Influencia de rhBMP-2 en la reparación ósea de defectos críticos con diferentes biomateriales / Influence of rhBMP-2 on bone repair of critical size defects with different biomaterials

La regeneración de defectos óseos críticos requiere la utilización de biomateriales óseos. Así, se han utilizados agentes osteogénicos como la proteína morfogenética (rhBMP-2). El objetivo fue describir la formación ósea de defectos óseos críticos en calota de ratas utilizando rhBMP-2 con distintos biomateriales. Se realizaron dos defectos óseos críticos de 5 mm en 15 calotas de ratas machos adultas divididos en grupo control (sin tratamiento) (C); autoinjerto + rhBMP-2 (A); fosfato tricálcico + rhBMP-2 (BTCP); xenoinjerto de bovino + rhBMP-2 (B) y hidroxihapatita + rhBMP-2 (HA). A las ocho semanas post tratamiento, se realizó la eutanasia y posterior análisis histológico de los defectos. El grupo C no presentó formación de tejido óseo en el defecto. En el resto de los grupos, se formó abundante tejido óseo en los márgenes, por lo tanto, el defecto presentó menor tamaño. El grupo HA presentó formación ósea trabecular con amplios espacios medulares y abundante tejido adiposo. El grupo B-TCP también presentó formación ósea trabecular y la mayoría de las muestras presentaron puente óseo en el defecto. El grupo B presentó partículas de material injertado rodeado por trabéculas óseas y tejido conectivo. En el grupo A, todas las muestras presentaban puente óseo formado por bloques de autoinjerto rodeado por tejido conectivo y óseo. Es posible concluir que los defectos óseos de 5 mm en calota de rata son defectos críticos que requieren utilizar biomateriales para la reparación del defecto. El grupo B-TCP presentó características histológicas más próximas a la regeneración ósea lograda con el Grupo A.

The regeneration of bone critical size defects requires the use of bone biomaterials. Therefore, an osteogenic agent such as bone morphogenetic protein (rhBMP-2) has been used. The objective was to describe the bone formation of bone critical size defects in the rat calvaria using rhBMP-2 with different biomaterials. Two critical bone defects of 5 mm were made in 15 calvaria of adult male rats divided into a control group (without treatment) (C); autograft + rhBMP-2 (A); tricalcium phosphate + rhBMP-2 (B-TCP); bovine xenograft + rhBMP-2 (B) and hydroxyhapatite + rhBMP-2 (HA). At eight weeks post treatment, euthanasia and subsequent histological analysis of the defects were performed. Group C did not show bone tissue formation in the defect. In the rest of the groups, abundant bone tissue formed in the margins, therefore, the defect was smaller. The HA group presented trabecular bone formation with large medullary spaces and abundant adipose tissue. The B-TCP group also presented trabecular bone formation and most of the samples formed a bone bridge across the defect. Group B presented grafted material particles surrounded by bone trabeculae and connective tissue. In group A, all samples presented a bone bridge formed by autograft blocks surrounded by connective and bone tissue. It is possible to conclude that 5 mm bone defects in rat calvaria are critical size defects that require the use of biomaterials for defect repair. The B-TCP group presented histological characteristics similar to the bone regeneration achieved with Group A.

Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation

Abstract The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.

Biologic Response of Degenerative Living Human Nucleus Pulposus Cells to Treatment with Cytokines

