Regenerative potential and healing dynamics of the periodontium: a critical‐size supra‐alveolar periodontal defect study

Objectives: The nature and characteristics of the newly formed periodontium obtained following regenerative procedures remain a matter of controversy. The objective of this study was to evaluate the regenerative potential of the periodontal attachment and healing dynamics as observed from the spatial distribution of newly formed cementum, periodontal ligament (PDL) and alveolar bone following optimal circumstances for wound healing/regeneration in a discriminating animal model. Material and Methods: Critical‐size, 6‐mm, supra‐alveolar, periodontal defects were surgically created in six young adult Beagle dogs. Space‐providing ePTFE devices with 300‐μm laser‐drilled pores were implanted to support wound stability and space provision in one jaw quadrant/animal. Treatments were alternated between left and right jaw quadrants in subsequent animals. The gingival flaps were advanced to submerge the defect sites for primary intention healing. Histometric analysis followed an 8‐week healing interval. Results: Healing was uneventful in all animals. The histometric analysis showed that cementum regeneration (2.99 ± 0.22 mm) was significantly greater than PDL (2.54 ± 0.18 mm, p=0.03) and bone regeneration (2.46 ± 0.26 mm, p=0.03). The wound area showed significant positive non‐linear effect on cementum (log β=1.25, p<0.001), PDL (log β=1.24, p<0.001) and new bone formation (log β=1.36, p<0.001). A high degree of concordance and significant linear relationship was observed between cementum, PDL and bone regeneration indicating that their formation virtually occurred in parallel. Conclusions: Cementum, PDL and alveolar bone virtually regenerate in parallel under optimal circumstances for periodontal wound healing/regeneration. Moreover, space provision positively influences the extent of periodontal regeneration.     

Filifactor alocis‐derived extracellular vesicles inhibit osteogenesis through TLR2 signaling

Filifactor alocis, an asaccharolytic anaerobic Gram‐positive rod (AAGPR), is an emerging marker of periodontitis. Severe periodontitis causes destruction of the alveolar bone that supports teeth and can even lead to tooth loss. Based on our previous report that F. alocis‐derived extracellular vesicles (FA EVs) contain various effector molecules and have immunostimulatory activity, we investigated the effect of FA EVs on osteogenesis using mouse bone‐derived mesenchymal stromal cells (BMSCs). FA EVs dramatically inhibited bone mineralization similar to whole bacteria and reduced the expression levels of osteogenic marker genes. The osteogenic differentiation of TLR2‐deficient BMSCs was not inhibited by FA EVs, suggesting that their inhibitory effect on osteogenesis is dependent on TLR2 signaling. FA EVs effectively activated TLR2 downstream signaling of the MAPK and NF‐κB pathways. In addition, FA EVs regulated RANKL and OPG gene expression, increasing the RANKL/OPG ratio in BMSCs in a TLR2‐dependent manner. Our study suggests that F. alocis‐derived EVs interfere with bone metabolism via TLR2 activation, providing insight into the pathogenesis of bone loss associated with periodontitis.     

Comparative osteogenesis of maxilla and iliac crest human bone marrow stromal cells attached to oxidized titanium: a pilot study

Objectives: Severe alveolar bone loss affects dental implant placement. Bone augmentation by grafting iliac crest bone rich in osteoprogenitor cells such as bone marrow stromal cells (BMSCs) requires a second surgical procedure in non‐orofacial bone. Skeletal site‐specific osteogenesis indicates maxilla and mandible BMSCs are highly proliferative and exhibit osteogenic properties superior to iliac crest BMSCs. Alveolar bone can be easily obtained during routine dental surgery, but it is unclear if titanium‐attached alveolar BMSCs will retain their superior osteogenic properties. This study evaluated and compared in vitro osteogenic properties of titanium‐attached maxilla and iliac crest BMSCs in same individuals. Material and methods: Primary culture of maxilla and iliac crest BMSCs from four normal healthy volunteers was expanded in culture. In 24‐well plates, first passage BMSCs were seeded directly (1 × 10⁴ cells/well) on oxidized titanium disks (1.27 cm diameter and 2 mm thickness) or tissue culture plate. Each cell type was assessed for affinity for titanium, post‐attachment survival and osteogenic differentiation based on alkaline phosphatase and osteopontin expressions. Results: There was no difference in the affinity of maxilla and iliac crest BMSCs to titanium. However, titanium‐attached maxilla BMSCs were apparently more osteogenically responsive than iliac crest cells based on calcium accumulation and gene expression of alkaline phosphatase and osteopontin. But these differences were not statistically significant in this small patient sample. Conclusion: Maxilla and iliac crest BMSCs have similar attachment affinity for titanium. This pilot study indicates that titanium‐attached maxilla BMSCs are more osteogenically responsive and may be a viable and more readily available donor graft material in implant dentistry.     

Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein-1 and human transforming growth factor-β3 in Class II furcation defects of Papio ursinus

Teare JA, Petit J‐C, Ripamonti U. Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein‐1 and human transforming growth factor‐β ₃ in Class II furcation defects of Papio ursinus. J Periodont Res 2012; 47: 336–344. © 2011 John Wiley & Sons A/S Background and Objective: Binary applications of recombinant human osteogenic protein‐1 (hOP‐1) and transforming growth factor‐β3 (hTGF‐β3) synergize to induce pronounced bone formation. To induce periodontal tissue regeneration, binary applications of hOP‐1 and hTGF‐β₃ were implanted in Class II furcation defects of the Chacma baboon, Papio ursinus. Material and Methods: Defects were created bilaterally in the furcation of the first and second mandibular molars of three adult baboons. Single applications of 25 μg hOP‐1 and 75 μg hTGF‐β₃ in Matrigel^® matrix were compared with 20:1 binary applications, i.e. 25 μg hOP‐1 and 1.25 μg hTGF‐β₃. Morcellated fragments of autogenous rectus abdominis striated muscle were added to binary applications. Sixty days after implantation, the animals were killed and the operated tissues harvested en bloc. Undecalcified sections were studied by light microscopy, and regenerated tissue was assessed by measuring volume and height of newly formed alveolar bone and cementum. Results: The hOP‐1 and hTGF‐β₃ induced periodontal tissue regeneration and cementogenesis. Qualitative morphological analysis of binary applications showed clear evidence for considerable periodontal tissue regeneration. Quantitatively, the differences in the histomorphometric values did not reach statistical significance for the group size chosen for this primate study. The addition of morcellated muscle fragments did not enhance tissue regeneration. Binary applications showed rapid expansion of the newly formed bone against the root surfaces following fibrovascular tissue induction in the centre of the treated defects. Conclusion: Binary applications of hOP‐1and hTGF‐β₃ in Matrigel^® matrix in Class II furcation defects of P. ursinus induced substantial periodontal tissue regeneration, which was tempered, however, by the anatomy of the furcation defect model, which does not allow for the rapid growth and expansion of the synergistic induction of bone formation, particularly when additionally treated with responding myoblastic stem cells.     

Effect of Enamel Matrix Derivative on Periodontal Wound Healing and Regeneration in an Osteoporotic Model

Background : Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis‐related periodontal defects. Methods: Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD‐treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD‐treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro‐CT) analysis. The number of osteoclasts was determined by tartrate‐resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. Results: OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by micro‐CT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165μm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP‐positive osteoclasts did not vary between untreated and EMD‐treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMD‐treated samples. Conclusions: The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis.     

慢性牙周炎中肿瘤坏死因子ɑ对骨髓间充质干细胞成骨分化的调控作用

骨髓间充质干细胞（BMSCs）在重构牙周组织结构和功能、促进牙周炎好转乃至愈合方面发挥重要作用,因此BMSCs的特性尤其是其成骨分化的调控机制是目前的研究热点。肿瘤坏死因子α（TNF-α）是牙周组织炎症微环境中的主要促炎因子,与BMSCs的成骨分化密切相关。探究TNF-α调控BMSCs成骨分化的机制有助于明确牙周炎的发病机制,寻找牙周疾病新的治疗靶点,改善牙周炎的治疗效果。本文将针对TNF-α在牙周炎发生发展过程中发挥的重要作用尤其是调控BMSCs成骨分化的可能机制作一综述。     

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

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. 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. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S 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.     

Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor–Associated Genes in PDLSCs

Background: This study investigates the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential, and expression of growth factor–associated genes in periodontal ligament stem cells (PDLSCs). Methods: Mesenchymal stem cells (MSCs) from PDL tissue were isolated from human premolars (n = 3). The MSCs’ identity was confirmed by immunophenotyping and trilineage differentiation assays. Cell proliferation activity was assessed through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Polymerase chain reaction array was used to profile the expression of 84 growth factor–associated genes. Pathway analysis was used to identify the biologic functions and canonic pathways activated by ASA treatment. The osteogenic potential was evaluated through mineralization assay. Results: ASA at 1,000 μM enhances osteogenic potential of PDLSCs. Using a fold change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and seven downregulated genes. Fibroblast growth factor 9 (FGF9), vascular endothelial growth factor A (VEGFA), interleukin‐2, bone morphogenetic protein‐10, VEGFC, and 2 (FGF2) were markedly upregulated (FC range, 6 to 15), whereas pleotropin, FGF5, brain‐derived neurotrophic factor, and Dickkopf WNT signaling pathway inhibitor 1 were markedly downregulated (FC 32). Of the 84 growth factor–associated genes screened, 35 showed high cycle threshold values (≥35). Conclusions: ASA modulates the expression of growth factor–associated genes and enhances osteogenic potential in PDLSCs. ASA upregulated the expression of genes that could activate biologic functions and canonic pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration, and differentiation. These findings suggest that ASA enhances PDLSC function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.     

