Effects of Vitamin and Amino Acid-Enriched Hyaluronic Acid Gel on the Healing of Oral Mucosa: In Vivo and In Vitro Study

Background and Objectives: Wound healing is a dynamic process that can be compromised in patients with chronic and metabolic conditions or unhealthy lifestyles. Numerous medical substances designed for topical use, charged with compounds that promote the healing process, have been developed to improve wound healing, especially in compromised subjects. The present study aimed to extend our understanding of the in vivo effects of a hyaluronic acid gel charged with amino acids (HAplus gel, Aminogam gel® Errekappa Euroterapici spa, Milan, Italy) and study the in vitro effects of the same gel charged with additional substances in an attempt to optimize its formulation. Materials and Methods: In a randomized controlled split-mouth clinical and histological trial, HAplus gel was tested on the gingival tissue of the lower third molar post-extraction socket. The gingiva was collected at the time of extraction (T0) and ten days after the extraction (T1) to be histologically analyzed. During the second stage of the study, culture media with HAplus gel and vitamin C and E at different concentrations (TEST) were tested on human gingival fibroblasts and compared to the HAplus-enriched medium (HA-Control). Results: Histological and immunohistochemical analysis of collected gingiva showed higher microvascular density and collagen fibers organized in closely packed and well-oriented bundles in sites treated with HAplus gel. In the in vitro study, all TEST groups showed an increased viability from 24 h to 48 h. After 24 h, the viability percentage in all experimental groups was below 100% of the HA-Control, demonstrating a mild toxicity. After 48 h from seeding, the TEST groups’ viability grew significantly compared to HA-Control. Conclusions: These encouraging preliminary results suggest that the use of HAplus gel enriched with vitamins C and E may be beneficial in patients with conditions that impair soft tissue healing

Polyblend Nanofibers to Regenerate Gingival Tissue: A Preliminary In Vitro Study

Aim: The regeneration of small periodontal defects has been considered an important divide and challenging issue for dental practitioners. The aim of this preliminary in vitro study was to analyze the effects of polycaprolactone (PCL) nanofibers enriched with hyaluronic acid and vitamin E vs. nude nanofibers on gingival fibroblasts activity, an innovative graft for periodontal soft tissue regeneration purposes. Methods: Nanofibers were produced in PCL (NF) or PCL enriched with hyaluronic acid and vitamin E (NFE) by electrospinning technique. NF and NFE were stereologically and morphologically characterized by scanning electron microscope (SEM), and composition was analyzed by infrared spectroscopy. Human fibroblasts were obtained from one gingival tissue fragment (HGF) and then seeded on NF, NFE, and plastic (CT). Cell adhesion and morphology were evaluated using SEM at 24 h and cell viability after 24, 48, and 72 h by alamarBlue® assay. Gene expression for COL-I, LH2b, TIMP-1, PAX, and VNC was analyzed by real-time RT-PCR in samples run in triplicate and GAPDH was used as housekeeping gene. Slot blot analysis was performed and immunoreactive bands were revealed for MMP-1 and COL-I. YAP and p-YAP were analyzed by Western blot and membranes were reprobed by α-tubulin. Statistical analysis was performed. Results: IR spectrum revealed the presence of PCL in NF and PCL and vitamin E and hyaluronic acid in NFE. At 24 h, HGF adhered on NF and NFE conserving fibroblast like morphology. At 72 h from seeding, statistically significant differences were found in proliferation of HGF cultured on NF compared to NFE. Expression of genes (LH2b, TIMP-1, and MMP-1) and proteins (COL-I) related to collagen turnover revealed a reduction of COL-1 secretion in cells cultured on NF and NFE compared to CT; however, NFE stimulated cross-linked collagen deposition. Mechanosensor genes (PAX, VNC, and YAP) were upregulated in HGF on NF while they were decreased in cells grown on NFE. Conclusion: Preliminary data suggest that PCL-enriched nanofibers could represent a support to induce HGF proliferation, adhesion, collagen cross-linking, and to reduce collagen degradation, therefore favoring collagen deposition in gingival connective tissue

