PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (µ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by µ-XRF. Interestingly, the hygroscopic 10-30µm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Infection of apical dentin and root-end cavity disinfection.

INTRODUCTION: The purpose of this study was to assess Enterococcus faecalis penetration into the dentin of the apical 3 mm and bacterial death after the application of either chlorhexidine or laser to root-end cavities. METHODS: Root canals of 60 single-rooted teeth were prepared. In part 1, cementum was removed semicircumferentially from 21 roots, and the smear layer was removed from 15 roots using 17% EDTA/cetrimide. Teeth were inoculated and incubated with E. faecalis for 10 days, rinsed, and live/dead stained. The effect of cementum and smear on bacterial penetration was assessed by confocal laser scanning microscopy (CLSM). In part 2, 39 teeth had root ends resected and cavities ultrasonically prepared. Inoculated roots were assigned to 1 of the following 3 groups: (1) root-end cavities irrigated with 0.2 % chlorhexidine, (2) root-end cavities irradiated with a laser for 20 seconds at 1.5 W, or (3) root-end cavities that received no treatment. Roots were live/dead stained, sectioned, and examined by CLSM. The depth of the bacterial penetration and bacterial survival were compared using the Mann-Whitney U test. RESULTS: The presence of a smear layer and/or cementum did not significantly affect bacterial penetration. In root-end cavities, chlorhexidine was more effective than laser (P < .001), reducing bacterial viability by 93% versus 70% with a laser. CONCLUSIONS: E. faecalis invaded the entire width of dentin in the apical 3 mm irrespective of the smear layer and/or cementum. Chlorhexidine was more effective than laser in disinfecting root-end cavities.

The combined use of Nd:YAG laser and enamel matrix proteins in the treatment of periodontal infrabony defects.

BACKGROUND: The objective of regenerative periodontal therapy is the reconstitution of lost periodontal structures, such as cementum, periodontal ligament, and alveolar bone. Enamel matrix proteins (EMP) are used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The aim of this split-mouth study evaluates and compares the healing of intrabony defects after treatment with an EMP with or without neodymium-doped:yttrium-aluminum-garnet (Nd:YAG) laser application for root surface conditioning. METHODS: Forty-two intrabony defects in 21 patients with chronic periodontitis were randomly assigned to an access flap surgery with application of Nd:YAG laser (1 W, 10 Hz, 100 mJ, 1064 nm) and EMP (test group), and on the contralateral defect to an access flap surgery with application of EDTA and EMP alone (control group). Clinical periodontal parameters were assessed at baseline and after 6 and 12 months. RESULTS: Both treatments yielded significant improvements in terms of decrease in probing depth (PD) and gain in clinical attachment level (CAL) compared to baseline values. At 12 months after therapy, in the test group, the mean PD value was reduced from 7.3 ± 0.6 to 3.3 ± 0.4 mm and the mean CAL value changed from 9.5 ± 0.7 to 6.9 ± 0.7 mm (P <0.001). The sites treated with EMP (control) showed a reduction in mean PD value from 7.3 ± 0.7 to 3 ± 0.4 mm and a change in mean CAL value from 9.3 ± 0.8 to 6.4 ± 0.5 mm (P <0.001). The control group showed a greater reduction in PD and gain in CAL compared to the test group (P <0.05). CONCLUSION: Within the limits of the present study, it may be concluded that both therapies led to improvements of the clinical parameters, and Nd:YAG laser root conditioning as used in this study compared to EDTA root conditioning did not improve the outcome of EMP use.

The effect of calcium sulfate on hard-tissue healing after periradicular surgery.

