Molecular Dynamic Simulations for Biopolymers with Biomedical Applications.

Computational modeling (CM) is a versatile scientific methodology used to examine the properties and behavior of complex systems, such as polymeric materials for biomedical bioengineering. CM has emerged as a primary tool for predicting, setting up, and interpreting experimental results. Integrating in silico and in vitro experiments accelerates scientific advancements, yielding quicker results at a reduced cost. While CM is a mature discipline, its use in biomedical engineering for biopolymer materials has only recently gained prominence. In biopolymer biomedical engineering, CM focuses on three key research areas: (A) Computer-aided design (CAD/CAM) utilizes specialized software to design and model biopolymers for various biomedical applications. This technology allows researchers to create precise three-dimensional models of biopolymers, taking into account their chemical, structural, and functional properties. These models can be used to enhance the structure of biopolymers and improve their effectiveness in specific medical applications. (B) Finite element analysis, a computational technique used to analyze and solve problems in engineering and physics. This approach divides the physical domain into small finite elements with simple geometric shapes. This computational technique enables the study and understanding of the mechanical and structural behavior of biopolymers in biomedical environments. (C) Molecular dynamics (MD) simulations involve using advanced computational techniques to study the behavior of biopolymers at the molecular and atomic levels. These simulations are fundamental for better understanding biological processes at the molecular level. Studying the wide-ranging uses of MD simulations in biopolymers involves examining the structural, functional, and evolutionary aspects of biomolecular systems over time. MD simulations solve Newton's equations of motion for all-atom systems, producing spatial trajectories for each atom. This provides valuable insights into properties such as water absorption on biopolymer surfaces and interactions with solid surfaces, which are crucial for assessing biomaterials. This review provides a comprehensive overview of the various applications of MD simulations in biopolymers. Additionally, it highlights the flexibility, robustness, and synergistic relationship between in silico and experimental techniques.

In Vitro Bone Differentiation of 3D Microsphere from Dental Pulp-Mesenchymal Stem Cells.

Bone defects lead to the structural loss of normal architecture, and those in the field of bone tissue engineering are searching for new alternatives to aid bone regeneration. Dental pulp-mesenchymal stem cells (DP-MSC) could provide a promising alternative to repair bone defects, principally due to their multipotency and capacity to fabricate three-dimensional (3D) spheroids. The present study aimed to characterize the 3D DP-MSC microsphere and the osteogenic differentiation capacity potential cultured by a magnetic levitation system. To achieve this, the 3D DP-MSC microsphere was grown for 7, 14, and 21 days in an osteoinductive medium and compared to 3D human fetal osteoblast (hFOB) microspheres by examining the morphology, proliferation, osteogenesis, and colonization onto PLA fiber spun membrane. Our results showed good cell viability for both 3D microspheres with an average diameter of 350 µm. The osteogenesis examination of the 3D DP-MSC microsphere revealed the lineage commitment, such as the hFOB microsphere, as evidenced by ALP activity, the calcium content, and the expression of osteoblastic markers. Finally, the evaluation of the surface colonization exhibited similar patterns of cell-spreading over the fibrillar membrane. Our study demonstrated the feasibility of forming a 3D DP-MSC microsphere structure and the cell-behavior response as a strategy for the applications of bone tissue guiding.

Citalopram improves vasomotor syndrome and urogenital syndrome of menopause in Mexican women: a randomized clinical trial.

