Regeneration of rat periodontium by cementum protein 1-derived peptide.

BACKGROUND AND OBJECTIVE: Cementum protein 1 (CEMP1) has the capacity to promote differentiation of periodontal ligament (PDL) cells toward a cementoblastic phenotype in vitro and bone regeneration in vivo. In this study, we tested the capabilities of a synthetic cementum protein 1-derived peptide, MGTSSTDSQQAGHRRCSTSN (CEMP1-p1), to promote regeneration of periodontal structures in a periodontal fenestration defect in rats. MATERIAL AND METHODS: Fenestration defects were created using an extra-oral approach in the buccal aspect of the mandibular first molar roots. Eighteen male Wistar rats were divided into three groups. Two controls (defects non-treated or defects treated with a gelatin matrix scaffold [GMS] only) and the experimental group treated with 5 µg/dose of CEMP1-p1 embedded in GMS. After 28 days, the animals were sacrificed, and the mandibles processed for histopathological examination. Expression of cementum proteins, cementum attachment protein (CAP), CEMP1, integrin binding sialoprotein (IBSP), and osteocalcin (OCN), was assessed using immunofluorescence. The formation of new cementum, bone, and PDL fibers were compared between control and experimental groups. RESULTS: The histological analysis revealed that the control group without any treatment new cementum or oriented PDL fibers were not observed. However, the presence of newly bone was detected. In the control group treated with GMS, new cementum formation was not detectable, the PDL fibers were oriented parallel to the longitudinal root axis, and new bone formation was observed. The experimental group showed deposit of acellular extrinsic fiber cementum (AEFC) in a lamellae-like feature with inserted Sharpey's fibers, formation of cellular mixed stratified cementum (CMSC) with the presence of cementocytes, and newly formed bone close to the cementum-enamel junction. Cementoblast cells adjacent to new cementum expressed CAP, CEMP1, IBSP, and OCN. CONCLUSION: These studies show that CEMP1-p1 promotes the formation of AEFC, CMSC, new PDL with Sharpey's fibers inserted in cementum and bone, thus providing strong evidence that the synthetic peptide CEMP1-p1 promotes periodontal regeneration in a rat fenestration model.

Cementum protein 1-derived peptide (CEMP 1-p1) modulates hydroxyapatite crystal formation in vitro.

A cementum protein 1-derived peptide (CEMP1-p1) consisting of 20 amino acids from the CEMP1's N-terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell-free system, was characterized. CEMP1-p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1-p1 were analyzed by scanning electron microscopy, Fourier-transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1-p1 lacks secondary structure, forms nanospheres that organize into three-dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1-p1 promotes the formation of spherical structures composed by densely packed prism-like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR-ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1-p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.

Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

The use of recombinant proteins has revolutionized the development of biologic pharmaceuticals; however, they are not free of complications. Some have very high molecular weight, some demonstrate in vivo instability, and the high cost of producing them remains a major problem. On the other hand, it has been shown that peptides derived from active domains keep their biologic activity and can trigger events, such as osteogenesis and bone regeneration. Small peptides are advantageous because of their ease of synthesis and handling and their low immunogenic activity. The purpose of this study was to investigate the functions of a synthetic peptide, cementum protein 1-peptide1 (CEMP-1-p1), both in vitro and in vivo. Our results show that CEMP-1-p1 significantly enhanced the proliferation and differentiation of human periodontal ligament cells toward a mineralizing-like phenotype, as evidenced by increasing alkaline phosphatase (ALP)-specific activity and osterix, runt-related transcription factor (RUNX)-2, integrin binding sialoprotein, bone morphogenetic protein-2, osteocalcin, and cementum protein (CEMP)-1 expression at mRNA and protein levels. In vivo assays performed through standardized critical-size calvarial defects in rats treated with CEMP-1-p1 resulted in newly formed bone after 30 and 60 d. These data demonstrate that CEMP-1-p1 is an effective bioactive peptide for bone tissue regeneration. The application of this bioactive peptide may lead to implementing new strategies for the regeneration of bone and other mineralized tissues.-Correa, R., Arenas, J., Montoya, G., Hoz, L., López, S., Salgado, F., Arroyo, R., Salmeron, N., Romo, E., Zeichner-David, M., Arzate, H. Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

CEMP1 Induces Transformation in Human Gingival Fibroblasts.

