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    Sequential controlled-released dual-drug loaded scaffold for guided bone regeneration in a rat fenestration defect model. Guo Zhenzhao,Bo Dongying,He Ping,Li Hong,Wu Gang,Li Zhizhong,Zhou Changren,Li Qiyan Journal of materials chemistry. B A microbially-induced inflammatory periodontal disease is the main initiator to disrupt the periodontium. It is desirable to develop a newly guided bone regeneration (GBR) scaffold to accomplish the periodontal tissue regeneration for the concurrent control of inflammation. A novel therapeutic solution for GBR based on 3D multifunctional scaffolds, which combines the merits of osseous regeneration and local anti-inflammatory drug delivery, has been developed. The 3D dual-drug delivery scaffold (DDDS) loaded with parthenolide and naringin was successfully developed by thermally-induced phase separation techniques. The DDDS was hierarchically interconnected to the porous PLLA scaffold loaded with the hydrophobic parthenolide. In addition, the hydrophilic naringin loaded in chitosan microspheres was embedded in the scaffold. In vitro drug release profile results revealed that the DDDS showed an efficient sequential controlled release pattern with parthenolide delivered rapidly, followed by naringin delivered in a more sustained manner. Cell viability of MC3T3-E1 showed a combined effect of dual-drug delivery. Hemolysis of the DDDS was 1.84 ± 0.44%, which is less than that of the pure PLLA scaffold. To further evaluate the in vivo guided bone regeneration effect of the DDDS, a rat fenestration defect model was generated. The defects were harvested after 4 and 8 weeks for micro-CT and histological observation. The results suggested that the DDDS group had significantly increased the regenerated bone volume fraction compared to both the control and PLLA groups at 8 weeks, which was in parallel with the reduced expression of IL-6. This DDDS, as a GBR scaffold, might be utilized as a novel adjunctive treatment in periodontitis. 10.1039/c7tb00909g
    Platelet-rich plasma, low-level laser therapy, or their combination promotes periodontal regeneration in fenestration defects: a preliminary in vivo study. Nagata Maria J H,de Campos Natália,Messora Michel R,Pola Natália M,Santinoni Carolina S,Bomfim Suely R M,Fucini Stephen E,Ervolino Edilson,de Almeida Juliano M,Theodoro Letícia H,Garcia Valdir G Journal of periodontology BACKGROUND:This study histomorphometrically analyzes the influence of platelet-rich plasma (PRP), low-level laser therapy (LLLT), or their combination on the healing of periodontal fenestration defects (PFDs) in rats. METHODS:PFDs were surgically created in the mandibles of 80 rats. The animals were randomly divided into four groups: 1) C (control) and 2) PRP, defects were filled with blood clot or PRP, respectively; 3) LLLT and 4) PRP/LLLT, defects received laser irradiation, were filled with blood clot or PRP, respectively, and then irradiated again. Animals were euthanized at either 10 or 30 days post-surgery. Percentage of new bone (NB), density of newly formed bone (DNB), new cementum (NC), and extension of remaining defect (ERD) were histomorphometrically evaluated. Data were statistically analyzed (analysis of variance; Tukey test, P <0.05). RESULTS:At 10 days, group PRP presented ERD significantly lower than group C. At 30 days, group PRP presented NB and DNB significantly greater than group C. Groups LLLT, PRP, and PRP/LLLT showed significant NC formation at 30 days, with collagen fibers inserted obliquely or perpendicularly to the root surface. NC formation was not observed in any group C specimen. CONCLUSIONS:LLLT, PRP, or their combination all promoted NC formation with a functional periodontal ligament. The combination PRP/LLLT did not show additional positive effects compared to the use of either therapy alone. 10.1902/jop.2013.130318
