Characterization of aTiO2 surfaces functionalized with CAP-p15 peptide.

Functionalization of Titanium implants using adequate organic molecules is a proposed method to accelerate the osteointegration process, which relates to topographical, chemical, mechanical, and physical features. This study aimed to assess the potential of a peptide derived from cementum attachment protein (CAP-p15) adsorbed onto aTiO2 surfaces to promote the deposition of calcium phosphate (CaP) minerals and its impact on the adhesion and viability of human periodontal ligament cells (hPDLCs). aTiO2 surfaces were synthesized by magnetron sputtering technique. The CAP-p15 peptide was physically attached to aTiO2 surfaces and characterized by atomic force microscopy, fluorescence microscopy, and water contact angle measurement. We performed in vitro calcium phosphate nucleation assays using an artificial saliva solution (pH 7.4) to simulate the oral environment. morphological and chemical characterization of the deposits were evaluated by scanning electronic microscopy (SEM) and spectroscopy molecular techniques (Raman Spectroscopy, ATR-FTIR). The aTiO2 surfaces biofunctionalized with CAP-p15 were also analyzed for hPDLCs attachment, proliferation, and in vitro scratch-healing assay. The results let us see that the homogeneous amorphous titanium oxide coating was 70 nanometers thick. The CAP-p15 (1 µg/mL) displayed the ability to adsorb onto the aTiO2 surface, increasing the roughness and maintaining the hydrophilicity of the aTiO2 surfaces. The physical adsorption of CAP-p15 onto the aTiO2 surfaces promoted the precipitation of a uniform layer of crystals with a flake-like morphology and a Ca/P ratio of 1.79. According to spectroscopy molecular analysis, these crystalline deposits correspond to carbonated hydroxyapatite. Regarding cell behavior, the biofunctionalized aTiO2 surfaces improved the adhesion of hPDLCs after 24 h of cell culture, achieving 3.4-fold when compared to pristine surfaces. Moreover, there was an increase in cell proliferation and cell migration processes. Physical adsorption of CAP-p15 onto aTiO2 surfaces enhanced the formation of carbonate hydroxyapatite crystals and promoted the proliferation and migration of human periodontal ligament-derived cells in in vitro studies. This experimental model using the novel bioactive peptide CAP-p15 could be used as an alternative to increasing the osseointegration process of implants.

Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process.

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Characterization of pulp calcifications and changes in their composition after treatments with citric acid and ethylenediaminetetraacetic acid solutions.

The ectopic calcifications of non-mineralized tissues can occur in several forms throughout life, such as pulpal calcification. The presence of pulp stones is a challenge in endodontic treatment because they partially or fully obliterate the pulp chamber hindering access to root canals and their subsequent shaping. This study aimed to determine their crystallographic properties and evaluate the capacity of citric acid (CA) and ethylenediaminetetraacetic acid (EDTA) to promote the demineralization of pulp calcifications. The samples were obtained from patients with indications of endodontic treatment, and the radiographic examination was suggestive of pulp stone in at least one permanent tooth. The samples were isolated and analyzed by scanning electron microscopy/energy-dispersive x-ray spectroscopy (SEM/EDX). The Fourier Transform by high resolution-transmission electron microscopy, Raman microscopy, and X-ray diffraction (XRD) were used to identify the mineral phase and crystallographic characteristics. To evaluate the effect of CA and EDTA on the crystallinity of calcifications, they were submerged into these two individual solutions and the changes were assessed in situ by Raman spectroscopy. The SEM images obtained from calcifications demonstrated irregular morphologies. EDX of sample surfaces shows a high presence of oxygen, carbon, calcium, and phosphorous, however, other elements such as sodium, magnesium, nitrogen, chlorine, potassium, sulfur, and zinc were identified in less quantity. According to Raman, XRD, and high-resolution transmission electron microscopy, the predominant mineral phase identified in the pulpal calcification was a poor crystallinity apatite. According to in situ analyses, the effect of CA and EDTA was observed on the signals of PO4 3- and CH2 groups corresponding to inorganic and organic components. The changes with CA were evident at 7 min while the effect of EDTA was observed until 15 min of treatment. All results indicate that pulp stones have a heterogeneous composition principally composed of apatite with low crystallinity. The solubility of these pathological minerals is adequate using solutions such as EDTA or CA; however, the effectivity depends on the mineralization grade of calcifications, time, and concentration of exposition to this chemical.

Human oral mucosa-derived neural crest-like stem cells differentiate into functional osteoprogenitors that contribute to regeneration of critical size calvaria defects.

