Periodontal Fenestration Research Articles

Effect of two delivery systems for recombinant human bone morphogenetic protein-2 on periodontal regeneration in vivo

Resorbable collagen membranes for guided tissue regeneration in periodontal therapy have shown promise but are not osteoinductive. As recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to have an affinity for collagen, the use of this osteoinductive agent incorporated into a collagen vehicle may act as a suitable carrier to promote periodontal regeneration. The aim of this study was to investigate the effects of two different collagen delivery systems for rhBMP-2 in rat periodontal fenestration defects. Using the collagen membrane delivery system, 3 groups of adult Wistar rats which had surgical defects created on the right side of the mandible involving the removal of bone and exposure of the molar roots were treated with either rhBMP-2 in collagen membrane (BMPm) (n= 12 animals), or collagen membrane only (COLm) (n = 12), or were left untreated (UN) (n= 14). Using the collagen gel delivery system, surgical defects were treated with either rhBMP-2 incorporated in a collagen gel carrier (BMPg) (n = 5) or had collagen gel only (COLg) (n = 6). Animals were killed 10 d postoperatively and tissues processed for histology. New bone formation was significantly greater in BMPg compared with both BMPm and controls (p<0.05). However, new cementum formation was significantly greater in BMPm (721 ± 166 μm2, mean ± SE) compared with COLm, COLg and UN (p<0.02) (190±44μm2, 327±114 μm2 and 172 ± 33 μm2, respectively) and more than 1.5 times BMPg (451 ± 158 μm2). In conclusion, both carrier systems for rhBMP-2 significantly increased new bone formation compared with controls during the early stages of periodontal wound healing. However, the more slowly dissolving collagen membrane carrier system for rhBMP-2 produced significantly greater new cementum compared with the collagen gel carrier, suggesting that a more prolonged exposure of rhBMP-2 is required to increase cementogenesis.

Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats

The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue. To obtain the cell sheet, human PDL cells were cultured on temperature-responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing. Most of the experimental group exhibited a new cementum-like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed. Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.

Periodontal tissue engineering by transplantation of multilayered sheets of phenotypically modified gingival fibroblasts

In periodontal tissue engineering, the sourcing of most of the relevant cells is limited by poor accessibility, whereas the use of readily available gingival fibroblasts is hampered because of their inhibitory effects on bone formation. To address the latter drawback, we developed a new graft composed of fibronectin (FN) matrix-based multilayered cell sheets of human gingival fibroblasts modified to express alkaline phosphatase (ALP). This study was undertaken to investigate the effects of this graft, called the FN-ALP transplant, on the healing of periodontal defects in a rat model. The FN-ALP transplants were grafted into periodontal fenestration bone defects in immunosuppressed rats. The process of periodontal healing was examined by histology, histomorphometry and immunohistochemistry. Grafted cells were tracked by immunostaining with human-specific antibodies. Control groups included non-transplanted empty defects and defects to which cell sheets without ALP induction had been grafted. After implantation, the FN-ALP transplants healed alveolar bone defects by intramembranous ossification, with formation of cementum and periodontal ligament. Moreover, FN-ALP transplants increased new bone formation, by endochondral ossification, on the mandibular cortex adjacent to the defect. Grafted fibroblasts were located near host osteoblasts and chondrocyte precursor cells early in the ossification process but were undetectable on and in newly formed bone and cartilage. These results indicate that the FN-ALP transplants support alveolar bone regeneration within the defect and augment bone formation outside the defect through the recruitment of host osteo/chondrogenic cells, suggesting their potential for periodontal tissue engineering applications.

