Mineral Trioxide Aggregate (MTA) Upregulates the Expression of DMP1 in Direct Pulp Capping in the Rat Molar.

Mineral trioxide aggregate (MTA) is an alternative endodontic material that predicts conductive or inductive calcified tissue formation from immature pulp mesenchymal stem cells (IPMSCs). The purpose of this study was to investigate whether MTA could promote reparative odontoblast differentiation via IPMSCs in the early phase of regeneration and compare with calcium hydroxide (CH). Direct pulp capping using calcium hydroxide (CH), MTA, and MTA with platelet-rich plasma (MTA + PRP) was performed on maxillary first molars of 8-week-old male Wistar rats (n = 36). After 3, 7, or 14 days, the teeth were analyzed for mineral density (MD) and volume of MD (VMD) via micro-focusing computed tomography (µCT), nestin, dentin matrix acidic phosphoprotein 1 (DMP1) immunohistochemistry, and real-time PCR for DMP1 mRNA expression. MTA stimulated the early phase differentiation of the IPMSCs into odontoblasts, with positive results for nestin and DMP1 compared with CH. Moreover, MTA + PRP stimulated calcified granule and dentin bridge formation through calcium mineral deposition, following the induction of DMP1 mRNA expression in IPMSCs. Our results suggested that the combination of MTA and PRP is an effective and clinically applicable method for activating endogenous dental pulp stem cells into odontoblasts in the early stages of pulp regeneration.

A Novel Regenerative Technique Combining Bone Morphogenetic Protein-2 With Fibroblast Growth Factor-2 for Circumferential Defects in Dog Incisors.

BACKGROUND: Periodontal regeneration of incisors is necessary for esthetic recovery. A novel regenerative method combining bone morphogenetic protein (BMP)-2 and fibroblast growth factor (FGF)-2 was developed. The purpose of this study is to evaluate periodontal healing, including root coverage, in circumferential defects of incisors. METHODS: Fifty incisors in five beagles were used. After circumferential defects were surgically created, each group, consisting of ten recipient sites, received: 1) a double layer with FGF-2 (2 µg)/collagen as inner layer and BMP-2 (4 µg)/collagen as outer layer (FB-DL group); 2) collagen impregnated with both FGF-2 (2 µg) and BMP-2 (4 µg) (FB-M group); 3) BMP-2 (4 µg)/collagen (B group); 4) FGF-2 (4 µg)/collagen (F group); or 5) collagen (C group). Dogs were sacrificed 8 weeks post-surgery, and healing was evaluated histologically. RESULTS: The three groups treated with BMP-2 showed enhanced new bone formation compared with control and F groups (P < 0.05). Furthermore, connective tissue attachment with cementum regeneration in the FB-DL group was significantly greater than in FB-M and B groups (P <0.05). Ankylosis in the FB-DL group was significantly less than in FB-M and B groups (P <0.05). Gingival recession was inhibited significantly better in FB-DL and FB-M groups compared with control and B groups. CONCLUSION: These data support development of a double-layer method combining BMP-2 and FGF-2 as a therapeutic approach to periodontal regeneration at incisors with horizontal circumferential defects.

Coverage of gingival recession defects using acellular dermal matrix allograft with or without beta-tricalcium phosphate.

The aim of this study was to investigate the effect of beta-tricalcium phosphate (ß-TCP) particles in combination with acellular dermal matrix (ADM) allograft in gingival recession. Experimental gingival recession defects were created in beagle dogs and randomly assigned to one of the following groups: ADM, ADM + ß-TCP, or coronally positioned flap (CPF; control). Tissues were histologically examined at 4, 8, or 16 weeks following treatment. A greater thickness of gingiva was observed at the sites treated in both the ADM + ß-TCP and ADM groups than in the CPF group. The ADM + ß-TCP group showed a statistically significant increase in both new bone and cementum formations compared to the ADM group. The results suggest that the combination of ß-TCP and ADM is more effective in promoting new bone and cementum formations than ADM graft alone.

Clinical case report of long-term follow-up in type-2 diabetes patient with severe chronic periodontitis and nifedipine-induced gingival overgrowth.

