Ability Of Human Gingival Fibroblasts Research Articles

Synthetic Calcium Phosphate Ceramics as a Potential Treatment for Bisphosphonate-Related Osteonecrosis of the Jaw.

(1) Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is one of the most often seen side effects in patients treated with nitrogen-containing bisphosphonates (BPs), a post-surgical non-healing wound condition. Since calcium phosphate (CP) compounds are able to adsorb zoledronate (ZOL) when used as a drug delivery vehicle, we aimed to verify if these ceramics might have a potential protective effect for soft tissues surrounding surgical osseous wounds. (2) Methods: The chemical reaction between ZOL and CP compounds was evaluated through ultraviolet-visible spectroscopy and elemental analysis. A primary culture of human gingival fibroblasts (HGF) was established as a model to evaluate the cytotoxicity of the association of ZOL (5–500 μM) and of ZOL/biphasic calcium phosphates (BCP). Metabolic activity, cell viability, types of cell death, the cell cycle through, and the migration ability of human gingival fibroblasts were evaluated. (3) Results: ZOL was adsorbed by biphasic calcium phosphate compounds in an aqueous solution. The HGF were sensitive to ZOL toxicity; nevertheless, ZOL/BCP showed a significant protective effect regarding metabolic activity, cell viability, and cell migration. (4) Conclusions: BCP interaction with ZOL reduces or abolishes its toxicity in HGF. This finding represents a potential solution for BRONJ in the case of patients undergoing therapy with ZOL.

Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg.

Composite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.

Effects of methacrilyc thermosets coated with Silver-polysaccharide nanocomposite on HGFs adhesion in a S. mitis co-culture system

Silver based medical products have been proven to be effective in retarding and preventing bacterial growth, being silver reported to control infections since ancient times (1). In the field of dentistry, the use of silver ions/nanoparticles has been explored to counteract bacteria in resins and implants, as silver can destroy bacterial cell walls by reacting with the thiol groups (–SH) of proteins exposed to the extracellular portion of the bacterial membrane. Conversely, eukaryotic cells lack these exterior binding sites, so nanoparticles are supposed to interact with them only upon metal internalization (2). To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized silver nanoparticles with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a co-culture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rised up to 20%. Moreover, the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKC α translocated into nuclei. These data confirm that Chitlac-nAg thermosets have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.This work was supported by grants from MIUR FIRB 2010 and MIUR PRIN-2009.

Effects of Cigarette Smoke Condensate and Nicotine on Human Gingival Fibroblast‐Mediated Collagen Degradation

Members of the matrix metalloproteinase (MMP) family have been shown to be involved in periodontal disease. Risk factors for periodontal disease include tobacco smoking. Cigarette smoke condensate (CSC) is comprised of thousands of chemicals. Nicotine is one of the active components in tobacco. This study compares the effects of CSC and nicotine at the level in CSC on the collagen-degrading ability of human gingival fibroblasts (HGFs) and the expression of selected MMPs and tissue inhibitors of metalloproteinases (TIMPs). HGFs were seeded in six-well collagen-coated plates, exposed to 100 μg/mL (2.4 μg/mL nicotine) of CSC or 2.4 μg/mL nicotine for 3 days, and then collagen degradation was analyzed. After 3 days exposure to CSC or nicotine, the conditioned media from HGFs was collected and the membrane proteins were extracted for gelatin zymography and Western blot analyses. The mRNA levels of MMP-2, MMP-14, and TIMP-2 were measured by reverse transcription-polymerase chain reaction. The CSC increased collagen degradation, and increased the levels of TIMP-2, MMP-14, and the active MMP-2 in the membrane extracts, and their mRNA levels. CSC also increased the level of active MMP-2 in the conditioned media. Nicotine at the level in CSC (2.4 μg/mL) had little influence on collagen degradation, as well as on the protein and mRNA levels of MMP-2, MMP-14, and TIMP-2. CSC may increase HGF-mediated collagen degradation by affecting membrane-associated MMPs and TIMPs.