BACKGROUND AND OBJECTIVE: Regeneration of large bony defects is an unmet medical need. The therapeutic effect of fully developed bony constructs engineered in vitro from mineralized scaffold and adult stem cells is hampered by deficient long-term graft integration. The purpose of the present study was to investigate the regenerative capacity of a bony primordial construct consisting of human oral mucosa stem cells (hOMSC)-derived osteoprogenitors and absorbable Gelfoam® sponges. METHODS: Gingiva and alveolar mucosa-derived hOMSC were differentiated into osteoprogenitors (Runx2 and osterix positive) and loaded into Gelfoam® sponges to generate primordial hOMSC constructs. These were implanted into critical size calvaria defects in the rat. Defects treated with human dermal fibroblasts (HDF) constructs; Gelfoam® sponges and untreated defects served as controls. RESULTS: After 120-day post-implantation defects treated with hOMSC constructs, HDF constructs and gelatin and untreated defects exhibited 86%, 30%, 21%, and 9% of new bone formation, respectively. Immunofluorescence analysis for human nuclear antigen (HNA), bone sialoprotein (BSP), and osteocalcin (OCN) revealed viable hOMSC-derived osteoblasts and osteocytes that formed most of the cell population of the newly formed bone at 30 and 120 days post surgery. Few HNA-positive HDF that were negative for BSP and OCN were identified together with inflammatory cells in the soft tissue adjacent to new bone formation only at 30 days post implantation. CONCLUSION: Collectively, the results demonstrate that primordial in vitro engineered constructs consisting of hOMSC-derived osteoprogenitors and absorbable gelatin almost completely regenerate critical size defects in an immunocompetent xenogeneic animal by differentiating into functional osteoblasts that retain the immunomodulatory ability of naïve hOMSC.

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.

Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.

Nucleation and growth inhibition of biological minerals by cementum attachment protein-derived peptide (CAP-pi).

Biomineralization is a highly regulated process where proteins/peptides-crystal interactions contribute to the shaping, phasing and aggregation of minerals. We have identified and synthesized a cementum attachment protein-derived peptide (CAP-pi), which corresponds to amino acids 40-53 of the N-terminal CAP domain (MASSDEDGTNGGAS) and its phosphorylated variant (MASpSpDEDGTNGGASp) (CAP-pip). The peptide is composed of polar and negatively charged amino acids, which are disordered, according to in silico analysis. Our results show that CAP-pi inhibits hydroxyapatite (HA) formation and growth. However, it possesses low capacity to inhibit calcium oxalate crystal growth. CAP-pip showed a stronger inhibitory effect on the formation and growth of HA. As well as a high capacity to inhibit calcium oxalate monohydrate growth, mainly due to adsorption on specific growth faces. Small peptides have many advantages over the full-size protein, including low-cost production and modulation characteristics that allow for structural changes. Our findings suggest that CAP-pip-derived peptide could possess therapeutic potential to prevent or treat pathological calcifications such as renal stones and vascular calcification.

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/ß-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.

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.

Aqueous extract of Sedum oxypetalum induces mineralization and osteogenic differentiation by human Periodontal Ligament-Derived cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plant Sedum oxypetalum Kunth (Crassulaceae), locally known as Jiote or in general Siempreviva (always alive) has been traditionally used by people of the Mexican community of Tenango del Valle as a home remedy to treat periodontal diseases, inducing teeth strengthening. Consequently, the aim of this work was to investigate its capacity directed to mineralized tissues regeneration. MATERIALS AND METHODS: The aerial parts of the plant were processed and its aqueous extract (AE) was chemically characterized. The AE and its components sedoheptulose and syngenite were tested for either osteogenic differentiation or mineral-nucleation induction respectively. RESULTS: The AE and one of its components (sedoheptulose) were shown to promote the proliferation and/or osteogenic differentiation by Human Periodontal Ligament-Derived Cells (hPDLs), while inducing the mineralization process. The AE also promoted the nucleation of octacalcium phosphate and its component syngenite, the hydroxyapatite crystals formation in vitro. CONCLUSION: The findings reported herein support the traditional use of S. oxypetalum due to its potential capacity to promote the regeneration of mineralized tissues.

High-level expression and characterization of a glycosylated human cementum protein 1 with lectin activity.