In this case report, we describe the clinical course over a 14-year follow-up in a 47-year-old diabetes patient with severe chronic periodontitis and nifedipine-induced gingival overgrowth. The patient had a history of hypertension for over 5 years and uncontrolled type 2 diabetes. Overgrown gingiva was observed in most of the teeth and was marked in the upper and lower anterior teeth. A probing pocket depth of ≥ 4 mm and bleeding on probing (BOP) were observed in 94 and 90% of sites examined, respectively. At baseline, his hemoglobin A1c (HbA1c) was 8.5%. The patient received periodontal and diabetic treatment simultaneously. Medication was changed from nifedipine chloride to an angiotensin-converting enzyme inhibitor. After initial therapy and subsequent periodontal surgery, gingival overgrowth disappeared and probing depth and BOP showed a significant improvement. No recurrence was observed during supportive periodontal therapy (SPT). The HbA1c level improved from 8.5 to 6.3% after periodontal treatment, subsequently remaining at a good level during SPT over 10 years. This study demonstrated that periodontal treatment, withdrawal of medication and control of diabetes can result in remarkable improvements in type 2 diabetes patients with chronic periodontitis and nifedipine-induced gingival overgrowth. These results suggest that comprehensive periodontal treatment in combination with treatment for diabetes mellitus can exert a positive influence on blood glucose levels and periodontal condition in diabetic patients.

Coverage of gingival recession defects using guided tissue regeneration with and without adjunctive enamel matrix derivative in a dog model.

The aim of this study was to clarify the adjunctive effect of enamel matrix derivative (EMD) on expanded polytetrafluoroethylene membrane guided tissue regeneration (GTR)-based root coverage by creating gingival recessions in beagle dogs. Recessions were treated with GTR + EMD, GTR alone, or neither GTR nor EMD (control). The control group was characterized by long junctional epithelium and little bone formation. The GTR + EMD group showed a statistically significant increase (P < .01) in new bone and cementum formation compared with the GTR group. The results of the present investigation suggest that the adjunctive use of EMD with GTR promotes formation of new bone and cementum without root resorption in recession-type defects in dogs.

Application of 4-META/MMA-TBB resin for fixation of membrane to tooth in guided tissue regeneration in dog.

The aim of this study was to evaluate 4-META/MMA-TBB resin as an alternative to sling sutures for fixation of membrane to tooth in GTR in terms of its effect on tissue regeneration. Dehiscence periodontal defects were created in 6 dogs which were divided at random into two groups, 3 dogs in each group: an experimental group, in which non-absorbable or absorbable membrane was fixed to the teeth with 4-META/MMA-TBB resin; and a control group, in which sling sutures were applied to fix the two types of membrane. Histologic and histometric evaluation was carried out at 8 weeks post-operatively to determine healing response in each group. Both methods of achieving membrane fixation to tooth were effective in inhibiting epithelial migration and encouraging formation of regenerated periodontal tissues around the root surfaces. These results suggest that 4-META/MMA-TBB resin is as effective as sling sutures in achieving membrane fixation in GTR and is, moreover, easier to apply.

Effect of ß tricalcium phosphate particle size on recombinant human platelet-derived growth factor-BB-induced regeneration of periodontal tissue in dog.

The aim of this study was to investigate the effect of ß tricalcium phosphate (ß-TCP) particle size on recombinant human platelet-derived growth factor-BB (rhPDGF-BB)-induced regeneration of periodontal tissue in dog. The control group (rhPDGF-BB alone) was characterized by incomplete, newly formed bone. The large-particle ß-TCP (L-TCP(O))/rhPDGF-BB group showed a statistically significant increase in both new bone and cementum formation compared to the small-particle ß-TCP (S-TCP(G))/rhPDGF-BB group. These findings suggest that L-TCP(O)-particle promotes rhPDGF-BB-induced formation of bone and cementum.

Effect of recombinant basic fibroblast growth factor on reimplanted teeth in beagle dogs.