Effects of Tobacco and P. gingivalis on Gingival Fibroblasts

Cigarette smoke condensate (CSC) is the particulate matter of cigarette smoke. Porphyromonas gingivalis (P. gingivalis) is an opportunistic pathogen involved in periodontitis. It was hypothesized that the combination of CSC and P. gingivalis would increase the collagen-degrading ability of human gingival fibroblasts (HGFs). In this study, HGFs were exposed to CSC, P. gingivalis supernatant, and CSC plus P. gingivalis supernatant. The collagen-degrading ability and protein/mRNA levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) of HGFs were examined. The combined treatment increased collagen degradation, protein levels of active forms of MMP-1, MMP-2, MMP-3, and MMP-14 in conditioned media, and the low-molecular-weight fragment of MMP-14 in membrane extracts, as well as mRNA levels of MMP-1, MMP-2, and MMP-14. In conclusion, the combined effects of CSC and P. gingivalis increased HGF-mediated collagen degradation by destroying the balance between MMPs and TIMPs at multiple levels.

Cigarette smoke condensate affects the collagen‐degrading ability of human gingival fibroblasts

Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen-degrading ability of human gingival fibroblasts and its mechanism. Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water-soluble tetrazolium-1 and lactate dehydrogenase, respectively. The collagen-degrading ability of human gingival fibroblasts was evaluated in collagen-coated six-well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 microg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 microg/mL. Cigarette smoke condensate increased the collagen-degrading ability of human gingival fibroblasts, especially at a concentration of 100 microg/mL (1.5-fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP-1, proMMP-2, MMP-14 and TIMP-1, and decreased the production of TIMP-2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP-2, MMP-14 and TIMP-2 in membrane extracts, especially at concentrations of 50-100 microg/mL. Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of >or= 400 microg/mL. Cigarette smoke condensate can increase the collagen-degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.

Effects of Porphyromonas gingivalis on human gingival fibroblasts from healthy and inflamed tissues

This study compared the ability of human gingival fibroblasts (HGFs) isolated from healthy and inflamed gingival tissues to degrade collagen in the presence and absence of Porphyromonas gingivalis supernatant. Human gingival fibroblasts were cultured from explants of 21 healthy and 21 inflamed periodontal tissues. The HGFs that grew out of the explants were seeded in the center of six-well plates coated with collagen in the presence and absence of 10% P. gingivalis supernatant. An inflamed and a healthy cell line were also evaluated with Arg-gingipain. After 6 days, Coomassie Blue was used to visualize the collagen cleavage. The collagen was totally cleaved in 12 (aggressive) of the 21 cell lines isolated from the inflamed tissues in the presence of P. gingivalis. The remaining nine cell lines (non-aggressive) cleaved only the collagen underneath the cell colonies in the presence of P. gingivalis. Of the healthy tissues, five (aggressive) of the 21 cell lines cleaved all the collagen and 16 cell lines (non-aggressive) only cleaved the collagen underneath the cell colonies in the presence of P. gingivalis. All the collagen was cleaved by an aggressive cell line and only the collagen underneath the cell colonies was cleaved by a non-aggressive cell line in the presence of Arg-gingipain. The collagen in the wells was more readily cleaved by the inflamed than by the healthy cell lines, and the difference was statistically significant (p = 0.0278). Arg-gingipain gave identical results to the P. gingivalis supernatant.

Nicotine increases the collagen‐degrading ability of human gingival fibroblasts

The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.

Porphyromonas gingivalisaffects host collagen degradation by affecting expression, activation, and inhibition of matrix metalloproteinases