    Harmine promotes periodontal ligament cell-induced tissue regeneration. Lim H-C,Cha B-Y,Song S U,Yun J-H Oral diseases OBJECTIVE:to investigate whether harmine has a promotive effect on human periodontal ligament cells (hPDLCs)-induced tissue regeneration. MATERIALS AND METHODS:Various concentrations of harmine on hPDLCs proliferation were tested. Osteogenic and cementogenic characteristics were examined in hPDLC/rhBMP-2 and hPDLC/harmine by alizarin red S staining, real-time PCR, and Western blotting assay. The activity of harmine was investigated in an ectopic transplantation nude mouse model. RESULTS:We determined that 10 μM of harmine was the threshold concentration. hPDLC/harmine showed similar mineralized nodule formation in alizarin S staining compared to hPDLC/rhBMP-2. In real-time PCR, the highest gene expression level was observed for Runx2 in hPDLC/harmine at all time points. The level of CEMP-1 in hPDLC/harmine was higher at 7 days than hPDLCs alone. Thicker band of Runx2 in hPDLC/harmine was observed than in hPDLC/rhBMP-2 at 7 days by Western blotting. The band for CEMP-1 in hPDLC/harmine was thicker than hPDLCs alone at both 7 and 14 days. In ectopic transplantation, hPDLCs with harmine showed a comparable amount of mineralized tissue formation compared to rhBMP-2. hPDLCs with harmine or rhBMP-2 formed both bone and cementum-like tissue with Sharpey's fiber-like collagen insertion. CONCLUSION:Harmine can be a potential candidate for promoting hPDLCs-induced tissue regeneration. 10.1111/odi.12770
    A Graded Multifunctional Hybrid Scaffold with Superparamagnetic Ability for Periodontal Regeneration. Sprio Simone,Campodoni Elisabetta,Sandri Monica,Preti Lorenzo,Keppler Tobias,Müller Frank A,Pugno Nicola M,Tampieri Anna International journal of molecular sciences The regeneration of dental tissues is a still an unmet clinical need; in fact, no therapies have been completely successful in regenerating dental tissue complexes such as periodontium, which is also due to the lack of scaffolds that are able to guide and direct cell fate towards the reconstruction of different mineralized and non-mineralized dental tissues. In this respect, the present work develops a novel multifunctional hybrid scaffold recapitulating the different features of alveolar bone, periodontal ligament, and cementum by integrating the biomineralization process, and tape casting and electrospinning techniques. The scaffold is endowed with a superparamagnetic ability, thanks to the use of a biocompatible, bioactive superparamagnetic apatite phase, as a mineral component that is able to promote osteogenesis and to be activated by remote magnetic signals. The periodontal scaffold was obtained by engineering three different layers, recapitulating the relevant compositional and microstructural features of the target tissues, into a monolithic multifunctional graded device. Physico-chemical, morphological, and ultrastructural analyses, in association with preliminary in vitro investigations carried out with mesenchymal stem cells, confirm that the final scaffold exhibits a good mimicry of the periodontal tissue complex, with excellent cytocompatibility and cell viability, making it very promising for regenerative applications in dentistry. 10.3390/ijms19113604
    Periodontal tissue engineering by transplantation of multilayered sheets of phenotypically modified gingival fibroblasts. Nakajima K,Abe T,Tanaka M,Hara Y Journal of periodontal research BACKGROUND AND OBJECTIVE:In periodontal tissue engineering, the sourcing of most of the relevant cells is limited by poor accessibility, whereas the use of readily available gingival fibroblasts is hampered because of their inhibitory effects on bone formation. To address the latter drawback, we developed a new graft composed of fibronectin (FN) matrix-based multilayered cell sheets of human gingival fibroblasts modified to express alkaline phosphatase (ALP). This study was undertaken to investigate the effects of this graft, called the FN-ALP transplant, on the healing of periodontal defects in a rat model. MATERIAL AND METHODS:The FN-ALP transplants were grafted into periodontal fenestration bone defects in immunosuppressed rats. The process of periodontal healing was examined by histology, histomorphometry and immunohistochemistry. Grafted cells were tracked by immunostaining with human-specific antibodies. Control groups included non-transplanted empty defects and defects to which cell sheets without ALP induction had been grafted. RESULTS:After implantation, the FN-ALP transplants healed alveolar bone defects by intramembranous ossification, with formation of cementum and periodontal ligament. Moreover, FN-ALP transplants increased new bone formation, by endochondral ossification, on the mandibular cortex adjacent to the defect. Grafted fibroblasts were located near host osteoblasts and chondrocyte precursor cells early in the ossification process but were undetectable on and in newly formed bone and cartilage. CONCLUSION:These results indicate that the FN-ALP transplants support alveolar bone regeneration within the defect and augment bone formation outside the defect through the recruitment of host osteo/chondrogenic cells, suggesting their potential for periodontal tissue engineering applications. 10.1111/j.1600-0765.2007.01072.x