Clinical, histological, immunohistochemical and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: a randomized, split‐mouth trial

Background: Hyaluronic acid (HA) exerts a fundamental role in tissue repair. In vitro and animal studies demonstrated its ability to enhance wound healing. Nevertheless, in vivo human studies evaluating mechanisms involved in oral soft tissue repair are lacking. The aim of this study was to evaluate the in vivo effect of HA on early wound healing of human gingival tissues (G). Methods: In the present randomized, split-mouth, double-blind, clinical trial, G biopsies were obtained in eight patients 24 hours after surgery after HA application (treatment group-HA) and compared with those obtained from the same patients without HA application (no treatment group-NT). Clinical response was evaluated through Early Wound Healing Score (EHS). Microvascular density (MVD), collagen content and cellular proliferation were evaluated through Sirius red, Masson trichrome staining and Ki67 immunohistochemistry, respectively. To assess collagen turnover, MMP-1, MMP-2, MMP-9, TGF-β1 protein levels and LOX, MMP-1, TIMP-1, TGF-β1 gene expression were analysed by Western Blot and Real Time PCR. Results: Twenty-four hours after surgery, EHS was significantly higher in HA group. MVD, collagen content and cell proliferation were not affected. LOX mRNA, MMP-1 protein and TIMP-1 gene expression were significantly up-regulated in HA compared to NT group. Conclusions: The additional use of 0.8% HA gel does not modify the new blood vessels growth in the early phase of gingival wound healing. Concerning the secondary outcomes, HA seems to enhance extracellular matrix remodelling and collagen maturation, that could drive the early wound healing of gingival tissues to improve clinical parameters. This article is protected by copyright. All rights reserved

Understanding the Role of Surface Modification of Randomized Trabecular Titanium Structures in Bone Tissue Regeneration: An Experimental Study

Background and Objectives: Three-dimensional (3D) metallic trabecular structures made by additive manufacturing (AM) technologies promote new bone formation and osteointegration. Surface modifications by chemical treatments can improve the osteoconductive properties of metallic structures. An in vivo study in sheep was conducted to assess the bone response to randomized trabecular titanium structures that underwent a surface modification by chemical treatment compared to the bone response to the untreated specimens. Material and Methods: Sixteen specimens with a randomized trabecular titanium structure were implanted in the spongious bone of the distal femur and proximal tibia and the cortical bone of the tibial diaphysis of two sheep. Of them, eight implants had undergone a chemical treatment (treated) and were compared to eight implants with the same structure but native surfaces (native). The sheep were sacrificed at 6 weeks. Surface features of the lattice structures (native and treated) were analyzed using a 3D non-contact profilometer. Compression tests of 18 lattice cubes were performed to investigate the mechanical properties of the two structures. Excellent biocompatibility for the trabecular structures was demonstrated in vitro using a cell mouse fibroblast culture. Histomorphometric analysis was performed to evaluate bone implant contact and bone ingrowth. Results: A compression test of lattice cubic specimens revealed a comparable maximum compressive strength value between the two tested groups (5099 N for native surfaces; 5558 N for treated surfaces; p > 0.05). Compared to native surfaces, a homogenous formation of micropores was observed on the surface of most trabeculae that increased the surface roughness of the treated specimens (4.3 versus 3.2 µm). The cellular viability of cells seeded on three-dimensional structure surfaces increased over time compared to that on plastic surfaces. The histomorphometric data revealed a similar behavior and response in spongious and cortical bone formation. The percentage of the implant surface in direct contact with the regenerated bone matrix (BIC) was not significantly different between the two groups either in the spongious bone (BIC: 27% for treated specimens versus 30% for native samples) or in the cortical bone (BIC: 75% for treated specimens versus 77% for native samples). Conclusions: The results of this study reveal rapid osseointegration and excellent biocompatibility for the trabecular structure regardless of surface treatment using AM technologies. The application of implant surfaces can be optimized to achieve a strong press-fit and stability, overcoming the demand for additional chemical surface treatments