Application of Autologous Cryopreserved Bone Marrow Mesenchymal Stem Cells for Periodontal Regeneration in Dogs

Background: Stem cell-based therapies have shown great promise in regenerative medicine and continue to generate wide interest in future clinical applications. However, the issue of storage and preservation of stem cells, for future clinical applications, still requires extensive investigation. Objective: The purpose of this study was to evaluate the effect of cryopreservation on the regenerative capacity of bone marrow stem cells in periodontal defects in dogs. Materi als and Methods: Bone marrow mesenchymal stem cells (BMSCs) were obtained from 5 beagle dogs. After cryopreservation for 1 month, cell viability, surface adherence ability, alkaline phosphatase activity and mineralized nodule formation were assessed. Twenty-six periodontal fenestration defects (5 × 5 mm) were created at a location 5 mm apical to the cemento-enamel junction in experimental teeth. Cryopreserved BMSCs were transplanted into the defects using a collagen scaffold carrier. Freshly isolated BMSCs and collagen scaffold alone were used as controls. All animals were sacrificed 8 weeks after surgery, and specimens were evaluated by histomorphometry. Results: Cryopreservation had no discernible negative effect on BMSC growth and differentiation in vitro. Both freshly isolated and cryopreserved BMSC transplantations induced significantly better periodontal regeneration with newly formed cementum, alveolar bone and periodontal ligament compared with the application of collagen scaffold alone. Conclusion: Cryopreserved BMSCs showed no altered regenerative capacity compared with freshly isolated BMSCs in the application of periodontal regeneration.

Experimental Intrabony and Periodontal Defects Treated With Natural Mineral Combined With a Synthetic Cell‐Binding Peptide in the Canine: Morphometric Evaluations

A synthetic peptide (P-15) analog of collagen added to anorganic bovine bone mineral (ABM) has recently been used as an enhanced bone graft material (ABM/P-15). The objective of this study was to test the contribution of ABM/P-15 in a new putty form (PEP) in two experimental membrane-protected defects: periodontal and intrabony. Its efficacy as filler biomaterial in guided tissue regeneration (GTR) and guided bone regeneration (GBR) procedures was evaluated histologically and morphometrically. In the maxillary canines, a facial mucoperiosteal flap was raised bilaterally in nine dogs. Two circular defects, 5 mm in diameter and 2 mm in depth, were made on each side: a fenestrated periodontal on the canine root and an intrabony in the alveolar diastema, anteriorly. PEP particles filled both defects on one side; the contralateral side was blood filled (control). All surgical sites were covered with a bioabsorbable membrane. Histologically, at 4 months, tissue blocks were made using the cutting/grinding non-decalcification method followed by morphometric analysis. In the periodontal fenestration root surface, the linear percentage of new cementum (%CEM), area percentage of new bone (%NB), and residual biomaterial particles (%PEP) were calculated. These same measurements were calculated at the intrabony sites, except cementum. The amount of direct NB to PEP contact was measured to assess the osteoconductivity level (OSC). The Pearson correlation test was used to evaluate any significant relationship between the different measured parameters. In the grafted and non-grafted fenestration root surface defects, %CEM averaged 59.5% and 73.9% (P <0.02), respectively; %NB averaged 36.1% and 31.4%, respectively; and %PEP averaged 20.6%. The mean percentage of OSC was 52.4%. In the intrabony grafted and non-grafted sites, %NB averaged 50.7% and 60.1%, respectively (P <0.02). Residual %PEP averaged 26.1%, and OSC averaged 35.6%. At the intrabony sites, higher %NB and lower %OSC were found compared to the fenestration sites (P <0.001 and P <0.03, respectively). Correlation analysis showed a negative correlation between %NB and %PEP at the fenestration defects. In between the two defect types, %OSC was significantly correlated (P <0.05). ABM/P-15 putty showed osteoconductive and biocompatible qualities. However, at 4 months in this model, no enhanced regeneration was present compared to a higher CEM and NB growth detected at non-grafted membrane-protected sites.

