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.

Cross-linked hyaluronic acid slows down collagen membrane resorption in diabetic rats through reducing the number of macrophages.

OBJECTIVES: We previously showed that accelerated degradation of collagen membranes (CMs) in diabetic rats is associated with increased infiltration of macrophages and blood vessels. Since pre-implantation immersion of CMs in cross-linked high molecular weight hyaluronic acid (CLHA) delays membrane degradation, we evaluated here its effect on the number of macrophages and endothelial cells (ECs) within the CM as a possible mechanism for inhibition of CM resorption. MATERIALS AND METHODS: Diabetes was induced with streptozotocin in 16 rats, while 16 healthy rats served as control. CM discs were labeled with biotin, soaked in CLHA or PBS, and implanted under the scalp. Fourteen days later, CMs were embedded in paraffin and the number of macrophages and ECs within the CMs was determined using antibodies against CD68 and transglutaminase II, respectively. RESULTS: Diabetes increased the number of macrophages and ECs within the CMs (∼2.5-fold and fourfold, respectively). Immersion of CMs in CLHA statistically significantly reduced the number of macrophages (p < 0.0001) in diabetic rats, but not that of ECs. In the healthy group, CLHA had no significant effect on the number of either cells. Higher residual collagen area and membrane thickness in CLHA-treated CMs in diabetic animals were significantly correlated with reduced number of macrophages but not ECs. CONCLUSIONS: Immersion of CM in CLHA inhibits macrophage infiltration and reduces CM degradation in diabetic animals. CLINICAL RELEVANCE: The combination of CLHA and CM may represent a valuable approach when guided tissue regeneration or guided bone regeneration procedures are performed in diabetic patients.

Hyaluronic acid slows down collagen membrane degradation in uncontrolled diabetic rats.

AIM: To examine the in vitro biokinetics of hyaluronic acid (HA) from a collagen membrane (CM) and to evaluate the in vivo effect of immersion of the CM in HA solution on its degradation in streptozotocin (STZ)-induced diabetes conditions in a rat calvaria subcutaneous model. BACKGROUND: CM degradation is accelerated in uncontrolled diabetic rats. Immersion of CM in HA has been suggested to decrease their resorption rate without interfering with their tissue integration and structural degradation. However, it is unknown to what extent CM degradation may be influenced by its immersion in HA solution under a condition mimicking a medically compromised situation with an increased inflammatory level such as diabetes. MATERIALS AND METHODS: CMs were soaked in cross-linked HA. Protein adsorption and the HA release were quantified by ELISA. Diabetes was induced in sixteen rats, while 16 healthy rats served as control. CM was prepared and labeled prior to implantation with Biotin. Seventeen CM were immersed in HA and 17 CM in PBS. In each animal, one test or one control disk was implanted. In order to compare the collagen content, two similar non-implanted CM were used as baseline. Fourteen days after surgery, thirty-two animals were sacrificed. The entire calvaria including the skin above, was chemically fixed, decalcified, and embedded in paraffin. Five-µm-thick sections were analyzed histologically and histomorphometrically using H&E and avidin-peroxidase staining. RESULTS: The in vitro results demonstrated that the CM adsorbed roughly 80% of the total HA content. After 10 days, 36.3% of the initial HA remained on the CM. The in vivo results demonstrated that diabetes significantly reduced the thickness of the CM, while HA had a significant effect on keeping the membrane thickness. HA increased the residual collagen content in the diabetic group (P < 0.0001) but no such effect was observed in the healthy group. CONCLUSION: Immersion of CM in HA prior to the implantation delays membrane degradation in uncontrolled diabetic compared with normoglycemic rats.

Accelerated degradation of collagen membranes in diabetic rats is associated with increased infiltration of macrophages and blood vessels.

OBJECTIVES: Increased collagenolytic activity in diabetes may compromise collagen membrane (CM) survival. Tetracycline (TTC) possesses anti-collagenolytic properties and delays CM degradation. This study evaluated macrophage and capillary infiltration within CMs in diabetic rats. MATERIALS AND METHODS: Diabetes was induced in 20 Wistar rats by streptozotocin and 20 served as controls. Biotin-labeled CM discs were immersed in either TTC (50 mg/ml) or PBS. In each animal, 2 discs (TTC and control) were implanted under the parietal periosteum and rats were sacrificed at 2 or 4 weeks post-implantation. The area and thickness of the residual disc collagen were measured following staining with streptavidin, and the number of macrophages and blood vessels within the membranes was determined using specific antibodies (to CD68 and transglutaminase II, respectively). RESULTS: Diabetes significantly reduced the area and thickness of the CMs, while TTC increased CM thickness significantly in both groups of rats at 2 and 4 weeks. Diabetes increased the number of macrophages (∼eightfold at 2 weeks and ∼fourfold at 4 weeks), but TTC had no significant effect. Finally, diabetes increased the number of blood vessels within the discs (∼threefold at 2 weeks and ∼twofold at 4 weeks), while TTC had no effect. CONCLUSIONS: Diabetes increases degradation of native CMs and the number of blood vessels and macrophages within them. TTC immersion delays CM degradation without an apparent effect on macrophage and blood vessel penetration. CLINICAL RELEVANCE: Enhanced CM degradation in diabetic conditions which impair guided regenerative procedure outcome is apparently related to increased blood vessel formation and macrophage infiltration.