OBJECTIVES: This study assessed the effect of recombinant fibroblast growth factor (FGF-2) on reimplanted teeth. STUDY DESIGN: Premolars were reimplanted in dogs. Roots on the left side of the mandible were treated with FGF-2, whereas roots on right side served as controls. At 2, 4, or 8 weeks after treatment, animals were humanely killed and specimens collected and processed for histopathologic examination. RESULTS: After 2 weeks, proliferating cell nuclear antigen-positive cell ratios in the experimental group were significantly greater than those in the control group (P < .01). After 4 and 8 weeks, the experimental group showed formation of new cementum. Moreover, incidence of replacement resorption in the experimental group was significantly lower than that in the control group (P < .01). CONCLUSION: The results indicate that FGF-2 promotes formation of new periodontal ligament and prevents ankylosis and root resorption following reimplantation of teeth.

Beta-tricalcium phosphate and basic fibroblast growth factor combination enhances periodontal regeneration in intrabony defects in dogs.

The combined effect of beta-tricalcium phosphate and basic fibroblast growth factor was observed in the regeneration of periodontal tissue in dogs. For this purpose, either beta-TCP and FGF-2 (beta-TCP/FGF-2 group) or FGF-2 alone (FGF-2 group) was applied in intrabony defects. The control group received beta-TCP alone. The tissues were histologically examined at 2, 4, or 8 weeks following treatment. The control group was characterized by incomplete, newly formed bone around beta-TCP particles. The beta-TCP/FGF-2 group showed a statistically significant increase in both new bone and cementum formation compared to the FGF-2-alone group (76.3% vs. 65.3%, p < 0.01; 81.0% vs. 68.3%, p < 0.01, respectively). These findings suggested that beta-TCP may be a suitable scaffold for FGF-2 and that the combination of beta-TCP and FGF-2 can enhance bone and cementum formation.

C-Graft in regeneration of periodontal tissue in intrabony periodontal defect in dog.

The aim of this study was to investigate the effect of C-Graft particles on formation of new cementum and bone in periodontal bone defects in dog. Healing and tissue change were histologically determined at 2, 4, 8, and 16 weeks. Little bone or cementum formation was observed in the control group. A statistically significant increase in bone and cementum formation was seen in the C-Graft group compared to the control group (75.4% vs. 44.9%, p < 50.01, 80.4% vs. 46.7%, p < 50.05, respectively). These findings suggest that C-Graft particles provide a scaffold for the regeneration of new bone and cementum.

Transplantation of human tooth with proliferating periodontal tissue--long-term clinical follow-up study--.

A case report is presented on transplantation of a left third molar to replace a right second molar lost due to large furcation perforation. Two weeks after surgery, a reentry surgical procedure was performed on the furcation defect of the second molar to remove membrane and retrieve proliferating periodontal tissue. The left third molar and proliferating periodontal tissue were then transplanted into the bone defect of the right second molar. Radiographically, the transplanted tooth showed no root resorption over a 3-year period. The results indicate that teeth replanted with proliferating periodontal tissue have a favorable long-term prognosis.

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis.

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

Age-related differences in expression of vascular endothelial growth factor by periodontal ligament cells in vitro.

The purpose of this study was to evaluate age-related differences in expression of vascular endothelial growth factor (VEGF) by periodontal ligament (PDL) cells. PDL cells were obtained from Wistar male rats weighing approximately 150 g each in the young group and 350 g each in the old group. PDL cells derived from upper and lower incisors were seeded in 35-mm culture dishes after primary culture. For cell proliferation assays, cells were detached and counted at 1, 3, 5, 7, 11 and 14 days after culture. VEGF mRNA expression was analyzed with TaqMan. The number of cells in both groups increased day by day, but the rate of increase in the young group was higher than that in the old group. VEGF mRNA expression in the young group increased from 3 to 14 days, but in the old group increased only slightly over the same time period. Expression ratios in the young group were higher than those in the old group, and there were significant differences between the young and old groups at 7 and 14 days of culture. In conclusion, the data revealed that PDL cells varied with age, and suggest that in view of such changes in cell proliferation and VEGF mRNA expression, age should be taken into consideration in periodontal treatment.

Combination of bovine-derived xenografts and enamel matrix derivative in the treatment of intrabony periodontal defects in dogs.

