Single-cell transcriptomic analysis of oral masticatory and lining mucosa-derived mesenchymal stromal cells.

AIM: To reveal the heterogeneity of ex vivo-cultured human mesenchymal stromal cells derived from either masticatory or lining oral mucosa. MATERIALS AND METHODS: Cells were retrieved from the lamina propria of the hard palate and alveolar mucosa of three individuals. The analysis of transcriptomic-level differences was accomplished using single-cell RNA sequencing. RESULTS: Cluster analysis clearly distinguished between cells from the masticatory and lining oral mucosa, and revealed 11 distinct cell sub-populations, annotated as fibroblasts, smooth muscle cells or mesenchymal stem cells. Interestingly, cells presenting a mesenchymal stem cell-like gene expression pattern were predominantly found in masticatory mucosa. Although cells of masticatory mucosa origin were highly enriched for biological processes associated with wound healing, those from the lining oral mucosa were highly enriched for biological processes associated with the regulation of epithelial cells. CONCLUSIONS: Our previous work had shown that cells from the lining and masticatory oral mucosae are phenotypically heterogeneous. Here, we extend these findings to show that these changes are not the result of differences in averages but rather represent two distinct cell populations, with mesenchymal stem cells more common in masticatory mucosa. These features may contribute to specific physiological functions and have relevance for potential therapeutic interventions.

Fibroblasts from the oral masticatory and lining mucosa have different gene expression profiles and proliferation rates.

AIMS: To compare the gene expression profiles and proliferation rates of fibroblasts from the oral lining and masticatory mucosae. MATERIALS AND METHODS: Primary human fibroblasts were retrieved from the posterior masticatory hard palate and the lining alveolar mucosa of five individuals. The gene expression profile was evaluated using total RNA sequencing. The proliferation rate was determined colorimetrically. RESULTS: Substantial differences in specific gene groups and pathways were observed between fibroblasts from the two tissues. Significantly enriched gene ontology processes were focused on the extracellular components. Lining mucosa fibroblasts exhibited significantly higher expression of the principal structural collagens, cranial neural crest markers, and homeobox genes associated with positional memory. Masticatory mucosa fibroblasts showed greater expression of genes related to transforming growth factor-ß signalling, which may be associated with fibrosis. In addition, they expressed higher levels of the EP2 prostaglandin E2 receptor and Toll-like receptor 1. Finally, masticatory mucosa fibroblasts exhibited a 10%-30% higher proliferation rate. CONCLUSIONS: Fibroblasts from the lining and masticatory oral mucosae are phenotypically heterogeneous, presenting distinct gene expression profiles and proliferation rates. These features may contribute to their specific physiological functions and have relevance for potential therapeutic applications.

Effect of root surface conditioning on gingival recession coverage with a connective tissue graft. A retrospective comparative study of three different agents.

OBJECTIVE: Different root modifiers have been proposed in the literature with an attempt to improve the healing process and the success rate of root coverage procedures. The aim of the present retrospective study was to evaluate the effect of three different types of root surface conditioning, namely, tetracycline (TTC), ethylene-di-amino-tetra-acetic acid (EDTA) and saline, on the outcome of root coverage procedures applying the same surgical technique. MATERIALS AND METHODS: Twenty-nine patients with 60 Classes I, II, or III recession defects were treated using connective tissue with a partial-thickness double-pedicle graft. In 21 recession defects root surface was treated with TTC and, in other 21, with EDTA, while in the remaining, saline solution was applied. Statistical analysis consisted of descriptive statistics and Kruskal-Wallis, Mann-Whitney, and chi-square tests. RESULTS: Differences between pre- and postoperative values were statistically significant only within but not between groups. Mean root coverage was 73.25%, 69.19%, and 82.17% in the TTC, the EDTA, and the saline groups, respectively. The study revealed no statistically significant differences for all evaluated parameters between groups. CONCLUSION: Within the limits of this study, root conditioning, prior to root coverage procedures, does not significantly affect their outcome. CLINICAL SIGNIFICANCE: Clinical outcome of root coverage procedures is not related to the type of root surface conditioning.

