Intermittent parathyroid hormone promotes cementogenesis via ephrinB2-EPHB4 forward signaling.

Intermittent parathyroid hormone (PTH) promotes periodontal repair, but the underlying mechanisms remained unclear. Recent studies found that ephrinB2-EPHB4 forward signaling mediated the anabolic effect of PTH in bone homeostasis. Considering the similarities between cementum and bone, we aimed to examine the therapeutic effect of PTH on resorbed roots and explore the role of forward signaling in this process. In vivo experiments showed that intermittent PTH significantly accelerated the regeneration of root resorption and promoted expression of EPHB4 and ephrinB2. When the signaling was blocked, the resorption repair was also delayed. In vitro studies showed that intermittent PTH promoted the expression of EPHB4 and ephrinB2 in OCCM-30 cells. The effects of PTH on the mineralization capacity of OCCM-30 cells was mediated through the ephrinB2-EPHB4 forward signaling. These results support the premise that the anabolic effects of intermittent PTH on the regeneration of root resorption is via the ephrinB2-EPHB4 forward signaling pathway.

Yes-associated protein promotes tumour necrosis factor α-treated cementoblast mineralization partly by inactivating NF-κB pathway.

Cementum regeneration, as one of the most difficult challenges of periodontal regeneration, is influenced by inflammatory factors. Inflammation may hamper or promote periodontal tissue repair under different circumstances, as it is found to do in dentin-pulp complex and bone tissue. Our team demonstrated that YAP promotes mineralization of OCCM, a cementoblast cell line. However, the effect of YAP on its mineralization under inflammatory microenvironment is unclear. In this study, cementogenesis in vitro was up-regulated after transient TNF-α treatment for 30 minutes. YAP expression also was increased by TNF-α treatment. YAP overexpression promoted OCCM mineralization after the cells were transiently treated with TNF-α because YAP overexpression inhibited NF-κB pathway activity, while YAP knockdown elevated it. The inhibited mineralization potential and activated NF-κB pathway activity by YAP knockdown also were partly rescued by the application of the NF-κB inhibitor Bay 11-7082. These results demonstrated that YAP plays a positive role in the mineralization of TNF-α transiently treated cementoblast, partly by inhibiting the NF-κB pathway activity.

Synergistic acceleration in the osteogenic and angiogenic differentiation of human mesenchymal stem cells by calcium silicate-graphene composites.

Recent exciting findings of the biological interactions of graphene materials have shed light on potential biomedical applications of graphene-containing composites. Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of biocomposites. In the present study, various ratios of graphene (0.25wt%, 0.5wt% and 1.0wt%) were reinforced into calcium silicate (CS) for bone graft application. Results show that the graphene was embedded in the composites homogeneously. Adding 1wt% graphene into CS increased the young's modulus by ~47.1%. The formation of bone-like apatite on a range of composites with graphene weight percentages ranging from 0 to 1 has been investigated in simulated body fluid. The presence of a bone-like apatite layer on the composites surface after immersion in simulated body fluid was considered by scanning electron microscopy. In vitro cytocompatibility of the graphene-contained CS composites was evaluated using human marrow stem cells (hMSCs). The proliferation and alkaline phosphatase, osteopontin and osteocalcin osteogenesis-related protein expression of the hMSCs on the 1wt% graphene-contained specimens showed better results than on the pure CS. In addition, the angiogenesis-related protein (vWF and ang-1) secretion of cells was significantly stimulated when the graphene concentration in the composites was increased. These results suggest that graphene-contained CS bone graft are promising materials for bone tissue engineering applications.

Redefining the induction of periodontal tissue regeneration in primates by the osteogenic proteins of the transforming growth factor-ß supergene family.

