OBJECTIVE

Interleukin-<em>10</em> (IL-<em>10</em>) is a pleiotropic immunoregulatory cytokine with a chondroprotective effect that is elevated in cartilage and synovium in patients with osteoarthritis. However, the role of IL-<em>10</em> during endochondral <em>bone</em> formation and its mechanism of action have not been elucidated.

METHODS

IL-<em>10</em>(-/-) mice and IL-<em>10</em>-treated tibial organ cultures were used to study loss and gain of IL-<em>10</em> functions, respectively, during endochondral <em>bone</em> formation. Primary chondrocytes from the long <em>bone</em>s of mouse embryos were cultured with and without IL-<em>10</em>. To assess the role of IL-<em>10</em> in chondrogenic differentiation, we conducted mesenchymal cell micromass cultures.

RESULTS

The lengths of whole skeletons from IL-<em>10</em>(-/-) mice were similar to those of their wild-type littermates, although their skull diameters were smaller. The tibial growth plates of IL-<em>10</em>(-/-) mice showed shortening of the proliferating zone. Treatment with IL-<em>10</em> significantly increased tibial lengths in organ culture. IL-<em>10</em> also induced chondrocyte proliferation and hypertrophic differentiation in primary chondrocytes in vitro. Mechanistically, IL-<em>10</em> activated STAT-3 and the Smad1/5/8 and ERK-1/2 MAP kinase pathways and induced the expression of <em>bone</em> morphogenetic protein 2 (BMP-2) and BMP-6 in primary chondrocytes. Furthermore, the blocking of BMP signaling attenuated the IL-<em>10</em>-mediated induction of cyclin D1 and RUNX-2 in primary chondrocytes and suppressed Alcian blue and alkaline phosphatase staining in mesenchymal cell micromass cultures.

CONCLUSIONS

These results indicate that IL-<em>10</em> acts as a stimulator of chondrocyte proliferation and chondrogenic or hypertrophic differentiation via activation of the BMP signaling pathway.

BACKGROUND

The unique action of bone morphogenetic proteins (BMPs) on mineralised tissue formation indicates that BMPs are good candidates for use in stimulating periodontal regeneration. Relatively little is known about the mechanisms of actions of BMPs during periodontal regeneration, although recent evidence from our laboratory suggests that the effects of BMPs may be profoundly influenced by various factors including root surface conditioning, delivery systems and masticatory forces.

OBJECTIVE

The aim of this study was to investigate the effect of rhBMP-2 on cell recruitment during periodontal regeneration using a pulse-chase technique where cells are labelled with a thymidine analogue (BrdU) (pulse) and the migration of their progeny is followed (chase) during early wound healing. The relationship between the rhBMP-2 influence on cell recruitment from the periodontal ligament (PDL) and its ability to stimulate cementogenesis was also evaluated.

METHODS

The buccal aspect of the distal root of the first molar was denuded of its PDL, cementum and superficial dentine through a bony window created in the mandible of 64 Wistar rats under general anaesthesia. Test animals were treated with 10 microL of 500 microg/ml rhBMP-2 in a collagen membrane sponge (n=32) and control defects received 10 microl of saline in a collagen sponge (n=32). All animals received an intraperitoneal single pulse injection of 40 mg/kg BrdU label 2 days postoperatively. Groups of test and control animals (n=8) were killed 2 hours later on day 2 and at 4, 7 and 10 days postoperatively. Mandibles were processed for histological examination.

RESULTS

The results show that rhBMP-2 had a profound effect on proliferation and migration of cells in the adjacent and deeper aspects of the PDL at 7 and 10 days post periodontal wounding (p<0.05). Significantly greater new cementum formation occurred in the test group at 10 days (p=0.03).

CONCLUSIONS

This study shows that following periodontal wounding rhBMP-2 stimulates cell recruitment by increasing proliferation and migration of cells from the adjacent unwounded PDL into the wounded area, thus promoting periodontal regeneration by increasing new cementum formation.

OBJECTIVE

The aims of this study were to evaluate the rate of bone formation and osseointegration after topical gene delivery with a liposomal vector system carrying bone morphogenetic protein (BMP)-2 cDNA in combination with a collagen carrier and autologous bone as a carrier in freshly created peri-implant bone defects.

METHODS

Eight domestic pigs received nine calvariae defects each (10 x 7 mm). A dental implant was inserted into the centre of each defect. In the test groups, the remaining space was filled with the liposomal vector/BMP-2 complex combined with a collagen carrier (n=18) or an autologous bone graft (n=18). Control groups were collagen only (n=18) and autologous bone graft only (n=18).

