In vitro and in vivo biocompatibility of a porcine cholecystic extracellular matrix (CECM) membrane for tissue regeneration

Some prior reports have suggested that guided tissue regeneration (GTR) procedures achieve only partial regeneration and induces the ankylosis rather than true attachment. Accordingly, others have developed an alternative procedure employing gelatine membrane compounded with bovine cementum particles (CGM) which has proven effective in stimulating a more physiologic form of attachment. This study was undertaken to perform a direct comparison of histological results when CGM and GTR membrane were used at comparable sites in the same monkey. Three monkeys with no periodontal disease were used. Following flap surgery, recession type defects were created on the buccal side of the maxillary lateral incisors and second premolars, and the cementum was removed from the root surface at an area corresponding to the bone crest. The right and left lateral incisors and second premolars were covered with CGM and GTR membrane, respectively. The GTR membranes were removed after 4 weeks. At 6 wks, the animals were sacrificed, and specimens were prepared for histological examination. More coronally placed true new attachment was observed following application of CGM to the planed root surfaces. Application of the GTR membrane resulted in formation of bone-like cementum and ankylosis, whereas CGM established true periodontal regeneration.

Biomaterials are used in the field of bone and tissue engineering, orthopaedics and dentistry. Dental biomaterials including commercially available biodegradable materials act as physical barriers to help quicker healing while stimulating the regeneration of periodontal tissues, which is defined as Guided Tissue Regeneration (GTR). Amongst natural and synthetic biomaterials, collagen and aliphatic polyesters, such as polylactic acid (PLA) and poly (lactic-co-glycolic) acid (PLGA) are the most frequently used biomaterials for regenerative therapies due to their excellent biocompatibility and biodegradability. Due to their resorption in the body and interaction with biological systems, the GTR membranes must be sterile and pyrogen free. The sterility and apyrogenicity of the GTR membranes before human use is a regulatory requirement, however the sterilization of biomaterials is challenging due to the physicochemical changes and toxic residues with the commonly used sterilization techniques. The purpose of the present study was to evaluate the effect of gamma radiation and ethylene oxide sterilization on dental biomaterials with analytical, microbiological and histological examinations. PLGA-based GTR dental biomaterial is selected as the most gamma stable membrane according to the FTIR, DSC, TGA, and SEM results. This dental membrane was sterilized with ethylene oxide (EtO) and the effect of sterilization method on PLGA-based membrane was also investigated. Animal experiments were carried out to evaluate the regenerative properties and inflammatory responses of gamma and EtO sterilized PLGA-based GTR membrane after implantation. Histological examinations showed that resorption and bone formation of gamma sterilized PLGA-based GTR membrane was completed in 12 weeks without any inflammatory response; while only 60.095 ± 2.019% of new bone formation was observed with EtO sterilized one. Gamma sterilized PLGA membrane had significantly faster (P < 0.05) resorption and bone formation in comparison with EtO sterilization. In conclusion, the PLGA-based biomaterials could be sterilized safely and time- and cost-effectively with validated radiation doses for the tissue engineering applications.