This work aims to contribute to the knowledge of human cementum protein 1 (CEMP1), its conformational characteristics and influence during the biomineralization process. The results revealed that hrCEMP1 expressed in Pichia pastoris is a 2.4% glycosylated, thermostable protein which possesses a molecular mass of 28,770 Da. The circular dichroism spectrum indicated a secondary structure content of 28.6% of alpha-helix, 9.9% of beta-sheet and 61.5% of random-coil forms. Biological activity assays demonstrated that hrCEMP1 nucleates and regulates hydroxyapatite crystal growth. Hereby, it is demonstrated for the first time that CEMP1 has a (C-type) lectin-like activity and specifically recognizes mannopyranoside. The information produced by this biochemical and structural characterization may contribute to understand more fully the biological functions of CEMP1.

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.

Periodontal Specific Differentiation of Dental Follicle Stem Cells into Osteoblast, Fibroblast, and Cementoblast.

The dental follicle is a source of dental follicle stem cells (DFCs), which have the potential to differentiate into the periodontal lineage. DFCs therefore are of value in dental tissue engineering. The purpose of this study was to evaluate the effect of growth factor type and concentration on DFC differentiation into periodontal specific lineages. DFCs were isolated from the human dental follicle and characterized for the expression of mesenchymal markers. The cells were positive for CD-73, CD-44, and CD-90; and negative for CD-33, CD-34, and CD-45. The expression of CD-29 and CD-31 was almost negligible. The cells also expressed periodontal ligament and cementum markers such as periodontal ligament-associated protein-1 (PLAP-1), fibroblast growth factor-2 (FGF-2), and cementum protein-1 (CEMP-1), however, the expression of osteoblast markers was absent. Further, the DFCs were cultured in three different induction medium to analyze the osteoblastic, fibroblastic, and cementoblastic differentiation. Runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP) activity, alizarin staining, calcium quantification, collagen type-1 (Col-1), and osteopontin (OPN) expression confirmed the osteoblastic differentiation of DFCs. DFCs cultured in recombinant human FGF-2 (rhFGF-2) containing medium showed enhanced PLAP-1, FGF-2, and COL-1 expression with increasing concentration of rhFGF-2 which thereby confirmed periodontal ligament fibroblastic differentiation. Similarly, DFCs cultured in recombinant human cementum protein-1 (rhCEMP-1) containing medium showed enhanced bone sialoprotein-2 (BSP-2), CEMP-1, and COL-1 expression with respect to rhCEMP-1 which confirmed cementoblastic differentiation. The expression of osteoblast, fibroblast, and cementoblast-related genes of DFCs cultured in induction medium was enhanced in comparison to DFCs cultured in noninduction medium. Thus, growth factor-dependent differentiation of DFCs into periodontal specific lineages was proved by quantitative analysis.

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.

Producción y caracterización de una proteína recombinante del cemento (CEMP 1) en células de Drosophila melanogaster (DML-2-23) / Production and characterization of a cement (CEMP1) recombining protein in Drosophila melanogaster (DML-2-23) cells

Los factores celulares y moleculares que regulan los procesos de formación y mineralización del cemento son poco conocidos hasta la fecha. Principalmente esto se debe a que no existe un marcador biológico de este tejido. Nosotros recientemente hemos aislado, clonado y expresado una proteína derivada del cemento radicular humano llamada proteína del cemento. Esta proteína es expresada por cementoblastos y células progenitoras del periodonto. El objetivo de este trabajo fue el de expresar la proteína del cemento en células de Drosophila melanogaster para en un futuro, determinar sus modificaciones postraduccionales. Nuestros resultados muestran que hemos establecido una línea celular que expresa constitutivamente y de forma estable la proteína del cemento. Esto es de singular importancia, ya que en un futuro mediato, estas células serán el vehículo para la producción de la proteína del cemento en cantidades suficientes para determinar su papel durante el proceso de la formación del periodonto de novo en modelos animales y de un modo predecible.

To the present date, molecular and cellular factors which regulate cement formation and mineralization processes are not well known. This is mainly due to the lack of a biological marker for this type of tissue. We have recently isolated, cloned and expressed a protein derived from human radicular (root) cement, called cement protein. This protein is expressed by cementoblasts as well as periodontium progenitor cells. The aim of the present work was to express cement protein in Drosophila melanogaster cells, so as to determine, in the future, its post-translational modifications. Our results show we have established a cellular line which expresses protein cement in an essential and stable fashion. This fact is of unique importance, since in the mediate future these cells will become the vehicle for cement protein production in sufficient amounts to determine in a predictable manner, its role during the process of de novo periodontium formation process in animal models.