Periodontal regeneration via chemoattractive constructs

Aim

Chemoattractants, such as stromal cell‐derived factor‐1α (SDF‐1α), can offer an advantage for periodontal regeneration by recruiting the patient’s own stem cells to stimulate self‐repair. We here developed a chemoattractive construct for periodontal regeneration using SDF‐1α and evaluated its efficacy in vivo.

Materials and Methods

SDF‐1α was loaded on gelatin sponge and tested in vitro for SDF‐1α release. Subsequently, SDF‐1α constructs were implanted into rat periodontal defects for 1 and 6 weeks, with unloaded materials and empty defects as controls. The regenerative efficacy was evaluated by micro‐CT, histological and histomorphometrical analyses.

Results

In vitro results showed limited SDF‐1α release up to 35 days. In contrast, SDF‐1α constructs significantly improved periodontal defect regeneration in terms of alveolar bone height, new bone area and functional ligament length. Additionally, SDF‐1α constructs decreased the inflammatory response at Week 6.

Conclusion

Chemoattractive constructs significantly improved periodontal regeneration in terms of alveolar bone height, new bone area and functional ligament length.     

Induction of cementogenesis and periodontal ligament regeneration by recombinant human transforming growth factor-beta3 in Matrigel with rectus abdominis responding cells

Background and Objective: In primates and in primates only, the transforming growth factor‐β proteins induce endochondral bone formation. Transforming growth factor‐β3 also induces periodontal tissue regeneration. Two regenerative treatments using human recombinant transforming growth factor‐β3 were examined after implantation in mandibular furcation defects of the nonhuman primate, Papio ursinus. Material and Methods: Class III furcation defects were surgically created bilaterally in the mandibular first and second molars of two adult Chacma baboons (P. ursinus). Different doses of recombinant transforming growth factor‐β3 reconstituted with Matrigel^® matrix were implanted in the rectus abdominis muscle to induce heterotopic ossicles for subsequent transplantation to selected furcation defects. Twenty days after heterotopic implantation, periodontal defects were re‐exposed, further debrided and implanted with minced fragments of induced heterotopic ossicles. Contralateral class III furcation defects were implanted directly with recombinant transforming growth factor‐β3 in Matrigel^® matrix with the addition of minced fragments of autogenous rectus abdominis muscle. Treated quadrants were not subjected to oral hygiene procedures so as to study the effect of the direct application of the recombinant morphogen in Matrigel^® on periodontal healing. Histomorphometric analyses on undecalcified sections cut from specimen blocks harvested on day 60 measured the area of newly formed alveolar bone and the coronal extension of the newly formed cementum along the exposed root surfaces. Results: Morphometric analyses showed greater alveolar bone regeneration and cementogenesis in furcation defects implanted directly with 75 μg of transforming growth factor‐β3 in Matrigel^® matrix with the addition of minced muscle tissue. Conclusion: Matrigel^® matrix is an optimal delivery system for the osteogenic proteins of the transforming growth factor‐β superfamily, including the mammalian transforming growth factor‐β3 isoform. The addition of minced fragments of rectus abdominis muscle provides responding stem cells for further tissue induction and morphogenesis by the transforming growth factor‐β3 protein.     

Serotonin Inhibits Osteoblast Differentiation and Bone Regeneration in Rats

Background: Although increasing evidence indicates that serotonin (SER; 5‐hydroxytrypamine [5‐HT]) is involved in the regulation of bone metabolism, conflicting data exist regarding whether SER promotes or inhibits osteoblast differentiation and bone formation. Regeneration of functional bone is required for proper osseointegration of dental implants. Noticeably, the use of selective SER reuptake inhibitors was recently associated with the failure of osseointegrated dental implants. The present study examines the direct role of peripheral SER on the regulation of bone regeneration. Methods: The effect of SER on osteoblast differentiation and bone regeneration was examined using rat calvarial cell cultures in vitro and a rat critical‐sized calvarial defect model in vivo. Results: Rat calvarial cells expressed SER receptors Htr1 (5‐HT1) and Htr2 (5‐HT2), which are known to transmit signals in bone cells. In vitro, SER significantly reduced osteogenic differentiation and mineralization of rat calvarial cells with concomitant reduction of osteoblast marker genes including alkaline phosphatase (Alpl), osterix (Sp7), and osteocalcin (Bglap). Histologic and radiologic analyses using the rat critical‐sized calvarial defect model revealed that the existence of SER significantly inhibited β‐phase tricalcium phosphate–induced bone regeneration. Conclusion: Results suggest that SER in the local bone microenvironment might play a negative role in osteoblast differentiation and bone formation in rats.     