    Tissue engineering of cementum/periodontal-ligament complex using a novel three-dimensional pellet cultivation system for human periodontal ligament stem cells. Yang Zhenhua,Jin Fang,Zhang Xiaojun,Ma Dandan,Han Chun,Huo Na,Wang Yinxiong,Zhang Yunfei,Lin Zhu,Jin Yan Tissue engineering. Part C, Methods Limitations of conventional regeneration modalities underscore the necessity of recapitulating development for periodontal tissue engineering. In this study, we proposed a novel three-dimensional pellet cultivation system for periodontal ligament stem cells (PDLSCs) to recreate the biological microenvironment similar to those of a regenerative milieu. Monodispersed human PDLSCs were cultured in medium with ascorbic acid and conditioned medium from developing apical tooth germ cells and were subsequently harvested from culture plate as a contiguous cell sheet with abundant extracellular matrix. The detached cell-matrix membrane spontaneously contracted to produce a single-cell pellet. The PDLSCs embedded within this cell-matrix complex exhibited several phenotypic characteristics of cementoblast lineages, as indicated by upregulated alkaline phosphatase activity, accelerated mineralization, and the expression of bone sialoprotein and osteocalcin genes. When this PDLSC pellets were transplanted into immunocompromised mice, a regular aligned cementum/PDL-like complex was formed. These results suggest that the combination of apical tooth germ cell-conditioned medium and endogenous extracellular matrix could maximally mimic the microenvironment of root/periodontal tissue development and enhance the reconstruction of physiological architecture of a cementum/PDL-like complex in a tissue-mimicking way; on the other hand, such PDLSC pellet may also be a promising alternative to promote periodontal defect repair for future clinical applications. 10.1089/ten.tec.2008.0561
    Effects of concomitant use of fibroblast growth factor (FGF)-2 with beta-tricalcium phosphate (β-TCP) on the beagle dog 1-wall periodontal defect model. Anzai Jun,Kitamura Masahiro,Nozaki Takenori,Nagayasu Toshie,Terashima Akio,Asano Taiji,Murakami Shinya Biochemical and biophysical research communications The effects of concomitant use of fibroblast growth factor-2 (FGF-2) and beta-tricalcium phosphate (β-TCP) on periodontal regeneration were investigated in the beagle dog 1-wall periodontal defect model. One-wall periodontal defects were created in the mesial portion of both sides of the mandibular first molars, and 0.3% FGF-2 plus β-TCP or β-TCP alone was administered. Radiographic evaluation was performed at 0, 3, and 6 weeks. At 6 weeks, the periodontium with the defect site was removed and histologically analyzed. Radiographic findings showed that co-administration of FGF-2 significantly increased bone mineral contents of the defect sites compared with β-TCP alone. Histologic analysis revealed that the length of the regenerated periodontal ligament, the cementum, distance to the junctional epithelium, new bone height, and area of newly formed bone were significantly increased in the FGF-2 group. No abnormal inflammatory response or ankylosis was observed in either group. These findings indicate the efficacy of concomitant use of FGF-2 and β-TCP as an osteoconductive material for periodontal regeneration following severe destruction by progressive periodontitis. 10.1016/j.bbrc.2010.11.032
    Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model. Tsumanuma Yuka,Iwata Takanori,Washio Kaoru,Yoshida Toshiyuki,Yamada Azusa,Takagi Ryo,Ohno Takahiro,Lin Konghua,Yamato Masayuki,Ishikawa Isao,Okano Teruo,Izumi Yuichi Biomaterials Cytotherapeutic approaches have been investigated to overcome the limitations of existing procedures for periodontal regeneration. In this study, cell sheet transplantation was performed using three kinds of mesenchymal tissue (periodontal ligament, alveolar periosteum, and bone marrow)-derived cells to compare the differences between cell sources in a canine severe defect model (one-wall intrabony defect). Periodontal ligament cells (PDLCs), iliac bone marrow mesenchymal stromal cells (BMMSCs), and alveolar periosteal cells (APCs) were obtained from each dog; a total of four dogs were used. Three-layered cell sheets of each cell source supported with woven polyglycolic acid were autologously transplanted to the denuded root surface. One-wall intrabony defects were filled with a mixture of β-tricalcium phosphate (β-TCP) and collagen. Eight weeks after the transplantation, periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the PDLC group than in the other groups. In addition, nerve filament was observed in the regenerated PDL tissue only in the PDLC group. The amount of alveolar bone regeneration was highest in the PDLC group, although it did not reach statistical significance among the groups. These results indicate that PDLC sheets combined with β-TCP/collagen scaffold serve as a promising tool for periodontal regeneration. 10.1016/j.biomaterials.2011.04.071
    Maturation of periodontal tissues following implantation of rhGDF-5/β-TCP in one-wall intra-bony defects in dogs: 24-week histological observations. Lee Jung-Seok,Wikesjö Ulf M E,Park Jung-Chul,Jang Yong-Ju,Pippig Susanne D,Bastone Patrizia,Choi Seong-Ho,Kim Chong-Kwan Journal of clinical periodontology OBJECTIVE:Although a previous study reported that recombinant human growth/differentiation factor-5 (rhGDF-5) coated onto a β-tricalciumphosphate (β-TCP) significantly enhanced periodontal regeneration, the long-term stability/maturation of the regenerated tissues has not been demonstrated. The objective of this study was to evaluate periodontal regeneration/maturation following application of rhGDF-5/β-TCP using an established periodontal defect model and a 24-week healing interval. MATERIAL & METHODS:Unilateral, surgically created, 4 × 4 × 5 mm (length × width × height), one-wall, critical-size, intra-bony periodontal defects at the mandibular second and fourth premolar teeth in five young adult Beagle dogs received rhGDF-5/β-TCP. Bilateral sites at the fourth premolar in the other four dogs served as pristine controls receiving mucogingival flap surgery without defect induction. The animals were euthanized at 24 weeks for histological analysis. Unpublished data from the previous 8-week study were used to compare tissue maturation between 8 and 24 weeks. RESULTS:Linear histometric observations of cementum and alveolar regeneration showed no significant differences between the 8- and 24-week observation intervals. However, parameters of periodontal tissue maturation showed significant differences between the observation intervals including increased fraction mineralized tissue and lamellar bone (p < 0.05) and decreased osteocyte counts (p < 0.05) at 24 weeks compared with 8 weeks. Although the count inserting Sharpey's fibre did not significantly change, regenerated cementum remote from the intact periodontal ligament appeared more highly mineralized and thicker at 24 weeks compared with 8 weeks, and compared with the pristine cementum. Minimal β-TCP remained. CONCLUSIONS:These 24-week observations suggest that regenerated periodontal tissues in sites receiving rhGDF-5/β-TCP undergo progressive maturation without debilitating aberrant tissue reactions. 10.1111/j.1600-051X.2012.01862.x