PURPOSE: This study aimed to determine the efficacy of non-hormonal therapy with citalopram vs fluoxetine for treating vasomotor syndrome (VMS) and urogenital syndrome of menopause (GSM) in Mexican women. METHODS: A parallel prospective randomized clinical trial was conducted in 91 postmenopausal women with a total score on the Menopause Rating Scale (MRS) ≥ 17 and with the clinical diagnosis of VSM and GSM. Patients were randomly assigned to receive citalopram (n = 49) or fluoxetine (n = 42). Follow-up was carried out at 3 and 6 months. RESULTS: The citalopram group experienced a significant improvement compared to the fluoxetine group in the MRS total score (p < 0.01), as well as in the psychological (p < 0.001) and somatic (p < 0.0001) domains at 3 and 6 months of follow-up. After 6 months of follow-up, the group that received citalopram decreased the relative risk (RR) to present VMS symptoms (RR = 0.30, CI 0.19-0.5, p = 0.0001), depressed mood (RR = 0.31, CI 0.15-0.6, p = 0.0002), irritability (RR = 0.40, CI 0.22-0.73, p = 0.002), anxiety (RR = 0.30, CI 0.13-0.69, p = 0.003), physical and mental exhaustion (RR = 0.35, CI 0.18-0.67, p = 0.001), sexual problems (RR = 0.18, CI 0.06-0.48, p = 0.0001), vaginal dryness (RR = 0.34, CI 0.14-0.80, p = 0.01), and urinary problems (RR = 0.36, CI 0.14-0.92, p = 0.043). CONCLUSION: We conclude that citalopram tends to improve VSM and GSM symptoms in postmenopausal Mexican women. Thus, we recommend the daily use of citalopram 20 mg. However, further studies will be required to support the results of the present work. These should include a larger number of patients and a placebo group. CLINICAL TRIAL REGISTRATION: This clinical trial was retrospectively registered by the United States National Library of Medicine in the www. CLINICALTRIALS: gov database on 04/20/2022. The given test Registration Number is NCT05346445.

3D-Printed Tubular Scaffolds Decorated with Air-Jet-Spun Fibers for Bone Tissue Applications.

The fabrication of instructive materials to engineer bone substitute scaffolds is still a relevant challenge. Current advances in additive manufacturing techniques make possible the fabrication of 3D scaffolds with even more controlled architecture at micro- and submicrometric levels, satisfying the relevant biological and mechanical requirements for tissue engineering. In this view, integrated use of additive manufacturing techniques is proposed, by combining 3D printing and air-jet spinning techniques, to optimize the fabrication of PLA tubes with nanostructured fibrous coatings for long bone defects. The physicochemical characterization of the 3D tubular scaffolds was performed by scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry, profilometry, and mechanical properties. In vitro biocompatibility was evaluated in terms of cell adhesion, proliferation, and cell-material interactions, by using human fetal osteoblasts to validate their use as a bone growth guide. The results showed that 3D-printed scaffolds provide a 3D architecture with highly reproducible properties in terms of mechanical and thermal properties. Moreover, nanofibers are collected onto the surface, which allows forming an intricate and interconnected network that provides microretentive cues able to improve adhesion and cell growth response. Therefore, the proposed approach could be suggested to design innovative scaffolds with improved interface properties to support regeneration mechanisms in long bone treatment.

3D spheroid cell cultures and their role in bone regeneration: a systematic review / Cultivos de esferoides 3D y su papel en la regeneración ósea: una revisión sistemática

Abstract Recently, the 3D spheroid cell culture application has been extensively used in the treatment of bone defects. A wide variety of methodologies have been used, which has made the comparison of results complex. Therefore, this systematic review has two aims: (i) to perform an analysis focused on the role of 3D spheroid cell culture in bone regeneration strategies; and (ii) address the main challenges in clinical application. A search of the following keywords "3D cell culture", "spheroid", and "bone regeneration" was carried out in the PubMed, Scopus, and ScienceDirect databases and limited to the years 2010-2020. Studies were included if their primary objective was the behavior of cell aggregates to formed spheroids structures by different 3D cell culture techniques focused on the regeneration of bone tissue. To address the risk of bias for in vitro studies, the United States national toxicology program tool was applied, and descriptive statistics of the data were performed, with the SPSS V.22 program. A total of 16 studies were included, which met the established criteria corresponding to in vitro and in vitro/in vivo studies; most of these studies used stem cells for the 3D cell spheroids. The most often methods used for the 3D formation were low adherence surface and rotational methods, moreover, mesenchymal stem cells were the cell line most frequently used because of their regenerative potential in the field of bone tissue engineering. Although the advances in research on the potential use of 3D spheroids in bone regeneration have made great strides, the constant innovation in cell spheroid formation methodologies means that clinical application remains in the future as strategy for 3D tissue bioprinting.