Tetracycline impregnation affects degradation of porcine collagen matrix in healthy and diabetic rats.

OBJECTIVES: The present study evaluated the degradation of collagen matrix (CM) immersed in tetracycline (TTC) or phosphate-buffered saline (PBS) in diabetic and normoglycemic rats. MATERIALS AND METHODS: Diabetes was induced in 15 rats by systemic streptozotocin (STZ) (experimental); 15 healthy rats served as controls. One day before implantation 60 CM disks, 5 mm in diameter, were labeled with biotin: 30 were immersed in tetracycline (TTC) and 30 in PBS. One disk of each type was implanted subdermally in each rat. Animals were euthanized after 3 weeks, and tissue specimens containing the disks were prepared for histologic analysis. Horseradish peroxidase (HRP)-conjugated streptavidin was used to detect the remaining biotinylated collagen. Residual collagen area within the CM disks was analyzed and compared to baseline. RESULTS: Diabetes significantly increased the CM degradation. Immersion of the CM disks in a 50-mg/mL TTC solution before implantation decreased its degradation both in diabetic and normoglycemic rats. CONCLUSIONS: Diabetes significantly increases collagen matrix degradation; immersion of collagen matrix in TTC before implantation decreases its degradation in both diabetic and normoglycemic conditions. CLINICAL RELEVANCE: Immersion of medical collagen devices in TTC may be an effective means to decrease their resorption rate and increase their effectiveness, especially in situations with increased degradation such as diabetes.

In vitro models for evaluation of periodontal wound healing/regeneration.

Periodontal wound healing and regeneration are highly complex processes, involving cells, matrices, molecules and genes that must be properly choreographed and orchestrated. As we attempt to understand and influence these clinical entities, we need experimental models to mimic the various aspects of human wound healing and regeneration. In vivo animal models that simulate clinical situations of humans can be costly and cumbersome. In vitro models have been devised to dissect wound healing/regeneration processes into discrete, analyzable steps. For soft tissue (e.g. gingival) healing, in vitro models range from simple culture of cells grown in monolayers and exposed to biological modulators or physical effectors and materials, to models in which cells are 'injured' by scraping and subsequently the 'wound' is filled with new or migrating cells, to three-dimensional models of epithelial-mesenchymal recombination or tissue explants. The cells employed are gingival keratinocytes, fibroblasts or endothelial cells, and their proliferation, migration, attachment, differentiation, survival, gene expression, matrix production or capillary formation are measured. Studies of periodontal regeneration also include periodontal ligament fibroblasts or progenitors, osteoblasts or osteoprogenitors, and cementoblasts. Regeneration models measure cellular proliferation, attachment and migration, as well as gene expression, transfer and differentiation into a mineralizing phenotype and biomineralization. Only by integrating data from models on all levels (i.e. a single cell to the whole organism) can various critical aspects of periodontal wound healing/regeneration be fully evaluated.

Evaluation of a topical herbal patch for soft tissue wound healing: an animal study.

AIM: This study evaluated the effects of a topical herbal patch (PerioPatch®) for gingival wound healing in a rat model. MATERIALS AND METHODS: A mid-crestal incision was performed on each side of the edentulous anterior maxilla in 48, 6-month-old, Wistar rats. Full-thickness flaps were raised, repositioned and sutured. Four experimental groups were established: herbal patch, placebo patch, no patch and no patch and no surgery. Patches were placed immediately after surgery and replaced every 12 h for the following 3 days. Half of the animals were killed after 5 and the remaining ones after 12 days. Tissue blocks were retrieved and processed for histological and immunohistochemical evaluation. Epithelial gap, collagen contents, amount of macrophages, cellular proliferation and vascular contents were evaluated in the central incision area. Statistical analysis consisted of two-way anova. RESULTS: The herbal patch group presented the smallest epithelial gap at 12 days, the highest collagen content both at 5 and 12 days, a larger number of proliferating cells at day 5 and more numerous blood vessels at day 12. Macrophage number was similar in all groups. CONCLUSION: Herbal patch improved wound healing in this animal model.

