Amino acid composition of nanofibrillar self-assembling peptide hydrogels affects responses of periodontal tissue cells in vitro.

BACKGROUND: The regeneration of tissue defects at the interface between soft and hard tissue, eg, in the periodontium, poses a challenge due to the divergent tissue requirements. A class of biomaterials that may support the regeneration at the soft-to-hard tissue interface are self-assembling peptides (SAPs), as their physicochemical and mechanical properties can be rationally designed to meet tissue requirements. MATERIALS AND METHODS: In this work, we investigated the effect of two single-component and two complementary ß-sheet forming SAP systems on their hydrogel properties such as nanofibrillar architecture, surface charge, and protein adsorption as well as their influence on cell adhesion, morphology, growth, and differentiation. RESULTS: We showed that these four 11-amino acid SAP (P11-SAP) hydrogels possessed physico-chemical characteristics dependent on their amino acid composition that allowed variabilities in nanofibrillar network architecture, surface charge, and protein adsorption (eg, the single-component systems demonstrated an ~30% higher porosity and an almost 2-fold higher protein adsorption compared with the complementary systems). Cytocompatibility studies revealed similar results for cells cultured on the four P11-SAP hydrogels compared with cells on standard cell culture surfaces. The single-component P11-SAP systems showed a 1.7-fold increase in cell adhesion and cellular growth compared with the complementary P11-SAP systems. Moreover, significantly enhanced osteogenic differentiation of human calvarial osteoblasts was detected for the single-component P11-SAP system hydrogels compared with standard cell cultures. CONCLUSION: Thus, single-component system P11-SAP hydrogels can be assessed as suitable scaffolds for periodontal regeneration therapy, as they provide adjustable, extracellular matrix-mimetic nanofibrillar architecture and favorable cellular interaction with periodontal cells.

Comparative evaluation of the antimicrobial susceptibility and cytotoxicity of husk extract of Cocos nucifera and chlorhexidine as irrigating solutions against Enterococcus Faecalis, Prevotella Intermedia and Porphyromonas Gingivalis - An in-vitro study.

AIM AND BACKGROUND: The aim of the present study is to evaluate and compare the antimicrobial susceptibility and cytotoxicity of Cocos nucifera and chlorhexidine (CHX) as irrigating solutions against Enterococcus faecalis, Prevotella intermedia, and Porphyromonas gingivalis. MATERIALS AND METHODS: The ethanolic extract of husk of C. nucifera was prepared. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract were determined using the serial broth dilution method and its cytotoxicity was evaluated against human periodontal fibroblasts using 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay. Antibacterial susceptibility for two irrigating solutions, namely 2% CHX gluconate irrigant (Group I) and 1.5% C. nucifera husk irrigant (Group II), was tested against P. gingivalis, P. intermedia, and E. faecalis. RESULTS: The MIC and MBC of C. nucifera husk extract for P. gingivalis were 468.75 µg/ml and 1562.5 µg/ml, for P. intermedia were 48.8 µg/ml and 1875 µg/ml, and for E. faecalis were 1562.5 µg/ml and 3750 µg/ml, respectively. The extract was nontoxic to the human periodontal fibroblast. Both the materials have shown similar antibacterial susceptibility and no difference was observed at baseline, 10, 30, and 60 min using two-way repeated measures of ANOVA. However, a statistically significant difference was observed between different time points for P. gingivalis and P. intermedia using Bonferroni multiple comparison test (f = 826.1390, P ≤ 0.05). CONCLUSION: 1.5% of ethanolic husk extract of C. nucifera has a significant antibacterial action against polymicrobial dental biofilm and its activity is comparable to that of 2% CHX which validates its use as a future irrigating solution for overcoming bacterial resistance with synthetic agents.

[Effects of Bushenguchiwan on expression of matrix metalloproteinase-13 in rats' periodontium].

OBJECTIVE: To investigate the influence of Bushenguchiwan on expression of matrix metalloproteinase-13 (MMP-13) in periodontium of rats with experimental periodontitis. METHODS: The model of experimental periodontitis of rats was established and treated by Bushenguchiwan with different doses. The periodontal tissues from groups of different doses were immunohistochemically stained by antibody of MMP-13. The expression of MMP-13 was examined and semi-quantitative analysis of signals performed by integrated absorbance. RESULTS: MMP-13 was intensely positive in gingival epithelial cells and periodontal fibroblasts in periodontitis models and negative in normal rat periodontal tissues. After 30 days of Bushenguchiwan treatment with high dose, middle dose and low dose, the expression of MMP-13 (2.9103 ± 0.5534, 3.6588 ± 0.4330, 4.4550 ± 0.4255) was down-regulated respectively compared with model rats (5.3233 ± 0.7993), P < 0.05. After 60 days of treatment the expression of MMP-13 (2.1855 ± 0.5381, 2.8558 ± 0.4759, 3.8980 ± 0.5885) was down-regulated more significantly. with model rats (6.2693 ± 0.4538), P < 0.05. CONCLUSIONS: Bushenguchiwan could down-regulate the expression of MMP-13 in rats' periodontium and the high dose group had better effect.

