sprague-Dawley rats were injected with Ad-BMP-9 or Ad-beta-Gal (3.75 x <em>10</em>(9) particles) in their thigh musculature and light microscopic, electron microscopic, and computerized tomography analysis was performed 3, 6, 9, 12, 15, 18, 21, and <em>10</em>0 days later. To assess early mesenchymal cell proliferation, a bromodeoxyuridine (BrdU) immunohistochemical analysis was also performed 48, 60, and 72 hr postinjection in athymic nude rats. All animals demonstrated extensive endochondral <em>bone</em> formation at the Ad-BMP-9 treatment sites within 3 weeks. The Sprague-Dawley rats also exhibited a massive, acute inflammatory infiltrate during the first week. Proliferating mesenchymal stem cells were clearly evident as early as 2 days after treatment, which differentiated into small or hypertrophied chondrocytes during the next week. During the third week, the cartilaginous matrix mineralized and formed woven <em>bone</em>, which converted to lamellar <em>bone</em> by 3 months. No evidence of <em>bone</em> formation was demonstrated at the Ad-beta-Gal injection sites in the athymic nude or Sprague-Dawley rats. In addition, no cellular proliferation was seen at the Ad-beta-Gal treatment sites in the athymic nude animals as assessed by light microscopy and BrdU immunohistochemistry. The extensive <em>bone</em> formation induced by Ad-BMP-9 suggests that BMP gene therapy may have potential utility in the treatment of degenerative, rheumatic, or traumatic <em>bone</em> pathology.

We carried out a histological analysis on <em>bone</em> defects grafted with mineral trioxide aggregate (MTA) treated or not with laser, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP), and guided <em>bone</em> regeneration (GBR). Benefits of the use of MTA, laser, BMPs, and GBR on <em>bone</em> repair are well known, but there is no report on their association with laser light. Ninety rats were divided into <em>10</em> groups each subdivided into 3. Defects on G II and I were filled with the blood clot. G II was further irradiated with LED. G III and IV were filled with MTA; G IV was further irradiated with laser. G V and VI, the defects filled with MTA and covered with a membrane (GBR). G VI was further irradiated with laser. G VII and VIII, BMPs were added to the MTA and group VIII further irradiated with laser. G IX and X, the MTA + BMP graft was covered with a membrane (GBR). G X was further irradiated with laser. Laser light (λ = 850 nm, 150 mW, 4 J/cm(2) ) was applied over the defect at 48-h intervals and repeated for 15 days. Specimens were processed, cut and stained with H&E and Sirius red and underwent histological analysis. Subjects on group X were irradiated. The results showed different tissue response on all groups during the experimental time. Major changes were seen on irradiated subjects and included marked deposition of new <em>bone</em> in advanced maturation. It is concluded that near infrared laser phototherapy improved the results of the use of the MTA on <em>bone</em> defects.

A hospital-based case-control study was conducted to identify interactions between the 538(T->>C) polymorphic site of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 gene (BMP4T538C) and exposures in pregnancy with nonsyndromic cleft lip, with or without cleft palate (nsCL/P). Associations between offspring polymorphism of BMP4T538C, paternal smoking, paternal high-risk drinking, maternal passive smoking, and maternal multivitamin supplement with nsCL/P were analyzed by logistic regression analysis. BMP4T538C polymorphism, maternal passive smoking exposures and maternal multivitamin use were associated with the risk of nsCL/P but paternal smoking and paternal high-risk drinking were not. Gene-environment interactions were analyzed using the multifactor dimensionality reduction (MDR) method. The two-factor model including maternal passive smoking and BMP4T538C, was the best for predicting nsCL/P risk with a maximum cross-validation consistency (<em>10</em>/<em>10</em>) and a maximum average testing accuracy(0.605; P<0.0001). The findings suggested that: BMP4T538C could be used as a genetic susceptibility marker for nsCL/P; maternal passive smoking exposure is a risk factor for nsCL/P; maternal multivitamin supplements are a protective factor; the synergistic effect of BMP4T538C and maternal passive smoking could provide a new tool for identifying individuals at high risk of nsCL/P, and provides additional evidence that nsCL/P is determined by genetic and environmental factors.

Our strategy entails investigating the influence of varied concentrations (0, <em>10</em>, <em>10</em>0 and <em>10</em>00ng/ml) of human recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with <em>10</em>00ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for <em>bone</em> regeneration.

BACKGROUND

Bone morphogenetic proteins (BMPs) have shown considerable promise as a therapeutic agent to enhance periodontal regeneration although the optimal characteristics of a suitable release system are not known.

OBJECTIVE

The aim of this study was to compare the effects of slow and fast degrading gelatin carriers on BMP-2-induced periodontal healing.

METHODS

Recombinant human bone morphogenetic protein-2 (rhBMP-2) was incorporated into gelatin and subsequently differentially cross-linked to produce slow and fast release carrier systems. Release kinetics were confirmed in vitro, by measuring release of 125I-growth hormone from similar gelatin plugs. Effects of BMP were evaluated in surgically created rat periodontal fenestration defects which were processed for histology 10 days post-operatively. The rats were divided into 4 groups and the control defects were treated with either slow or fast degrading gelatin (CONs or CONf respectively), whilst test groups were treated with 1.25 microg rhBMP-2 in the slow or fast degrading gelatin (BMPs or BMPf respectively).

