Bone regeneration in rat cranium critical-size defects induced by Cementum Protein 1 (CEMP1).

Gene therapy approaches to bone and periodontal tissue engineering are being widely explored. While localized delivery of osteogenic factors like BMPs is attractive for promotion of bone regeneration; method of delivery, dosage and side effects could limit this approach. A novel protein, Cementum Protein 1 (CEMP1), has recently been shown to promote regeneration of periodontal tissues. In order to address the possibility that CEMP1 can be used to regenerate other types of bone, experiments were designed to test the effect of hrCEMP1 in the repair/regeneration of a rat calvaria critical-size defect. Histological and microcomputed tomography (µCT) analyses of the calvaria defect sites treated with CEMP1 showed that after 16 weeks, hrCEMP1 is able to induce 97% regeneration of the defect. Furthermore, the density and characteristics of the new mineralized tissues were normal for bone. This study demonstrates that hrCEMP1 stimulates bone formation and regeneration and has therapeutic potential for the treatment of bone defects and regeneration of mineralized tissues

Essential Role for NFI-C/CTF Transcription-Replication Factor in Tooth Root Development

The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and Nfix. In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic(−/−) mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic(−/−) mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic(−/−) animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development

CEMP1 Induces Transformation in Human Gingival Fibroblasts

<div><p>Cementum Protein 1 (CEMP1) is a key regulator of cementogenesis. CEMP1 promotes cell attachment, differentiation, deposition rate, composition, and morphology of hydroxyapatite crystals formed by human cementoblastic cells. Its expression is restricted to cementoblasts and progenitor cell subpopulations present in the periodontal ligament. CEMP1 transfection into non-osteogenic cells such as adult human gingival fibroblasts results in differentiation of these cells into a “mineralizing” cell phenotype. Other studies have shown evidence that CEMP1 could have a therapeutic potential for the treatment of bone defects and regeneration of other mineralized tissues. To better understand CEMP1’s biological effects in vitro we investigated the consequences of its expression in human gingival fibroblasts (HGF) growing in non-mineralizing media by comparing gene expression profiles. We identified several mRNAs whose expression is modified by CEMP1 induction in HGF cells. Enrichment analysis showed that several of these newly expressed genes are involved in oncogenesis. Our results suggest that CEMP1 causes the transformation of HGF and NIH3T3 cells. CEMP1 is overexpressed in cancer cell lines. We also determined that the region spanning the CEMP1 locus is commonly amplified in a variety of cancers, and finally we found significant overexpression of CEMP1 in leukemia, cervix, breast, prostate and lung cancer. Our findings suggest that CEMP1 exerts modulation of a number of cellular genes, cellular development, cellular growth, cell death, and cell cycle, and molecules associated with cancer.</p></div

Time series expression pattern of key genes.

<p><b>Expression of CDH1 before and after RNAi of CEMP1.</b> Expression patterns of the key genes were obtained in an independent time series experiment at 3, 6, 12, 24, 48, hours and 3, 7 and 14 days. The major expression changes as a result of CEMP1 overexpression initiated 24hrs (A). The relationship between CEMP1 and CDH1 was evaluated by RT-qPCR before and after knockdown of CEMP1 expression by RNAi. The RNAi directed against CEMP1 resulted in a significant decrease of CDH1 expression (B).</p

Western Blot validation of microarray results for key genes.

<p>Validation at the protein level by Western Blot was made at different time terms (basal, 24 hr, 3, and 7 days). Western blot demonstrated protein expression was higher in CEMP1 expressing cells than in controls.</p

Histological sections of the rat calvaria critical-size defect treated with <i>hr</i>CEMP1 and stained with Masson’s trichrome.

<p>The pictures show bonny tissue islands limited by dense fibrous connective tissue (FCT) (A and B). The bone-like tissue formed showed lamellar features with the presence of, blood vessels (BV) and embedded osteocytes (OCT) from (C and D). All microphotographs were taken at 400x.</p

Crystal morphological characterization.

<p>(A) SEM image of spheres-like structures formed by <i>hr</i>CEMP1. (B) Crystals irradiate from a mineralized core. EDS of apatite crystals showing a Ca/P ratio of 1.33, as expected for OCP (insert in B). SEM image of organized flattened-like crystals nucleated in the presence of 20 µg of <i>hr</i>CEMP1(C). Microribbon-like crystals nucleated in the presence of <i>hr</i>CEMP (D and E). Plate-like crystals induced by control protein (BSA) (F) and the EDS revealed a Ca/P ratio of 1.06 (Insert in F).</p

Expression of CEMP1 in HGF/CEMP1.

<p>The expression of CEMP1 after transfection in HGF and HGF/CEMP1 was evaluated using quantitative real time PCR at 3, 7 and 14 days. Fold increase of CEMP1 expression are represented, the graph shows that expression was higher in HGF/CEMP1 in all time points. * p = 0.0001 (A). CEMP1 protein expression levels were corroborated by western blot assays (B).</p

Histomorphometric quantitation of the bone matrix area within the rat calvaria critical-size defect.

<p>As can be seen in the graph, <i>hr</i>CEMP1 promoted regeneration of the rat calvarial defects up to 97% (A). This can also be seen in the three dimensional µCT of the not treated defect (B), control gelatin matrix scaffold (C) and experimental gelatin matrix scaffold containing <i>hr</i>CEMP1 (D) after 16 weeks post-surgery. Image reconstructions were performed at a resolution of 18 µm. The circles indicate the edges of the cranial defect.</p