The aim of this study was to investigate the effect of a combination of enamel matrix derivative (EMD) and bovine-derived xenograft (BDX). Intrabony defects were created in dogs and treated with BDX plus EMD, with BDX alone, or with neither (control group). Control group defects were characterized by a long junctional epithelium and little bone formation. The BDX+EMD sites showed a statistically significant increase (P < .05) in new bone and cementum formation compared with the BDX-only sites. These findings suggest that the use of BDX with EMD is effective in enhancing new bone and cementum formation and that this combination is effective in the treatment of intrabony defects.

Effectiveness of proliferating tissues in combination with bovine-derived xenografts to intrabony defects of alveolar bone in dogs.

The aim of this study was to investigate the effect of proliferating tissue used in combination with bovine-derived xenograft (BDX) on the formation of new cementum and bone in dogs. Intrabony defects were treated with either BDX in conjunction with autogenous proliferating tissues (BDXplus-proliferating tissues: BDX-P group) or BDX alone (BDX-alone group). The control group received no BDX or proliferating tissues. The animals were sacrificed after 2, 4, and 8 weeks of the treatment, and tissues were histologically examined. Specimens from the control group were characterized by long junctional epithelium and little bone formation. The BDX-P group showed a statistically significant increase in new bone and cementum formation compared to the BDX-alone group (30.9% vs. 18.7, p < 0.01 and 87.8% vs. 61.8, p < 0.01). The ratio of proliferating cell nuclear antigen (PCNA)-positive cells in the newly formed connective tissue of the BDX-P group was significantly greater than that in the BDX-alone group. These findings suggest that the use of proliferating tissues in combination with BDX enhances new bone and cementum formation, offering potential as therapeutic material in periodontal regeneration.

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling.

The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild-type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh(-/-) embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh(-/-)/Gli3(-/-) mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3(R) in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild-type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin-expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components.

Periodontal regeneration following transplantation of proliferating tissue derived from periodontal ligament into class III furcation defects in dogs.

The aim of this study was to evaluate the healing of class III furcation defects following transplantation of proliferating tissue derived from periodontal ligament (pPDL). Two weeks after removing alveolar bone, pPDL was excised. Class III furcation defects were created in the mandibular premolars. pPDL was transplanted into the furcation defects in the experimental group, while no treatment was performed on the furcation defects in the controls. Two, four and eight weeks after surgery, histologic examination, quantitative RT-PCR, and immunohistochemistry were carried out. bFGF and VEGF mRNA showed a significant increase in pPDL. In the pPDL treatment group, new cementum regenerated around almost the entire circumference of the furcation, with new bone filling most of the defect, while the control group presented epithelial downgrowth and defects filled with connective tissue. These results provide histological evidence that pPDL plays an important role in wound healing by promoting periodontal regeneration in class III furcation defects.

Cellular and molecular mechanisms of synovial joint and articular cartilage formation.

Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri-joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri-joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF-5, Wnt-14, and CD-44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C-1-1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C-1-1 (ERGp55Delta81) and conditionally expressed it in transgenic mice under cartilage-specific Col2 gene promotor-enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular-like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild-type littermate. Together, our studies reveal that peri-joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

Effect of proliferating tissue on transplanted teeth in dogs.

OBJECTIVE: The objective of this study was to investigate the effect of transplanted proliferating tissue on the regeneration of periodontal ligaments (PDL) in tooth transplantation. STUDY DESIGN: Two weeks after removing the alveolar bone, proliferating tissues were excised and examined for expression of basic fibroblast growth factor (bFGF) and alkaline phosphatase (ALP) using a LightCycler. Next, the teeth to be transplanted (first premolars) were extracted and PDLs were removed by scaling. After that, the first premolar teeth and proliferating tissues for the experimental group were transplanted into bone cavities created at the sites of the third and fourth premolars, while the control sites received teeth only. Finally, 1, 2, and 4 weeks later, the animals were sacrificed and specimens were collected and processed for histopathologic examination. RESULTS: bFGF and ALPmRNA showed a significant increase in the transplanted proliferating tissue. Transplantation of the proliferating tissues positively affected the formation of new cementum and PDL. Moreover, application of the transplanted proliferating tissues decreased the occurrence and extent of ankylosis and root resorption at the root surface. CONCLUSION: These results indicate that transplanted proliferating tissue may promote the regeneration of periodontal tissue and prevent ankylosis and root resorption following the transplantation of teeth.