Isolation and characterization of postnatal stem cells from human dental tissues.

It was reported that postnatal stem cells are present in adult tissues such as bone marrow, liver, muscle, dental pulp, and periodontal ligament. We isolated postnatal stem cells from human dental tissues such as dental pulp (DPSC), periodontal ligament (PDLSC), periapical follicle (PAFSC), and the surrounding mandibular bone marrow (MBMSC) to ascertain their properties. Immunocytochemistry proved the existence of stem cells in these cell populations using STRO-1 as a stem cell marker. These cells also expressed the mesenchymal stem cell (MSC) markers CD29 and CD44. The isolated cells showed self-renewal capabilities and colony-forming efficiency. Almost all of the dental stem cells showed optimal growth when they were cultured in alpha modification of Eagle's medium (alpha-MEM) supplemented with 10% fetal calf serum (FCS) and 100 microM ascorbic acid. Only the PAFSC showed increased proliferation in 20% FCS and 50 microM ascorbic acid. All of the dental stem cells were capable of differentiating into adipocytes and mineral nodule forming cells. MBMSC, in particular, showed much better mineralization compared to the others. These results indicate that MSCs exist in various tissues of the teeth and can differentiate into osteoblasts, adipocytes, and other kinds of cells with varying efficiency.

Effect of mechanical loading on periodontal cells.

Mechanical loading is an important regulatory factor in alveolar bone homeostasis, and plays an essential role in maintaining the structure and mass of the alveolar processes throughout lifetime. A better understanding of the cellular and molecular responses of periodontal cells is a prerequisite for further improvements of therapeutic approaches in orthodontics, periodontal and alveolar bone repair and regeneration, implantology, and post-surgical wound healing. The purpose of this review is to provide an insight into some cell culture and animal models used for studying the effects of mechanical loading on periodontal cells, and into the recent developments and utilization of new in vivo animal models. There has been an increased awareness about the need for improvement and development of in vivo models to supplement the widely used cell culture models, and for biological validation of in vitro results, especially in the light of evidence that developmental models may not always reflect bone homeostasis in an adult organism. Due to the limitations of in vivo models, previous studies on mechanical regulation of alveolar bone osteoblasts and cementoblasts mostly focused on proliferative responses, rather than on the stimulation of cell differentiation. To address this problem, we have recently characterized and implemented a mouse osteoinductive tooth movement model for studying mechanically induced regulation of osteoblast- and cementoblast-associated genes. In this model, a defined and reproducible mechanical osteogenic loading is applied during a time course of up to two weeks. Regulation of gene expression in either wild-type or transgenic animals is assessed by a relative quantitative measurement of the level of target mRNAs directly within the subpopulations of periodontal cells. To date, results demonstrate a defined temporal pattern of cell-specific gene regulation in periodontal osteoblasts mechanically stimulated to differentiate and deposit bone matrix. The responses of osteoblast-associated genes to mechanical loading were 10- to 20-fold greater than the increase in the numbers of these cells, indicating that the induction of differentiation and an increase of cell function are the primary responses to osteogenic loading. The progression of the osteoblast phenotype in the intact mouse periodontium was several-fold faster compared with that in cultured cells, suggesting that the mechanical signal may be targeting osteoblast precursors in the state of readiness to respond to an environmental challenge, without the initial proliferative response. An early response of alkaline phosphatase and bone sialoprotein genes was detected after 24 hrs of treatment, followed by a concomitant stimulation of osteocalcin and collagen I between 24 and 48 hrs, and deposition of osteoid after 72 hrs. Although cementoblasts constitutively express biochemical markers similar to those of osteoblasts, distinct responses of osteocalcin, collagen I, and bone sialoprotein genes to mechanical loading were observed in the two cell phenotypes. This finding indicates that differential genetic responses to mechanical loading provide functional markers for distinction of the cementoblast and osteoblast phenotypes.

Enhanced production of mineralized nodules and collagenous proteins in vitro by calcium ascorbate supplemented with vitamin C metabolites.

BACKGROUND: Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate). METHODS: Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence. RESULTS: In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate. CONCLUSIONS: In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.