Background: Periodontitis leads to tissue deterioration, prompting the need for effective regenerative therapies. Conventional barrier membranes, face challenges in mechanical strength and support. This study explores a novel formulation combining Astaxanthin’s antioxidant and anti-inflammatory properties, Aloe vera’s healing effects, chitosan nanoparticles biocompatibility and antimicrobial benefits to enhance periodontal tissue regeneration. Aim: This study aimed to develop a Guided Tissue Regeneration (GTR) membrane incorporating Astaxanthin, Aloe vera, and chitosan nanoparticles and evaluate its in vitro properties. Methods: Astaxanthin and Aloe vera extracts were dissolved in deionized water and boiled for complete incorporation. Chitosan nanoparticles were created by dissolving chitosan in acetic acid, followed by the drop wise addition of sodium tripolyphosphate while stirring. The nanoparticles were collected via centrifugation, washed, and re-suspended in deionized water. A chitosan solution was then prepared with glycerol as a plasticizer, and a cross-linking agent was added for stabilization. Finally, the chitosan nanoparticles and bioactive extracts were blended to form the membrane and later the same was analyzed for Scanning Electron Microscopic (SEM), Fourier Transform Infrared Spectroscopy (FTIR), cytotoxic, anti-oxidant, and degradation test activities. Results: The GTR membrane demonstrated a homogeneous nanoparticle distribution and initial degradation signs in SEM analysis, while FTIR confirmed the integration of Astaxanthin, Aloe vera, and Chitosan. Cytotoxicity tests showed low toxicity, with significant MG-63 cell viability at 15, 20, and 25 μg/mL. Antioxidant activity was dose-dependent, peaking at nearly 50% scavenging at 25 μg/mL, though still lower than ascorbic acids 65%. The degradation test revealed an increasing rate with concentration, reaching approximately 40% at 25 μg/mL. Conclusion: Thus the formulated GTR membrane effectively integrates Astaxanthin, Aloe vera, and chitosan nanoparticles, demonstrating promising in vitro properties for periodontal tissue regeneration with low cytotoxicity and significant antioxidant activity suitable for regenerative therapies. Major Findings: The developed GTR membrane successfully incorporated with Astaxanthin, Aloe vera, and chitosan nanoparticles, exhibited favourable in vitro characteristics for periodontal tissue regeneration. It demonstrated minimal cytotoxicity and notable antioxidant activity, highlighting its potential for periodontal therapeutic applications.

Bacterial adhesion to antibiotic-loaded guided tissue regeneration membranes – A scanning electron microscopy study

Background:Marginal tissue recession is a frequent clinical scenario that creates substantial compromise in esthetic appearance of the patient. The current randomized, double-blind interventional trial aims to evaluate the effectiveness of the combination of “platelet rich fibrin (PRF)” membrane with bioresorbable guided tissue regeneration (GTR) membrane as compared to GTR membrane alone utilizing “double lateral sliding bridge flap (DLSBF) technique” for the management of simultaneous GR defects in human subjects.Materials and Methods:Twenty subjects were randomly allotted in two groups: Group 1 (test): 10 subjects treated with PRF + GTR membrane using DLSBF technique and Group 2 (control): 10 subjects treated with GTR membrane alone using DLSBF technique. Clinical measurements such as relative gingival marginal level, “relative clinical attachment level (R-CAL),” “probing pocket depth (PPD),” “gingival recession (GR),” and “width of keratinized gingiva (WKG) and gingival thickness (GT)” were evaluated at the initiation of the study and 6 months thereafter.Results:Two groups showed statistically significant differences with respect to probing depth reduction, CAL gain, and increase in WKG and GT. No significant result was observed with mean root coverage (RC) and complete RC for test (84.80% ± 19.53% and 54.99% ± 38.53%) and control group (75.69% ± 18.86% and 35.83% ± 39.29%), respectively.Conclusions:The combination of PRF membrane used along with GTR membrane provides no additional benefits over GTR membrane alone. However, the DLSBF technique convincingly shows simultaneous elimination of multiple problems associated with GR, shallow vestibule, hypersensitivity, and aberrant frenum pull in a single stage with meticulous performance.

Early exposure of a guided tissue regeneration (GTR) membrane in the oral cavity results in bacterial contamination, which may lead to failure or incomplete regeneration. Incorporation of antimicrobial agents in GTR membranes may be valuable to control membrane-associated infection during GTR therapy. The purpose of this study was to evaluate whether the incorporation of chlorhexidine into various GTR membranes improves the attachment of periodontal ligament cells in the presence of Actinobacillus actinomycetemcomitans. The possible effects of chlorhexidine on the viability of primary human periodontal ligament (PDL) cells were determined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT), which measures cellular metabolic activity. An expanded polytetrafluoroethylene (ePTFE) membrane, glycolide fiber membrane, and collagen membrane were loaded with chlorhexidine and characterized. Attachment of PDL cells to the chlorhexidine-loaded membranes with or without A. actinomycetemcomitans was examined using scanning electron microscopy (SEM) analysis. Relative cellular viability of PDL cells was reduced to approximately 50% when 15 microg/ml (0.0015%) of chlorhexidine was used. Chlorhexidine released from the coated GTR membranes inhibited the growth of A. actinomycetemcomitans. At the concentration used in this study, chlorhexidine incorporated into the GTR membranes did not interfere with the attachment of PDL cells. The inhibitory effects of A. actinomycetemcomitans on cellular attachment were reduced using chlorhexidine-loaded membranes, including ePTFE, glycolide fiber, and collagen membranes. These results suggest that incorporation of chlorhexidine into GTR membranes is beneficial in reducing bacterial effects on cellular attachment. The future application of chlorhexidine-loaded membranes during GTR therapy may be of value.