RESULTS

BMPf greatly increased bone formation compared with the control (CONf) (1.67 +/- 0.65 versus 0.34 +/- 0.11 x 10(-4) m2), but no significant differences were observed with BMPs and CONs. In contrast, new cementum formation was significantly greater in the BMPs group compared with all other groups (p<0.05).

CONCLUSIONS

Release kinetics of BMP may have important effects on the outcome of BMP-induced periodontal regeneration. New bone formation may be affected by rapid-release kinetics although further investigation is necessary to confirm this. In contrast, new cementum formation is promoted by slow release of BMP.

In therapeutic <em>bone</em> repairs, autologous <em>bone</em> grafts, conventional or vascularized allografts, and biocompatible artificial <em>bone</em> substitutes all have their shortcomings. The <em>bone</em> formed from peptides [recombinant human <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs)], demineralized <em>bone</em> powder, or a combination of both is small in size. Tissue engineering may be an alternative for cranial <em>bone</em> repair. In this study, the authors developed an animal model to test the hypothesis that replication-defective, adenovirus-mediated human BMP-2 gene transfer to <em>bone</em> marrow stromal cells enhances the autologous <em>bone</em> formation for repairing a critical-size craniofacial defect. The mesenchymal stromal cells of miniature swine were separated from the iliac crest aspirate and expanded in monolayer culture 1 month before implantation. The cultured mesenchymal stromal cells were infected with recombinant, replication-defective human adenovirus BMP-2, 7 days before implantation. Bilateral 2 x 5-cm2 cranial defects were created, leaving no osteogenic periosteum and dura behind. Mesenchymal stromal cells at 5 x <em>10</em>(7)/ml were mixed with collagen type I to form mesenchymal stromal cell/polymer constructs. Mesenchymal stromal cells used for the control site were infected with adenovirus beta-Gal under the same conditions. After 6 weeks and 3 months, <em>10</em> miniature swine were euthanized and the cranium repair was examined. Near-complete repair of the critical-size cranial defect by tissue-engineered mesenchymal stromal cell/collagen type I construct was observed. The new <em>bone</em> formation area (in square centimeters) measured by three-dimensional computed tomography demonstrated that the improvement from 6 weeks to 3 months was significantly greater on the experimental side than on the control side (2.15 cm2 versus 0.54 cm2, p < 0.001) and significantly greater at 3 months than at 6 weeks (2.13 cm2 versus 0.52 cm2, p < 0.001). The difference between the experimental and control groups was significant at 3 months (mean difference, 2.13 cm2; p < 0.001). The maximal compressive strength of the new <em>bone</em> was similar to that of the normal cranial <em>bone</em> when evaluated by biomechanical testing (cranium <em>bone</em> versus tissue-engineered <em>bone</em>, 88.646 +/- 5.121 MPa versus 80.536 +/- 19.302 MPa; p = 0.227). Adenovirus was absent from all constructs by immunochemical staining at 6 weeks and 3 months after implantation. The successful repair of cranial defects in this experiment demonstrates the efficacy of the integration of the autologous stem cell concept, gene medicine, and polymers in producing tissue-engineered <em>bone</em>.

BACKGROUND

Bone morphogenetic proteins (BMPs) may play significant roles in bone formation. The ability of BMP-6 to promote wound healing has been chosen as the subject of this investigation. In this study, a synthetic rat BMP-6 polypeptide was applied to a periodontal fenestration defect in rats to elucidate the effects of BMP-6 on periodontal wound healing.

METHODS

Following surgery to create a bony window on the buccal aspects of mandibular molar roots, 24 male Sprague Dawley rats were divided into four groups according to BMP application (0, 1, 3 and 10 microg, respectively). Animals were killed after 28 days and the mandible taken for histological examination. Histometric measurements were performed on sections selected from three levels (coronal, middle and apical levels; with 240 microm apart from the central) of the defect. New bone and cementum formation (including area and thickness) were analyzed and compared.

RESULTS

In general, minimal new bone was observed on the surgically created defects in the non-BMP group, whereas a complete osseous healing occurred in all BMP-6 treated animals. New bone formation (both in area and thickness) was significantly influenced by both the dosage and the examining level, whereas new cementum formation was affected by dosage only. An increase in bone and cementum formation was noted in all three BMP groups when compared with the control group at all examined levels. Among the BMP groups, greatest new bone and cementum formation were noted in the 3 microg group. New cementum thickness increased on the cementum surfaces of the defects compared with the dentinal surfaces in all study groups.

CONCLUSIONS

An increase in new bone and cementum formation was noted after applying a synthetic BMP-6 polypeptide to a periodontal fenestration defect in rats. Therefore, we suggest that BMP-6 may play a certain role in periodontal regeneration.