Studies have shown that Porphyromonas gingivalis and host matrix metalloproteinases (MMPs) play important roles in the tissue destruction associated with periodontal disease. It is still unclear which MMPs or their inhibitors are regulated by P. gingivalis at the transcriptional and/or at the protein levels. Therefore, this study was conducted to determine what effects P. gingivalis supernatant has on the collagen degrading ability of human gingival fibroblasts (HGFs) and how it regulates the activation, mRNA expression, and inhibition of MMPs. Culture supernatant from P. gingivalis ATCC 33277 was added to HGFs cultured in six-well plates coated with Type I collagen. At certain time intervals, the cell conditioned media was collected for zymography and/or western blot analyses to determine the MMP and tissue inhibitor of MMPs (TIMP) protein levels. The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. The mRNA expression of multiple MMPs and TIMPs by the treated and untreated HGFs was determined by reverse transcription-polymerase chain reaction. The collagen in the six-well plates was degraded more rapidly by the HGFs treated with 10% v/v P. gingivalis supernatant. More active MMP-1, MMP-2, MMP-3, and MMP-14 were detected in the conditioned media from the HGFs treated with the P. gingivalis supernatant. TIMP-1, but not TIMP-2, was decreased in the presence of the P. gingivalis supernatant. MMP-1 mRNA expression by the treated HGFs increased more than two-fold over the untreated HGFs. MMP-3 mRNA was unchanged, MMP-2 mRNA had a slight increase, MMP-14 mRNA decreased, and MMP-15 increased. MMP-12 mRNA was induced in the P. gingivalis treated HGFs. TIMP-1 and TIMP-2 mRNA had a slight increase with P. gingivalis treatment. Porphyromonas gingivalis increased the collagen degrading ability of HGFs, in part, by increasing MMP activation and by lowering the TIMP-1 protein level, as well as by affecting the mRNA expression of multiple MMPs and TIMPs.

Migration of Human Gingival Fibroblasts Over Guided Tissue Regeneration Barrier Materials

The purpose of this investigation was to determine the ability of human gingival fibroblasts, in vitro, to migrate along a chemotactic gradient over 3 different guided tissue regeneration barrier materials; i.e., polytetrafluoroethylene, polylactic acid, and sterile calcium sulfate. Forty petri dishes were divided into 4 equal groups. In each group of 10 dishes, a different barrier material served as the fibroblast substrate with the polystyrene floor of one group of Petri dishes serving as the control. The under agarose technique of measuring cell migration was employed using platelet derived growth factor-BB homodimer as the chemoattractant and Hanks balanced salt solution to test random migration. In addition, fibroblasts were directly cultured on triplicate sets of barrier materials and the control surface for 24 hours and examined by scanning electron microscopy. Comparative analysis of the fibroblast migration data showed the mean migration distance (adjusted for random migration) for controls to be significantly greater than any of the three barrier materials. Further, mean migration distance over calcium sulfate was significantly greater when compared to that of the polylactic acid barrier group. All other comparisons between groups were not statistically significant. Scanning electron microscopic examination fibroblasts cultured directly on barrier membranes and compared to controls indicated that the calcium sulfate substrate appeared to facilitate cell attachment and spreading whereas cells on polytetrafluoroethylene and polylactic acid barriers exhibited a morphology not conducive to migration or, in many cases, cell health. Based on these limited in vitro results and, given the 3 barrier materials considered, it would appear that calcium sulfate offers the greater potential for guided tissue regeneration in surgical sites where primary wound closure cannot be obtained.

Interleukin‐6 production by human gingival fibroblasts

The ability of human gingival fibroblasts to synthesize interleukin-6 (IL-6) was studied using in vitro and immunohistochemical techniques. Culture supernatants of human gingival fibroblasts contained significant quantities of IL-6 activity which could be stimulated by fetal calf serum, recombinant interleukin-1 beta and lipopolysaccharide. The activity in the supernatants was specifically attributed to IL-6 since up to 97% of the activity could be inhibited by an anti-IL-6 antibody. Immunohistochemical studies on low-density human gingival fibroblast cultures indicated that the cells were associated with material reactive to the anti-IL-6 antibody. This localization was seen on the cell surface and in the cytoplasm of the cells. Immunoreactivity towards IL-6 was also noted in sections of human gingivae. Moderate staining was seen in the connective tissues and lower portions of the gingival epithelium, while intense staining was seen at foci of inflammation. The identification of IL-6 with human gingival tissues and cells implicates this lymphokine in the molecular events associated with the inflammatory periodontal diseases.

Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens

We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens. HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined. HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens. When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (~85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin. That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types. HGF were also capable of attaching to all of the collagen types in the complete absence of serum. In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological. However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.