Bone remodeling at implants with different configurations and placed immediately at different depth into extraction sockets. Experimental study in dogs.

OBJECTIVES: This study evaluated the effect of implant macrodesign and position, related to the bone crest, on bone-to-implant contact (BIC) and crestal bone (CB) in immediate implants. MATERIAL AND METHODS: The study comprised of six foxhound dogs in which 48 immediate implants were placed. Three types of implants from the same manufacturer with similar surface characteristics but different macrodesigns were randomly placed: Group A (external hex with no collar microthreads), Group B (internal hex and collar microrings), and Group C (internal conical connection and collar microrings). Half of the implants were placed leveled with the bone crest (control) and the remaining, 2 mm subcrestally (test). Block sections were obtained after 12 weeks and processed for mineralized ground sectioning. Statistical analysis consisted of nonparametric Friedman and Wilcoxon test. RESULTS: All implants were clinically stable and histologically osseointegrated. Mean BIC percentage within the control group was as follows: A: 42.52 ± 8.67, B: 35.19 ± 18.12, and C: 47.46 ± 11.50. Within the test group: A: 47.33 ± 5.23, B: 48.38 ± 11.63, and C: 54.88 ± 11.73. Differences between each subgroup in the test and the control groups were statistically significant. BIC was statistically significantly higher in the test (50.588 ± 8.663) than in the control (43.317 ± 9.851) group. Within both groups, differences between group C and the other 2 were statistically significant. Distance from the implant shoulder to the buccal CB was statistically significantly larger in the control than in the test group and between subgroups B and C in the control and test groups. Within the test groups, relative bone gain was noticed. CONCLUSIONS: Subcrestal immediate implant positioning may lead to a relatively reduced CB resorption and increased BIC. Implants macrodesign with crestal microrings may enhance BIC in post-extraction implants.

Addition of Resolvins D1 or E1 to Collagen Membranes Mitigates Their Resorption in Diabetic Rats.

Uncontrolled diabetes is characterized by aberrant inflammatory reactions and increased collagenolysis. We have reported that it accelerates the degradation of implanted collagen membranes (CM), thus compromising their function in regenerative procedures. In recent years, a group of physiological anti-inflammatory agents called specialized pro-resolving lipid mediators (SPMs) have been tested as a treatment for various inflammatory conditions, either systemically or locally, via medical devices. Yet, no study has tested their effect on the fate of the biodegradable material itself. Here, we measured the in vitro release over time of 100 or 800 ng resolvin D1 (RvD1) incorporated into CM discs. In vivo, diabetes was induced in rats with streptozotocin, while buffer-injected (normoglycemic) rats served as controls. Resolvins (100 or 800 ng of RvD1 or RvE1) were added to biotin-labeled CM discs, which were implanted sub-periosteally over the calvaria of rats. Membrane thickness, density, and uniformity were determined by quantitative histology after 3 weeks. In vitro, significant amounts of RvD1 were released over 1-8 days, depending on the amount loaded. In vivo, CMs from diabetic animals were thinner, more porous, and more variable in thickness and density. The addition of RvD1 or RvE1 improved their regularity, increased their density, and reduced their invasion by the host tissue significantly. We conclude that addition of resolvins to biodegradable medical devices can protect them from excessive degradation in systemic conditions characterized by high degree of collagenolysis.

Enzyme-Responsive Nanoparticles for Dexamethasone Targeted Delivery to Treat Inflammation in Diabetes.