The molecular bases of periodontal tissue induction and regeneration are the osteogenic proteins of the transforming growth factor-ß (TGF-ß) supergene family. These morphogens act as soluble mediators for the induction of tissues morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented ligament fibers into newly formed cementum. Human TGF-ß3 (hTGF-ß3 ) in growth factor-reduced Matrigel® matrix induces cementogenesis when implanted in class II mandibular furcation defects surgically prepared in the non-human primate Chacma baboon, Papio ursinus. The newly formed periodontal ligament space is characterized by running fibers tightly attached to the cementoid surface penetrating as mineralized constructs within the newly formed cementum assembling and initiating within the mineralized dentine. Angiogenesis heralds the newly formed periodontal ligament space, and newly sprouting capillaries are lined by cellular elements with condensed chromatin interpreted as angioblasts responsible for the rapid and sustained induction of angiogenesis. The inductive activity of hTGF-ß3 in Matrigel® matrix is enhanced by the addition of autogenous morcellated fragments of the rectus abdominis muscle potentially providing myoblastic, pericytic/perivascular stem cells for continuous tissue induction and morphogenesis. The striated rectus abdominis muscle is endowed with stem cell niches in para/perivascular location, which can be dominant, thus imposing stem cell features or stemness to the surrounding cells. This capacity to impose stemness is morphologically shown by greater alveolar bone induction and cementogenesis when hTGF-ß3 in Matrigel® matrix is combined with morcellated fragments of autogenous rectus abdominis muscle. The induction of periodontal tissue morphogenesis develops as a mosaic structure in which the osteogenic proteins of the TGF-ß supergene family singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. In primates, the presence of several homologous yet molecularly different isoforms with osteogenic activity highlights the biological significance of this apparent redundancy and indicates multiple interactions during embryonic development and bone regeneration in postnatal life. Molecular redundancy with associated different biological functionalities in primate tissues may simply represent the fine-tuning of speciation-related molecular evolution in anthropoid apes at the early Pliocene boundary, which resulted in finer tuning of the bone induction cascade.

Periodontal healing with a preameloblast-conditioned medium in dogs.

BACKGROUND AND OBJECTIVE: The predictability of conventional periodontal treatments for damaged periodontal tissue is limited, particularly on the regeneration of new cementum. As signaling molecules, a range of growth factors has been used to promote periodontal regeneration on periodontal ligament (PDL) and cementum defects. A preameloblast-conditioned medium (PA-CM) was prepared from cultured murine apical bud cells, which can differentiate into ameloblasts. We examined the effect of PA-CM on PDL cells and cementoblasts in vitro and evaluated histologically the effects of PA-CM on the regeneration of experimentally induced periodontal defects in vivo. MATERIAL AND METHODS: In vitro, the effects of PA-CM on the migration of human PDL cells were examined using a scratch wound healing assay and a transwell assay. The differentiation and mineralization potential of PA-CM-treated human PDL cells and murine cementoblastic OCCM-30 cells was examined by real-time polymerase chain reaction and Alizarin red-S staining. In vivo, six mongrel dogs (12-16 kg; 6-8 mo old) were used. Twenty-four roots were replanted with either, (i) only periodontal defects (n = 12; control group), or (ii) periodontal defects and PA-CM treatment (n = 12; experimental group). In the experimental group, the PDL and cementum between notches was removed using a Gracey curette and soaked in 0.08 mL water containing 80 µg of a PA-CM for 2 min. The dogs were killed at 4 and 8 wk post-surgery. RESULTS: The in vitro results showed that PA-CM stimulated the migration of PDL cells and promoted the differentiation and mineralization of PDL cells and cementoblasts. Real-time polymerase chain reaction analysis revealed stronger expression of Runx2, Osx, OC, Bsp and Cap mRNAs in the PA-CM-treated PDL cells and cementoblasts than those in the control cells. In vivo, newly formed PDL-like tissue and cementum-like tissue were observed partially between the root surfaces and newly formed bone in the experimental group. The regenerated PDL-like tissue in the experimental group was significantly higher than that in the control group at 8 wk (p < 0.05). The replacement resorption on the experimental group was significantly lower than that in the control group at 8 wk (p < 0.05). In addition, the amount of newly formed cementum-like tissue in the experimental group was significantly higher than that in the control group at 4 and 8 wk (p < 0.05). CONCLUSION: These results suggest that PA-CM has the potential to regenerate periodontal tissues in PDL and cementum defects.

Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells.

The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.

Fibroblast growth factor-2 promotes healing of surgically created periodontal defects in rats with early, streptozotocin-induced diabetes via increasing cell proliferation and regulating angiogenesis.