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.

Differences in crestal bone-to-implant contact following an under-drilling compared to an over-drilling protocol. A study in the rabbit tibia.

OBJECTIVES: The objective of this study is to compare bone-to-implant contact (BIC) between implants inserted at high torque due to under-drilling of the crestal bone to those inserted at low torque due to over-drilling of the crestal bone. MATERIALS AND METHODS: Forty implants with diameters of 3.75 mm (group A) or 3.55 mm (group B) were inserted in the proximal tibiae of NZW rabbits in two separate surgeries on day 0 or 21. Osteotomy of the crestal bone was finalized with a 3.65-mm drill. In group A, implants were inserted at torque ≥35 Ncm (under-drilling) and in group B with torque <10 Ncm (over-drilling). Implants and their surrounding bone were retrieved on day 42, thus creating 3- and 6-week observation periods, processed for non-decalcified histology and stained with toluidine blue. Crestal BIC (c-BIC) and total BIC (t-BIC) were measured. Wilcoxon test was used to evaluate differences between groups. RESULTS: Three weeks post-surgery, the mean c-BIC in group A was 16.3 ± 3.3 vs 31.5 ± 3.4 % in group B (P < 0.05). At 6 weeks, a similar trend was observed (group A: 28.7 ± 3.6 %; group B: 38.4 ± 4.9 %) (P > 0.05). No differences in t-BIC were noted at 3 weeks and at 6 weeks between the groups. CONCLUSIONS: Insertion of implants with an over-drilling protocol of the crestal aspect of the osteotomy resulted in increased short-term crestal bone-to-implant contact. CLINICAL RELEVANCE: Insertion of implants with a high torque following an under-drilling protocol, commonly used for immediate loading, may reduce crestal bone-to-implant contact at early healing stages.

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.

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.

The effect of surface treatments on the adhesion of electrochemically deposited hydroxyapatite coating to titanium and on its interaction with cells and bacteria.

The effect of different mechanical and chemical pre-treatments on the adhesion strength of hydroxyapatite (HAp) coating on a commercially pure titanium (CP-Ti) substrate was studied by means of a standard tensile test followed by microscopic and chemical analysis to determine the locus of fracture. In addition, the effects of either these pre-treatments or post-treatment by low-energy electron irradiation, which allowed tuning the wettability of the surface, on both osteoblast progenitor attachment and S. aureus bacteria attachment were investigated. A dedicated program was developed for unambiguous identification and count of stained cells. A single-phase HAp coating was formed by electrodeposition. A series of surface pre-treatments consisted of grinding down to P1000, etching in HNO3/HF solution, grit blast, soaking in NaOH and subsequent heat treatment provided the highest adhesion strength to the HAp coating. Osteoblast progenitors derived from rats may be attached preferentially to a hydrophilic surface (post-treatment to θ = 30°), while the bacteria seemed to be less attached to hydrophobic surfaces (post-treatment to θ = 105°). However, the results were not statistically different. The bacteria seemed to be less attached to the smoother, uncoated surfaces.

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.

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.

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.

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac.

Elevated levels of prostaglandins such as PGE(2) in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE(2) inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE(2) effect. GFs derived from healthy human gingiva were treated with PGE(2) and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE(2) inhibited the proliferation of hGFs dose-dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP-breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE(2) and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti-proliferative effect of PGE(2) is mediated via the EP(2) receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE(2) involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho-ERK in hGFs by approximately 300%, PGE(2) decreased it by approximately 50%. Finally, the PGE(2) effect does not require endogenous production of prostaglandins since it was not abrogated by two COX-inhibitors. In conclusion, in human gingival fibroblasts PGE(2) activates the EP(2)-cAMP-Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease.

Human parathyroid hormone 1-34 prevents bone loss in experimental biliary cirrhosis in rats.