Diabetes is a global epidemic accompanied by impaired wound healing and increased risk of persistent infections and resistance to standard treatments. Therefore, there is an immense need to develop novel methods to specifically target therapeutics to affected tissues and improve treatment efficacy. This study aims to use enzyme-responsive nanoparticles for the targeted delivery of an anti-inflammatory drug, dexamethasone, to treat inflammation in diabetes. These nanoparticles are assembled from fluorescently-labeled, dexamethasone-loaded peptide-polymer amphiphiles. The nanoparticles are injected in vivo, adjacent to labeled collagen membranes sub-periosteally implanted on the calvaria of diabetic rats. Following their implantation, collagen membrane resorption is linked to inflammation, especially in hyperglycemic individuals. The nanoparticles show strong and prolonged accumulation in inflamed tissue after undergoing a morphological switch into microscale aggregates. Significantly higher remaining collagen membrane area and less inflammatory cell infiltration are observed in responsive nanoparticles-treated rats, compared to control groups injected with free dexamethasone and non-responsive nanoparticles. These factors indicate improved therapeutic efficacy in inflammation reduction. These results demonstrate the potential use of enzyme-responsive nanoparticles as targeted delivery vehicles for the treatment of diabetic and other inflammatory wounds.

Histologic and Histomorphometric Evaluation of a Connective Tissue Graft Retrieved with a Novel Deepithelialization Procedure: A Case Series.

Various free connective tissue graft (CTG) harvesting techniques have been reported. The lining epithelium of the palatal graft may be retrieved either intra- or extraorally. This report presents a series of root coverage cases where deepithelialization was intraorally performed before harvesting the graft with a round diamond bur mounted on a low-speed handpiece. Ten single-tooth gingival recession defects in five patients were treated, applying a surgical procedure based on a coronally advanced flap combined with a free CTG that was deepithelialized in situ by the same method. Recession and probing depths and keratinized tissue and recession widths were recorded at baseline and the follow-up evaluations. Follow-up was between 7 and 21 months (mean: 12.1 ± 5.04 months). Clinical, esthetic, and histologic evaluations were performed. Mean root coverage was 89% ± 24.86% (range: 25% to 100%), and complete root coverage was observed in 80% of cases; the esthetic score range was 6 to 9 (mean: 7.44 ± 1.01). Epithelial remnants, although different in proportions, were evident in all samples (range of prevalence: 4.57% to 29.12%). Within the limitations of the small number of clinical cases, the presented in situ deepithelialization technique for CTG seems to be valuable and may accordingly be routinely applied.

Simultaneous versus two-stage implant placement and guided bone regeneration in the canine: histomorphometry at 8 and 16 months.

AIM: To compare the effect of timing of implant placement and guided bone regeneration (GBR) procedure on osseointegration and newly formed bone at 8 and 16 months. MATERIAL AND METHODS: In seven dogs, four different sites were bilaterally established: (1) an implant placed in a 6-month healed (6m-GBR) bovine bone mineral (BBM) grafted site; (2) a simultaneously placed implant with the grafted BBM (Si-GBR) followed by a membrane coverage; (3) an implant placed in a membrane-protected non-grafted defect; and (4) an implant placement in a naturally healed site (Cont). Histomorphometry was obtained at 8 and 16 months post-implant placement. Bone-implant contact (BIC), crestal bone resorption (CBR), vertical intra-bony (VIB) defect, bone (BAF) and particle (PAF) area fractions, and osteoconductivity (CON) levels were measured. RESULTS: In all sites, BIC ranged between 62% and 79% with no significant differences. PAF ranged from 17% to 27%, with no effect of time. At 8 and 16 months, BAF was significantly smaller at the Si-GBR site when compared with all other sites, CON was significantly greater at the 6m-GBR site, and CBR and VIB were significantly smaller at the 6m-GBR when compared with the Si-GBR sites. CONCLUSIONS: The simultaneous and delayed techniques both showed a similar osseointegration level over time. However, the staged approach showed enhanced newly formed bone, higher osteoconduction around the grafted mineral, less CBR, and smaller vertical bone defect over time compared with the combined approach.

Clinical evaluation of particulate allogeneic with and without autogenous bone grafts and resorbable collagen membranes for bone augmentation of atrophic alveolar ridges.