Resumen Recientemente, la aplicación del cultivo 3D de esferoides se ha utilizado ampliamente en el tratamiento de defectos óseos. La variedad de metodologías para lograr los cultivos 3D de esferoides ha hecho compleja la comparación de resultados. Por tanto, esta revisión sistemática tiene dos objetivos: (i) realizar un análisis centrado en el papel de los cultivos 3D de esferoides en las estrategias de regeneración ósea; y (ii) abordar los principales desafíos en la aplicación clínica. Se realizó una búsqueda de las siguientes palabras clave "cultivo celular 3D", "esferoide" y "regeneración ósea" en las bases de datos PubMed, Scopus y ScienceDirect y se limitó a los años 2010-2020. Se incluyeron los estudios si su principal objetivo era el comportamiento de agregados celulares para generar las estructuras esferoidales desarrollados por diferentes técnicas de cultivo celular 3D enfocadas a la regeneración del tejido óseo. Para abordar el riesgo de sesgo de los estudios in vitro, se aplicó la herramienta del programa nacional de toxicología de Estados Unidos y se realizaron estadísticas descriptivas de los datos, con el programa SPSS V.22. Se incluyeron un total de 16 estudios, que cumplieron con los criterios establecidos correspondientes a estudios in vitro e in vitro/in vivo; la mayoría de estos estudios utilizaron células troncales para generar los esferoides celulares 3D. Los métodos más utilizados para la formación de los esferoides 3D fueron la superficie de baja adherencia y los métodos de rotación, asimismo, la línea celular de células troncales mesenquimales fueron las más utilizadas debido a su gran potencial regenerativo en el campo de la ingeniería de tejidos óseos. Aunque los avances en la investigación sobre el uso potencial de los cultivos celulares de esferoides 3D en la regeneración ósea han logrado grandes avances, la constante innovación en las metodologías de la generación de esferoides 3D deja claro que la aplicación clínica de estos permanecerá en el futuro como estrategia en la bioimpresión tisular.

Composite Fiber Spun Mat Synthesis and In Vitro Biocompatibility for Guide Tissue Engineering.

Composite scaffolds are commonly used strategies and materials employed to achieve similar analogs of bone tissue. This study aims to fabricate 10% wt polylactic acid (PLA) composite fiber scaffolds by the air-jet spinning technique (AJS) doped with 0.5 or 0.1 g of zirconium oxide nanoparticles (ZrO2) for guide bone tissue engineering. ZrO2 nanoparticles were obtained by the hydrothermal method and characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). SEM and fourier-transform infrared spectroscopy (FTIR) analyzed the synthesized PLA/ZrO2 fiber scaffolds. The in vitro biocompatibility and bioactivity of the PLA/ZrO2 were studied using human fetal osteoblast cells. Our results showed that the hydrothermal technique allowed ZrO2 nanoparticles to be obtained. SEM analysis showed that PLA/ZrO2 composite has a fiber diameter of 395 nm, and the FITR spectra confirmed that the scaffolds' chemical characteristics are not affected by the synthesized technique. In vitro studies demonstrated that PLA/ZrO2 scaffolds increased cell adhesion, cellular proliferation, and biomineralization of osteoblasts. In conclusion, the PLA/ZrO2 scaffolds are bioactive, improve osteoblasts behavior, and can be used in tissue bone engineering applications.

Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering.

Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-ß1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.

Antimicrobial effect of gold nanoparticles in the formation of the Staphylococcus aureus biofilm on a polyethylene surface.

The main of this study was to evaluate the inhibitory effect on the in vitro formation of the Staphylococcus aureus biofilm formed on a polyethylene (PE) surface with a nanostructured Gold (Au) coating for medical devices. An experimental in vitro study was carried out using PE discs with an Au nanoparticle coating (AuNPs) on one side (experimental group) and without coating on the other (control group); the discs were mounted in the CDC biofilm reactor adding broth of yeast-dextrose-peptone (YPD) sterile culture inoculated with S. aureus in a cell suspension (5 × 108 cells/ml). The specimens were evaluated at different times (6, 12, 24, 48, 72 h) and stained with the Live/Dead Bacterial Viability Kit (Invitrogen) for observation, analysis, and quantification with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that as evaluation time passed an increasing of S. aureus biofilm formation was observed in the control group, in the experimental group, a statistically significant biofilm inhibition was observed with respect to the AuNPs uncoated specimens (p ≤ 0.05) and showed a ratio of almost 4:1 viable/nonviable in the biofilm of the uncoated surfaces, with a difference > 5 Log10 in the CFU counts. The PE with AuNP coating showed an inhibitory effect on the biofilm formation of S. aureus.

