The effect of experimental diabetes and membrane occlusiveness on guided bone regeneration: A proof of principle study.

OBJECTIVES: To evaluate the effect of membrane occlusiveness and experimental diabetes on early and late healing following guided bone regeneration. MATERIAL AND METHODS: A total of 30 Wistar rats were randomly allocated to three groups: healthy (H), uncontrolled diabetic (UD) and controlled diabetic (CD). A critical size calvarial defect (CSD) was created at the mid-portion of one parietal bone, and it was treated with a double layer of e-PTFE membrane presenting 0.5 mm perforations. The animals were killed at 7 and 30 days of healing, and qualitative and quantitative histological evaluations were performed. Data were compared with the ones previously obtained from other 30 animals (10H, 10UD, 10 CD), where two CSDs were randomly treated with a double-layer e-PTFE occlusive membrane or left empty. RESULTS: Following application of cell occlusive or cell permeable membranes, significant regeneration can be observed. However, at 30 days in the H group occlusive compared to cell permeable membranes promoted enhanced bone regeneration (83.9 ± 7.3% vs. 52.5 ± 8.6%), while no significant differences were observed within the CD and UD groups. UD led to reduced regeneration compared to H when an occlusive barrier was applied, whereas comparable outcomes to H and CD were observed when placing perforated membranes. CONCLUSION: The application of cell permeable membranes may have masked the potentially adverse effect of experimental UD on bone regeneration. CLINICAL RELEVANCE: Membrane porosity might contribute to modulate the bone regenerative response in UD conditions. Future studies are needed to establish the degree of porosity associated with the best regenerative outcomes as well as the underlying molecular mechanisms.

The effect of experimental diabetes and glycaemic control on guided bone regeneration: histology and gene expression analyses.

OBJECTIVES: To investigate the effect of experimental diabetes and metabolic control on intramembranous bone healing following guided bone regeneration (GBR). MATERIAL AND METHODS: Ninety-three Wistar rats were allocated to three experimental groups, healthy (H), uncontrolled diabetes (D) and controlled diabetes (CD). Twenty one days following diabetes induction, a standardised 5-mm defect was created at the mid-portion of each parietal bone. In 75 animals (25H, 25D, 25CD), one defect was treated with an intracranial and extracranial membrane according to the GBR principle, and one defect was left empty (control); five animals per group were then randomly sacrificed at 3, 7, 15, 30 and 60 days and processed for decalcified histology. In 18 animals (6H, 6D, 6CD), both defects were treated according to the GBR principle; three animals from each group were then randomly sacrificed at 7 and 15 days of healing and employed for gene expression analysis. RESULTS: Application of the GBR therapeutic principle led to significant bone regeneration even in the D group. However, at 15 and 30 days, the osteogenesis process was impaired by uncontrolled diabetes, as shown by the significant reduction in terms of defect closure (38-42%) and newly formed bone (54-61%) compared to the healthy group. The comparison of the D vs. H group at 15 days of healing yielded the largest number of genes with significantly differential expression, among which various genes associated with the ossification process (bmp4, ltbp4, thra and cd276) were identified. CONCLUSIONS: Uncontrolled diabetes seems to affect early phases of the bone regeneration following GBR. A misregulation of genes and pathways related to cell division, energy production, inflammation and osteogenesis may account for the impaired regeneration process in D rats. Further studies are warranted to optimise the GBR process in this medically compromised patient population.

Microarray gene expression during early healing of GBR-treated calvarial critical size defects.

OBJECTIVES: To investigate the gene expression and molecular pathways implicated in the regulation of the osseous healing process following guided bone regeneration (GBR). MATERIAL AND METHODS: Six 6-month-old Wistar male rats were used. Standardized 5-mm critical size defects were created in the parietal bones of each animal and treated with an extracranial and intracranial ePTFE membrane, according to the GBR principle. Three animals were randomly sacrificed after 7 and 15 days of healing. Total RNA was extracted from each sample and prepared for gene expression analysis. RNA quality and quantity were assessed, followed by hybridization of the cRNA to Affymetrix GeneChip Rat Genome 230 2.0 Arrays. The Affymetrix data were processed, and first-order analysis, quality control and statistical analysis were performed. Biological interpretation was performed via pathway and Gene Ontology (GO) analysis. RESULTS: Between the 7- and 15-day samples, 538 genes were differently regulated. At day 7, inflammatory and immune responses were clearly upregulated. In addition, GO terms related to angiogenesis and cell cycle regulation were overexpressed. At day 15, a more complex cellular activity and cell metabolism were evident. The bone formation processes were significantly overexpressed, with several genes encoding growth factors, enzyme activity, and extracellular matrix formation found as upregulated. Remarkably, a negative regulation of Wnt signalling pathway was observed at 15 days. DISCUSSION: The gene expression profile of the cells participating in osseous formation varied depending on the healing stage. A number of candidate genes that seem differentially expressed during early stages of intramembranous bone regeneration was suggested.