AIM: To evaluate the effects of fibroblast growth factor (FGF)-2 on the healing of surgical periodontal defects in rats with early, streptozotocin-induced diabetes. MATERIALS AND METHODS: Fifty Wistar rats were assigned to streptozotocin-induced diabetes or non-diabetes group. Periodontal defects were surgically created at maxillary first molars. Defects were treated with hydroxypropyl cellulose (HPC) or FGF-2 with HPC. Defect fill was evaluated by microcomputed tomography. Histological and immunohistochemical analyses were performed. RESULTS: Compared to vehicle alone, FGF-2 treatment yielded significantly greater bone volume and trabecular thickness in diabetes group. Diabetes group displayed reduced new bone formation and significantly longer epithelial down-growth compared to non-diabetes group. In diabetes group, FGF-2 treatment increased PCNA-positive cells and new bone formation after 2 weeks and suppressed epithelial down-growth, but new cementum formation was minimal even after 4 weeks. In diabetes group, overexpression of vascular endothelial growth factor was evident in cells within connective tissue, and no significant enhancement was observed by FGF-2 treatment. FGF-2 increased the expression of α-smooth muscle actin in diabetes group. CONCLUSIONS: Treatment of surgical periodontal defects in diabetic rats with the single application of FGF-2 provided beneficial effects primarily on new bone formation via increasing cell proliferation and regulating angiogenesis.

Gene expression and cytokine release during odontogenic differentiation of human dental pulp stem cells induced by 2 endodontic biomaterials.

INTRODUCTION: Mineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) have shown osteogenic/cementogenic/dentinogenic activities; however, their mechanism of action is not fully understood. We aimed to evaluate the effect of these biomaterials on odontogenic differentiation of human dental pulp stem cells (DPSCs). METHODS: Flow cytometry with stem cell markers for the confirmation of stemness and homogeneity was first performed. Then isolated DPSCs were seeded on prepared discs of MTA, CEM, differentiation medium (DM), and growth medium (GM) and incubated up to 14 days. Concentrations of transforming growth factor-ß1, bone morphogenetic protein (BMP)2, BMP4, and fibroblast growth factor 4 were measured at each interval using an enzyme-linked immunosorbent assay reader. Gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and the cytokines were evaluated by reverse-transcription polymerase chain reaction. To evaluate the cell morphology, scanning electron micrographs were taken; mineralization potential was evaluated using alizarin red S staining. RESULTS: Scanning electron micrographs showed that DPSCs spread/adhered/proliferated similarly on MTA and CEM. On day 14, alizarin red S staining confirmed that mineralization occurred in all groups except GM. Expressions of dentin matrix protein 1 and dentin sialophosphoprotein genes were similar in the CEM, MTA, and DM groups; they were significantly higher compared with the GM group (P < .05). A greater amount of transforming growth factor-ß1 gene was expressed in MTA compared with the other groups (P < .05). However, the expression of fibroblast growth factor 4 and BMP2 genes was significantly greater in the CEM group (P < .05). In all the tested groups, the expression of BMP4 was less than GM (P < .01); however, CEM and DM were similar but more than MTA (P < .05). Concentrations of protein product detected using an enzyme-linked immunosorbent assay reader confirmed these gene expressions. CONCLUSIONS: MTA and CEM can induce osteo-/odontogenic-like phenotype differentiation of human DPSCs; however, they stimulate different gene expressions and growth factor release.

Acemannan sponges stimulate alveolar bone, cementum and periodontal ligament regeneration in a canine class II furcation defect model.