RESULTS

There was a significant difference in mineralisation rate in the BMP-2/bone graft (29.9%+/- 4.8 and 68.3%+/- 7.2) and bone graft only (22.6%+/- 2.6 and 49.4%+/- 13.9) groups after 7 and 28 days. Mineralisation values were also significantly higher in the BMP-2/collagen group (21.2%+/- 16.2 and 53.1%+/- 12.5) compared with the collagen-only group (8.2%+/- 7 and 41%+/- 8.1) in two different regions after 28 days. Also the bone-to-implant contact was significantly increased in the BMP-2/bone graft group after 28 days and in the BMP-2/collagen group after 7 and 28 days compared with their control groups.

CONCLUSIONS

The results of this study show a significantly positive effect of liposomal vector/BMP-2 on bone regeneration and osseointegration in bony circumferential peri-implant defects.

The purpose of this study was to investigate in vivo biocompatibility and osteogenesis as well as degradability of the porous strontium-doped calcium polyphosphate (SCPP) scaffolds as a biomaterial for <em>bone</em> substitute applications. The evaluation was performed on a rabbit model over a period of 16 weeks by histology combined with image analysis, X-ray microradiography and immunohistochemistry methods. The histological and X-ray microradiographic results showed that the SCPP scaffold exhibited good biocompatibility and extensive osteoconductivity with host <em>bone</em>. Moreover, a significant more <em>bone</em> formation was observed in the SCPP group compared with that in the CPP group, especially at the initial stage after implantation. New <em>bone</em> volumes (NBVs) of the SCPP group determined at week 4, 8 and 16 were 14, 27 and 45%, respectively. Accordingly, NBVs of the CPP group were <em>10</em>, 19 and 40%. Immunohistochemical results revealed that both the expression of collagen type I and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> in the SCPP group were higher than that in the CPP group, which might be associated with the release of strontium ions during the implantation. In addition, during 16 weeks implantation the SCPP scaffold exhibited similar degradability with the CPP scaffold in vivo. Both scaffolds showed the greatest degradation rate for the first 4 weeks, and then the degradation rate gradually decreased. The results presented in this study demonstrated that SCPP scaffold can be considered as a biocompatible material, making it attractive for <em>bone</em> substitute application purposes.

Salivary pleomorphic adenomas are often associated with chondroid tissue formation. We investigated the relationship between chondroid tissue formation and the expression of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), which are strong inducers of ectopic <em>bone</em> and cartilage formation. Fifteen pleomorphic adenomas and seven normal salivary glands were examined genetically and immunohistochemically. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that BMP-1, BMP-2, BMP-3, BMP-4, and BMP-7 mRNAs were overexpressed in <em>10</em> (66.7%), 9 (60.0%), 1 (6.7%), 8 (53.3%), and 12 (80.0%), respectively, of the 15 pleomorphic adenomas. Overexpression of BMP-2 mRNA was observed in pleomorphic adenomas. Marked chondroid formation or expression of type II collagen was frequently observed in pleomorphic adenomas that overexpressed BMP-2 mRNA. Immunohistochemically, BMP-2 was detected in modified myoepithelial cells aroud chondroid tissue and basement membranes. These results suggest that BMPs, and expecially BMP-2, have a role in chondroid formation in pleomorphic adenomas.

BACKGROUND

Synovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system.

METHODS

The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II, aggrecan, SOX9, link-protein, collagen type X and BMP receptor II.

RESULTS

Cells isolated under the optimized culturing density (10(4)/60 cm(2)) showed clonogenicity and multi-differentiation potential. These cells were positive >> 99%) for CD44, CD90, CD105 and negative (< 10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type II was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type II and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone.

CONCLUSIONS

SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

The object of this research is to investigate the influence of interleukin-1 (IL-1) on heterotopic ossification (HO) induced by <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP). Adult mice were implanted with doses of 1, 2, 5, and <em>10</em> mg of BMP. Several local injections of <em>10</em>, <em>10</em>0, and <em>10</em>00 units of a recombinant IL-1 beta (rIL-1 beta) were administered during the <em>morphogenetic</em> phase of development, starting a day before operation until one week postoperation. While IL-1 acts principally on cell proliferation, BMP primarily shows cell differentiation in the form of HO. BMP-induced HO is quantitated by computer X-ray image scanning, <em>bone</em> ash weight, alkaline phosphatase activity, and histological methods. The area of human BMP-induced HO was completely abolished by injections of polyclonal and monoclonal anti-IL-1 antibody. Monoclonal antibody did not cross-react with the same efficiency as polyclonal with bovine BMP. Polyclonal anti-IL-1 antibody totally neutralizes bovine BMP activity. IL-1-enhanced BMP induced HO and increased the volume of new <em>bone</em> in a statistically significant increment.