    The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model. Kim Y-T,Park J-C,Choi S-H,Cho K-S,Im G-I,Kim B-S,Kim C-S Journal of periodontal research BACKGROUND AND OBJECTIVE:Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well-standardized assessment tool that excludes the innate healing factors from the animals. MATERIAL AND METHODS:Human periodontal ligament stem cells that were isolated and characterized from teeth (n=12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n=20). Animals were killed after several different healing periods: 3 d (n=4), 1 (n=4), 2 (n=4), 4 (n=4) and 8 wk (n=4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson's trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue-specific markers associated with periodontal regeneration. RESULTS:The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid-like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey's fibers, was associated with active mineralization of cementum-like tissues, and in the fourth phase, the maturation of cementum-like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well-orchestrated time order. Interestingly, well-organized cementum-like and periodontal ligament fiber-like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell-rich zone. CONCLUSION:The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell-based treatment techniques. 10.1111/j.1600-0765.2011.01463.x
    Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering. Wu Chengtie,Zhou Yinghong,Lin Chucheng,Chang Jiang,Xiao Yin Acta biomaterialia To achieve the ultimate goal of periodontal tissue engineering, it is of great importance to develop bioactive scaffolds which can stimulate the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for the favorable regeneration of alveolar bone, root cementum and periodontal ligament. Strontium (Sr) and Sr-containing biomaterials have been found to induce osteoblast activity. However, there has been no systematic report about the interaction between Sr or Sr-containing biomaterials and PDLCs for periodontal tissue engineering. The aims of this study were to prepare Sr-containing mesoporous bioactive glass (Sr-MBG) scaffolds and investigate whether the addition of Sr could stimulate osteogenic/cementogenic differentiation of PDLCs in a tissue-engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nanopore volume and nanopore distribution) of Sr-MBG scaffolds were characterized. The proliferation, alkaline phosphatase (ALP) activity and osteogenesis/cementogenesis-related gene expression (ALP, Runx2, Col I, OPN and CEMP1) of PDLCs on different kinds of Sr-MBG scaffolds were systematically investigated. The results show that Sr plays an important role in influencing the mesoporous structure of MBG scaffolds in which high contents of Sr decreased the well-ordered mesopores as well as their surface area/pore volume. Sr(2+) ions could be released from Sr-MBG scaffolds in a controlled way. The incorporation of Sr into MBG scaffolds has significantly stimulated ALP activity and osteogenesis/cementogenesis-related gene expression of PDLCs. Furthermore, Sr-MBG scaffolds in a simulated body fluid environment still maintained excellent apatite-mineralization ability. The study suggests that the incorporation of Sr into MBG scaffolds is a viable way to stimulate the biological response of PDLCs. Sr-MBG scaffolds are a promising bioactive material for periodontal tissue-engineering applications. 10.1016/j.actbio.2012.06.023
    Regeneration of periodontal tissues using allogeneic periodontal ligament stem cells in an ovine model. Mrozik Krzysztof Marek,Wada Naohisa,Marino Victor,Richter Ward,Shi Songtao,Wheeler Donna L,Gronthos Stan,Bartold P Mark Regenerative medicine AIM:To investigate the capacity of allogeneic periodontal ligament stem cells (PDLSCs) to regenerate periodontal tissues using an ovine periodontal defect model. MATERIALS & METHODS:Surgically created zero-wall dehiscence periodontal defects created in Merino sheep were filled with 1 × 10(7) allogeneic PDLSCs attached to Gelfoam(®), Gelfoam alone or left untreated. After 4 weeks, histological analysis was performed to assess periodontal regeneration. RESULTS:Allogeneic PDLSCs were well tolerated by recipient animals. The mean area of new alveolar bone was significantly greater in the PDLSC + Gelfoam treatment group compared with the defect-alone group. The PDLSC + Gelfoam and Gelfoam-only treatment groups displayed significantly greater length of new cementum and percentage of cementum regrowth compared with the defect-alone group. New Sharpey's fibers were generally more organized and significantly thicker within the PDLSC + Gelfoam treatment group. The PDLSC + Gelfoam treatment group also showed a trend of increased Sharpey's fiber attachment length compared with the Gelfoam-only and defect-alone groups. CONCLUSION:These studies support the potential use of allogeneic PDLSC preparations as viable therapies for periodontal regeneration in the clinical setting. 10.2217/rme.13.66