Localización de las proteínas específicas del cemento radicular CEMP1 y CAP en células neoplásicas / Immunolocalization of cementum specific proteins CEMP1 and CAP in tumor cells

Introducción: Las proteínas CEMP1 y CAP presentes en los cementoblastos y sus progenitores contribuyen a los procesos de mineralización en tejidos del ligamento periodontal, incluyendo la migración y la proliferación de fibroblastos gingivales; sin embargo su papel y relación con procesos neoplásicos no se han estudiado a profundidad. Para lograr un mejor entendimiento de la posible contribución de estas proteínas en los procesos tumorales, particularmente en las metástasis óseas, se investigó su expresión y localización en tejidos y líneas celulares de cáncer humano. Materiales y métodos: Trece casos de cáncer de próstata y mama que desarrollaron enfermedad metastásica ósea fueron analizados por medio de inmunohistoquímica; mientras que la expresión de las proteínas en dos líneas celulares de carcinoma de próstata (PC-3) y mama (MCF-7) se estudió por medio de ensayos de Western Blot. Resultados: Los tejidos de cáncer revelaron expresión citoplasmática y ocasionalmente nuclear de CAP en células tumorales y estructuras glandulares pequeñas, así como en el citoplasma de los fibroblastos estromales adyacentes al frente de invasión tumoral. En lo correspondiente a CEMP1, su expresión se localizó en el citoplasma de las células tumorales de 5 casos, pero no en el estroma. Ensayos de Wester Blot mostraron expresión de CEMP1 en las células PC-3 y MCF-7; y de CAP en las MCF-7. Conclusiones: Los resultados muestran que las proteínas de cemento radicular CEMP1 y CAP se expresan en tejidos neoplásicos y células neoplásicas, y que posiblemente contribuyen en ciertas condiciones patológicas como el cáncer metastásico en humanos.

Introduction: CEMP1 and CAP are recognized as cementum proteins, they appear to be limited to cementoblasts and their progenitors, and participate in the mineralization process of periodontal ligament tissues, including the proliferation and migration of periodontal ligament fibroblasts. However, their contribution in neoplastic processes had not been explored. In the present study, we investigated their protein expression and localization in cancer tissues and cells. Materials and Methods: CEMP1 and CAP expressions were analyzed immunohistochemically in 13 cancer cases with bone metastasis. In addition, Wester Blot essays were use to detect expression of the proteins in the prostate (PC-3) and mama (MCF-7) cancer cell lines. Results: CAP expression was detected in all tissues examined. Strong cytoplasmatic and rarely nuclear staining was found in small tumor nests, glandular structures and, in the stromal fibroblasts at the immediate vicinity of the tumor nests. CEMP1 was found in the cytoplasm of tumor cells in 5 cases, but its expression was negative in the stromal tissues. Also, cancer lines PC-3 and MCF-7 showed CEMP1 expression; however, CAP expression was observed only in MCF-7 cells. Conclusions: The results suggest that CEMP1 and CAP are present in tissues other that cementum and possibly contribute to pathological conditions such as metastatic cancer.

Stem cells of the lamina propria of human oral mucosa and gingiva develop into mineralized tissues in vivo.

AIMS: To characterize the mineralized tissue formed constitutively in the supracalvarial region of scid mice by a primitive stem cell population (hOMSC) derived from the lamina propria of the human oral mucosa and gingiva. MATERIAL AND METHODS: Fibrin-hOMSC constructs were cultured for 14 days at which time point they were analysed for the expression of osteoblastic/cementoblastic markers and implanted between the skin and calvaria bones into scid mice. After 8 weeks, the animals were sacrificed and the implantation sites analysed. RESULTS: Two-week-old cultures of fibrin-hOMSC constructs expressed osteogenic/cementogenic markers at the gene level. Macroscopic and radiographic examinations revealed mineralized masses at the implantation sites of fibrin-hOMSC constructs. Histology, histochemistry and immunofluorescence showed mineralized masses consisting of avascular cellular and acellular matrices that stained positively for collagen, Ca, cementum attachment protein, cementum protein 1, bone sialoprotein, alkaline phosphatase, osteocalcin, amelogenin and ameloblastin. Positive anti-human nuclear antigen indicated the human origin of the cells. Atomic force microscopy depicted long prismatic structures organized in lamellar aggregates. CONCLUSIONS: Within the limitation of this study, the results indicate for the first time that fibrin-hOMSC constructs are endowed with the constitutive capacity to develop into mineralized tissues that exhibit certain similarities to cementum and bone.