This study examined the effect of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 on several parameters of growth, differentiation, and matrix synthesis and on the endogenous production of mRNA of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 2 and 4 by growth plate chondrocytes in culture. Chondrocytes from resting and growth zones were obtained from rat costochondral cartilage and cultured for 24 or 48 hours in medium containing 0.05-<em>10</em>0 ng/ml recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and <em>10</em>% fetal bovine serum. Incorporation of [3H]thymidine, cell number, alkaline phosphatase specific activity, incorporation of [3H]proline into collagenase-digestible <em>protein</em> and noncollagenase-digestible <em>protein</em>, and incorporation of [35S]sulfate were assayed as indicators of cell proliferation, differentiation, and extracellular matrix synthesis. mRNA levels for <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 2 and 4 were determined by Northern blot analysis. Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 increased the incorporation of [3H]thymidine by quiescent resting-zone and growth-zone cells in a similar manner, whereas it had a differential effect on nonquiescent cultures. At 24 and 48 hours, 12.5-<em>10</em>0 ng/ml recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 caused a dose-dependent increase in cell number and DNA synthesis in resting-zone chondrocytes. No effect was seen in growth-zone cells. Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 stimulated alkaline phosphatase specific activity in resting-zone chondrocytes in a bimodal manner, causing significant increases between 0.2 and 0.8 ng/ml and again between 25 and <em>10</em>0 ng/ml. In contrast, alkaline phosphatase specific activity in growth-zone chondrocytes was significantly increased only between 12.5 and <em>10</em>0 ng/ml. Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 increased the production of both collagenase-digestible <em>protein</em> and noncollagenase-digestible <em>protein</em> by resting-zone and growth-zone cells, but incorporation of [35S]sulfate was unaffected. Administration of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 also increased incorporation of [3H]uridine in both resting-zone and growth-zone chondrocytes; these cells produced mRNA for <em>bone</em> <em>morphogenetic</em> <em>proteins</em> 2 and 4. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-2 mRNA levels in both resting-zone and growth-zone chondrocytes increased in the presence of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2; however, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4 mRNA levels in growth-zone cells decreased under its influence, and those in resting-zone cells were upregulated only with a dose of <em>10</em> ng/ml. This indicates that recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 regulates chondrocyte proliferation, differentiation, and matrix production, and the effects are dependent on the stage of cell maturation. Resting-zone chondrocytes were more sensitive, suggesting that they are targeted by <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and that this growth factor may have autocrine effects on these cells.

A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase astacin, the prototype of the astacin family and of the metzincin superfamily of metallo<em>protein</em>ases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-P ro-Leu-Val, binds to astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1.s-1) and dissociation (koff = 4.1 x <em>10</em>(-6) s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-Pro-O-methyl, Ki = 14 microM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stöcker (1996) Nature Struct. Biol. 3, 671-675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique 'tyrosine switch' in the structure of astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like <em>bone</em> <em>morphogenetic</em> <em>protein</em> 1 (BMP1; i.e. the procollagen C-<em>protein</em>ase), have become targets of pharmacological research.

Resorbable collagen membranes for guided tissue regeneration in periodontal therapy have shown promise but are not osteoinductive. As recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) is known to have an affinity for collagen, the use of this osteoinductive agent incorporated into a collagen vehicle may act as a suitable carrier to promote periodontal regeneration. The aim of this study was to investigate the effects of two different collagen delivery systems for rhBMP-2 in rat periodontal fenestration defects. Using the collagen membrane delivery system, 3 groups of adult Wistar rats which had surgical defects created on the right side of the mandible involving the removal of <em>bone</em> and exposure of the molar roots were treated with either rhBMP-2 in colagen membrane (BMPm) (n = 12 animals), or collagen membrane only (COLm) (n = 12), or were left untreated (UN) (n = 14). Using the collagen gel delivery system, surgical defects were treated with either rhBMP-2 incorporated in a collagen gel carrier (BMPg) (n = 5) or had collagen gel only (COLg) (n = 6). Animals were killed <em>10</em> d postoperatively and tissues processed for histology. New <em>bone</em> formation was significantly greater in BMPg compared with both BMPm and controls (p < 0.05). However, new cementum formation was significantly greater in BMPm (721 +/- 166 micron2, mean +/- SE) compared with COLm, COLg and UN (p < 0.02) (190 +/- 44 micron2, 327 +/- 114 micron2 and 172 +/- 33 micron2, respectively) and more than 1.5 times BMPg (451 +/- 158 micron2). In conclusion, both carrier systems for rhBMP-2 significantly increased new <em>bone</em> formation compared with controls during the early stages of periodontal wound healing. However, the more slowly dissolving collagen membrane carrier system for rhBMP-2 produced significantly greater new cementum compared with the collagen gel carrier, suggesting that a more prolonged exposure of rhBMP-2 is required to increased cementogenesis.