BACKGROUND AND OBJECTIVE: Periodontal disease is a common infectious disease, found worldwide, causing the destruction of the periodontium. The periodontium is a complex structure composed of both soft and hard tissues, thus an agent applied to regenerate the periodontium must be able to stimulate periodontal ligament, cementum and alveolar bone regeneration. Recent studies demonstrated that acemannan, a polysaccharide extracted from Aloe vera gel, stimulated both soft and hard tissue healing. This study investigated effect of acemannan as a bioactive molecule and scaffold for periodontal tissue regeneration. MATERIAL AND METHODS: Primary human periodontal ligament cells were treated with acemannan in vitro. New DNA synthesis, expression of growth/differentiation factor 5 and runt-related transcription factor 2, expression of vascular endothelial growth factor, bone morphogenetic protein-2 and type I collagen, alkaline phosphatase activity, and mineralized nodule formation were determined using [(3)H]-thymidine incorporation, reverse transcription-polymerase chain reaction, enzyme-linked immunoabsorbent assay, biochemical assay and alizarin red staining, respectively. In our in vivo study, premolar class II furcation defects were made in four mongrel dogs. Acemannan sponges were applied into the defects. Untreated defects were used as a negative control group. The amount of new bone, cementum and periodontal ligament formation were evaluated 30 and 60 d after the operation. RESULTS: Acemannan significantly increased periodontal ligament cell proliferation, upregulation of growth/differentiation factor 5, runt-related transcription factor 2, vascular endothelial growth factor, bone morphogenetic protein 2, type I collagen and alkaline phosphatase activity, and mineral deposition as compared with the untreated control group in vitro. Moreover, acemannan significantly accelerated new alveolar bone, cementum and periodontal ligament formation in class II furcation defects. CONCLUSION: Our data suggest that acemannan could be a candidate biomolecule for periodontal tissue regeneration.

Intermittent parathyroid hormone administration improves periodontal healing in rats.

BACKGROUND: Intermittent administration of parathyroid hormone (PTH) promotes new bone formation in patients with osteoporosis and bone fractures. It was shown previously that PTH also reduces periodontitis-related bone loss. The aim of this study is to evaluate the effect of treatment with PTH on periodontal healing in rats. METHODS: Fenestration defects were created at the buccal surface of the distal root of the mandibular first molars, and both periodontal ligament (PDL) and cementum were removed. Animals were then assigned to two groups (eight animals per group): group 1: control, placebo administration; and group 2: test, human PTH (hPTH) 1-34 administration at a concentration of 40 µg/kg. For both groups, the animals were injected every 2 days, and the animals were sacrificed at 14 and 21 days after surgery. Specimens were harvested and processed for routine decalcified histologic sections. The following parameters were assessed: 1) remaining bone defect extension (RBDE); 2) newly formed bone density (NFBD); 3) total callus area (TCA); 4) osteoclast number (ON) in the callus region; and 5) newly formed dental cementum-like tissue (NFC). Birefringence of root PDL reattachment was also evaluated. RESULTS: Birefringence analysis showed root PDL reattachment for both groups 21 days after treatment. Intermittent hPTH 1-34 administration decreased RBDE (P <0.01) and increased NFBD (P <0.01), TCA (P <0.01), area of NFC (P <0.01), and ON in the callus region (P <0.01). CONCLUSION: Within the limits of the present study, intermittent administration of hPTH 1-34 led to an enhanced periodontal healing process compared with non-treated animals.

Periodontal healing after application of enamel matrix derivative in surgical supra/infrabony periodontal defects in rats with streptozotocin-induced diabetes.

BACKGROUND AND OBJECTIVE: Epidemiologic and clinical studies have indicated that diabetes is a risk factor for periodontal disease progression and healing. The aim of the present study was to evaluate short-term healing after enamel matrix derivative (EMD) application in combined supra/infrabony periodontal defects in diabetic rats. MATERIAL AND METHODS: Thirty male Wistar rats were initially divided into two groups, one with streptozotocin-induced diabetes and another one with healthy (non-diabetic) animals. Bony defects were surgically created on the mesial root of the first maxillary molars. After root surface planing and EDTA conditioning, EMD was applied to the roots at one side of the maxillae, while those on the contralateral sides were left untreated. Animals were killed 3 wk after surgery, and block sections were prepared for histologic and histomorphometric analysis. RESULTS: There was statistically significant more gingival recession in diabetic animals than in non-diabetic animals. The length of the junctional epithelium was significantly shorter in the EMD-treated sites in both diabetic and normoglycemic rats. Sulcus depth and length of supracrestal soft connective tissue showed no statistically significant differences between groups. In all animals, new bone formation was observed. Although new bone occurred more frequently in healthy animals, the extent of new bone was not significantly different between groups. In none of the teeth, a layer of new cementum was detectable. EMD had no influence on bone or cementum regeneration. Adverse reactions such as excessive inflammation due to bacterial root colonization, ankylosis and bone fractures were exclusively observed in diabetic animals, irrespective of EMD treatment. CONCLUSION: Within the limits of the present study, it can be concluded that periodontal healing was impaired in streptozotocin-induced diabetic rats. EMD had no beneficial effects on new bone and cementum formation during short-term healing in this defect model and could not ameliorate the adverse effects in the systemically compromised animals.