OBJECTIVES: The evaluate the clinical outcome of bone augmentation with the use of particulate mineralized freeze-dried bone allograft (FDBA) with or without the addition of autogeneous bone chips, applied in a bi-layered (BL) technique, covered by a resorbable cross-linked collagen membrane. MATERIAL AND METHODS: Fifty patients presenting with a vertical and/or lateral ridge deficiency of at least 3 mm were included: Group FDBA, N=27 patients, particulate FDBA was the only graft; and Group BL, N=23 patients, a BL bone grafting procedure where autogenous bone chips were the inner layer and FDBA the outer. Bone graft was covered with a ribose cross-linked collagen barrier membrane. Ridge dimensions were clinically or radiographically (computerized tomography scan) measured at the time of the bone augmentation procedure and at implant placement or uncovering and the maximum linear vertical or horizontal calcified tissue gain was calculated. Statistical analysis consisted of linear regression analysis, with maximum bone gain being the dependent variable. RESULTS: In the FDBA group, mean vertical bone gain was 3.47 mm (SD 1.25) and the horizontal, 5 mm (SD 1.28), while in the BL values were 3.5 mm (SD 1.2) and 3.6 mm (SD 1.72), respectively. Addition of autogenous bone does not appear to statistically significantly enhance the results. Spontaneous membrane exposure occurred in 24% of the cases and was the only variant that significantly influenced results (P<0.001). CONCLUSIONS: Large vertical and/or horizontal ridge deficiencies may be treated with FDBA and ribose cross-linked collagen barrier membranes with good clinical outcome. No added effect of the application of a layer of autogenous bone in these bone augmentation procedures could be demonstrated. Spontaneous membrane exposure was the only parameter to affect the degree of new calcified tissue formation.

Accelerated degradation of collagen membranes in type 1 diabetic rats is associated with increased expression and production of several inflammatory molecules.

BACKGROUND: Membrane durability is critical for regenerative procedures. We reported previously that type 1-like diabetes in rats accelerates the degradation of collagen membranes and we tested here whether this is associated with increased local production of inflammatory molecules as part of a diabetes-induced chronic inflammation around and within the membranes. METHODS: Collagen membrane discs were implanted under the scalp in diabetic (streptozotocin-induced) and control rats, which were sacrificed after 2 or 3 weeks. Total RNA and proteins were isolated from the membrane and its surrounding tissues and the expression and production of six inflammatory molecules (interleukin-6 [IL-6], tumor necrosis factor alpha [TNFα], matrix metalloproteinase [MMP]-9, macrophage migration inhibitory factor [MIF], MIP-1α, and MIP-2α) was measured using real-time PCR and western blotting, respectively. Minimal histological analysis of the membranes was conducted to conform to previous studies. RESULTS: Hyperglycemia resulted in reduced membrane thickness (by 10% to 25%) and increased mononuclear infiltrate inside the membrane. mRNA and protein levels of IL-6, TNFα, and MMP-9 were elevated in diabetic rats both 2 and 3 weeks post-surgery. The levels (both mRNA and protein) of MIF were increased at 2 weeks post-surgery and those of MIP-1α and MIP-2α at 3 weeks. There was a very good match in the temporal changes of all examined genes between the mRNA and protein levels. CONCLUSIONS: Elevated local production of inflammatory cytokines and MMPs, together with apparent mononuclear infiltrate and increased collagenolysis confirm that hyperglycemia leads to a chronic inflammation in and around the implanted collagen membranes, which reduces membrane longevity.

Porphyromonas gingivalis lipopolysaccharide and glycated serum albumin increase the production of several pro-inflammatory molecules in human gingival fibroblasts via NFκB.