Many calcium oxalate (CaOx) kidney stones develop attached to renal papillary sub-epithelial deposits of calcium phosphate (CaP), called Randall's plaque (RP). Pathogenesis of the plaques is not fully understood. We hypothesize that abnormal urinary environment in stone forming kidneys leads to epithelial cells losing their identity and becoming osteogenic. To test our hypothesis male rats were made hyperoxaluric by administration of hydroxy-l-proline (HLP). After 28days, rat kidneys were extracted. We performed genome wide analyses of differentially expressed genes and determined changes consistent with dedifferentiation of epithelial cells into osteogenic phenotype. Selected molecules were further analyzed using quantitative-PCR and immunohistochemistry. Genes for runt related transcription factors (RUNX1 and 2), zinc finger <em>protein</em> Osterix, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP2 and 7), <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor (BMPR2), collagen, osteocalcin, osteonectin, osteopontin (OPN), matrix-gla-<em>protein</em> (MGP), osteoprotegrin (OPG), cadherins, fibronectin (FN) and vimentin (VIM) were upregulated while those for alkaline phosphatase (ALP) and cytokeratins <em>10</em> and 18 were downregulated. In conclusion, epithelial cells of hyperoxaluric kidneys acquire a number of osteoblastic features but without CaP deposition, perhaps a result of downregulation of ALP and upregulation of OPN and MGP. Plaque formation may additionally require localized increases in calcium and phosphate and decrease in mineralization inhibitory potential.

Repair and regeneration of periodontal tissues by tissue engineering is dependent on the use of biodegradable polymer scaffolds which serve as a carrier for cells or bioactive substances. There is a need to understand how a specific biomaterial may influence gene expression. The aim of this investigation was to develop and to optimize an in vitro technique for the adherance and proliferation of primary human gingivaL cells on implantable and biodegradable matrices. Square pieces of Bio-Gide matrix (BG) and slices of Ethisorb tamponade (ET) were coated with poly-L-lactide. The stability of coated and uncoated scaffolds was investigated by incubation in standard culture medium. Various concentrations of the cells were seeded onto coated and uncoated polymer matrices in tissue culture dishes without shaking ("static seeding") or continuous shaking ("agitated seeding"). Cultures were grown for 4 week and were then evaluated by light and scanning electron microscopy. After a culture period of <em>10</em> d, BG-carriers showed a delicate consistency which made histological processing difficult. Cells were grown only sparsely in coated and non-coated BG-scaffolds. Contrary. ET-specimens were stable during a 4 week culture period. After "static seeding" a significantly higher number of cells resulted in comparison to those in "agitated" cultures. The cells were evenly distributed throughout the ET-carriers and produced extracellular matrix compounds as well. Furthermore, the examination with RT-PCR (reverse transcription-polymerase chain reaction) revealed that the cells synthesized and secreted type I collagen, and expressed genes implicated in transducing <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signals. Messenger RNAs for BMP-2, -4, -7, the BMP type I receptors Act R-1 (alk 2, activin-like kinase receptor), BMPR-IA (alk 3), -IB (alk 6), and the type II receptor BMPR-II were detected. These data reveal that static seeding favors the adherence and proliferation of primary gingival cells on polyglactin matrices. This system may serve as a valuable tool for periodontal tissue engineering.