Histomorphometry of Bone after Intentionally Exposed Non-Resorbable d-PTFE Membrane or Guided Bone Regeneration for the Treatment of Post-Extractive Alveolar Bone Defects with Implant-Supported Restorations: A Pilot Randomized Controlled Trial

Aim: The aim of the present study was to investigate quantitative histological examination of bone reconstructed with non-resorbable high-density polytetrafluoroethylene membrane (d-PTFE), left intentionally exposed in post extraction sockets grafted with anorganic bone material, and removed after four weeks, versus extraction and guided bone regeneration (GBR), performed two months later. Materials and Methods: This study was designed as a multicenter randomized controlled trial of parallel-group design. Patients were selected and consecutively treated in three centers in Italy. Patients randomly received intentionally exposed non-resorbable d-PTFE membrane (group A), or guided bone regeneration (group B), to treat post-extractive alveolar bone defects with implant-supported restorations. Outcomes were: the implant failure, any mechanical and biological complications, patient satisfaction, and qualitative and histomorphometric evaluation of the collected bone samples. Results: Eighteen patients were consecutively enrolled in the trial. Of these, six out of 18 patients were male. All the included patients were treated according to the allocated interventions, and no drop out occurred. No implant failure and no complications were experienced, and all the patients were fully satisfied with the function and aesthetic of their implant-supported restoration, without difference between groups. Morphological analysis revealed no sign of tissue reaction, such as fibrosis or necrosis. Regenerated bone was well mineralized in both groups, but it seemed more mature in group B than in group A. Three samples showed a minimal number of lymphocytes. Several blood vessels of small size occupied the medullary spaces, where the tissue resulted in more maturity, indicating the activity of the tissue in progress. The histomorphometric evaluation showed no statistically significant differences in the tissue volume fractions between the two groups of patients. Conclusions: With the limitation of the present study, buccal plate reconstruction with an intentionally exposed non-resorbable membrane is an effective and easy procedure for regenerating a resorbed buccal bone plate, reducing the need for guided bone regeneration

Immunohistochemical evaluation of pro-inflammatory molecules in the soft tissue surrounding switching platform implants

Introduction. Switching platform implants have been proposed to reduce the peri-implant bone loss by shifting the implant abutment interface internally and away from the bone tissue. Aim of the present study was to characterize the inflammatory infiltrate in the soft tissue surrounding switching and matching platform implants and to evaluate the expression of pro-inflammatory molecules involved in the bone loss. Material and methods. A total of 32 implants with diameters of 3.8mm, 4.3mm, 4.8mm and 5.5mm were all restored with abutment of 3.8mm of diameter, thus resulting the following implant-abutment mismatches: 0mm (n=10) (control group), 0.25mm (n=7) (test group1), 0.5mm (n=8) (test group2), and 0.85mm (n=7) (test group3). Four years after loading, peri-implant soft tissue samples were harvested and processed for immunohistochemical analysis. Total amount of lymphocytes T (LyT) -B (LyB) infiltrated, and expression of IL-17 and RANKL were detected. Results. At the harvesting time all sites were clinically healthy. No significant differences were found between groups (p>0.05) in terms of infiltrated T and B cells amount, IL-17 and RANKL expression. In all samples lymphocytes T and B were mainly localized close to the junctional epithelium and sparsely detected in the surrounding connective tissue. The distribution of IL-17 and RANKL staining resulted strictly correlated to the inflammatory infiltrated. When pooled data were analyzed, amount of lymphocytes T and IL-17 were higher than respectively lymphocytes B and RANKL. Amount of LyT and LyB were highly correlated (Pearson’s r>0.7) and IL-17 was moderately correlated (Pearson’s r>0.4, <0.7) to LyT and LyB. Conclusions. In the prolonged exposure of the abutment at the oral cavity the configuration of the implant abutment interface may do not affect the inflammatory cellular and molecular pattern responsible for bone loss

Endocannabinoid system and periodontal diseases: a histological preliminary study