Effects of bone morphogenetic protein‐6 on periodontal wound healing in a fenestration defect of rats

Bone morphogenetic proteins (BMPs) may play significant roles in bone formation. The ability of BMP-6 to promote wound healing has been chosen as the subject of this investigation. In this study, a synthetic rat BMP-6 polypeptide was applied to a periodontal fenestration defect in rats to elucidate the effects of BMP-6 on periodontal wound healing. Following surgery to create a bony window on the buccal aspects of mandibular molar roots, 24 male Sprague Dawley rats were divided into four groups according to BMP application (0, 1, 3 and 10 microg, respectively). Animals were killed after 28 days and the mandible taken for histological examination. Histometric measurements were performed on sections selected from three levels (coronal, middle and apical levels; with 240 microm apart from the central) of the defect. New bone and cementum formation (including area and thickness) were analyzed and compared. In general, minimal new bone was observed on the surgically created defects in the non-BMP group, whereas a complete osseous healing occurred in all BMP-6 treated animals. New bone formation (both in area and thickness) was significantly influenced by both the dosage and the examining level, whereas new cementum formation was affected by dosage only. An increase in bone and cementum formation was noted in all three BMP groups when compared with the control group at all examined levels. Among the BMP groups, greatest new bone and cementum formation were noted in the 3 microg group. New cementum thickness increased on the cementum surfaces of the defects compared with the dentinal surfaces in all study groups. An increase in new bone and cementum formation was noted after applying a synthetic BMP-6 polypeptide to a periodontal fenestration defect in rats. Therefore, we suggest that BMP-6 may play a certain role in periodontal regeneration.

In situ tissue engineering of periodontal tissues by seeding with periodontal ligament-derived cells.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

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Our strategy for tissue regeneration is to induce maximum intrinsic healing potential at the site of a tissue defect, applying the elements of tissue engineering. Regeneration of periodontal tissues occurs through the combined application of a collagen sponge scaffold and gelatin microspheres incorporating basic fibroblast growth factor (bFGF) for controlled release. This “sandwich membrane” with or without bFGF (100μg) was applied to a three-walled alveolar bone defect (3 x 4 x 4 mm) in nine dogs. Periodontal tissues, both hard and soft, treated with bFGF were effectively regenerated four weeks after the operation with functional recovery of the periodontal ligament in parts. Next, the effect of combining cells with the treatment was evaluated. Periodontal fenestration defects (6 x 4 mm) were created bilaterally in the maxillary canines of six dogs. One of these was filled with the collagen sponge scaffold seeded with autologous periodontal ligament-derived cells (3 x 105), and the other was left empty. After four weeks, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surfaces, indicating that the seeded cells had formed new cementum. Our findings suggest a promising new approach to periodontal regeneration that is based upon in situ tissue engineering.

Healing Effects of Demineralized Freeze-Dried Bone Allograft and Deproteinized Bovine Bone Mineral on Periodontal Fenestration Defect in Rats

Healing Effects of Demineralized Freeze-Dried Bone Allograft and Deproteinized Bovine Bone Mineral on Periodontal Fenestration Defect in Rats

Regenerative Effects of Alloplastic Grafts in Rat Periodontal Fenestration Defects

Regenerative Effects of Alloplastic Grafts in Rat Periodontal Fenestration Defects

Effect of allogenic freeze-dried demineralized bone matrix on guided tissue regeneration in dogs.