Effect of Wnt3a delivery on early healing events during guided bone regeneration.

OBJECTIVE: To evaluate the effect of recombinant Wnt3a delivery on the bone regeneration potential following application of the guided bone regeneration (GBR) principle. MATERIALS AND METHODS: A critical-size calvarial defect was created on each parietal bone of 14 Wistar strain rats. One defect was used as the test side and was treated with a collagen sponge carrying 2.0 µg of recombinant Wnt3a protein, whereas the contralateral side served as sham-operated control. Both defects were covered at both the extracranial and intracranial aspects with ePTFE non-resorbable membranes, according to the GBR principle. Following healing periods of 4 and 7 days, qualitative histological and histomorphometric evaluation of undecalcified sections was performed in subgroups of seven animals. The primary outcome parameter was the mean percentage of defect closure in the test and control defects. RESULTS: At 4 days of healing, a network of coagulum and fibrin was observed and initial signs of granulation tissue formation were evident with no apparent differences between the test and control groups. At 7 days of healing, the test group presented newly formed woven bone, originating from the borders of the defect, as opposed to the control group, whereby woven bone formation was not observed in any of the specimens. CONCLUSIONS: The delivery of mouse recombinant Wnt-3a protein in combination with GBR may promote woven bone formation in critical-size calvarial defects at 7 days of healing.

Transcriptional profiling of "guided bone regeneration" in a critical-size calvarial defect.

OBJECTIVES: Guided bone regeneration (GBR) is a commonly utilized surgical technique in the craniofacial region. The transcriptional mechanisms associated with this type of bone regeneration are not well understood. The aim of this study was to characterize the transcriptome associated with GBR of a critical-size calvarial defect in the rat. MATERIAL AND METHODS: Critical-size calvarial defects were created in six Wistar strain rats and treated according to the principles of GBR. The tissue filling the regenerating defect was harvested at 7 and 14 days. Total RNA was extracted and microarray analysis was carried out to identify the differences in the transcriptome between days 7 and 14. RESULTS: Gene ontology (GO) analysis of the genes up-regulated at day 7 showed that immature wound healing-related mechanisms, such as protein metabolism and cell proliferation, were up-regulated at this time point. Furthermore, the immuno-inflammatory process was also up-regulated at the earlier time point. In contrast, by day 14, GO groups consistent with wound maturation, such as extracellular matrix formation, anatomical structure development and cell differentiation, were up-regulated. Furthermore, the functionally important GO categories of skeletal development, ossification and bone mineralization were up-regulated at day 14. Genes of interest that belonged to this group and were up-regulated at day 14 included growth and differentiation factors (Bmp2, Bmp3, Tgfb3), extracellular matrix proteins (osteocalcin, osteomodulin, stenniocalcin 1) and transcription factors (Runx2, Sox6, Satb2). Furthermore, a number of genes associated with Tgfß/Bmp and Wnt signalling were also up-regulated. Besides skeletogenesis, genes associated with angiogenesis and neurogenesis were also up-regulated at day 14. CONCLUSIONS: The transcriptome associated with a maturing GBR-treated craniofacial bone defect is characterized by the down-regulation of the immuno-inflammatory response and up-regulation of skeletogeneis-, angiogenesis- and neurogenesis-associated genes. The Tgfß/Bmp and Wnt signalling pathways play an important role in the regenerative process.

In vivo gene expression profile of guided bone regeneration associated with a microrough titanium surface.