    A tissue engineering approach for periodontal regeneration based on a biodegradable double-layer scaffold and adipose-derived stem cells. Requicha João F,Viegas Carlos A,Muñoz Fernando,Azevedo Jorge M,Leonor Isabel B,Reis Rui L,Gomes Manuela E Tissue engineering. Part A Human and canine periodontium are often affected by an inflammatory pathology called periodontitis, which is associated with severe damages across tissues, namely, in the periodontal ligament, cementum, and alveolar bone. However, the therapies used in the routine dental practice, often consisting in a combination of different techniques, do not allow to fully restore the functionality of the periodontium. Tissue Engineering (TE) appears as a valuable alternative approach to regenerate periodontal defects, but for this purpose, it is essential to develop supportive biomaterial and stem cell sourcing/culturing methodologies that address the complexity of the various tissues affected by this condition. The main aim of this work was to study the in vitro functionality of a newly developed double-layer scaffold for periodontal TE. The scaffold design was based on a combination of a three-dimensional (3D) fiber mesh functionalized with silanol groups and a membrane, both made of a blend of starch and poly-ɛ-(caprolactone). Adipose-derived stem cells (canine adipose stem cells [cASCs]) were seeded and cultured onto such scaffolds, and the obtained constructs were evaluated in terms of cellular morphology, metabolic activity, and proliferation. The osteogenic potential of the fiber mesh layer functionalized with silanol groups was further assessed concerning the osteogenic differentiation of the seeded and cultured ASCs. The obtained results showed that the proposed double-layer scaffold supports the proliferation and selectively promotes the osteogenic differentiation of cASCs seeded onto the functionalized mesh. These findings suggest that the 3D structure and asymmetric composition of the scaffold in combination with stem cells may provide the basis for developing alternative therapies to treat periodontal defects more efficiently. 10.1089/ten.TEA.2013.0360
    Effect of a tunnel-structured β-tricalcium phosphate graft material on periodontal regeneration: a pilot study in a canine one-wall intrabony defect model. Matsuura T,Akizuki T,Hoshi S,Ikawa T,Kinoshita A,Sunaga M,Oda S,Kuboki Y,Izumi Y Journal of periodontal research BACKGROUND AND OBJECTIVE:Tissue regeneration is affected by the porosity, chemical properties and geometric structure of graft materials. Regeneration of severe periodontal defects, such as one-wall intrabony defects, is difficult because of reduced tissue support, and bone grafts are commonly used in such cases. In the present study, a tunnel-structured β-tricalcium phosphate (tunnel β-TCP) graft material designed to stimulate bone formation was fabricated. The objective of this pilot study was to evaluate the effect of this graft material on periodontal regeneration in one-wall intrabony defects in dogs. MATERIAL AND METHODS:Six male beagle dogs were used in this study. First, the mandibular second and third incisors were extracted. Experimental surgery was performed 12 wk after tooth extraction. Bilateral 4 × 8 mm (width × depth) one-wall intrabony defects were created in the mesial side of the mandibular canines. At the experimental sites, the defects were filled with tunnel β-TCP, whereas the control defects were left empty. Twelve weeks after surgery, qualitative and quantitative histological analyses were performed. RESULTS:There were no signs of clinical inflammation 12 wk after surgery. Coronal extension indicative of new bone formation was higher at the experimental sites than at the control sites, although the differences between both the sites in the newly formed cementum and connective tissue attachment were not significant. Newly formed periodontal ligament and cementum-like tissue were evident along the root surface at the experimental sites. The inner surface of the tunnels was partially resorbed and replaced with new bone. New blood vessels were observed inside the lumens of tunnel β-TCP. CONCLUSION:Tunnel β-TCP serves as a scaffold for new bone formation in one-wall intrabony defects. 10.1111/jre.12213
    Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs. Nagayasu-Tanaka Toshie,Anzai Jun,Takaki Shu,Shiraishi Noriko,Terashima Akio,Asano Taiji,Nozaki Takenori,Kitamura Masahiro,Murakami Shinya PloS one Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL. 10.1371/journal.pone.0131870
    Standardized in vivo model for studying novel regenerative approaches for multitissue bone-ligament interfaces. Padial-Molina Miguel,Rodriguez Juan C,Volk Sarah L,Rios Hector F Nature protocols The regeneration of the original structure and function of bone-ligament interfaces remains a major challenge in biomedical research. A preclinical model that maintains physiologic mechanical loads and controls for other external factors, such as microbial influence, is of great value for testing novel regenerative materials, provided that studies are performed by highly trained researchers with proper regard for animal welfare. The tooth root fenestration preclinical model is an ideal tool for hard tissue evaluation by micro-computed tomography, histological techniques and RNA analyses. The procedure starts with an extraoral incision lateral to the mandible and reflection of the masseter muscle. Superficial lateral mandibular bone is removed with standardized dimensions to expose the roots of the teeth and to eliminate periodontal ligament and cementum to expose the tooth dentin. The testing material can subsequently be applied to the defect and the flap can be repositioned and secured back in place. At specific time points, samples are collected and processed according to the subsequent analyses to be performed, which can include descriptive histology, histomorphometry, immunostaining, 3D bone imaging, electron microscopy, gene expression analyses and safety assessments. 10.1038/nprot.2015.063
    Cell-Based Approaches in Periodontal Regeneration: A Systematic Review and Meta-Analysis of Periodontal Defect Models in Animal Experimental Work. Yan Xiang-Zhen,Yang Fang,Jansen John A,de Vries Rob B M,van den Beucken Jeroen J J P Tissue engineering. Part B, Reviews Various cell types have been assessed for experimental periodontal tissue regeneration in a variety of animal models. Nonetheless, the efficacy of cell-based approaches for periodontal regeneration is still controversial. Therefore, the purpose of this study was to systematically review cell-based approaches for periodontal regeneration in animal studies including a meta-analysis to obtain more clarity on their efficacy. The results of this systematic review and meta-analysis revealed that cell-based approaches have a favorable effect on periodontal tissue regeneration, as displayed by the positive effect of cell-based approaches on new bone, cementum, and periodontal ligament (PDL) formation in periodontal defects. Moreover, subgroup analysis showed a favorable effect on PDL formation by PDL-derived cells, but not by bone marrow mesenchymal stem cells (BMSCs). However, meta-analysis did not show any statistically significant differences in effect between PDL-derived cells and BMSCs. These results provide important information for the implementation of cell-based approaches in clinical practice as a routine treatment for periodontal regeneration in the future. 10.1089/ten.TEB.2015.0049
    Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway. Mao Lixia,Liu Jiaqiang,Zhao Jinglei,Chang Jiang,Xia Lunguo,Jiang Lingyong,Wang Xiuhui,Lin Kaili,Fang Bing International journal of nanomedicine The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration. 10.2147/IJN.S90343
    Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration. Akita Daisuke,Kano Koichiro,Saito-Tamura Yoko,Mashimo Takayuki,Sato-Shionome Momoko,Tsurumachi Niina,Yamanaka Katsuyuki,Kaneko Tadashi,Toriumi Taku,Arai Yoshinori,Tsukimura Naoki,Matsumoto Taro,Ishigami Tomohiko,Isokawa Keitaro,Honda Masaki Frontiers in physiology Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration. 10.3389/fphys.2016.00050