Periodontitis is an inflammatory disease which can cause the destruction of the supporting tissues of the tooth leading to tooth loss. The guided tissue regeneration is considered as a gold standard for its treatment but the re-infection of surgical site limits its overall success. Objective: To synthesize novel monolayer guided tissue regeneration (GTR) membrane containing drug loaded chitosan nanoparticles and to evaluate the drug release from the synthesized GTR membranes. Methods: The chitosan nanoparticles containing ciprofloxacin were synthesized by the ionotropic gelation method and these synthesized nanoparticles were added into chitosan GTR membrane fabricated by the freeze gelation method. For comparison GTR membrane was prepared as a control by freeze gelation method in which the drug was added directly. The prepared membranes were characterized by the SEM and FTIR. The drug release was measured from the membrane samples in the phosphate buffer saline (PBS) at 1, 3, 5, 7 and 9 days. Results: The GTR membrane containing the ciprofloxacin loaded chitosan nanoparticles showed fast drug release as compared to the membrane in which the ciprofloxacin was added directly. Conclusions: The inclusion of antibiotic loaded chitosan nanoparticles can increase the drug release from GTR membrane.

The aim of this study is to investigate the bacterial adherence on 5, 10 and 15 nm nanosilver-impregnated resorbable guided tissue regeneration (GTR) membranes. GTR membranes were incorporated with 5 (NS-GTR-5), 10 (NS-GTR-10), and 15 nm (NS-GTR-15) nanosilver in the test group. The control group contained GTR membranes incorporated with 25% metronidazole (M-GTR) and plain membranes (GTR-C). Qualitative evaluation of microbial adherence to the GTR membranes was performed using four organisms-Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans-since these organisms are reported to have strong adherent capabilities to collagen membranes. The main results have been obtained: The mean bacterial adherence scores were significantly greater in the GTR-C group when compared to those in the M-GTR and NS-GTR-5, NS-GTR-10, and NS-GTR-15 groups. M-GTR showed higher adherence scores than NS-GTR-5, NS-GTR-10, and NS-GTR-15 for all microorganisms; the differences were statistically significant. We have concluded that collagen membranes impregnated with 5- and 15 nm nanosilver exhibited an apparent antibacterial effect against anaerobic oral pathogenic bacteria and aerobic bacteria. This outcome indicates that nanosilver-impregnated GTR membranes may be useful in periodontal regeneration therapy.

Microbial colonization on barrier materials used in guided tissue regeneration (GTR) may adversely affect treatment outcomes. The purposes of this study were: 1) to compare the invasion of Streptococcus mutans and Actinobacillus actinomycetemcomitans through 3 GTR membranes, composed of expanded polytetrafluoroethylene (ePTFE; non-resorbable), a glycolide fiber composite, and type I collagen (both bioabsorbable), and 2) to explore the effects of bacteria on the attachment of periodontal ligament (PDL) fibroblasts onto these membranes. Bacterial permeability was analyzed using a tube capped with a GTR membrane as a septum and filled with media. The tube was then placed in a bigger tube inoculated with S. mutans or A. actinomycetemcomitans. The passage of bacteria through the membranes into the inner tube was monitored. For cellular attachment experiments, primary human PDL cells were placed onto the GTR membranes with or without bacteria. Attached cells were analyzed by scanning electron microscopy (SEM) analysis. The ePTFE membrane had the best barrier effects followed by the collagen membrane and then the glycolide fiber composite membrane. Moreover, S. mutans passed through these membranes faster than A. actinomycetemcomitans. The attachment of PDL cells on the 3 membranes was also varied. The ePTFE membrane was the worst substrate for PDL fibroblast attachment. Moreover, both bacteria influenced the cellular attachment on the GTR membranes. Differences in the behavior of 3 GTR membranes penetrated by S. mutans and A. actinomycetemcomitans were demonstrated. The results suggest that attachment of PDL cells was affected on bacterial-contaminated GTR membranes, which may alter healing following membrane exposure.