The purpose of this study was to determine the effect of calcium sulfate (CS) on cementum deposition and osseous healing after periradicular surgery. The root canals of 24 mandibular premolars in four 2-yr-old beagle dogs were endodontically treated, followed 2 weeks later by periradicular surgery. Mineral trioxide aggregate (MTA) was used as root-end-filling material. The right or left side was assigned at random to receive CS alpha-hemihydrate or no material in the osteotomy sites before closure. The animals were killed after 4 months. Hard-tissue healing was analyzed histomorphometrically. All samples displayed evidence of cementum deposition adjacent to the root-end fillings and bone regeneration in the osteotomy sites. The data was analyzed using the Mann-Whitney U test, comparing the scores for cementum and osseous healing of the two groups at significance level of alpha = 0.05. The results indicated that placement of CS in osteotomy sites after periradicular surgery does not significantly affect periradicular healing.

Effect of mechanical loading on periodontal cells.

Mechanical loading is an important regulatory factor in alveolar bone homeostasis, and plays an essential role in maintaining the structure and mass of the alveolar processes throughout lifetime. A better understanding of the cellular and molecular responses of periodontal cells is a prerequisite for further improvements of therapeutic approaches in orthodontics, periodontal and alveolar bone repair and regeneration, implantology, and post-surgical wound healing. The purpose of this review is to provide an insight into some cell culture and animal models used for studying the effects of mechanical loading on periodontal cells, and into the recent developments and utilization of new in vivo animal models. There has been an increased awareness about the need for improvement and development of in vivo models to supplement the widely used cell culture models, and for biological validation of in vitro results, especially in the light of evidence that developmental models may not always reflect bone homeostasis in an adult organism. Due to the limitations of in vivo models, previous studies on mechanical regulation of alveolar bone osteoblasts and cementoblasts mostly focused on proliferative responses, rather than on the stimulation of cell differentiation. To address this problem, we have recently characterized and implemented a mouse osteoinductive tooth movement model for studying mechanically induced regulation of osteoblast- and cementoblast-associated genes. In this model, a defined and reproducible mechanical osteogenic loading is applied during a time course of up to two weeks. Regulation of gene expression in either wild-type or transgenic animals is assessed by a relative quantitative measurement of the level of target mRNAs directly within the subpopulations of periodontal cells. To date, results demonstrate a defined temporal pattern of cell-specific gene regulation in periodontal osteoblasts mechanically stimulated to differentiate and deposit bone matrix. The responses of osteoblast-associated genes to mechanical loading were 10- to 20-fold greater than the increase in the numbers of these cells, indicating that the induction of differentiation and an increase of cell function are the primary responses to osteogenic loading. The progression of the osteoblast phenotype in the intact mouse periodontium was several-fold faster compared with that in cultured cells, suggesting that the mechanical signal may be targeting osteoblast precursors in the state of readiness to respond to an environmental challenge, without the initial proliferative response. An early response of alkaline phosphatase and bone sialoprotein genes was detected after 24 hrs of treatment, followed by a concomitant stimulation of osteocalcin and collagen I between 24 and 48 hrs, and deposition of osteoid after 72 hrs. Although cementoblasts constitutively express biochemical markers similar to those of osteoblasts, distinct responses of osteocalcin, collagen I, and bone sialoprotein genes to mechanical loading were observed in the two cell phenotypes. This finding indicates that differential genetic responses to mechanical loading provide functional markers for distinction of the cementoblast and osteoblast phenotypes.

GTR treatment of degree III furcation defects following application of enamel matrix proteins. An experimental study in dogs.

