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Constructs intended for bone tissue engineering are influenced by the initial cell seeding procedure. The seeding method should be rapid, convenient, improve cell spatial distribution, and have no negative effects on cellular viability and differentiation. This study aimed to compare the effect of short-run seeding methods (centrifuge and vortex) with a static method on the scaffolds prepared from poly(L-lactide-co-1,5-dioxepan-2-one) by solvent-casting particulate-leaching (SCPL) technique. Human osteoblast-like cells (HOB) were seeded by the three methods described above. The seeding efficiency was determined by attached cell numbers. Cellular proliferation was analyzed by WST-1 and dsDNA assay. Cell distribution was examined by scanning electron (SEM) and fluorescence microscopy. Expression of Alkaline Phosphatase (ALP), Collagen type I (Col I), Osteocalcin (OC) and Proliferating Cell Nuclear Antigen (PCNA) were determined by real time RT-PCR. Results indicated that centrifuge and vortex increased seeding efficiency and had no negative effects on cellular viability. The data obtained by the fluorescence microscope confirmed the SEM results that the vortex method improved cell distribution through the scaffolds more than the other two methods (p

In this in vitro study, the initial response of human periodontal ligament (PDL) cells and alveolar osteoblast-like cells (HOB) to three biodegradable polymers with varying pore size and different mechanical properties were evaluated. Scaffolds were synthesized from poly(L-lactide), [poly(LLA)], poly(L-lactide-co-1,5-dioxepan-2-one), [poly(LLA-co-DXO)], poly(L-lactide-co-ε-caprolactone), and [poly(LLA-co-CL)] with pore sizes greater or less than 90 µm by salt leaching. Cells were obtained from patients undergoing routine oral surgery. After 2—4 passages, the cells were grown on scaffolds and in culture plates (control) for 3 h (PDL cells), 3 days (PDL cells and HOB), 10 and 14 days (HOB), respectively. The cellular morphology and spreading were determined by scanning electron microscopy (SEM) and the attachment and proliferation were evaluated by MTT assays. The SEM images revealed heterogeneous cellular morphology and good spreading. Cellular attachment and proliferation were significantly higher on poly(LLA-co-DXO) and poly(LLA-co-CL) than on poly(LLA) scaffolds (p = 0.003) and highest for poly(LLA-co-DXO). Expression of bone formation markers, collagen-I (COL-I), transforming growth factor-β 1 (TGF-β1), and osteocalcin (OCN), was determined by ELISA. The expression of COL-1 was similar for HOB and PDL cells, but significantly higher for pore size >90 µm while the HOB expression of TGFβ 1 and OCN was greater on poly(LLA-co-CL) and poly(LLA-co-DXO) than on poly(LLA) scaffolds.

The toxicity of formaldehyde, a monomer released from certain polymeric dental materials, was studied in cultured human oral fibroblasts and epithelial cells. The influences of growth conditions were evaluated for both cell types, as well as the role of the internal and external thiol states. A one-hour exposure to formaldehyde decreased the colony-forming efficiency (CFE) of both cell types in a concentration-dependent manner, although the toxicity varied up to 100-fold with the conditions. Clearly, the presence of serum and the thiol cysteine counteracted the toxicity in fibroblasts. Similarly, pituitary extract and cysteine, or a mixture of amino acids and ethanolamines, counteracted the formaldehyde toxicity in serum-free cultures of epithelial cells. In contrast, a growth-promoting surface matrix of fibronectin and collagen did not influence the formaldehyde toxicity, as shown by both the CFE assay and a dye reduction assay. Further, a short-term change to the various growth media per se with or without the supplements serum or cysteine did not significantly alter the CFE. Analysis of the thiol state demonstrated significant differences between epithelial cells and fibroblasts, i.e., comparatively lower cellular levels of the free low-molecular-weight thiols glutathione and cysteine in fibroblasts. This result correlated to significantly higher formaldehyde toxicity in the fibroblasts than in the epithelial cells. Taken together, the results indicated the cytoprotective function of both intracellular and extracellular thiols toward formaldehyde, as well as the usefulness of thiol-free and chemically defined conditions for toxicity assessments in oral epithelial cells and fibroblasts. We conclude that the combined use of a controlled external milieu and the presumed target cell type may be advantageous in evaluations of oral toxicity mechanisms or the toxic potency of dental materials, particularly those which, like formaldehyde, may react with thiols or amines.