The present study was undertaken to determine whether ex vivo <em>bone</em> <em>morphogenetic</em> <em>protein</em>-9 (BMP-9) gene therapy using human mesenchymal stem cells (hMSCs) can induce endochondral <em>bone</em> formation in athymic nude rats. An in vitro study was initially performed on hMSCs to evaluate morphological changes and osteoblastic differentiation induced by replication-defective adenovirus type 5 with the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or beta-galactosidase (Ad-beta-gal) gene. In vivo, athymic nude rats received an injection (<em>10</em>(6) hMSCs transduced with recombinant adenovirus at 50 PFU/cell) into the anterior thigh musculature: Ad-BMP-9 on the left and Ad-beta-gal (control) on the right. Computed tomography scans and histological analysis were obtained 7, 14, 28, 42, 56, and 84 days postinjection. In vitro, human mesenchymal stem cells treated with Ad-BMP-9 (50 PFU/cell) showed signs of differentiation, whereas hMSCs treated with 250 and 1250 PFU/cell showed cytotoxicity. In vivo, computed tomography and histological analysis clearly demonstrated ectopic <em>bone</em> at hMSC/Ad-BMP-9 treatment sites, whereas the hMSC/Ad-beta-gal treatment sites showed no evidence of osteogenesis. None of the animals showed clinical evidence of toxicity. Ex vivo gene therapy with hMSC/BMP-9 may be efficacious for promoting <em>bone</em> formation for a variety of <em>bone</em> pathologies and certainly warrants further investigations.

Transcription of the hepcidin (Hamp) gene is controlled by iron stores and the rate of erythropoiesis. Functional hierarchy between these two stimuli has not yet been completely established. It is also not known whether the erythropoiesis-related downregulation of Hamp expression utilises the <em>bone</em> <em>morphogenetic</em> <em>protein</em>/hemojuvelin (Bmp/Hjv) pathway. Hemojuvelin-mutant (Hjv-/-) mice treated with erythropoietin (EPO) at 50IU/mouse/day for three days displayed marked decrease in Hamp mRNA, demonstrating that hemojuvelin is not an indispensable component in EPO-induced Hamp gene downregulation. Irradiation of Hjv-/- mice prevented the EPO-induced decrease of Hamp mRNA, highlighting the role of erythropoiesis in Hamp gene regulation by EPO. After a single injection of EPO, Hamp mRNA levels were not significantly changed at 6h, but decreased at <em>10</em> and 24h. Chronic bleeding decreased hepatic Bmp6 mRNA levels; however, repeated EPO treatment did not change Bmp6 mRNA, suggesting that the erythropoietic regulator(s) act independently of the Bmp/Hjv pathway. Pretreatment of C57BL/6 mice with iron (5mg/mouse) almost completely inhibited the EPO-induced decrease of Hamp mRNA. This result suggests that administration of EPO to patients with transfusional iron overload is probably not associated with the risk of additional absorption of substantial amounts of iron from the diet.

Neurofibromatosis type 1 (NF1) is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue-specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1(flox/flox) and Nf1(flox/-) mice. The effects of the presence of Nf1(null) cells were extensively examined. Cultured Nf1(null)-committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) treatment. Similarly, Nf1(null)-inducible osteoprogenitors obtained from Nf1 MyoDnull mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1(flox/flox) and Nf1(flox/-) mice, local AdCre injection significantly impaired <em>bone</em> healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1(flox/flox) and Nf1(flox/-) mice. Histological analyses showed invasion of the Nf1(null) fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of <em>10</em>-fold in Nf1(null) fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1(null) fractures, tartrate-resistant acid phosphatase-positive (TRAP+) cells were frequently observed within the fibrous tissue, not lining a <em>bone</em> surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.

One human body is composed of 6 x <em>10</em>(13) cells, and eyes are also composed of many cells of different functions. The cellular functions and intercellular interaction are regulated by many regulators including cytokines and growth factors to maintain the homeostasis. The transforming growth factor-beta (TGF-beta) superfamily, a large family of multifunctional factors, regulates various cellular functions, including cellular proliferation, migration, differentiation, apoptosis and extracellular matrix production. The TGF-beta superfamily contains about 30 multifunctional factors, and is divided into several families according to the sequence homology. The TGF-beta family, the activin family, and <em>bone</em> morphogenic <em>proteins</em> belong to the TGF-beta superfamily. TGF-beta superfamily members transduce signals through type I and type II serine/threonine type transmembrane receptors. The signals are transduced from receptors through nuclei by Smad family members, which are phosphorylated by the activated type I receptors and translocate from cytoplasm into nuclei. TGF-beta family members and the TGF-beta superfamily receptor family are expressed in ocular tissues including the cornea, ciliary epithelium, lens epithelium, retina, and blood vessels. This observation suggests the importance of the TGF-beta superfamily in eyes. Smad family members (Smad 1, Smad 2, Smad 3 and Smad 4) are expressed in the cultured retinal pigmant epithelial cell line (D407), in which TGF-beta and activin A stimulate the translocation of Smad 2, but not Smad 1 into nuclei, whereas <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) stimulates that of Smad 1, but not Smad 2. TGF-beta superfamily members play important roles in the pathogenesis of retinal neovascularization and in the wound healing process of corneal tissue. TGF-beta inhibits the endothelial functions, but, stimulates angiogenesis in vivo. TGF-beta is involved in the formation of abnormal connective tissue in corneal wound healing. In these processes, many cytokines and growth factors are involved, interacting with each other and forming networks. It is mandatory to clarify the networks to investigate molecular pathogenesis and new therapeutic agents.

