Solution mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSC) on PLGA by promote the formation of a calcium phosphate rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, we tested whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSC, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell derived factors which promoted distant MSC differentiation. Thus PLGABG composites demonstrates significant potential as a bone replacement material

Smad-Runx interactions during chondrocyte maturation

BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. Clinical Relevance: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy

Does Gender Bias Influence Awards Given by Societies?

AGU is a participant in a U.S. National Science Foundation (NSF)–funded project called Advancing Ways of Awarding Recognition in Disciplinary Societies (AWARDS), which seeks to examine whether gender bias affects selection of recipients of society awards. AGU is interested in learning why there is a higher proportion of female recipients of service and education awards over the past 2 decades. Combined with a lower rate of receipt of research awards, these results suggest that implicit (subconscious) bias in favor of male candidates still influences awardee selection. Six other professional societies (American Chemical Society, American Mathematical Society, American Society of Anesthesiologists, Mathematical Association of America, Society for Neuroscience, and Society for Industrial and Applied Mathematics) are participating in the project. Volunteers from each participant society attended an Association for Women in Science (AWIS)–sponsored workshop in May 2010 to examine data and review literature on best practices for fair selection of society awardees. A draft proposal for implementing these practices will be brought before the AGU Council and the Honors and Recognition Committee at their upcoming meetings

Utilization of Bone Morphogenetic Protein Receptors during Chondrocyte Maturation.

Cartilage from the upper, cephalic portion of embryonic chick sternums undergoes hypertrophy, while the lower, caudal portion of the sternum remains as cartilage. Bone morphogenetic proteins (BMPs) induce type X collagen (colX) in cultured upper but not lower sternal chondrocytes (LSCs). We have examined the utilization of BMP receptors (BMPRs) by upper sternal chondrocytes (USCs) and LSCs both by analyzing receptor expression and by overexpressing mutant BMPRs. Reverse-transcription polymerase chain reaction (RT-PCR) analyses indicate that both upper and lower chondrocytes produce messenger RNA (mRNA) for all three receptors: BMPR type IA (BMPR-IA), BMPR type IB (BMPR-IB), and BMPR type II (BMPR-II). Infection of USC with retroviral vectors expressing constitutively active (CA) BMPRs showed that CA-BMPR-IB, like exogenous BMP-4, induced both colX mRNA and elevated alkaline phosphatase (AP), while CA-BMPR-IA was markedly less potent. However, expression of activated receptors in LSC cultures resulted in only minimal induction of hypertrophic markers. Consistent with the results seen for CA receptors, dominant negative (DN) BMPR-IB blocked BMP-induced hypertrophy in USCs more effectively than DN-BMPR-IA. These results imply that the major BMPR required for BMP induction of chondrocyte hypertrophy is BMPR-IB, and that difference between permanent and prehypertrophic chondrocytes is not caused by absence of receptors required for BMP signaling

Authentic matrix vesicles contain active metalloproteases (MMP): A role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-ß

Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization. Although many components in these microstructures have been identified, the specific function of each component is still poorly understood. In this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV isolated from growth plate cartilage. In addition, we provide evidence that MV contain transforming growth factor-ß (TGF-ß) and that MV-associated MMP-13 is capable of activating latent TGF-ß. To determine whether MMPs are associated directly with MV, vesicles isolated from growth plate cartilage were sequentially treated with hyaluronidase, NaCl, and bacterial collagenase to remove matrix proteins and other components attached to their outer surface. Finally, the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymography, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were still retained in the pellet fraction even after detergent treatment, suggesting that the gelatinases, MMP-2 and -9, are integral components of MV. In addition, MV contain TGF-ß in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-ß. Since the amount of TGF-ß activated by hypertrophic chondrocytes increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-ß seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage

MMP-13 is induced during chondrocyte hypertrophy

During development, mRNA for matrix metalloproteinase-13 (MMP-13) is found associated with cartilage undergoing hypertrophy, suggesting that this collagenase plays a role in cell enlargement and/or cartilage calcification. Using chondrocytes from prehypertrophic cartilage of chick embryo sternae, we have examined the relationship between MMP-13 expression and the transition to hypertrophy. When hypertrophy was induced by serum-free culture with ascorbate and bone morphogenetic protein-2 (BMP-2), MMP-13 mRNA levels paralleled those for type X collagen. Chondrocytes from the caudal, nonhypertrophying portion of chick sternae expressed neither type X collagen nor MMP-13, confirming that MMP-13 mRNA is a marker for hypertrophy. Zymography with conditioned medium yielded a proteinase band at 59 kDa, which was absent in nonhypertrophic chondrocytes. A polyclonal antibody raised against chick MMP-13 reacted with the 59-kDa protein, confirming that it is MMP-13. Although mRNA for MMP-13 peaked at days 4-5 of culture, only low levels of MMP-13 activity were present, and the activity increased gradually in parallel with later increases in MMP-2. These results suggest that MMP-13 is activated by MMP-2 during chondrocyte maturation, and that the combination of both proteinases is required to prepare cartilage matrix for subsequent calcification, before endochondral ossification. (C) 2000 Wiley-Liss, Inc