Periodontal disease is a chronic condition characterized by an opportunistic infection of the periodontal tissues by Gram- anaerobic bacteria (1). Tissue destruction can be limited by the production of anti-inflammatory substances and its final entity depends on the equilibrium between pro- and anti-inflammatory mechanisms. The endocannabinoid system (EC) is supposed to have an anti-inflammatory action. The two most extensively studied molecules of the EC are anandamide and 2-AG with their specific receptors, CB1 and CB2. Nakajima et al (2) showed the presence of CB1 and CB2 receptors in human gingival fibroblasts from healthy patients and subjects affected by periodontal inflammation and found that AEA significantly reduced the production of cytokines (IL-6, IL-8 and MCP-1) by gingival fibroblasts after bacteria stimulation, through CB1 and CB2 receptors. The aim of this project was to quantitatively and qualitatively evaluate the presence of CB1 and CB2 receptors in the gingival tissue from patients affected by periodontitis compared to healthy subjects. Ten patients were recruited, six healthy subjects (H) and four with chronic periodontitis (P). In each patient, a gingival biopsy was harvested during periodontal treatment and processed for immunohistochemistry to reveal CB1 and CB2 expression. Every sample was mapped at 200X total magnification and the presence of CB1 and CB2 was quantified using a standardized method with Adobe Photoshop PS5. The percentage of marked tissue in the connective tissue and in the epithelium was calculated. In P the mean CB1 percentage on the connective tissue (CB1-c) was 2.27% + 0.14% and CB2 (CN2-c) was 2.12% + 0.43%, while in H the values were 0.13% + 0.31% and 0.06% + 0.11% respectively. In P the mean CB1 value on the epithelium (CB1-e) was 5.08 + 2.27% and CB2 (CB2-e) was 4.07% + 1.64%, while in H the corresponding values were respectively 0.16% + 0.36% and 0.03% + 0.02%. CB1 and CB2 expression resulted significantly higher in P than in H samples in both epithelial and connective tissue (Wilcoxon sum rank test, p<0.01). Such data confirm that EC are involved in periodontal disease

Immunohistochemical and molecular analysis of bone remodelling pattern in alveolar socket

Following tooth extraction, the alveolar bone remodelling process starts. Bundle bone and buccal wall resorption occur early with horizontal and vertical bone crest reduction [1]. The use of bone substitutes has been proposed to limit bone resorption, thus allowing further dental rehabilitation [2]. Aim of this project was to characterize by a molecular and morphological approach the physiological remodelling of post-extractive alveolar socket and to compare it with the bone remodelling occurring after alveolar bone reconstruction with an alloplastic material. Thirty-six patients needing tooth extraction were enrolled and equally divided into three groups: A) baseline, B) spontaneous healing, C) biomaterial. In each group, 2 biopsies per site were harvested during tooth extraction (group A) or 4-6 months after tooth extraction (groups B and C). In group B, patients recovered spontaneously, while in group C the alveolar socket was filled with a magnesium-enriched hydroxyapatite. One biopsy was processed for immunohistochemistry to localise TNF-α, IL-6, RANK, RANKL and OPG. The second biopsy underwent a Real-Time PCR analysis for the same biomarkers in order to evaluate gene expression. In groups B and C, a third biopsy was retrieved and processed for ground section aiming to assess tissue composition. Differences between the three groups were investigated using Kruskal Wallis test (p<0,05) followed by post-hoc tests. All samples showed a normal structure without inflammatory infiltrate. At immunohistochemical analysis, all biomarkers except for OPG had increased. Significant differences were found between the three groups for TNF-α (p< 0,05), IL-6 (p<0,001), RANK (p< 0,01) and RANKL (p<0,001), between groups A and C for IL-6 (p≤ 0,001), RANK (p≤ 0,01), RANKL (p≤ 0,001) and between B and C for IL-6 (p≤ 0,01). Gene expression did not show statistical differences. Crumbles of biomaterial surrounded by regenerated bone were evident. A higher percentage of mineral component was obtained in group B than in C. The biomarkers selected in the current study were involved in the alveolar remodelling and the biomaterial used for socket preservation did not influence the process