OBJECTIVE: Diabetes increases the incidence/severity of periodontal diseases by inducing a chronic inflammation, driven by accumulation of AGEs (advanced glycation end products). We tested whether glycated human serum albumin (G-HSA, a form of AGE), representing a diabetic state, augments the pro-inflammatory response of human gingival fibroblasts (hGFs) to a bacterial challenge (Porphyromonas gingivalis Lipopolysaccharide (LPS)). METHODS: Primary hGFs were incubated with LPS (0.5-5 µg/mL) and G-HSA (50-200 µg/mL) and the production and gene expression of IL-1ß, IL-6, IL-8, MMP-1, MCP-1, and TNFα were analyzed by Magnetic Luminex Assay and real-time PCR, respectively. Non-glycated serum albumin (HSA) served as negative control. Cytotoxicity of the 2 agents was tested with an XTT assay. NFκB activation (p65 phosphorylation) was measured with an ELISA. RESULTS: P. gingivalis LPS and G-HSA were not toxic to hGFs and increased the amount of MMP-1, MCP-1, IL-6, and IL-8, (but not TNFα and IL-1ß) secreted into the medium at 24 h. Control HSA had no effect. Many LPS/G-HSA combinations displayed a synergistic stimulation of these molecules. Both agents increased mRNA levels of these 4 molecules at 6 h, 12 h or both (IL-6). NFκB activation at 6 h was caused by both agents with a possible synergism at the higher concentrations. CONCLUSIONS: glycated albumin augments the pro-inflammatory response of human gingival fibroblasts to P. gingivalis LPS. Thus, AGE accumulation in diabetes could aggravate periodontal inflammation by augmenting the pro-inflammatory response of host GFs to P. gingivalis, a well-recognized periopathogenic bacteria.

Dental implant placement is a possible risk factor for the development of multiple cracks in non-endodontically treated teeth.

The objective of this study was to evaluate potential risk factors, including the placement of dental implants, for the development of tooth cracks. A series of 212-patients, who were referred for endodontic treatment, were retrospectively screened, of which 72 (34%) patients had been diagnosed with 80-cracked teeth confirmed with an operating microscope. These patients had an average age of 53-years and were equally distributed between genders. Forty-one percent of the cracked teeth were diagnosed after the placement of dental implants, with an average of 3-implants per patient. Seventy percent of the cracks were diagnosed more than 1-year after implant loading. Implant placement was associated with higher odds of having multiple cracks (OR = 9.78, CI:2.320, 41.216)(p < 0.05). The proportion of cracked premolars was relatively high (30%), and most cracked teeth (79%) were vital and with a normal periapical diagnosis (86%). Most cracked teeth (71%) had an amalgam restoration, and teeth restored with amalgam were at a higher risk of having multiple cracks (p < 0.05). Clinicians should be aware of a common profile of endodontic patients with multiple cracks in a non-endodontically treated premolar, restored with an amalgam restoration, which was diagnosed with the cracks more than 1-year after reconstruction utilizing multiple implants.

Systemic tetracycline delays degradation of three different collagen membranes in rat calvaria.

OBJECTIVES: The aim of this study was to quantitatively evaluate the effect of systemic tetracycline (TTC) on the degradation of three different collagen membranes. MATERIALS AND METHODS: Collagen membranes were cut into 5 mm diameter membrane discs and labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester. One membrane disc each of a non-cross-linked [BioGide (BG)], glutaraldehyde cross-linked [BioMend Extend (BM)], and ribose cross-linked [Ossix (OS)] was implanted on the calvaria of 40 Wistar rats. Another 10 biotinylated collagen membrane discs from each membrane type were processed for histologic observation and served as baseline; half of them (five from each group) were also treated with formic acid to inspect possible interference with biotinilazation of collagen by formic acid used during the decalcification process. A 10 mg/kg dose of TTC (50% of the minimal recommended antibacterial dose) to the experimental (20 animals) and saline to the control (20 animals) group was administered intramuscularly every 3 days. From each group, block sections were retrieved in half of the animals after 14 days and in the remaining after 28 days. Decalcified tissue histology was stained with streptavidin horseradish peroxidase. A computer-assisted program measured the membranes' collagen contents. Statistical analysis consisted of analysis of variance (ANOVA) with repeated measures. RESULTS: No statistically significant differences in collagen contents were appreciated between biotinylated non-implanted membranes treated or not treated by formic acid. Systemic TTC had a different effect on the bio-degradation of the membranes: while it significantly decreased the resorption of two of the membranes (BG and BM), it had minimal influence on the ribose cross-linked membrane (OS). ANOVA with repeated measures, tests of within-subjects effects, showed a statistically significant difference between the membranes (P<0.001), within the membranes at the different time-points (P<0.001), a significant interaction between membranes and time and between the membranes and administered TTC (P<0.001). Test of between-subject effects revealed a statistically significant interaction with time and with TTC (P<0.001). CONCLUSIONS: Systemically administered TTC in sub-antibacterial doses may offer a possible treatment alternative to reduce bio-degradation and enhance bio-durability of certain collagen membranes. The findings of the present study could have clinical application in large non-self-contained bone defects, where prolonged membrane barrier functions are desirable.