OBJECTIVES: To determine the gene expression profile characteristic of "guided bone regeneration" associated with a microrough titanium surface. MATERIAL AND METHODS: Critical-size calvarial defects were treated with the principle of "guided bone regeneration," whereby the extracranial barriers were either polished (SMO) or microrough (SLA) titanium disks. After 7 and 14 days, the contents of the regenerating defect were collected, RNA was extracted and microarray analysis was carried out. At each time point, the healing associated with the microrough surface was compared with that associated with the polished titanium surface. RESULTS: On comparing the SLA and SMO profiles, there were few genes different at day 7 (∼250), whereas there were a large number of genes different at day 14 (∼6500). At day 14, the list of genes that were differentially regulated in response to the SMO and SLA surfaces had an over-representation of genes associated with the functionally relevant gene ontology categories of regeneration, skeletogenesis, mesenchymal cell differentiation, angiogenesis and neurogenesis. There were a greater number of genes within each of these functionally relevant categories that were up-regulated on the SLA surface compared with the SMO surface. The main signalling pathway that was differentially regulated between the two surfaces at day 14 was the Wnt signaling pathway. CONCLUSIONS: Minimal difference was observed between the SMO and the SLA samples at day 7, whereas significant differences were noted at day 14, including genes associated with a number of functionally relevant gene ontology groups. The differentially regulated biological processes provide an insight into the influence of surface topography on "guided bone regeneration" at the cellular and molecular level.

The effect of diabetes mellitus on osseous healing.

Diabetes mellitus and, in particular, type 1 diabetes has been associated with impaired osseous wound healing properties. The scope of the present review is to discuss the clinical evidence supporting a higher rate of complications during fracture healing in diabetic patients and the histological evidence indicating impaired potential for intramembranous and endochondral ossification in the presence of uncontrolled experimental diabetes. The article further provides a synthesis of our current understanding of the plausible molecular mechanisms underlying the diabetic bone healing pathophysiology and of the role of insulin treatment in promoting osseous healing in the diabetic status.

Comparison of gingival blood flow during healing of simplified papilla preservation and modified Widman flap surgery: a clinical trial using laser Doppler flowmetry.

AIM: This prospective randomized-controlled clinical trial compared the gingival blood flow responses following simplified papilla preservation (test) versus modified Widman flap (control). MATERIALS AND METHODS: Twenty contra-lateral upper sites with pocket depth > or = 5 mm after initial treatment in 10 chronic periodontitis patients were randomly assigned to either test or control treatment, using a split-mouth design. Laser Doppler flowmetry recordings were performed pre-operatively, following anaesthesia, immediately post-operatively and on days 1, 2, 3, 4, 7, 15, 30 and 60, at nine selected sites per flap. RESULTS: Significant ischaemia was observed at all sites following anaesthesia and immediately post-operatively. At the mucosal flap basis, a peak hyperaemic response was observed on day 1, which tended to resolve by day 4 at the test sites, but persisted until day 7 at the control sites. The buccal and palatal papillae blood perfusion presented the maximum increase on day 7 in both groups and returned to baseline by day 15. Both surgical modalities yielded significant pocket depth reduction, recession increase and clinical attachment gain. CONCLUSIONS: Periodontal access flaps represent an ischaemia-reperfusion flap model. The simplified papilla preservation flap may be associated with faster recovery of the gingival blood flow post-operatively compared with the modified Widman flap.

Gingival blood flow changes following periodontal access flap surgery using laser Doppler flowmetry.

AIM: To investigate the pattern of gingival blood flow changes following periodontal access flap surgery by laser Doppler flowmetry (LDF). MATERIAL AND METHODS: Fourteen patients with chronic periodontitis presenting upper anterior sites with pocket depth >or=5 mm after initial treatment were included in the study. Periodontal access flap surgery was performed on the experimental areas and LDF recordings were taken at baseline, following anaesthesia, immediately postoperatively and on days 1, 2, 3, 4, 7, 15, 30 and 60 of healing, at nine predetermined sites per flap. RESULTS: Significant ischaemia was observed at all flap sites following anaesthesia and immediately postoperatively. At the alveolar mucosal sites, a peak increase of the gingival blood flow was observed on postoperative day 1 (p<0.001), which persisted until day 7 (p=0.012) and resolved by day 15. The mucosal sites close to the flap periphery presented higher blood perfusion compared with the sites located centrally in the flap. The microcirculatory perfusion of the buccal and palatal papillae was maximum on postoperative day 7 (p=0.013 and <0.001, respectively) and returned to baseline by day 15. CONCLUSION: Topographically distinct areas of the periodontal access flap consistently present different patterns of microvascular blood flow alterations during the wound-healing period.