The occurrence and distribution of several neurochemical markers were investigated. Numerous nerve fibres were shown, using antibodies to protein gene product (PGP) 9.5, neurone-specific enolase, calcitonin gene-related peptide (CGRP), substance P, neurokinin A or protein S-100. The presence of vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI), neuropeptide tyrosine, dopamine-β-hydroxylase (DBH), cholecystokinin/gastrin, glutamate and galanin was more scarce. Nerve fibres containing these above-mentioned markers were found at several locations, i.e. in the epithelium, connective tissue, and around blood vessels. In the taste buds, numerous PGP 9.5-, neurone-specific enolase-, CGRP-, substance P-, neurokinin A- and protein S-100-containing structures were found, but few VIP and galanin ones. No immunoreactivity was found with antibodies against somatostatin, bombesin, enkephalin or dynorphin. These findings extend knowledge about the general as well as the neurochemical messenger-based innervation of rat fungiform papillae, forming a firm basis for future functional investigations of normal, experimental and also clinical materials.

In tissue engineering, the resorbable aliphatic polyester poly(L-lactide) (PLLA) is used as scaffolds in bone regeneration. Copolymers of poly(L-lactide)-co-(e-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)], with superior mechanical properties to PLLA, have been developed to be used as scaffolds, but the influence on the osteogenic potential is unclear. This in vitro study of test scaffolds of poly(LLA-co-CL) and poly(LLA-co-DXO) using PLLA scaffolds as a control demonstrates the attachment and proliferation of human osteoblast-like cells (HOB) as measured by SEM and a methylthiazol tetrazolium (MTT) colorimetric assay, and the progression of HOB osteogenesis for up to 3 weeks; expressed as synthesis of the osteoblast differentiation markers: collagen type 1 (Col 1), alkaline phosphatase, bone sialoprotein, osteocalcin (OC), osteopontin and runt related gene 2 (Runx2). Surface analysis disclosed excellent surface attachment, spread and penetration of the cells into the pores of the test scaffolds compared to the PLLA. MTT results indicated that test scaffolds enhanced the proliferation of HOBs. Cells grown on the test scaffolds demonstrated higher synthesis of Col 1 and OC and also increased bone markers mRNA expression. Compared to scaffolds of PLLA, the poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds enhanced attachment, proliferation, and expression of osteogenic markers by HOBs in vitro. Therefore, these scaffolds might be appropriate carriers for bone engineering. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010

Background Improved understanding of the interactions between bone cells and endothelial cells involved in osteogenesis should aid the development of new strategies for bone tissue engineering. The aim of the present study was to determine whether direct communication between bone marrow stromal cells (MSC) and human umbilical vein endothelial cells (EC) could influence the osteogenic potential of MSC in osteogenic factor-free medium. Methods After adding EC to MSC in a direct-contact system, cell viability and morphology were investigated with the WST assay and immnostaining. The effects on osteogenic differentiation of adding EC to MSC was systematically tested by the using Superarray assay and results were confirmed with real-time PCR. Results Five days after the addition of EC to MSC in a ratio of 1:5 (EC/MSC) significant increases in cell proliferation and cellular bridges between the two cell types were detected, as well as increased mRNA expression of alkaline phosphatase (ALP). This effect was greater than that seen with addition of osteogenic factors such as dexamethasone, ascorbic acid and β-glycerophosphate to the culture medium. The expression of transcription factor Runx2 was enhanced in MSC incubated with osteogenic stimulatory medium, but was not influenced by induction with EC. The expression of Collagen type I was not influenced by EC but the cells grown in the osteogenic factor-free medium exhibited higher expression than those cultured with osteogenic stimulatory medium. Conclusion These results show that co-culturing of EC and MSC for 5 days influences osteogenic differentiation of MSC, an effect that might be independent of Runx2, and enhances the production of ALP by MSC.

Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factor beta-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca2+ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia.

Background Current conflicting information on the innervation of the human oral cavity indicates technical problems such as different detectability of the neural structures according to the various staining methods used and difficulties in reproducibility. The possibility of intraoral regional differences has not been properly considered. Methods Human biopsies of mucosa from different intraoral regions were prepared for immunohistochemistry using protein gene product 9.5 (PGP 9.5; a marker for neuronal structures). Results Nerves were found consistently in all the biopsies. The neural pattern showed clear regional differences. Intraepithelial nerve fibers were found in the gingiva, labia, palate, within certain fungiform papillae, and in some salivary excretory ducts. Organized nerve endings were found in varying frequencies in all but one (sublingual) region, appearing as lamellar (Meissner-like), coiled or glomerular neural structures. Merkel cell-neurite complexes were observed in the buccal, gingival, and palatal epithelia. Immunoreactive cells with many similarities to Merkel cells but without a neural connection were also encountered. Conclusions Conflicting results from earlier innervation studies of the oral cavity could be attributed to regional innervation differences. The distribution of the nerves also casts doubt on some of the present theories concerning the function(s) of intraoral nerves, such as the free nerve endings and the Merkel cell-neurite complexes. © 1996 Wiley-Liss, Inc.