Experimental and clinical studies on regenerative periodontal therapy

The recognition of a periodontal therapy as a regenerative procedure requires the demonstration of new cementum, periodontal ligament, and bone coronal to the base of the defect. A diversity of regenerative strategies has been evaluated, including root surface conditioning, bone grafts and bone substitute materials, guided tissue regeneration, enamel matrix proteins, growth/differentiation factors, combined therapies and, more recently, tissue‐engineering approaches. The aim of this chapter of Periodontology 2000 is to review the research carried out in Latin America in the field of periodontal regeneration, focusing mainly on studies using preclinical models (animal models) and randomized controlled clinical trials. This review may help clinicians and researchers to evaluate the current status of the therapies available and to discuss the challenges that must be faced in order to achieve predictable periodontal regeneration in clinical practice.     

Dental Pulp Stem Cell Transplantation with 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside Accelerates Alveolar Bone Regeneration in Rats

Introduction

Although the therapeutic potential of human dental pulp stem cells (hDPSCs) has been studied for bone regeneration, the therapeutic efficiency needs further consideration and examinations for clinical applications. Thus, the aims of this study were to evaluate the effect of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) on the osteogenic differentiation of hDPSCs and to examine the therapeutic efficiency of the THSG-enhanced osseous potential of hDPSCs in alveolar bony defects of rats.

Sequential process in brain‐derived neurotrophic factor‐induced functional periodontal tissue regeneration

We recently demonstrated that brain‐derived neurotrophic factor (BDNF) promotes periodontal tissue regeneration. The purpose of this study was to establish an essential component of a rational approach for the clinical application of BDNF in periodontal regenerative therapy. Here, we assessed the sequence of early events in BDNF‐induced periodontal tissue regeneration, especially from the aspect of cementum regeneration. Brain‐derived neurotrophic factor was applied into experimental periodontal defects in Beagle dogs. The localization of cells positive for neurotrophic tyrosine kinase, receptor, type 2, proliferating cell nuclear antigen, osteopontin, integrin αVβ3, and integrin α2β1 was evaluated by immunohistochemistry. The effects of BDNF on adhesion of cultured human periodontal ligament cells was examined by an in vitro study. The results suggest that BDNF could induce rapid cementum regeneration by stimulating adhesion, proliferation, and differentiation of periodontal ligament cells in the early regenerative phase, resulting in enhancement of periodontal tissue regeneration.     

Dose- and time-dependent effects of recombinant human bone morphogenetic protein-2 on the osteogenic and adipogenic potentials of alveolar bone-derived stromal cells

Park J‐C, Kim J.C, Kim B‐K, Cho K‐S, Im G‐I, Kim B‐S, Kim C‐S. Dose‐ and time‐dependent effects of recombinant human bone morphogenetic protein‐2 on the osteogenic and adipogenic potentials of alveolar bone‐derived stromal cells. J Periodont Res 2012; 47: 645–654. © 2012 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a well‐known growth factor that can induce robust bone formation, and recent studies have shown that rhBMP‐2‐induced osteogenesis is closely related to adipogenesis. The aim of the present study was to determine the dose‐ and time‐dependent effects of rhBMP‐2 on the osteogenic and adipogenic differentiation of human alveolar bone‐derived stromal cells (hABCs) in vivo and in vitro. Material and Methods: hABCs were isolated and cultured, and then transplanted using a carrier treated either with or without rhBMP‐2 (100 μg/mL) into an ectopic subcutaneous mouse model. Comprehensive histologic and histometric analyses were performed after an 8‐wk healing period. To further understand the dose‐dependent (0, 10, 50, 200, 500 and 1000 ng/mL) and time‐dependent (0, 3, 5, 7 and 14 d) effects of rhBMP‐2 on osteogenic and adipogenic differentiation, in vitro osteogenic and adipogenic differentiation of hABCs were evaluated, and the expression of related mRNAs, including those for alkaline phosphatase, osteocalcin, bone sialoprotein, peroxisome‐proliferator‐activated receptor gamma‐2 and lipoprotein lipase, were assessed using quantitative RT‐PCR. Results: rhBMP‐2 significantly promoted the osteogenic and adipogenic differentiation of hABCs in vivo, and gradually increased both the osteogenic and adipogenic potential in a dose‐ and time‐dependent manner with minimal deviation in vitro. The expression of osteogenesis‐ and adipogenesis‐associated mRNAs were concomitantly up‐regulated by rhBMP‐2. Conclusion: The findings of the present study showed that rhBMP‐2 significantly enhanced the adipogenic as well as the osteogenic potential of hABCs in dose‐ and time‐dependent manner. The control of adipogenic differentiation of hABCs should be considered when regenerating the alveolar bone using rhBMP‐2.