The in vitro and in vivo cementogenesis of CaMgSi2O6 bioceramic scaffolds.

The goal of periodontal tissue engineering is to regenerate alveolar bone, root cementum and periodontal ligament. To achieve this goal, bioactive scaffolds play an important role in inducing in vitro osteogenic/cementogenic gene expression of periodontal ligament cells (PDLCs) and in vivo bone/cementum formation. Diopside (DIOP: CaMgSi2O6) ceramics have shown excellent in vitro bioactivity for potential bone repair application. However, there is no study about DIOP porous scaffolds for periodontal tissue engineering. The aim of this study is to prepare DIOP scaffolds and investigate their in vitro and in vivo osteogenesis/cementogenesis for periodontal regeneration application. DIOP scaffolds with highly porous architecture were prepared and ß-tricalcium phosphate (ß-TCP) scaffolds were used for the control. The interaction of DIOP scaffolds with PDLCs was studied by investigating cell attachment, proliferation and ostegenic/cementogenic differentiation of PDLCs. DIOP scaffolds were implanted into the periodontal defects of beagle dogs to evaluate their in vivo osteogenesis/cementogenesis by hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase staining, and immunohistochemistry (type I collagen: Col I; cementum attachment protein) analyses. The results have shown that DIOP scaffolds supported the attachment and proliferation of PDLCs. DIOP scaffolds significantly enhanced osteogenesis/cementogenesis-related gene expression (Col 1, Runx2, transforming growth factor beta 1, and bone morphogenetic protein 2) of PDLCs, compared to ß-TCP scaffolds. The in vivo study showed that DIOP scaffolds induced new bone and cementum regeneration of periodontal tissue defects. The rate of new bone and cementum in DIOP scaffolds is comparable to that in conventional ß-TCP scaffolds. Our results indicated that silicate-based DIOP ceramics could not only be used for bone tissue engineering, but also for periodontal tissue engineering due to their excellent in vitro and in vivo osteogeneis/cementogenesis.

Effects of bone morphogenetic protein-6 on periodontal wound healing/regeneration in supraalveolar periodontal defects in dogs.

OBJECTIVE: Application of a synthetic BMP-6 polypeptide in a rat periodontal fenestration defect model enhanced periodontal wound healing/regeneration including new bone and cementum formation. The purpose of this study was to translate the relevance of these initial observations into a discriminating large animal model. METHODS: Critical-size (4-5 mm) supraalveolar periodontal defects were created at the 2(nd) and 3(rd) mandibular premolar teeth in 11 Beagle dogs. Experimental sites received BMP-6 at 0.25, 1.0 and 2.0 mg/ml soak-loaded onto an absorbable collagen sponge (ACS) carrier or ACS alone (control) each condition repeated in four jaw quadrants. The animals were euthanized at 8 weeks when block biopsies were collected and processed for histologic/histometric analysis. RESULTS: BMP-6 at 0.25, 1.0 and 2.0 mg/ml soak-loaded onto the ACS yielded significantly enhanced new bone (0.99 ± 0.07 versus 0.23 ± 0.13 mm/BMP-6 at 0.25 mg/ml) and cementum (2.45 ± 0.54 versus 0.73 ± 0.15 mm/BMP-6 at 0.25 mg/ml) formation including a functionally oriented periodontal ligament compared with control (p < 0.05). A significant inverse linear association between BMP-6 dose and new bone (ß = -0.21 ± 0.09 mm, p = 0.016) and cementum height (ß = -0.34 ± 0.15 mm, p = 0.023) was observed. Minimal root resorption was observed without significant differences between groups. Ankylosis was not observed for any of the experimental groups. CONCLUSIONS: Surgical application of BMP-6/ACS onto critical-size supraalveolar defects enhanced periodontal wound healing/regeneration, in particular cementogenesis including a functionally oriented periodontal ligament; the low BMP-6 0.25 mg/ml concentration apparently providing the most effective dose.

Effects of EMD in combination with bone swaging and calcium phosphate bone cement on periodontal regeneration in one-wall intrabony defects in dogs.