Bio-degradation of a resorbable collagen membrane (Bio-Gide) applied in a double-layer technique in rats.

OBJECTIVE: The aim of this study was to evaluate histologically the bio-degradation of two layers of Bio-Gide((R)) (BG) membrane, as compared with that of a single layer. MATERIAL AND METHODS: Two circular calvarial bony defects, 5 mm in diameter, were made in 24 Wistar rats. BG membrane, labeled with biotin, was cut into 5-mm-diameter disks, and placed in defects either as a mono-layer membrane (MLM) or as a double-layer membrane (DLM). Rats were sacrificed after 4 or 9 weeks and histology was performed. Membranes were stained with horseradish peroxidase-conjugated streptavidin and aminoethyl carbazole as a substrate for detection of biotinylated collagen. The area of collagen and thickness of the residual membranes were measured by image analysis software. Statistical analysis was performed using the non-parametric Wilcoxon's signed-ranks test. RESULTS: At 4-week collagen area per measurement window within the DLM sites (0.09+/-0.05 mm(2)) was significantly greater (P<0.01) than that in the MLM sites (0.047+/-0.034 mm(2)). At 9 weeks, the collagen area was also greater in the DLM sites (0.037+/-0.026 mm(2)) compared with that of the MLM sites (0.025+/-0.016 mm(2)); however, this difference did not reach statistical significance. The rate of membrane degradation, calculated as percent membrane lost compared with baseline, was similar for the DLM and MLM at both time points ( approximately 60% at 4 weeks and approximately 80% at 9 weeks). In addition, the residual DLM thickness at 4 weeks (475.5+/-73.77 mum) was significantly (P<0.01) greater than that of MLM (262.38+/-48.01 mum). At 9 weeks, membrane thickness was also greater in the DLM sites (318.22+/-70.45 mum) compared with that of the MLM sites (183.32+/-26.72 mum); however, this difference did not reach statistical significance. The reduction in thickness between 4 and 9 weeks was 30% for MLM and 33% for DLM. DISCUSSION: The use of a double layer of BG membrane results in a barrier of increased collagen area and thickness, compared with application of a single layer.

Modified Tunnel Double Papilla Procedure for Root Coverage in the Anterior Mandible.

Free connective tissue grafts, barrier membranes, pedicle flaps, soft tissue allografts, and xenografts have been described for root coverage and augmenting the zone of attached gingiva. The present report evaluated a modified tunnel surgical procedure for root coverage of mandibular anterior teeth where a connective tissue graft was combined with a tunnel and double papilla flap. Fourteen patients with 18 consecutive Miller Class I or II gingival recession defects in the anterior mandible were treated with a connective tissue graft combined with a tunnel and double papilla flap procedure. The following parameters were recorded at baseline and every 6 months postsurgery for up to 19 months: probing depth (PD), vertical recession dimension (RD), keratinized tissue width (KT), and recession width (RW). Statistical analysis consisted of descriptive statistics, analysis of variance with repeated measures, and t test. Statistical analysis proved significant differences between pre- and postoperative values. Mean percentage of root coverage was 83.28% (standard deviation: 22.897), while complete root coverage was obtained in 55% of sites. Baseline values differed between Class I and II recession defects. Clinical attachment level gain, KT gain, and amount of root coverage were statistically significantly larger in Class II defects, while the degree of residual recession and percentage of root coverage were similar in both recession classes. A statistically significant interaction between recession class, independent variable, and pre- and postoperative vertical recession defects (dependent variables) was recorded (P = .004). Within the limitations of the sample size, the reported procedure showed predictable root coverage with color match combined with an increased zone of keratinized tissue.