Neural stem cells (NSCs) reside in the anterior portion of the forebrain subventricular zone (SVZa) and generate the progenitors which will differentiate into neurons, and via a tangential migratory pathway, known as the rostral migratory stream (RMS), migrate to the olfactory bulbs (OB). <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) play significant roles in neural development at different stages and locations, but their roles have not been determined in the SVZa. To explore possible roles of BMPs in SVZa NSCs, BMP4 at various concentrations were tested for their capacity to induce SVZa NSCs. The expression of BMP4 was also examined in living cells using a reportor vector, in which the BMP4 promotor was conjugated with red fluorescent <em>protein</em> (RFP). In the meantime, the differentiation of SVZa NSCs was dynamically monitored by using reportor vectors of the Nestin enhancer and the promoters of TH and GFAP. In the OB, high expression of BMP4 was found using both promoter activity analysis and in situ hybridization. However, low BMP4 expression was found in the RMS and only moderate expression of BMP4 was displayed in the SVZa. The results also demonstrated that low concentrations (1-5 ng/ml) of BMP4 promoted the proliferation of SVZa NSCs but high concentrations (<em>10</em>-<em>10</em>0 ng/ml) of BMP4 inhibited this proliferation. BMP4 enhanced neuron commitment before 4 days but inhibited it after 4 days. As the antagonist of BMP4, Noggin almost completely blocked all these BMP4 responses. Thus, our findings indicate that BMP4 promotes the exit from the cell cycle and triggers the differentiation of neuron progenitors in the OB. BMP4 also promotes the proliferation of the committed neuron progenitors in the RMS, but in the SVZa, BMP4 may facilitate the commitment of NSCs into astrocytes.

Natural and synthetic estrogens, Including diethylstilbestrol (DES), given during the critical period of newborn life Induce abnormalities in ovaries of mice. Induction of polyovular follicles (PFs) containing two or more oocytes in a follicle is one example. In this study, the involvement of estrogen receptor subtypes ERalpha and ERbeta in induction of PFs by neonatal treatment with DES was analyzed by using ERalpha knockout (alphaERKO) and ERbeta knockout (betaERKO) mice. Ovaries of mice injected with 3 microg DES for 5 days from the day of birth were examined histologically from <em>10</em> to 60 days of age, and the expression of genes involved in folliculogenesis was analyzed by real-time quantitative PCR. The PF Incidence (percent of PFs per <em>10</em>0 follicles greater than 50 microm in diameter) in the ovary of alphaERKO mice treated with DES was not different from that in the DES-treated wild-type mice. However, neonatal DES treatment did not increase the PF incidence in betaERKO mice, suggesting that PFs were induced by DES through ERbeta but not ERalpha. The expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15, growth differentiation factor 9, inhibin-alpha, Müllerian inhibiting substance, and other genes in the ovaries of DES-treated betaERKO mice was not different from that in the ovaries of DES-treated wild-type mice. These results indicate that ERbeta but not ERalpha is essential for DES to Induce PFs in mice.

BACKGROUND

Bone morphogenetic protein-2 (BMP-2) and Wnt are involved in the normal development and tumorigenesis of several organs, and in the development of skin and skin appendages as a morphogen. However, the crosstalk between BMP-2 and the Wnt/beta-catenin signaling pathway is not clear.

OBJECTIVE

We examined BMP-2-dependent expression of Wnt and its receptor frizzled in normal human keratinocytes.

METHODS

The mRNA expression of the Wnt and frizzled families was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or ribonuclease protection assay. beta-Catenin expression was measured using RT-PCR and Western blotting. T-cell factor/lymphoid enhancing factor activity was analyzed using the luciferase reporter assay.