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.

Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration.

Destruction of the periodontium is normally associated with periodontal disease, although many other factors, such as trauma, aging, infections, orthodontic tooth movement and systemic and genetic diseases, can contribute to this process. Strategies (such as guided tissue regeneration) have been developed to guide and control regeneration using bioresorbable membranes and bone grafts. Although effective to a certain point, these strategies have the problem that they are not predictable and do not completely restore the architecture of the original periodontium. To achieve complete repair and regeneration it is necessary to recapitulate the developmental process with complete formation of cementum, bone and periodontal ligament fibers. Detailed knowledge of the biology of cementum is key for understanding how the periodontium functions, identifying pathological issues and for developing successful therapies for repair and regeneration of damaged periodontal tissue. It is the purpose of this review to focus on the role of cementum and its specific components in the formation, repair and regeneration of the periodontium. As cementum is a matrix rich in growth factors that could influence the activities of various periodontal cell types, this review will examine the characteristics of cementum, its composition and the role of cementum components, especially the cementum protein-1, during the process of cementogenesis, and their potential usefulness for regeneration of the periodontal structures in a predictable therapeutic manner.

Human recombinant cementum attachment protein (hrPTPLa/CAP) promotes hydroxyapatite crystal formation in vitro and bone healing in vivo.

Cementum extracellular matrix is similar to other mineralized tissues; however, this unique tissue contains molecules only present in cementum. A cDNA of these molecules, cementum attachment protein (hrPTPLa/CAP) was cloned and expressed in a prokaryotic system. This molecule is an alternative splicing of protein tyrosine phosphatase-like A (PTPLa). In this study, we wanted to determine the structural and functional characteristics of this protein. Our results indicate that hrPTPLa/CAP contains a 43.2% α-helix, 8.9% ß-sheet, 2% ß-turn and 45.9% random coil secondary structure. Dynamic light scattering shows that this molecule has a size distribution of 4.8 nm and aggregates as an estimated mass of 137 kDa species. AFM characterization and FE-SEM studies indicate that this protein self-assembles into nanospheres with sizes ranging from 7.0 to 27 nm in diameter. Functional studies demonstrate that hrPTPLa/CAP promotes hydroxyapatite crystal nucleation: EDS analysis revealed that hrPTPLa/CAP-induced crystals had a 1.59 ± 0.06 Ca/P ratio. Further confirmation with MicroRaman spectrometry and TEM confirm the presence of hydroxyapatite. In vivo studies using critical-size defects in rat cranium showed that hrPTPLa/CAP promoted 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively. Although originally identified in cementum, PTPLa/CAP is very effective at inducing bone repair and healing and therefore this novel molecule has a great potential to be used for mineralized tissue bioengineering and tissue regeneration.

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.

Cementum protein 1 (CEMP1) induces differentiation by human periodontal ligament cells under three-dimensional culture conditions.