Although many different types of Guided Tissue Regeneration (GTR) membranes (resorbable/non-resorbable, including titanium mesh) have been used in the field of Periodontics till now, but this is the first and only clinical study testing the effectiveness of an ultra thin pure Titanium Membrane (Ultra Ti) as a GTR membrane in infra-bony periodontal defects. To compare the efficacy of GTR in intra-bony defects with newly introduced non-resorbable barrier membrane, made of titanium called "Ultra-Ti ® GTR Membrane" versus open flap debridement. A prospective, randomized, controlled, clinical split mouth study was designed wherein each patient received both the control and test treatment. Two similar defects were selected in each of the 12 patients and were randomly assigned to one of the two treatments. Both the surgeries consisted of identical procedures except for the omission of the barrier membrane in the control sites. Full mouth Plaque Index (PI), Gingival Index (GI), Pocket Probing Depth (PPD) and Relative Attachment Level (RAL) were recorded before surgery and after 6 months and 9 months along with hard tissue measurements at the time of surgery and then at re-entry after 9 months. Radiographs were also taken before surgery and 9 months post operatively. Student's paired t-test and unpaired t-test (SPSS software version 9) were used to analyze the results. Nine months after treatment, the test defects gained 4.375 ± 1.189mm of RAL, while the control defects yielded a significantly lower RAL gain of 3.417 ± 0.996mm. Pocket reduction was also significantly higher in the test group (4.917 ± 0.996mm) when compared with the controls (3.83 ± 0.718mm). There was a significant bone fill (54.69% of defect fill) obtained in the test site, unlike the control site (8.91%). The present study demonstrated that GTR with "Ultra-Ti® GTR Membrane" resulted in a significant added benefit in comparison with open flap debridement.

Fabrication of hydroxyapatite reinforced polymeric hydrogel membrane for regeneration

Objectives: The main objective of periodontal treatment is to resolve the inflammatory lesion in periodontal tissues. However, the contemporary goal has become regeneration of the lost attachment apparatus through guided tissue regeneration (GTR) technique. The GTR membranes currently available in the market are expensive, which in turn warrants the identification, development, and utilization of more cost-effective and biocompatible materials as barrier membranes for GTR. Materials and Methods: The present case-control study was conducted on patients with infrabony defects having minimal or no gingival recession and with a probing depth of >4 mm and controls (12 each). It highlights the use of a newly developed fish collagen-chitosan film as a barrier membrane for the management of human infrabony defects using the principles of GTR in periodontitis subjects. Results: Infrabony defects in the test and control sites were matched at baseline. The differences in probing pocket depth within the groups at baseline and 6 months after periodontal therapy were statistically significant. After 6 months, there was statistically significant difference in the probing pocket depth between the test and control sites. Conclusion : Within its limitations, the present study showed that the collagen-chitosan film is effective as a barrier membrane for GTR therapy in infrabony defects, as it resulted in improved clinical outcome with low incidence of gingival recession and device exposure.

The study was designed to compare the extent of bacterial colonisation on the surface of Polylactic acid/Polyglycolic acid copolymer and expanded Polytetrafluoroethylene based Guided tissue regeneration (GTR) membrane in an in vitro model by spot analysis and scanning electron microscopy. Earlier in vitro models have aimed to study the barrier function against the bacterial penetration across just one surface of the GTR membranes. No such study is present in the literature which aimed to quantify bacterial adhesion over both the surfaces of the membrane. Sterile Resorbable PLA/PGA copolymer and non-resorbable microporous ePTFE based GTR membrane were used in the study. Both groups were subdivided into two subgroup (n=10) based on incubation period of 24 and 48 hours. Incubated in Todd Hewitt broth with Streptococcus mutans, the samples were vortexed and analysed for bacterial count using spot test and scanning electron microscopy. Between PLA/PGA and ePTFE GTR membrane at 24 hours incubation time period, there was a significant difference in terms of Colony forming units (p = &lt;0.001), with the median Colony forming units being highest in the PLA/PGA GTR membrane. At 48 hours incubation time period, there was a significant difference in terms of Colony forming units (p = &lt;0.001), with the median Colony forming units being highest in the PLA/PGA GTR membrane. Our findings depict that ePTFE based GTR membrane showed significantly lesser bacterial colonisation on its surfaces as compared to PLA/PGA at both the incubation periods i.e., 24 hours and 48 hours as shown by both spot test and SEM.