RESULTS

We detected the expression of Wnt-2b/13, -4, -5a, -5b, -7a, -7b, and -10a, frizzled-1, -4, -5, -6, -8, -9, and -10, MFRP, and SFRP-1/SARP-2 in keratinocytes. BMP-2 increased Wnt-2b/13, -5b, and -7b, and frizzled-6, -8, and -10. Conversely, BMP-2 suppressed Wnt-10a and SFRP-1/SARP-2. Although Wnt-4 expression was not affected by BMP-2 in confluent conditioned keratinocytes, BMP-2 suppressed cell density-dependent Wnt-4 induction. The transcriptional activity of TCF/LEF, which is a target of the canonical Wnt pathway, was upregulated by BMP-2 in both time- and dose-dependent manners. However, BMP-2-dependent differentiation of keratinocytes suppressed TCF/LEF transcriptional activity.

CONCLUSIONS

These results suggest that BMP-2 modulates the expression of molecules involved in Wnt signaling, and activates the canonical Wnt pathway in normal human keratinocytes. Moreover, Wnt signaling may be influenced by the fate of keratinocytes, such as proliferation, migration, and differentiation.

OBJECTIVE

This study investigated the role of the bone marrow-derived CD34+ cell in a milieu of osteoprogenitor cells, bone marrow plasma cell adhesion molecules, recombinant human bone morphogenetic protein (rhBMP), and a matrix of crushed cancellous allogeneic bone in the clinical regeneration of functionally useful bone in craniomandibular reconstructions. The history and current concepts of bone marrow hematopoietic stem cells and mesenchymal stem cells are reviewed as they relate to bone regeneration in large continuity defects of the mandible.

METHODS

Patients with 6- to 8-cm continuity defects of the mandible with retained proximal and distal segments were randomized into two groups. Group A received an in situ tissue-engineered graft containing 54 ± 38 CD34+ cells/mL along with 54 ± 38 CD44+, CD90+, and CD105+ cells/mL together with rhBMP-2 in an absorbable collagen sponge (1 mg/cm of defect) and crushed cancellous allogeneic bone. Group B received the same graft, except the CD34+ cell concentration was 1,012 ± 752 cells/mL. The results were analyzed clinically, radiographic bone density was measured in Hounsfield units (HU), and specimens were analyzed histomorphometrically.

RESULTS

Forty patients participated (22 men and 12 women; mean age, 57 years). Eight of 20 group A patients (40%) achieved the primary endpoint of mature bone regeneration, whereas all 20 group B patients (100%) achieved the primary endpoint. CD34+ cell counts above 200/mL were associated with achievement of the primary endpoint. Bone density was lower in group A (424 ± 115 HU) than in group B (731 ± 98 HU). Group A bone showed a mean trabecular bone area of 36% ± 10%, versus 67% ± 13% for group B.

CONCLUSIONS

The CD34+ cell functions as a central signaling cell to mesenchymal stem cells and osteoprogenitor cells in bone regeneration. The mechanism of bone marrow-supported grafts requires a complete milieu to regenerate large quantities of functionally useful bone. CD34+ cell counts in a concentration of at least 200/mL in composite grafts are directly correlated to clinically successful bone regeneration.

A prospective animal study of posterolateral lumbar spine arthrodesis was performed to determine the temporal and spatial pattern of gene expression and to determine the effect of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 on the gene expression pattern of a healing spine fusion mass. In Group 1, 20 adult New Zealand rabbits underwent L4-L5 posterolateral intertransverse process arthrodesis using autograft alone. Two rabbits were euthanized at each of the following points: 0, 2, and 4 days, and 1, 2, 3, 4, 5, 6, and <em>10</em> weeks after surgery. The same surgical technique was used for 16 rabbits in Group II, except that the autograft first was soaked in a solution of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 before implantation. Ribonucleic acid was extracted from different regions of the fusion mass at each point and analyzed for expression of <em>bone</em> and cartilage related genes using reverse transcription polymerase chain reaction. A reproducible temporal sequence and spatial pattern of gene expression was found in healing spine fusions. In the central portion of the fusion mass a temporal lag in gene expression was observed that parallels the lag in healing within the central zone previously observed in histologic studies. Treatment of <em>bone</em> graft with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 resulted in an increase in the early expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6 which was associated with expression of higher levels of Type I collagen, osteocalcin, and other <em>bone</em> related genes. These findings suggest that central nonunion may be associated with delayed expression of osteoblast related genes in the central region of the forming fusion mass. The growth factor, recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2, increased the level of <em>bone</em> related gene expression throughout the fusion mass, eliminated the delay in healing within the central zone, and may decrease the likelihood of a nonunion.