PDL (periodontal ligament) is a source of multi-potent stem cells in humans and their differentiation potential to a cementoblast and osteoblast phenotypes has been shown. Tissue construction from PDL-derived cells could be considered as a valuable technique for periodontal regenerative medicine. On these basis, we determined the role of CEMP1 (cementum protein 1) as a factor to induce differentiation of human PDL cells in a 3D (three-dimensional) fashion. Human PDL cells were grown in an RCCS (rotary cell culture system) D-410 RWV (rotating wall vessel) bioreactor, and maintained in either experimental (CEMP1 2.5 µg/ml) or control media during 4 weeks. Cell proliferation in the presence of CEMP1 was determined. The tissue-like structure formed was analysed histologically, stained with Alizarin Red and Alcian Blue. ALP (alkaline phosphatase)-specific activity, immunostaining, RT-PCR (reverse transcription-PCR) and Western blotting were performed to determine the expression of BSP (bone sialoprotein), enamel [AMBN (ameloblastin) and AMEL (amelogenin)], cementum [CAP (cementum attachment protein) and CEMP1] and cartilage-related proteins (Sox9, aggrecan, types II and X collagens). Our results show that hrCEMP1 (human recombinant CEMP1) promoted cell proliferation by human PDL cells in 3D cultures and induced the formation of a tissue-like structure resembling bone and/or cementum and material similar to cartilage. The addition of hrCEMP1 to the 3D human PDL cell cultures increased ALP-specific activity by 2.0-fold and induced the expression of markers for the osteogenic, cementogenic and chondrogenic phenotypes at the mRNA and protein levels. Our data show that human PDL cells in 3D cultures with the addition of CEMP1 has the potential to be used for the bioengineering reconstruction of periodontal tissues and cartilage since our results suggest that CEMP1 stimulates human PDL cells to differentiate towards different phenotypes.

Characterization of recombinant human cementum protein 1 (hrCEMP1): primary role in biomineralization.

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M(r) 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of beta-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.

Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

Establishment and characterization of an immortomouse-derived odontoblast-like cell line to evaluate the effect of insulin-like growth factors on odontoblast differentiation.

Insulin-like growth factors (IGF-I and IGF-II) play important roles in regulating growth and differentiation of many different organs including teeth. The presence of these factors in the developing tooth has been demonstrated. In vitro studies using tooth explants grown in the presence of IGFs suggest that they promote differentiation of ameloblast and odontoblasts cells. This is achieved by inducing or repressing gene expression associated with these cells. Since some of the genes involved in tooth differentiation are expressed by both cells, to determine the effect of IGF on odontoblast cell differentiation we first need a cell line in which a controlled environment can be created. In this study, we report the establishment and characterization of an Immortomouse-derived odontoblast-like cell line. This conditional cell line can grow indefinitely under permissive conditions in the presence of INF-gamma at 33 degrees C, differentiate into odontoblast-like cells and produce a mineralized extracellular matrix when the INF-gamma is removed and cell maintained at 39 degrees C. Addition of exogenous IGFs to the media results in an accelerated production of a mineralized matrix. This is the result of increased transcription of genes associated with bone mineralization while down regulating genes associated with dentin formation like DSPP. This data suggest that IGFs induce dental papillae mesenchyme cells to produce a bone-like mineralized extracellular matrix.

Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23).

Cementum is a unique mineralized connective tissue that covers the root surfaces of the teeth. The cementum is critical for appropriate maturation of the periodontium, both during development as well as that associated with regeneration of periodontal tissues, IU; however, one major impediment to understand the molecular mechanisms that regulate periodontal regeneration is the lack of cementum markers. Here we report on the identification and characterization of one such differentially human expressed gene, termed "cementum protein-23" (CP-23) that appears to be periodontal ligament and cementum-specific. We screened human cementum tumor-derived cDNA libraries by transient expression in COS-7 cells and "panning" with a rabbit polyclonal antibody against a cementoblastoma conditioned media-derived protein (CP). One isolated cDNA, CP-23, was expressed in E. coli and polyclonal antibodies against the recombinant human CP-23 were produced. Expression of CP-23 protein by cells of the periodontium was examined by Northern blot and in situ hybridization. Expression of CP-23 transcripts in human cementoblastoma-derived cells, periodontal ligament cells, human gingival fibroblasts and alveolar bone-derived cells was determined by RT-PCR. Our results show that we have isolated a 1374-bp human cDNA containing an open reading frame that encodes a polypeptide with 247 amino acid residues, with a predicted molecular mass of 25.9 kDa that represents CP species. The recombinant human CP-23 protein cross-reacted with antibodies against CP and type X collagen. Immunoscreening of human periodontal tissues revealed that CP-23 gene product is localized to the cementoid matrix of cementum and cementoblasts throughout the entire surface of the root, cell subpopulations of the periodontal ligament as well as cells located paravascularly to the blood vessels into the periodontal ligament. Furthermore, 98% of putative cementoblasts and 15% of periodontal ligament cells cultured in vitro expressed CP-23 gene product. Cementoblastoma cells and periodontal ligament cells contained a 5.0 kb CP-23 mRNA. In situ hybridization showed strong expression of CP-23 mRNA on cementoblast, cell subpopulations of the periodontal ligament and cells located around blood vessels into the periodontal ligament. Our results demonstrate that CP-23 represents a novel, tissue-specific-gene product being expressed by periodontal ligament subpopulations and cementoblasts. These findings offer the possibility to determine the cellular and molecular events that regulate the cementogenesis process during root development. Furthermore, it might provide new venues for the design of translational studies aimed at achieving predictable new cementogenesis and regeneration of the periodontal tissues.