Background: There is still inadequate information available regarding the role of GTR techniques in such lesions with the presently available data suggesting that there is a possibility of healing of apico-marginal defects without use of GTR technique by using modern microsurgical procedures. Fewer randomized-clinical trials have evaluated the response of GTR membranes in the treatment of apico-marginal defects. Aim: The present prospective, controlled clinical trial was, therefore, planned to evaluate the role of collagen membrane as GTR material in the healing of apico-marginal defects. Subjects and Methods: Thirty patients meeting inclusion criteria were selected and allocated randomly to either the GTR membrane group or, the control group. Clinical and radiographic examination was done after one week for baseline measurements and then, during follow-ups at regular intervals of 3, 6, 9, and 12 months after the procedure. The criteria for success included the absence of clinical signs and symptoms and signs of radiographic healing. Statistical Analysis Used: The statistical analysis of the ordinal data was carried-out by using non-parametric methods. Mann-Whitney and Wilcoxon signed rank test were used for the unpaired and paired data respectively. Chi-square test was used to evaluate dichotomous data. Results: Significant reductions were observed in the periodontal pocket depth (PD), clinical attachment level (CAL), gingival margin position (GMP) and the size of the peri-apical lesion at 12-month follow-up (P < 0.05) in each treatment group except gingival margin position (GMP) in the GTR membrane group with the corresponding P value being 0.059. The results for the reduction in the size of the peri-apical lesion were, also, found to be statistically significant with the corresponding P value being <0.05. Furthermore, 83.33% of the patients showed complete healing in case of GTR membrane group while the same was found to be 90.9% in case of the control group although the difference in the percentage reduction in the size of the peri-apical lesion at different time intervals after surgery was found to be statistically insignificant between the two groups. Conclusion: The results of the present study indicated that there might not be any additional clinical advantage obtained from GTR membrane barriers in the surgical management of isolated apico-marginal defects of primary endodontic origin with absent or, minimal proximal bone loss.

Background: There is still inadequate information available regarding the role of GTR techniques in such lesions with the presently available data suggesting that there is a possibility of healing of apico-marginal defects without use of GTR technique by using modern microsurgical procedures. Fewer randomized-clinical trials have evaluated the response of GTR membranes in the treatment of apico-marginal defects. Aim: The present prospective, controlled clinical trial was, therefore, planned to evaluate the role of collagen membrane as GTR material in the healing of apico-marginal defects. Subjects and Methods: Thirty patients meeting inclusion criteria were selected and allocated randomly to either the GTR membrane group or, the control group. Clinical and radiographic examination was done after one week for baseline measurements and then, during follow-ups at regular intervals of 3, 6, 9, and 12 months after the procedure. The criteria for success included the absence of clinical signs and symptoms and signs of radiographic healing. Statistical Analysis Used: The statistical analysis of the ordinal data was carried-out by using non-parametric methods. Mann-Whitney and Wilcoxon signed rank test were used for the unpaired and paired data respectively. Chi-square test was used to evaluate dichotomous data. Results: Significant reductions were observed in the periodontal pocket depth (PD), clinical attachment level (CAL), gingival margin position (GMP) and the size of the peri-apical lesion at 12-month follow-up (P &lt; 0.05) in each treatment group except gingival margin position (GMP) in the GTR membrane group with the corresponding P value being 0.059. The results for the reduction in the size of the peri-apical lesion were, also, found to be statistically significant with the corresponding P value being &lt;0.05. Furthermore, 83.33% of the patients showed complete healing in case of GTR membrane group while the same was found to be 90.9% in case of the control group although the difference in the percentage reduction in the size of the peri-apical lesion at different time intervals after surgery was found to be statistically insignificant between the two groups. Conclusion: The results of the present study indicated that there might not be any additional clinical advantage obtained from GTR membrane barriers in the surgical management of isolated apico-marginal defects of primary endodontic origin with absent or, minimal proximal bone loss.