The aim of the present study was to evaluate the effect of enamel matrix proteins (EMD) on periodontal wound healing in degree III furcation defects in dogs. The experiment was performed in 5 foxhound dogs. 2 months prior to the start of the experiment, the 2nd and 4th lower premolars were extracted. Degree III furcation defects were created in the 3rd mandibular premolars (3P3). The furcation defects were subsequently exposed to reconstructive surgery. Buccal and lingual full thickness flaps were elevated in the lower premolar regions. The exposed root surfaces of the experimental teeth were planed. A notch was placed in the roots at the base of the defect. In one side of the mandible (Test group), phosphoric acid gel was applied over the root surfaces for 15 s. The acid was removed by flushing the root surfaces with sterile saline. Subsequently, a gel of EMD was applied to cover all instrumented root surfaces. Following gel application, a resorbable barrier membrane was adjusted to cover the buccal and lingual entrances of the furcation defect. The flaps were repositioned to cover the barrier and sutured. The contralateral premolar (Control group) received the same treatment, but acid etching was not performed and EMD was not applied prior to barrier installation. 4 months after reconstructive surgery, the animals were sacrificed and biopsies from the 3P3 regions harvested. The biopsies were placed in a fixative, demineralized in EDTA, dehydrated and embedded in paraffin. 3 mesiodistal sections, representing the central portion of the furcation site, were selected for histological analysis of the defect. The furcation defects of both the Test and Control groups were clinically closed and were found to harbor bone and periodontal ligament tissue which appeared to be in structural continuity with a newly formed root cementum. The relative amounts of mineralized bone, bone marrow and periodontal ligament tissue that had formed were similar in the Test and the Control group. In the Test group, however, the cementum that had formed in the apical portion of the furcation defect was different from the corresponding tissue in the coronal portion, and also different from the cementum observed in the Control group. In the apical portion of the test defect a thin (12 microm) acellular cementum had been laid down, while in the coronal portion a thick (32 microm) cellular cementum, similar to the cementum found in the Control group, could be observed. The current observation, hence, seems to confirm that EMD when applied onto an instrumented and acid etched dentine surface may create an environment conducive for the formation of acellular cementum.

GTR treatment of degree III furcation defects with 2 different resorbable barriers. An experimental study in dogs.

The aim of the present study was to describe the periodontal tissue that formed after GTR when different resorbable barriers were applied to degree III furcation defects. The study was performed in 5 foxhound dogs. The 2nd and 4th premolars in both sides of the mandible were extracted. Degree III furcation defects were produced in the 3rd mandibular premolars. 5 weeks later, GTR therapy using a barrier composed by a polylactide-glycolide copolymer was performed on one quadrant (group A). In the contralateral quadrant, a barrier made of polylactide and citric acid ester (group B) was used. The dogs were sacrificed 6 months after reconstructive therapy. Tissue blocks containing the experimental teeth were excised, demineralised in EDTA and embedded in paraffin. Serial sections were cut in the mesio-distal plane and parallel with the long axis of the roots. The microtome was set at 7 microm. The sections were stained in hematoxyline and eosin. From each biopsy, 3 sections representing the central part of the furcation were selected for light microscopic examination. In the healed furcation sites, descriptive histological analysis and histomorphometric measurements of the newly formed tissues were performed. In both groups the root surface of the healed furcation defects was covered by a cellular, extrinsic-intrinsic fibers type cementum. The composition of the newly formed periodontal ligament was similar in both groups. The proportions of bone, bone marrow and periodontal ligament, however, were substantially larger in group A than in group B. In Group B, an area in the previous furcation defect was consistently occupied by a granuloma. It is suggested that the presence of the granuloma in the healed furcation defect prevented bone regrowth.

Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins.

There is increasing evidence that cells of the epithelial root sheath synthesize enamel matrix proteins and that these proteins play a fundamental role in the formation of acellular cementum, the key tissue in the development of a functional periodontium. The purpose of the present study was to explore the effect of locally applied enamel matrix and different protein fractions of the matrix on periodontal regeneration in a buccal dehiscence model in monkeys. Buccal, mucoperiosteal flaps were raised from the canine to the 1st molar on each side of the maxilla. The buccal alveolar bone plate, the exposed periodontal ligament and cementum were removed. Various preparations of porcine enamel matrix with or without vehicles were applied before the flaps were repositioned and sutured. After 8 weeks, the healing was evaluated in the light microscope, and morphometric comparisons were made. Application of homogenized enamel matrix or an acidic extract of the matrix containing the hydrophobic, low molecular weight proteins, amelogenins, resulted in an almost complete regeneration of acellular cementum, firmly attached to the dentin and with collagenous fibers extending over to newly formed alveolar bone. After application of fractions obtained by neutral EDTA extraction containing the acidic, high molecular weight proteins of the enamel matrix, very little new cementum was formed and hardly any new bone. The results of the controls in which no test substance was applied before the repositioning of the flap, were very similar to those obtained with the EDTA extracted material. Propylene glycol alginate (PGA), hydroxyethyl cellulose and dextran were tried as vehicles for the enamel matrix preparations. Only PGA in combination with the amelogenin fraction resulted in significant regeneration of the periodontal tissues.