This randomized, split-mouth study was designed to evaluate the adjunctive effect of allogenic, freeze-dried, demineralized bone matrix (DBM) to guided tissue regeneration (GTR). Contralateral fenestration defects (6 x 4 mm) were created 6 mm apical to the buccal alveolar crest on maxillary canine teeth in 6 beagle dogs. DBM was implanted into one randomly selected fenestration defect. Expanded polytetrafluoroethylene (ePTFE) membranes were used to provide bilateral GTR. Tissue blocks including defects with overlying membranes and soft tissues were harvested following a four-week healing interval and prepared for histometric analysis. Differences between GTR+DBM and GTR defects were evaluated using a paired t-test (N = 6). DBM was discernible in all GTR+DBM defects with limited, if any, evidence of bone metabolic activity. Rather, the DBM particles appeared solidified within a dense connective tissue matrix, often in close contact to the instrumented root. There were no statistically significant differences between the GTR+DBM versus the GTR condition for any histometric parameter examined. Fenestration defect height averaged 3.7+/-0.3 and 3.9+/-0.3 mm, total bone regeneration 0.8+/-0.6 and 1.5+/-0.8 mm, and total cementum regeneration 2.0+/-1.3 and 1.6+/-1.7 mm for GTR+DBM and GTR defects, respectively. The histologic and histometric observations, in concert, suggest that allogenic freeze-dried DBM has no adjunctive effect to GTR in periodontal fenestration defects over a four-week healing interval. The critical findings were 1) the DBM particles remained, embedded in dense connective tissue without evidence of bone metabolic activity; and 2) limited and similar amounts of bone and cementum regeneration were observed for both the GTR+DBM and GTR defects.

Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs

The effect of a collagen gel matrix as a submembranous space-maintaining material was evaluated in guided tissue regeneration procedures. In 4 dogs, contralateral surgical circular fenestration defects, 5 mm in diameter, were produced at the midbuccal aspect of the alveolar bone in 8 maxillary canines. Removal of bone, PDL and cementum was complete. Experimental sites were filled with collagen gel and covered with collagen membranes; control sites were covered with collagen membranes and the underlying space was spontaneously filled with blood. Mucogingival flaps were repositioned. Histological and histomorphometric observations, 6 weeks post-surgery, indicated that defects covered by collagen membranes presented the most impressive regeneration with almost complete coverage of the denuded root by new cementum (98.4%) and new bone (63.2%). In the experimental defects, 83.5% coverage of new cementum with only 21.9% new bone regeneration was observed. These results suggest that collagen gel, interfered with healing by PDL and bone-derived cells in the submembranous space.

Impaired early bone formation in periodontal fenestration defects in dogs following application of insulin-like growth factor (II). Basic fibroblast growth factor and transforming growth factor beta 1.

Effects of a topically applied growth factor combination on fibroblast migration, collagen fiber formation and bone regeneration were studied in standardized periodontal defects in 4 beagle dogs. Following elevation of facial mucoperiosteal flaps, fenestration defects, 3 mm in diameter, were made through the cortical bone and into the dentin of maxillary and mandibular teeth. Collagen sponges, impregnated with 200 ng insulin-like growth factor II, 20 ng basic fibroblast growth factor and 6 ng transforming growth factor beta 1 were fitted to defects randomly in right or left quadrants and the flaps repositioned and sutured. Contralateral control defects received the collagen with vehicle only. Experimental procedures were staggered to allow observations of healing 3, 7, 10, and 14 days after surgery. Histometric analysis showed no differences in fibroblast and collagen density between control and growth factor defects. Bone regeneration was significantly greater in control than in growth factor defects 10 and 14 days after surgery. The rate of healing generally appeared more affected by intra-dog variations or procedural variations than by the growth factor combination.

Effect of a collagen matrix on healing in periodontal fenestration defects in dogs.

Contralateral periodontal fenestration defects in seven beagle dogs were used to evaluate influence of a collagen matrix on periodontal wound healing. The defects (6 x 4 mm) were created through the buccal cortical plates of the maxillary canine teeth following elevation of mucoperiosteal flaps. The collagen was fitted to the defects on one side. Contralateral defects served as controls. Flaps were repositioned and sutured. Dogs were sacrificed 4 weeks after surgery and block sections including teeth and surrounding structures were prepared for histometric analysis. No meaningful differences in cementum and bone regeneration were observed between treatments. There was seemingly more bone regeneration in the apical than in the coronal aspect of the defects and significantly more cementum regeneration. Root resorption was observed in one collagen and one control defect. Ankylosis was not observed. The results suggest that the maxillary canine periodontal fenestration defect can be used as a model to evaluate factors that may enhance cementum and bone regeneration. The collagen matrix neither enhanced nor inhibited periodontal wound healing in this model.