IGFs increase enamel formation by inducing expression of enamel mineralizing specific genes.

Insulin-like growth factors (IGF-I and IGF-II) have been shown to play an important role in growth and differentiation in a number of tissues including mineralizing bone. Little is known about their role in tooth mineralization. Previous work in our laboratory has shown the presence of IGFs ligands, their receptors, and their binding proteins during mouse tooth morphogenesis. The expression of IGF I coincides with the expression of amelogenin, ameloblastin and enamelin at the late bell and secretory stage. The objective of this study is to determine the mechanisms by which IGFs modulate enamel and dentin formation. Mouse first mandibular molars were dissected from E16 and E17 mouse embryos and placed in organ culture in the presence of IGF-I or IGF-II. The molars were harvested after 12 days for histological examination or 1 day for mRNA expression analysis by real-time RT-PCR. Our results show an increase in enamel deposition, and an induction of enamelin, amelogenin and collagen type I mRNA expression, while expression of DSPP was down-regulated. These results suggest that IGFs increase enamel formation by the induction of gene expression of enamel related genes. Studies are underway to determine a possible mechanism for these factors.

Role of Hertwig's epithelial root sheath cells in tooth root development.

During tooth development, after the completion of crown formation, the apical mesenchyme forms the developing periodontium while the inner and outer enamel epithelia fuse below the level of the crown cervical margin to produce a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS). The role of HERS cells in root formation is widely accepted; however, the precise function of these cells remains controversial. Functions suggested have ranged from structural (subdivide the dental ectomesenchymal tissues into dental papilla and dental follicle), regulators of timing of root development, inducers of mesenchymal cell differentiation into odontoblasts and cementoblasts, to cementoblast cell precursors. The characterization of the HERS phenotype has been hindered by the small amount of tissue present at a given time during root formation. In this study, we report the establishment of an immortal HERS-derived cell line that can be maintained in culture and then induced to differentiate in vitro. Characterization of the HERS phenotype using reverse transcriptase-polymerase chain reaction and Western blot immunostaining suggests that HERS cells initially synthesize and secrete some enamel-related proteins such as ameloblastin, and then these cells appear to change their morphology and produce a mineralized extracellular matrix resembling acellular cementum. These studies suggest that the acellular and cellular cementum are synthesized by two different types of cells, the first one by HERS-derived cementoblasts and the later by neural crest-derived cementoblasts.

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.

Immediate-loading dental endosteal implants and the elderly patient.

The concept of immediately loading dental implants has the potential of being a viable addition to treatment modalities. The major decision-making challenge in managing depleted dentitions and complete edentulism in an aging society lies in differentiating the treatment outcomes, especially patient-mediated assessments (including economic analyses) of the various prosthodontic options available for older adults. The ability to chew properly is of great importance to maintain a healthy nutrition and improve oral comfort and quality of life, particularly in the elderly years.