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Louis C Gerstenfeld

Publications and source records attributed to Louis C Gerstenfeld.

22 records · Page 2Linked to original sources

Transcriptional regulation restricting bone sialoprotein gene expression to both hypertrophic chondrocytes and osteoblasts.

This study identifies a cis-acting element that confers tissue-restricted expression to the bone sialoprotein (BSP) gene. Using both gain of function and loss-of function studies, we demonstrate that this element acts as a tissue specific enhancer of BSP expression in osteoblasts and hypertrophic chondrocytes but does not function in non-hypertrophic chondrocytes or fibroblasts. Furthermore, our data demonstrate that binding of this element occurs in correlation with active BSP expression. While Dlx5 has been implicated as the tissue-specific regulator of BSP expression through direct DNA binding at an element with homology to the one under study here, our results demonstrate that Dlx5 does not act as a positive regulator of BSP expression. Finally, mutational analyses of this element demonstrate that while there is homology to putative homeodomain binding elements, this site is unlikely to bind homeodomain factors including Dlx5. Thus, these studies identify an important cis-acting element in the BSP promoter that acts as a tissue-specific enhancer of BSP expression in both osteoblasts and hypertrophic chondrocytes. As such this is the first demonstration of a common regulatory mechanism utilized by both chondrocytes and osteoblasts for the tissue-restricted expression of the BSP gene.

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Induction of a neoarthrosis by precisely controlled motion in an experimental mid-femoral defect.

Bone regeneration during fracture healing has been demonstrated repeatedly, yet the regeneration of articular cartilage and joints has not yet been achieved. It has been recognized however that the mechanical environment during fracture healing can be correlated to the contributions of either the endochondral or intramembranous processes of bone formation, and to resultant tissue architecture. Using this information, the goal of this study was to test the hypothesis that induced motion can directly regulate osteogenic and chondrogenic tissue formation in a rat mid-femoral bone defect and thereby influence the anatomical result. Sixteen male Sprague Dawley rats (400 +/- 20 g) underwent production of a mid-diaphyseal, non-critical sized 3.0 mm segmental femoral defect with rigid external fixation using a custom designed four pin fixator. One group of eight animals represented the controls and underwent surgery and constant rigid fixation. In the treatment group the custom external fixator was used to introduce daily interfragmentary bending strain in the eight treatment animals (12 degree angular excursion), with a hypothetical symmetrical bending load centered within the gap. The eight animals in the treatment group received motion at 1.0 Hz, for 10 min a day, with a 3 days on, one day off loading protocol for the first two weeks, and 2 days on, one day off for the remaining three weeks. Data collection included histological and immunohistological identification of tissue types, and mean collagen fiber angles and angular conformity between individual fibers in superficial, intermediate, and deep zones within the cartilage. These parameters were compared between the treatment group, rat knee articular cartilage, and the control group as a structural outcome assessment. After 35 days the control animals demonstrated varying degrees of osseous union of the defect with some animals showing partial union. In every individual within the mechanical treatment group the defect completely failed to unite. Bony arcades developed in the experimental group, capping the termini of the bone segments on both sides of the defect in four out of six animals completing the study. These new structures were typically covered with cartilage, as identified by specific histological staining for Type II collagen and proteoglycans. The distribution of collagen within analogous superficial, intermediate, and deep zones of the newly formed cartilage tissue demonstrated preferred fiber angles consistent with those seen in articular cartilage. Although not resulting in complete joint development, these neoarthroses show that the induced motion selectively controlled the formation of cartilage and bone during fracture repair, and that it can be specifically directed. They further demonstrate that the spatial organization of molecular components within the newly formed tissue, at both microanatomical and gross levels, are influenced by their local mechanical environment, confirming previous theoretical models.

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Chondrocytes provide morphogenic signals that selectively induce osteogenic differentiation of mesenchymal stem cells.

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T1/2 mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T1/2 mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T1/2 cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T1/2 cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T1/2 cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T1/2 cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.

Alkaline Phosphatase↗

Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing.

Fracture healing is a unique postnatal repair process in which the events of endochondral and intramembranous bone formation follow a definable temporal sequence. The temporal patterns of messenger RNA (mRNA) expression for members of the transforming growth factor beta (TGF-beta) superfamily were examined over a 28-day period of fracture healing in mouse tibias. Bone morphogenetic protein 2 (BMP-2) and growth and differentiation factor 8 (GDF8) showed maximal expression on day 1 after fracture, suggesting their roles as early response genes in the cascade of healing events. Restricted expression of GDF8 to day 1, in light of its known actions as a negative regulator of skeletal muscle growth, suggests that it may similarly regulate cell differentiation early in the fracture healing process. GDF5, TGF-beta2, and TGF-beta3 showed maximal expression on day 7, when type II collagen expression peaked during cartilage formation. In contrast, BMP-3, BMP-4, BMP-7, and BMP-8 showed a restricted period of expression from day 14 through day 21, when the resorption of calcified cartilage and osteoblastic recruitment were most active. TGF-beta1, BMP-5 and BMP-6, and GDF10 were constitutively expressed from day 3 to day 21. However, during the same time period, GDF3, GDF6, and GDF9 could not be detected, and GDF1 was expressed at extremely low levels. These findings suggest that several members of the TGF-beta superfamily are actively involved in fracture healing and although they are closely related both structurally and functionally, each has a distinct temporal expression pattern and potentially unique role in fracture healing.

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