DEVELOPMENT OF AN EXPERIMENTAL MODEL OF INFECTED BONE VOID IN THE ULNA OF RABBITS

Objective: Develop a model that allowed the study of bone regeneration in infection conditions. Method: A 15 mm defect was surgically created in the rabbit ulna and inoculated with 5x10(8) colony-forming units (CFU) of S. aureus. Surgical debridement was performed two weeks after and systemic gentamicin was administered for four weeks. Animals were followed up to 12 weeks to evaluate infection control and bone regeneration. Result: Bone regeneration was inferior to 25% of the defect in radiological and histological analysis. Conclusion: Infected bone defect of 15 mm in the rabbit ulna was unable to achieve full regeneration without further treatment. Level of Evidence V, Experimental Study.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.

Newly Formed Bone in Mandible Decortication Experimental Model Using rhBMP-2 Evaluated by Densitometric Study

The purpose of this study was to evaluate the newly formed bone, comparing two different carriers for rhBMP-2, using the decortication and nondecorticatication surgical technique in Wistar rat mandibles, evaluated by radiographic densitometry method. It was used fifty six animals according to specific treatment, which were sacrificed after 3 and 6 weeks after this. It was concluded that the decortication surgical technique was able to optimize the osteoinduction properties of the rhBMP-2, independently of the material carrier used and the period of time, according to radiographic densitometry technique.

Morphometric evaluation of the repair of critical-size defects using demineralized bovine bone and autogenous bone grafts in rat calvaria

Objective: To evaluate the repair of critical-size bone defects in rats treated with demineralized bovine bone (DBB) compared with autogenous bone (AB). Material and method: A bone defect of 8 mm in diameter was created in the calvaria of 50 Rattus norvegicus, treated either with DBB or AB. Sub-groups of five rats of each group were killed at 7, 14, 21, 30 and 90 days post-operatively, and the skulls were removed and processed histologically. Histological sections were stained with hematoxylin and eosin. Result: Histological analysis showed complete closure of the defects with new bone at 90 days in group AB, and substitution of the biomaterial by fibrotic connective tissue in the DBB group at 21 days. Morphometric analysis showed that DBB was rapidly absorbed at 14 days, with its volume density decreasing from 47%+/- 0.8% at 7 days to 1.2%+/- 0.41% at 14 days. Subsequently, volume densities of the connective tissue and neoformed bone increased from 51.1%+/- 11.17% to 86.8%+/- 7.92% and from 1.9%+/- 1.13% to 12%+/- 8.02%, respectively, for the same time interval. The volume density of AB particles did not change throughout the experimental periods, but the amount of new bone increased markedly between 7 and 90 days, from 4.5%+/- 1.57% to 53.5%+/- 6.42% (P < 0.05). Conclusion: DBB did not provide complete repair of the defects, with significantly less new bone formation than in the AB group.

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep

Background Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures Purpose The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-D, L-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental Implants Materials and methods Nine adult female sheep were submitted to bilateral sinus-floor elevation In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest Three different implants (Branemark (R), 31 (R) and Straumann (R)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated Results BD and BIC were significantly higher at 12 weeks in the test group if the Implants were placed at the time of the sinus lift (p < 0 05) No difference was observed between the different implant systems or positions Conclusions The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone Different implant systems and positions of the implants had no effect on BIC or BD (C) 2010 European Association for Cranio-Maxillo-Facial Surgery

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Acellular dermal matrix allograft used alone and in combination with enamel matrix protein in gingival recession: Histologic study in dogs

Gingival recession was created in six mongrel dogs. The dogs were divided into two groups based on treatment: group 1-AlloDerm only, group 2-AlloDerm + Emdogain. The histologic results were compared. At the end of the study, the mean values were, for groups I and 2, respectively: 0.06 and 0.32 mm for cementum regeneration; -0.75 and -0.86 mm for bone regeneration; -2.15 and -3.11 mm for attachment level; and 4.90 and 5.51 mm for defect extent. The epithelial formation parameter was 2.88 mm in group 1 and 2.15 mm in group 2, which was a statistically significant difference. It could be concluded that Emdogain did not result in beneficial effects when associated with AlloDerm.