Histopathological observations of periapical repair in teeth with radiolucent areas submitted to two different methods of root canal treatment.

Dogs' teeth with induced chronic periapical periodontitis were treated endodontically by two different methods, and the results were compared. A total of 40 root canals from the upper and lower premolars of two dogs were prepared chemomechanically. In method 1, a high-concentration (5.25%) hypochlorite solution was used during the instrumentation of the root canal, and an antibacterial dressing was applied between sessions, followed by filling of the root canal. In method 2, a low-concentration (0.5%) sodium hypochlorite solution was used as an adjunct to mechanical debridement, and the root canal was filled during the same session. The histopathological results showed that method 1 led to better periapical repair than method 2.

Periodontal repair in dogs: recombinant human bone morphogenetic protein-2 significantly enhances periodontal regeneration.

This study evaluated bone and cementum regeneration following periodontal reconstructive surgery using recombinant human bone morphogenetic protein-2 (rhBMP-2) in six beagle dogs. Surgically created mandibular supraalveolar premolar tooth defects in contralateral jaw quadrants were randomly assigned to receive rhBMP-2 or control vehicle. Clinical defect height was prepared to 5 mm. rhBMP-2 was applied with synthetic bioerodable particles and autologous blood using 20 micrograms rhBMP-2 per 100 microliters implant volume. Flaps were advanced to submerge the teeth and sutured. The dogs were sacrificed 8 weeks postsurgery. Histometric recordings included defect height, height and area of alveolar bone regeneration, height of cementum regeneration, root resorption, and ankylosis. Group means, standard deviations, and P values are shown (Student t test; n = 6). Histometric defect height for rhBMP-2 and control defects was 3.7 +/- 0.3 and 3.9 +/- 0.4 mm, respectively (P = 0.446). Height of alveolar bone regeneration amounted to 3.5 +/- 0.6 and 0.8 +/- 0.6 mm for rhBMP-2 and control defects, respectively (P = 0.000). Corresponding values for bone area were 8.4 +/- 4.5 and 0.4 +/- 0.5 mm2, respectively (P = 0.006). Cementum regeneration was observed in all experimental defects (17/17) and in 15 out of 17 controls, averaging 1.6 +/- 0.6 and 0.4 +/- 0.3 mm for rhBMP-2 and control defects, respectively (P = 0.005). Small amounts of root resorption were seen in rhBMP-2 defects, whereas controls exhibited substantial resorption (0.2 +/- 0.1 and 1.1 +/- 0.3 mm, respectively; P = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Overview of effects of electrical stimulation on osteogenesis and alveolar bone.

One endpoint of periodontal therapy is to regenerate structure lost to periodontal disease. Periodontal regeneration requires both formation of a new connective tissue attachment to the tooth and formation of alveolar bone. Several procedural advances may support regeneration of the attachment, however, regeneration of alveolar bone does not occur consistently. Therefore, factors which stimulate bone repair are areas for research in periodontal reconstructive therapy. Effects of cytokines or growth factors on bone repair are examples of such areas. Another one is electrical stimulation which naturally occurs in bone, and as such bone may be particularly susceptible to electrical therapy. This overview describes the potential of electrical stimulation for bone regeneration and applications in alveolar and periodontal research.