The presence of distribution of several neurochemical markers in human fungiform papillae and taste buds were investigated by the immunohistochemical technique. The gustatory cells of the taste buds are in synaptic contact with sensory nerve endings, and considering the taste buds strictly as specialized sensory organs, the amounts and distribution of some of the neurochemical markers were different to what we expected. For example, few structures showed immunoreactivity to the tachykinins substance P (SP), calcitonin gene-related peptide (CGRP), and neurokinin A (NKA) also for the peptides vasoactive intestinal polypeptide (VIP), neuropeptide tyrosine (NPY) and galanin, low amounts of immunoreactivity occurred. On the other hand, using antibodies to protein gene product 9.5 (PGP 9.5), protein S-100, and glutamate, numerous nerve fibres and/or immunoreactive cells were found in the fungiform papillae, in the epithelium, in the connective tissue and around blood vessels, as well as in or near taste buds. Incubation with the antibodies against somatostatin, enkephalin, bombesin, peptide histidine isoleucine amide (PHI), cholecystokinin (CCK)/gastrin and dopamine-beta-hydroxylase (DBH) was negative for the fungiform papillae. In conclusion, the present study has shown several immunoreactive structures using antibodies against certain neurochemical markers. Further investigations will hopefully correlate these morphological findings with functional taste perception data. Future studies of patients with taste disorders or other pathological changes correlated with taste and tongue will also be of utmost importance.

With the aim of establishing conditions applicable to the testing of dental materials in human target cells, fibroblastic cell lines have been derived and grown from explants of human oral mucosa. Both a high-serum medium (termed "HSM") (CMRL 1066 supplemented with 10% fetal bovine serum) and a low-serum medium (termed "LSM") (a 1:1 mixture of M 199:MCDB 153 supplemented with 1.25% serum) supported radial outgrowths of cells from oral explants, as well as the subsequent transfer and growth of the cells in mass culture and at clonal density. Cells were typically fibroblastic in that they expressed vimentin uniformly, but did not express immunocytochemical markers of epithelial or endothelial cells. Cells derived in either LSM or HSM showed significantly higher colony-forming efficiency and clonal growth rate when transferred in LSM, as compared with HSM. Because cell migration occurred to a lesser extent in LSM, microscopic scoring of colony formation was also markedly facilitated. In both LSM and HSM, cellular low-molecular-weight thiols constituted about 30% of the total amount of sulfhydryls. Glutathione was present in about six- to seven-fold-higher amounts than cysteine-glutathione primarily in its reduced form and cysteine primarily in its oxidized form. A corrosion product of dental amalgam, i.e., Hg2+, decreased cell survival measured as colony-forming efficiency in a dose-dependent manner following either an acute (one h) exposure or continuous exposure (seven days). These studies demonstrated that human oral fibroblasts could be cultured at about one-tenth of the serum content that is commonly used. It is concluded that human oral fibroblasts grown at low-serum conditions provide a highly sensitive and easily applicable in vitro model for toxicity studies of dental materials.

Serial sections of 182 fungiform papillae, obtained at autopsy from 22 individuals aged 2 days to 90 years, were examined by light microscopy with regard to location and number of taste buds. The taste buds were always found on the convex, dorsal surface of the papillae but otherwise failed to display any preferential location pattern. A total of 262 taste buds, an average of 1.4 per papilla, were found. However, there was considerable variation in the occurrence of taste buds, both from papilla to papilla and from case to case. While the number of taste buds in a single papilla varied from 0 to 27, 63% of the papillae had no taste buds at all, 26% had 1-3 buds and the remainder 4 or more buds. The mean number of taste buds per papilla varied from 0 to 9 between individuals; no dependence upon sex or age could be demonstrated for this variation. The significance of these anatomical findings with regard to physiological studies on taste involving the fungiform papillae is discussed.

The number of basic taste qualities registered by single human fungiform papillae is correlated with the number of taste buds borne on these papillae. Multiple sensitivity was demonstrated both in single fungiform papillae and in single taste buds, with response to all four of the basic taste qualities occuring in a single taste bud.