Green synthesis of ZnO nanoparticles using a Dysphania ambrosioides extract. Structural characterization and antibacterial properties.

The Structural properties of Zinc oxide nanoparticles (ZnO-NPs) as well as their antibacterial properties against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa; as well as bacteria that are usually found in the mouth of humans and are related to dental conditions, such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans and Streptococcus sanguinis, are presented in this report. ZnO-NPs were grown by green synthesis, using the Mexican plant Dysphania ambrosioides known in Mexico as "epazote", which was used by native populations of Mexico as a dewormer, is currently used widely in traditional Mexican cuisine and is rich in organic compounds as flavonoids and terpenes which may favor the synthesis of nanoparticles (NPs). ZnO-NPs were synthesized by the mentioned technology and were compared with commercial ZnO-NPs as a reference. Synthesized and commercial ZnO-NPs were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), X-ray energy dispersive spectroscopy (EDS), Fourier transformed infrared spectroscopy (FTIR) and thermogravimetry (TG). Antibacterial properties were evaluated using a disc diffusion test (Kirby-Bauer method). The results indicate that ZnO-NPs were synthesized in the size range of 5-30 nm. The presence of the ZnO crystalline phase was identified by high resolution transmission electron microscopy (HRTEM) and XRD analysis. The commercial ZnO-NPs were in the size range of 15-35 nm. The antibacterial test indicates that most of the bacterial strains used in this study were sensitive to synthesized and commercial NPs, with Prevotella intermedia being the most sensitive to ZnO-NPs.

In vivo Regeneration of Mineralized Bone Tissue in Anisotropic Biomimetic Sponges.

In the last two decades, alginate scaffolds have been variously studied as extracellular matrix analogs for tissue engineering. However, relevant evidence is still lacking concerning their ability to mimic the microenvironment of hierarchical tissues such as bone. Hence, an increasing amount of attention has recently been devoted to the fabrication of macro/microporous sponges with pore anisotropy able to more accurately replicate the cell niche structure as a trigger for bioactive functionalities. This paper presents an in vivo study of alginate sponges with anisotropic microporous domains (MAS) formed by ionic crosslinking in the presence of different fractions (30 or 50% v) of hydroxyapatite (HA). In comparison with unloaded sponges (MAS0), we demonstrated that HA confers peculiar physical and biological properties to the sponge, depending upon the inorganic fraction used, enabling the sponge to bio-mimetically support the regeneration of newly formed bone. Scanning electron microscopy analysis showed a preferential orientation of pores, ascribable to the physical constraints exerted by HA particles during the pore network formation. Energy dispersive spectroscopy (EDS) and X-Ray diffraction (XRD) confirmed a chemical affinity of HA with the native mineral phase of the bone. In vitro studies via WST-1 assay showed good adhesion and proliferation of human Dental Pulp-Mesenchymal Stem Cells (hDP-MSC) that increased in the presence of the bioactive HA signals. Moreover, in vivo studies via micro-CT and histological analyses of a bone model (e.g., a rat calvaria defect) confirmed that the maximum osteogenic response after 90 days was achieved with MAS30, which supported good regeneration of the calvaria defect without any evidence of inflammatory reaction. Hence, all of the results suggested that MAS is a promising scaffold for supporting the regeneration of hard tissues in different body compartments.

Mesenchymal Stem/Stromal Cells Derived from Dental Tissues: A Comparative In Vitro Evaluation of Their Immunoregulatory Properties Against T cells.