PURPOSE: To investigate the molecular responses of various genes and proteins related to disc degeneration upon treatment with cytokines that affect disc-cell proliferation and phenotype in living human intervertebral discs (IVDs). Responsiveness to these cytokines according to the degree of disc degeneration was also evaluated. MATERIALS AND METHODS: The disc specimens were classified into two groups: group 1 (6 patients) showed mild degeneration of IVDs and group 2 (6 patients) exhibited severe degeneration of IVDs. Gene expression was analyzed after treatment with four cytokines: recombinant human bone morphogenic protein (rhBMP-2), transforming growth factor-beta (TGF-beta), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). Molecular responses were assessed after exposure of cells from the IVD specimens to these cytokines via real-time polymerase chain reaction and immunofluorescence staining. RESULTS: mRNA gene expression was significantly greater for aggrecan, type I collagen, type II collagen, alkaline phosphatase, osteocalcin, and Sox9 in group 1 than mRNA gene expression in group 2, when the samples were not treated with cytokines. Analysis of mRNA levels for these molecules after morphogen treatment revealed significant increases in both groups, which were much higher in group 1 than in group 2. The average number of IVD cells that were immunofluorescence stained positive for alkaline phosphatase increased after treatment with rhBMP-2 and TGF-beta in group 1. CONCLUSION: The biologic responsiveness to treatment of rhBMP-2, TGF-beta, TNF-alpha, and IL-1beta in the degenerative living human IVD can be different according to the degree of degeneration of the IVD.

Bone Morphogenetic Protein Receptor in the Osteogenic Differentiation of Rat Bone Marrow Stromal Cells

PURPOSE: Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of bone marrow stromal cells (BMSCs). Bone morphogenetic protein (BMP) signaling has important effects on the process of skeletogenesis. In the present study, we tested the role of bone morphogenetic protein receptor (BMPR) in the osteogenic differentiation of rat bone marrow stromal cells in osteogenic medium (OM) with or without BMP-2. MATERIALS AND METHODS: BMSCs were harvested from rats and cultured in OM containing dexamethasone, beta-glycerophosphate, and ascorbic acid, with or without BMP-2 in order to induce osteogenic differentiation. The alkaline phosphatase (ALP) activity assay and von kossa staining were used to assess the osteogenic differentiation of the BMSCs. BMPR mRNA expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The BMSCs that underwent osteogenic differentiation in OM showed a higher level of ALP activity and matrix mineralization. BMP-2 alone induced a low level of ALP activity and matrix mineralization in BMSCs, but enhanced the osteogenic differentiation of BMSCs when combined with OM. The OM significantly induced the expression of type IA receptor of BMPR (BMPRIA) and type II receptor of BMPR (BMPRII) in BMSCs after three days of stimulation, while BMP-2 significantly induced BMPRIA and BMPRII in BMSCs after nine or six days of stimulation, respectively. CONCLUSION: BMSCs commit to osteoblastic differentiation in OM, which is enhanced by BMP-2. In addition, BMP signaling through BMPRIA and BMPRII regulates the osteogenic differentiation of rat BMSCs in OM with or without BMP-2.

Msx2 mediates the inhibitory action of TNF-alpha on osteoblast differentiation

TNF-alpha, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-alpha signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-alpha-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-alpha down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-alpha induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-alpha. TNF-alpha activated the NF-kappaB and the JNK pathways. Inhibition of NF-kappaB or JNK activation reduced the inhibitory effect of TNF-alpha on ALP expression, whereas TNF-alpha-induced Msx2 expression was only suppressed by the inhibition of the NF-kappaB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-alpha on BMP2-regulated osteoblast differentiation and that the TNF-alpha-activated NF-kappaB pathway is responsible for Msx2 induction.

Use of Long-term Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2

PURPOSE: Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs. MATERIALS AND METHODS: hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed. RESULTS: Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (alphaMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS. CONCLUSION: We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.

Osteoinductive Potential of the rhBMP-2 in Soft Tissues / Potencial Osteoinductor de la rhBMP-2 en Tejidos Blandos

The rhBMP-2 is an osteoinductive protein used in the reconstructive with the objective to create newly formed bone. The aim of this study was to confirm the rhBMP-2 osteoinductive capability, when implanted in soft tissues. The results showed that the protein used in this study is highly osteoinductive.

La proteína oseoinductora rhBMP-2 es usada en cirugías reconstructoras, con la finalidad de crear tejido óseo neoformado. El objetivo de este estudio fue confirmar la capacidad oseoinductora de la rhBMP-2, cuando fue implantada en tejidos blandos. Los resultados mostraron que la proteína usada en este estudio es altamente oseoinductora.