BACKGROUND & AIMS: Reduced bone mass and increased fracture rate are complications of primary biliary cirrhosis (PBC). The effect of intermittent administration of human parathyroid hormone (hPTH) 1-34 on bone mass and architecture in bile duct-ligated (BDL) rats was studied. METHODS: Six-month-old male rats were subjected to BDL or sham operation (SO) and were treated from the second postoperative week intermittently with either hPTH 1-34 40 microg/kg per day, 80 microg/kg per day, or a vehicle for 4 weeks. Femoral and tibial bones were evaluated ex vivo by dual x-ray absorptiometry, microcomputed tomography, and histomorphometry. Serum osteocalcin and urinary deoxypyridinoline cross-links (DPD) were determined. RESULTS: BDL rats had decreased bone mass compared with SO rats as indicated by a 6% decrease in femoral and tibial bone mineral density (BMD), 18% reduction in femoral trabecular bone volume (bone volume/total volume [BV/TV]), 17% decrease in trabecular thickness, and 10% decrease in tibial cortical thickness. The administration of hPTH 1-34 at 40 microg/kg per day increased femoral and tibial BMD (9% and 9%), femoral trabecular BV/TV (50%), trabecular thickness (50%), tibial cortical thickness (17%), and serum osteocalcin (82%). On the other hand, hPTH 1-34 80 microg/kg per day had no effect on BMD and tibial cortical thickness, was associated with a smaller increase in trabecular BV/TV (24%), and had a higher osteoclast number and DPD compared with untreated BDL rats and the lower hPTH 1-34 dose treatment group. CONCLUSIONS: BDL rats exhibit loss of bone mass and structure, which can be prevented by the intermittent administration of hPTH 1-34, a potential therapy for osteoporosis in PBC.

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.

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.

Biodegradation of three different collagen membranes in the rat calvarium: a comparative study.

BACKGROUND: Collagen barrier membranes are commonly applied in periodontal and bone-regenerative procedures. Membranes differ in their resorption pattern following implantation, thus influencing clinical outcome. The purpose of this study was to quantitatively evaluate the biodegradation of three different commercially available collagen membranes. METHODS: Collagen membranes were cut into 5-mm-diameter disks and labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester. One membrane disk of each type (non-cross-linked [NCL], glutaraldehyde cross-linked [GCL], and ribose cross-linked [RCL]) was implanted on the calvaria of 20 Wistar rats. Block sections were retrieved after 2 days (baseline, two animals), 14 days (10 animals), or 28 days (eight animals). Decalcified histologic sections were stained with streptavidin horseradish peroxidase. Residual membrane thickness and area were measured. Statistical analysis consisted of analysis of variance (ANOVA) with repeated measures. RESULTS: Statistically significant differences in the amount of residual membrane material were recorded within each membrane (among different time points) and among different membranes at the same time points (P <0.001). At 28 days, the least amount of residual collagen area, expressed as the percentage of baseline, was observed in the NCL group (13.9% +/- 10.25%), followed by the GCL (24.7% +/- 35.11%) and RCL (91.3% +/- 10.35%) groups. Residual membrane thickness, expressed as the percentage of baseline thickness, presented a similar pattern (31% +/- 16.55%, 37% +/- 41.90%, and 94.1% +/- 12.22%, respectively). ANOVA with repeated measures showed a significant interaction between membranes and time (P <0.001). CONCLUSIONS: The tested membranes differed in their degradation patterns and collagen contents. Membranes should be chosen for each clinical case according to the desired biodegradation characteristics.

Histomorphometric evaluation of natural mineral combined with a synthetic cell-binding peptide (P-15) in critical-size defects in the rat calvaria.

PURPOSE: The objective of this study was to histomorphometrically evaluate the synthetic peptide analog P-15 bound to anorganic bovine mineral (Pepgen/P15) in critical-size defects in the rat calvaria. MATERIALS AND METHODS: A 5-mm-diameter critical-size defect was prepared in 48 rat skulls and divided into 4 equal groups: Pepgen/P15 particles covered by a membrane, Pepgen/P15 particles uncovered, nongrafted membrane-protected sites, and nongrafted uncovered control sites. At 12 weeks, histomorphometric measurements were made of the percentage area of newly formed bone and residual particles, the length of internal and external bone bridging, and linearly, the regenerated marginal and central total tissue augmentation height. RESULTS: Nongrafted, membrane-protected sites gained 60.6% of newly formed bone, followed by 50.6% and 44.2% (P < .05 versus membrane only) at the grafted covered and uncovered sites, respectively. All experimental sites contained significantly (P < .005) more bone than did control sites (19.9%). In both types of grafted sites, the percentage area of Pepgen/P15 particles was similar. Mean internal and external length of bone bridging at nongrafted membrane-protected sites (76.7% and 71.2%, respectively) was significantly greater (P < .005) than that of the grafted covered (43.95% and 51.8%, respectively), grafted uncovered (28.7% and 23.9%, respectively), and control (28% and 25.5%, respectively) groups, except for internal bone bridging in the grafted covered sites. Regenerated marginal and central augmentation heights (0.92 mm and 1.02 mm, respectively) were greatest in the grafted covered group, followed by the non-grafted membrane-protected (0.88 mm and 0.51 mm, respectively), and grafted uncovered (0.89 mm and 0.12 mm, respectively) groups, all of which were significantly greater (P < .001) than the control group (0.63 mm and 0.04 mm, respectively). CONCLUSION: While anorganic bovine mineral/cell-binding peptide contributes in volume, membrane application significantly increases the amount of bone regeneration.