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory properties and have been used as immune regulators for the treatment of graft-versus-host disease (GVHD). Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to BM-MSCs for potential clinical applications because of their accessibility and easy preparation. The aim of this in vitro study was to compare MSCs from dental pulp (DP-MSCs), gingival tissue (G-MSCs), and periodontal ligament (PDL-MSCs) in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells to determine which MSCs would be the most appropriate for in vivo immunoregulatory applications. BM-MSCs were included as the gold standard. Our results demonstrated, in vitro, that MSCs from DP, G, and PDL showed immunoregulatory properties similar to those from BM, in terms of the cellular proliferation inhibition of both CD4+- and CD8+-activated T-cells. This reduced proliferation in cell co-cultures correlated with the production of interferon-γ and tumor necrosis factor alpha (TNF-α) and the upregulation of programmed death ligand 1 (PD-L1) in MSCs and cytotoxic T-cell-associated Ag-4 (CTLA-4) in T-cells and increased interleukin-10 and prostaglandin E2 production. Interestingly, we observed differences in the production of cytokines and surface and secreted molecules that may participate in T-cell immunosuppression in co-cultures in the presence of DT-MSCs compared with BM-MSCs. Importantly, MSCs from four sources favored the generation of T-cell subsets displaying the regulatory phenotypes CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+. Our results in vitro indicate that, in addition to BM-MSCs, MSCs from all of the dental sources analyzed in this study might be candidates for future therapeutic applications.

Neuronal Transdifferentiation Potential of Human Mesenchymal Stem Cells from Neonatal and Adult Sources by a Small Molecule Cocktail.

Human mesenchymal stem cells (MSCs) are good candidates for brain cell replacement strategies and have already been used as adjuvant treatments in neurological disorders. MSCs can be obtained from many different sources, and the present study compares the potential of neuronal transdifferentiation in MSCs from adult and neonatal sources (Wharton's jelly (WhJ), dental pulp (DP), periodontal ligament (PDL), gingival tissue (GT), dermis (SK), placenta (PLAC), and umbilical cord blood (UCB)) with a protocol previously tested in bone marrow- (BM-) MSCs consisting of a cocktail of six small molecules: I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dbcAMP (ICFRYA). Neuronal morphology and the presence of cells positive for neuronal markers (TUJ1 and MAP2) were considered attributes of neuronal induction. The ICFRYA cocktail did not induce neuronal features in WhJ-MSCs, and these features were only partial in the MSCs from dental tissues, SK-MSCs, and PLAC-MSCs. The best response was found in UCB-MSCs, which was comparable to the response of BM-MSCs. The addition of neurotrophic factors to the ICFRYA cocktail significantly increased the number of cells with complex neuron-like morphology and increased the number of cells positive for mature neuronal markers in BM- and UCB-MSCs. The neuronal cells generated from UCB-MSCs and BM-MSCs showed increased reactivity of the neuronal genes TUJ1, MAP2, NF-H, NCAM, ND1, TAU, ENO2, GABA, and NeuN as well as down- and upregulation of MSC and neuronal genes, respectively. The present study showed marked differences between the MSCs from different sources in response to the transdifferentiation protocol used here. These results may contribute to identifying the best source of MSCs for potential cell replacement therapies.

Expresión de la osteocalcina en el ligamento periodontal al inducir fuerzas ortodóncicas / Osteocalcin expression in periodontal ligament when inducing orthodontic forces

La osteocalcina es una proteína no colágena presente en hueso alveolar, cemento radicular y subpoblaciones del ligamento periodontal. Esta proteína juega un papel importante en la biomineralización y en la matriz extracelular regulando la maduración de los cristales de hidroxiapatita y en el reclutamiento de los osteoclastos participando en la remodelación ósea. La remodelación y la nueva formación de tejido periodontal es parte esencial durante los movimientos ortodóncicos, los cuales al aplicar fuerzas causan tensión en las células provocando una adaptación que se traduce en respuestas celulares y moleculares que pueden afectar la matriz extracelular. Por ello, el propósito de esta investigación fue determinar la expresión de la osteocalcina asociada a la remodelación periodontal cuando se aplican fuerzas ortodóncicas. En primeros premolares superiores e inferiores se colocó aparatología fija prescripción Roth 0.022 con un arco NiTi 0.016, la cual se aplicó a todos los dientes de ambas arcadas con excepción de los premolares superiores e inferiores izquierdos. Los premolares sin aparatología (t = 0) y en presencia de aparatología para inducir movimientos ortodóncicos durante 1, 3, 5, 7 y 9 días; fueron extraídos para analizar la expresión de la osteocalcina en la matriz extracelular del ligamento periodontal. Para determinar la expresión temporal y espacial de los mensajeros de la osteocalcina en el ligamento periodontal se llevó a cabo la técnica RT-PCR. La expresión de la osteocalcina en el grupo experimental estuvo presente en todos los días de prueba, sugiriendo que los movimientos ortodónticos generan cambios que son susceptibles en las concentraciones del mensajero de la proteína osteocalcina.