Tetracyclines, including tetracycline (TC), minocycline (MINO), and doxycycline (DOXY), were widely used as topical therapy in treating periodontitis, which is an infectious disease of the gingival crevice caused by periodontopathic bacteria. In addition, TC is used during <em>bone</em> grafting procedures because of its anticollagenase activity, antibacterial effect, and fibroblast-stimulatory properties.In this study, the effects of TC on osteoprecursor cells were evaluated. The cytotoxic effect was determined by testing cell viability. Differentiation and mineralization were evaluated using an alkaline phosphatase activity test and alizarin red S staining. In addition, <em>protein</em> expressions related to <em>bone</em> formation, such as <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2), estrogen receptor α (ER-α), and estrogen receptor β (ER-β), were evaluated by using Western blot analysis.The morphology of the cells seemed normal, and their viability was not significantly affected when they were treated with <em>10</em> μM MINO and <em>10</em> μM DOXY. Cells treated with <em>10</em> μM DOXY showed alkaline phosphatase activity that was comparable to the control. Results from the Western blot analysis showed that TC, MINO, and DOXY reduced the expression of BMP-2 and ER-β. Normalization of the <em>protein</em> expressions showed that <em>10</em> μM DOXY retained 87% in BMP-2 and 85% in ER-β.Higher levels of these agents led to a dose-dependent decrease of cellular differentiation and <em>protein</em> expression. There are several commercially available agents for TC, which has to be considered when applying the TC in local delivery applications.

Scaffold design incorporating multiscale cues for clinically relevant, aligned tissue regeneration has potential to improve structural and functional integrity of multitissue interfaces. The objective of this preclinical study is to develop poly(ε-caprolactone) (PCL) scaffolds with mesoscale and microscale architectural cues specific to human ligament progenitor cells and assess their ability to form aligned <em>bone</em>-ligament-cementum complexes in vivo. PCL scaffolds are designed to integrate a 3D printed <em>bone</em> region with a micropatterned PCL thin film consisting of grooved pillars. The patterned film region is seeded with human ligament cells, fibroblasts transduced with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 genes seeded within the <em>bone</em> region, and a tooth dentin segment positioned on the ligament region prior to subcutaneous implantation into a murine model. Results indicate increased tissue alignment in vivo using micropatterned PCL films, compared to random-porous PCL. At week 6, 30 μm groove depth significantly enhances oriented collagen fiber thickness, overall cell alignment, and nuclear elongation relative to <em>10</em> μm groove depth. This study demonstrates for the first time that scaffolds with combined hierarchical mesoscale and microscale features can align cells in vivo for oral tissue repair with potential for improving the regenerative response of other <em>bone</em>-ligament complexes.

Over the past decades, the focus on the regenerative properties of platelets (PLTs) has intensified and many PLT-derived growth factors are readily used in medical settings. A general lack of standardization in the preparation of these growth factors remains, however, and this study therefore examines the dynamics of growth factors throughout the freeze-thaw procedure.

Plateletpheresis (PA) and PLT-poor plasma (PPP) samples were collected from <em>10</em> healthy donors. PA was lysed to produce PLT lysate (PL) for 1, 3, 5, <em>10</em>, and 30 freeze-thaw cycles. The resulting growth factor and cytokine concentrations from PPP, PA, and PL of different cycles were analyzed and compared using enzyme-linked immunosorbent assay and multiplex bead assays.

PL produced by the freeze-thaw procedure resulted in approximately four- to <em>10</em>-fold enrichment of transforming growth factor-β1, epidermal growth factor, PLT-derived growth factor (PDGF)-AB/BB, PLT factor-4, and fibroblast growth factor-2. The increase in concentrations plateaued at Cycles 3 and 5 and in some cases declined with further cycles. The concentrations of insulin-like growth factor-1, hepatocyte growth factor, vascular endothelial growth factor, and bone morphogenetic protein-2 in PL were essentially comparable to those in PPP.

Using the freeze-thaw method, optimal preparation of PL with regard to the concentration of growth factors was achieved at Cycles 3 to 5. Based on our findings, the clinical significance of using a greater number of cycles is likely limited.