BACKGROUND AND OBJECTIVE: Although the application of EMD is a widely accepted periodontal-regenerative therapy, its effects on noncontained intrabony defects are unpredictable because of the lack of a space-making property. The combined use of EMD and autogenous bone grafts reportedly stimulates significant periodontal regeneration in intrabony defects. The aim of the present study was to evaluate the effects of EMD in combination with bone swaging (BS) and injectable calcium phosphate bone cement (CPC), which was placed into the spaces between the grafted swaged bone and the proximal host bone, on periodontal healing in one-wall intrabony defects in dogs. MATERIAL AND METHODS: One-wall intrabony defects (3 mm wide and 5 mm deep) were surgically created on the mesial and distal sides of the bilateral mandibular premolars in four dogs. The 16 defects were assigned to one of the following treatments: EMD only, BS only, EMD with BS (EMD + BS), or EMD with BS and CPC (EMD + BS + CPC). The animals were killed 8 wk after surgery for histologic evaluation. RESULTS: The height of newly formed bone was significantly greater in the EMD + BS + CPC group (3.73 ± 0.30 mm) than in the BS-only (2.74 ± 0.33 mm; p < 0.05) and EMD + BS (2.88 ± 0.98 mm; p < 0.05) groups. The area of newly formed bone was significantly larger in the EMD + BS + CPC group (5.68 ± 1.66 mm(2)) than in the EMD-only (3.68 ± 0.33 mm(2); p < 0.05), BS-only (3.48 ± 1.26 mm(2); p < 0.05) and EMD + BS (3.38 ± 1.37 mm(2); p < 0.05) groups. The EMD-only (4.63 ± 0.42 mm), EMD + BS (4.67 ± 0.30 mm) and EMD + BS + CPC (4.78 ± 0.54 mm) groups showed significantly greater cementum formation than did the BS-only group (3.93 ± 0.56 mm; p < 0.05). CONCLUSION: These results indicate that treatment with EMD + BS + CPC promotes favorable periodontal healing in one-wall intrabony defects in dogs.

Comparison between a ß-tricalcium phosphate and an absorbable collagen sponge carrier technology for rhGDF-5-stimulated periodontal wound healing/regeneration.

BACKGROUND: The objective of this study is to compare a candidate ß-tricalcium phosphate (ß-TCP) carrier technology with the absorbable collagen sponge (ACS) benchmark to support recombinant human growth/differentiation factor-5 (rhGDF-5)-stimulated periodontal wound healing/regeneration. METHODS: Routine, bilateral, critical-size (5-mm), 1-wall, intrabony periodontal defects were surgically created in the mandibular premolar region in 10 beagle dogs. Five animals received rhGDF-5/ß-TCP and five animals received rhGDF-5/ACS, with a total of 20 µg rhGDF-5 per defect. The animals were euthanized for histologic and histometric analyses at 8 weeks postsurgery. RESULTS: Both rhGDF-5/ACS and rhGDF-5/ß-TCP stimulated the formation of functionally oriented periodontal ligament, cellular mixed fiber cementum, and woven/lamellar bone. Bone regeneration (height and area) was significantly greater for the rhGDF-5/ß-TCP construct than for the rhGDF-5/ACS (3.26 ± 0.30 mm versus 2.22 ± 0.82 mm, P <0.01; and 10.45 ± 2.26 mm(2) versus 5.62 ± 2.39 mm(2), P <0.01, respectively). Cementum formation ranged from 3.83 ± 0.73 mm to 3.03 ± 1.18 mm without significant differences between groups. Root resorption/ankylosis was not observed. CONCLUSIONS: The ß-TCP carrier technology significantly enhanced rhGDF-5-stimulated bone formation compared with the ACS benchmark in this discriminating periodontal defect model. The structural integrity of the ß-TCP carrier, preventing compression while providing a framework for bone ingrowth, may account for the observed results.

Wound healing/regeneration using recombinant human growth/differentiation factor-5 in an injectable poly-lactide-co-glycolide-acid composite carrier and a one-wall intra-bony defect model in dogs.