Osteocalcin is a non-collagenous protein located in alveolar bone, root cementum and subpopulations of periodontal ligament cells. This protein plays an important role in the biomineralization process and in the extra-cellular matrix, regulating maturation of hydroxyapatite and osteoclast recruitment which participate in bone remodeling. Periodontal tissue new formation and remodeling is a vital part of the process during orthodontic movements. These movements, when force is exerted, cause tension in the cells, provoking adaptation which results in molecular and cellular responses which, in turn, can affect the extracellular matrix. Due to the aforementioned facts, the aim of the present research was to determine osteocalcin expression associated to periodontal remodeling when orthodontic forces are applied. Roth 0.022 " fixed brackets with a NiTi 0.016" archwire were applied to first upper and lower bicuspids. This was applied to all teeth of both arches except to left lower and upper bicuspids. Bicuspids without brackets (t = 0) as well as with brackets to elicit orthodontic movements during 1, 3, 5, 7 and 9 days were extracted to assess osteocalcin expression in the extra-cellular matrix of the periodontal ligament. The RT-PCR technique was followed to determine temporal and spatial expression of osteocalcin messengers. Osteocalcin expression in the experimental group was present in all test days, suggesting thus the fact that orthodontic movements elicit changes that are susceptible in osteocalcin protein messenger concentrations.

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.

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.

Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line.

The effect of human anti-cementoblastoma-derived protein antibody during cementogenesis in vitro was investigated by using human cementoblastoma-derived cells. Cultures treated with 5 microg/ml of CP antibody from day 1 to day 15 revealed a significant decrease in alkaline phosphatase activity (ALP) 40% (p < 0.005), 44% (p < 0.001), 49% (p < 0.1), and 45% (p < 0.02) at 9, 11, 13, and 15 days, respectively. Immunoexpression of osteopontin revealed that in cultures treated with anti-CP antibody, the positive number of cementoblastoma cells was reduced by 87, 83, 69, and 52% at 5, 7, 9, and 11 days, respectively. Bone sialoprotein immunoexpression showed a decrease in positive cells of 82, 51, 60, 80, 83, and 87% at 5, 7, 9, 11, 13, and 15 days, respectively, as compared to controls. The Ca/P ratio of the mineral-like tissue deposited in vitro by cementoblastoma cells revealed that control cultures had a Ca/P ratio of 1.45 and 1.61 at 5 and 15 days, whereas experimental cultures revealed a Ca/P ratio of 0.50 and 0.79 at 5 and 15 days, respectively. Electron diffraction patterns showed inner double rings representing D-spacing that were consistent with those of hydroxyapatite in both control and experimental cultures. Examination of the crystallinity with high resolution transmission electron microscopy showed homogeneous and preferential spatial arrangement of hydroxyapatite crystallites in control and experimental cultures at 15 days. Atomic force microscopy images of control cultures at 5 and 15 days revealed small granular particles and grain agglomeration that favored the formation of crystalline plaques with a lamellar-like pattern of the mineral-like tissue. Experimental cultures at 5 and 15 days showed tiny and homogeneous granular morphology. The agglomerates maintained spherical morphology without organization of needle-like crystals to form plaque-like structures. Based on these findings, it is hypothesized that cementoblastoma-derived protein may be associated to crystal growth, compositional and morphological features during the mineralization process of cementum in vitro.