Tibial segmental bone defect treated with bone plate and cage filled with either xenogeneic composite or autologous cortical bone graft

Tibia segmental defect healing in sheep were clinically, radiographically and histologically evaluated. Twelve young sheep aged four to five months were divided into two groups, G1 and G2. A 3.5 cm long segmental defect was created in the right tibial diaphysis with maintenance of the periosteum. The bone defects in both groups were stabilized with a bone plate combined with a titanium cage. In G1 the cage was filled with pieces of autologous cortical bone graft. In G2 it was filled with a composite biomaterial which consisted of inorganic bovine bone, demineralized bovine bone, a pool of bovine bone morphogenetic proteins bound to absorbable ultra-thin powdered hydroxyapatiteand bone-derived denaturized collagen. Except for one G1 animal, all of them showed normal limb function 60 days after surgery. Radiographic examination showed initial formation of periosteal callus in both groups at osteo-tomy sites, over the plate or cage 15 days postoperatively. At 60 and 90 days callus remodeling occurred. Histological and morphometric analysis at 90 days after surgery showed that the quantity of implanted materials in G1 and G2 were similar, and the quantity of new bone formation was less (p = 0.0048) and more immature in G1 than G2, occupying 51 +/- 3.46% and 62 +/- 6.26% of the cage space, respectively. These results suggest that the composite biomaterial tested was a good alternative to autologous cartical bone graft in this experimental ovine tibial defect. However, additional evaluation is warranted prior to its clinical usage.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

doi: 10.1111/j.1741-2358.2011.00526.x Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model Objective: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Materials and methods: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 mu g of pure rhBMP-2, (2) 5 mu g of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 mu g of pure P-1, (5) 5 mu g of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. Result and conclusion: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm2). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 mu g of pure rhBMP-2; (b) 5 mu g of pure P-1 fraction; (c) 5 mu g of rhBMP-2/monoolein gel; (d) 5 mu g of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 mu g of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 mu g of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. (c) 2011 Wiley Periodicals, Inc.

Loss of inhibition over master pathways of bone mass regulation results in osteosclerotic bone metastases in prostate cancer

Prostate cancer is the most common cancer among men in industrialised countries. Most patients with prostate cancer, however, will not die of it. As a result, many of them will experience symptomatic metastasis during the course of the disease. Prostate cancer has a high propensity to metastasize to bone. Unlike many other cancers prostate cancer cells induce a rather osteosclerotic than osteolytic reaction in the bone marrow by interfering with physiological bone remodelling. A proper understanding of the mechanisms of tumour cell-induced bone alterations and exaggerated bone deposition in prostate cancer may open new and urgently needed therapeutic approaches in the field of palliative care for affected patients. In this review we focus on the central role of two major regulators of bone mass, the wingless type integration site family members (WNTs) and the bone morphogenetic proteins (BMPs), in the development of osteosclerotic bone metastases.

Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements The authors would like to thank Dr Marius Sudol for the hYAP plasmids (obtained through Addgene), Dr Pete Zammit for the pMSCV-IRES-eGFP plasmid, Dr Robert Judson for subcloning the hYAP cDNAs into the pMSCV-IRES-eGFP plasmid, Dr Lynda Erskine for the provision of mouse embryo samples, and Professor Jimmy Hutchison and the Orthopaedics Department at the Aberdeen Royal Infirmary for the provision of human tissue samples. The authors are also grateful to Denise Tosh and Susan Clark for excellent technical support. This work was funded by Arthritis Research UK (grant 19429).

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements The authors would like to thank Dr Marius Sudol for the hYAP plasmids (obtained through Addgene), Dr Pete Zammit for the pMSCV-IRES-eGFP plasmid, Dr Robert Judson for subcloning the hYAP cDNAs into the pMSCV-IRES-eGFP plasmid, Dr Lynda Erskine for the provision of mouse embryo samples, and Professor Jimmy Hutchison and the Orthopaedics Department at the Aberdeen Royal Infirmary for the provision of human tissue samples. The authors are also grateful to Denise Tosh and Susan Clark for excellent technical support. This work was funded by Arthritis Research UK (grant 19429).