Cross-linked and non-cross-linked collagen barrier membranes disintegrate following surgical exposure to the oral environment: a histological study in the cat.

BACKGROUND: Early barrier membrane degradation may result in decreased bone formation in guided bone regeneration (GBR) procedures. The aim of this study was to evaluate the bio-degradation of cross-linked (CLM) and non-cross-linked (NCLM) collagen membranes experimentally exposed to the oral environment of study animals. METHODS: In eight cats, 48 surgical procedures were performed, three along each side of the palate: 24 full-thickness soft tissue perforations were made and 24 full-thickness mini-flaps were raised. CLM or NCLM discs were placed either under the perforations and peripheral mucosa and left exposed (experimental) or covered by the flaps (controls). The four treatment modalities were equally distributed among the eight animals. Block sections were retrieved at 7 and 28 days post-operatively, providing histological specimens (6 each) at 7 and 28 days for each treatment modality. RESULTS: Histological observation revealed that CLM and NCLM remained intact in the control sites during the 28 days. At 7 and 28 days, CLM appeared interrupted in three and two experimental sites, respectively, and were undetected in the remaining sites. NCLM were interrupted in two sites each at 7 and 28 days, and were undetected in the other sites. There was no statistical difference between control specimens and between CLM and NCLM of the different treatment modalities at 7 or 28 days. CONCLUSIONS: Both cross-linked and non-cross-linked membranes were resistant to tissue degradation and maintained continuity throughout the study. However, none of the membranes was resistant to degradation when exposed to the oral environment

Histomorphometric assessment of bone formation in sinus augmentation utilizing a combination of autogenous and hydroxyapatite/biphasic tricalcium phosphate graft materials: at 6 and 9 months in humans.

OBJECTIVE: The aim of this study was to examine the efficacy of a new biphasic hydroxyapatite/tricalcium phosphate (HA/TCP) bone substitute in combination with particulate autogenous bone in sinus floor augmentation procedures. MATERIAL AND METHODS: A simultaneous or a two-stage sinus augmentation and implant placement were conducted in 28 patients. A mixture of HA/TCP and autogenous bone chips in a 1 : 1 ratio was used as the grafting biomaterial. Cylindrical specimen bone retrieval was performed in all patients except one. Specimens were harvested either at 6 (n=14) or 9 (n=13) months post-augmentation. For histologic and histomorphometric evaluations, the non-decalcified tissue processing (Donath's technique) was performed. RESULTS: Newly formed bone around the grafted particles was found in all samples. The encircling, highly cellular bone followed the outline of the grafted particles in direct contact. Both woven and lamellar types of bone were observed. Morphometrically, the total mean bone area fraction of all sections was 34.8+/-10.3%, increasing from 28.6+/-7.8% at 6 months to 41.6+/-8.3% at 9 months (P<0.001). Mean particle area fraction average was 25.5+/-11.6% and 23.5+/-9.3% at 6 and 9 months, respectively, with a total mean of 24.5+/-10.4%. The increase in bone area fraction was not significantly correlated to the decrease of the grafted particles area fraction. CONCLUSIONS: The biphasic HA/TCP showed biocompatible and osteoconductive properties. This alloplast as a composite with autogenous bone chips promotes newly formed bone, which increases in its fraction along an extended healing period.