OBJECTIVE: The purpose of this study was to evaluate the effect of recombinant human growth/differentiation factor-5 (rhGDF-5) on periodontal wound healing/regeneration using an injectable poly-lactide-co-glycolide-acid (PLGA) composite carrier and an established defect model. METHODS: Bilateral 4 × 5 mm (width × depth) one-wall, critical-size, intra-bony periodontal defects were surgically created at the second and the fourth mandibular pre-molar teeth in 15 Beagle dogs. The animals were randomized to receive (using a split-mouth design; defect sites in the same jaw quadrant getting the same treatment) rhGDF-5 high dose (188 µg/defect) versus sham-surgery control (five animals), rhGDF-5 mid dose (37 µg/defect) versus carrier control (five animals), and rhGDF-5 low dose (1.8 µg/defect) versus treatment reported elsewhere (five animals). The animals were euthanized for histometric analysis following an 8-week healing interval. RESULTS: Clinical healing was uneventful. The rhGDF-5/PLGA construct was easy to assemble and apply. The rhGDF-5 high dose supported significantly increased bone formation compared with the low-dose, sham-surgery, and carrier controls (p<0.05) and induced significantly increased cementum formation compared with the controls (p<0.05). Root resorption/ankylosis or other aberrant healing events were not observed. CONCLUSION: rhGDF-5 appears to effectively support periodontal wound healing/regeneration in a dose-dependent order; the PLGA composite appears to be an effective ease-of-use candidate for carrier technology.

Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia.

Hypophosphatasia (HPP) occurs from loss-of-function mutation in the tissue-non-specific alkaline phosphatase (TNALP) gene, resulting in extracellular pyrophosphate accumulation that inhibits skeletal and dental mineralization. TNALP-null mice (Akp2(-/-)) phenocopy human infantile hypophosphatasia; they develop rickets at 1 week of age, and die before being weaned, having severe skeletal and dental hypomineralization and episodes of apnea and vitamin B(6)-responsive seizures. Delay and defects in dentin mineralization, together with a deficiency in acellular cementum, are characteristic. We report the prevention of these dental abnormalities in Akp2(-/-) mice receiving treatment from birth with daily injections of a mineral-targeting, human TNALP (sALP-FcD(10)). sALP-FcD(10) prevented hypomineralization of alveolar bone, dentin, and cementum as assessed by micro-computed tomography and histology. Osteopontin--a marker of acellular cementum--was immuno-localized along root surfaces, confirming that acellular cementum, typically missing or reduced in Akp2(-/-) mice, formed normally. Our findings provide insight concerning how acellular cementum is formed on tooth surfaces to effect periodontal ligament attachment to retain teeth in their osseous alveolar sockets. Furthermore, they provide evidence that this enzyme-replacement therapy, applied early in post-natal life--where the majority of tooth root development occurs, including acellular cementum formation--could prevent the accelerated tooth loss seen in individuals with HPP.

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration.

BACKGROUND AND OBJECTIVE: Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP-2 on the in vitro and in vivo biologic activity of well-characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP-2. MATERIAL AND METHODS: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP-2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8-wk healing period. The effects of rhBMP-2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP-2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. RESULTS: In the present study, rhBMP-2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down-regulated following treatment with rhBMP-2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. CONCLUSION: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP-2 on cementum and PDL tissue regeneration by hPDLSCs.

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.

Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells.

During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts/cementoblasts when they are in contact with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation could occur in a co-culture model. To test this hypothesis, we investigated the characteristics of DFCs that are influenced by DPCs in an in vitro co-culture and in vivo heterotopic transplant model. One week into the co-culture, there were significant increases in the mRNA level of bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), and a decrease of the receptor activator of nuclear factor κB ligand (RANKL). Additionally, the number of BMP2-, OPG-, BSP- and OCN-positive DFCs increased whereas RANKL-positive DFCs decreased. Three weeks after co-culture, DFCs produced calcified nodules, accompanied with increased sub-cellular organelles for protein synthesis and secretion. In the heterotopic transplant model, the adult male rats were used as hosts, DFCs were transplanted into the omentum. In vivo 5-week growth of DFCs in the presence of DPCs led to the formation of bone-like tissues, positive for BSP, OCN and BMP2. In contrast, DFCs alone led to fibrous-like tissues. These results indicated that in the absence of pre-existing dentin, DPCs can stimulate osteogenesis and inhibit osteoclastogenesis in DFCs and suggested a novel strategy to promote DFCs differentiation.