    In vitro studies on human periodontal ligament stem cell sheets enhanced by enamel matrix derivative. Wang Zhongshan,Feng Zhihong,Wu Guofeng,Bai Shizhu,Dong Yan,Zhao Yimin Colloids and surfaces. B, Biointerfaces Numerous preclinical and clinical studies have focused on the periodontal regenerative functions of enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs) of developing porcine teeth. In this study, periodontal ligament (PDL) stem cells (PDLSCs) were isolated, and the effects of EMD on the extracorporeal induction process and the characteristics of PDLSC sheets were investigated for their potential as a more effective stem-cell therapy. EMD-enhanced cell sheets could be induced by complete medium supplemented with 50 μg/mL vitamin C and 100 μg/mL EMD. The EMD-enhanced cell sheets appeared thicker and more compact than the normal PDLSC sheets, demonstrated more layers of cells (3-7 layers), secreted richer extracellular matrix (ECM), showed varying degrees of increases in mRNA expression of periodontal tissue-specific genes (COL I, POSTN), calcification-related genes (RUNX2, OPN, OCN) and a cementum tissue-specific gene (CAP), and possessed a better mineralization ability in terms of osteogenic differentiation in vitro. These EMD-enhanced cell sheets may represent a potential option for stem-cell therapy for PDL regeneration. 10.1016/j.colsurfb.2016.01.036
    Periodontal healing with a preameloblast-conditioned medium in dogs. Yu S-J,Lee D-S,Kim B-O,Choi S-H,Park J-C Journal of periodontal research 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. 10.1111/jre.12307
    Periodontal regeneration with stem cells-seeded collagen-hydroxyapatite scaffold. Liu Zeping,Yin Xing,Ye Qingsong,He Wulin,Ge Mengke,Zhou Xiaofu,Hu Jing,Zou Shujuan Journal of biomaterials applications Re-establishing compromised periodontium to its original structure, properties and function is demanding, but also challenging, for successful orthodontic treatment. In this study, the periodontal regeneration capability of collagen-hydroxyapatite scaffolds, seeded with bone marrow stem cells, was investigated in a canine labial alveolar bone defect model. Bone marrow stem cells were isolated, expanded and characterized. Porous collagen-hydroxyapatite scaffold and cross-linked collagen-hydroxyapatite scaffold were prepared. Attachment, migration, proliferation and morphology of bone marrow stem cells, co-cultured with porous collagen-hydroxyapatite or cross-linked collagen-hydroxyapatite, were evaluated in vitro. The periodontal regeneration capability of collagen-hydroxyapatite scaffold with or without bone marrow stem cells was tested in six beagle dogs, with each dog carrying one sham-operated site as healthy control, and three labial alveolar bone defects untreated to allow natural healing, treated with bone marrow stem cells - collagen-hydroxyapatite scaffold implant or collagen-hydroxyapatite scaffold implant, respectively. Animals were euthanized at 3 and 6 months (3 animals per group) after implantation and the resected maxillary and mandibular segments were examined using micro-computed tomography scan, H&E staining, Masson's staining and histometric evaluation. Bone marrow stem cells were successfully isolated and demonstrated self-renewal and multi-potency in vitro. The porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite had average pore sizes of 415 ± 20 µm and 203 ± 18 µm and porosity of 69 ± 0.5% and 50 ± 0.2%, respectively. The attachment, proliferation and migration of bone marrow stem cells were satisfactory on both porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite scaffolds. Implantation of bone marrow stem cells - collagen-hydroxyapatite or collagen-hydroxyapatite scaffold in beagle dogs with experimental periodontal defects resulted in significantly enhanced periodontal regeneration characterized by formation of new bone, periodontal ligament and cementum, compared with the untreated defects, as evidenced by histological and micro-computed tomography examinations. The prepared collagen-hydroxyapatite scaffolds possess favorable bio-compatibility. The bone marrow stem cells - collagen-hydroxyapatite and collagen-hydroxyapatite scaffold - induced periodontal regeneration, with no aberrant events complicating the regenerative process. Further research is necessary to improve the bone marrow stem cells behavior in collagen-hydroxyapatite scaffolds after implantation. 10.1177/0885328216637978
    The differentiation potential of gingival mesenchymal stem cells induced by apical tooth germ cell‑conditioned medium. Chen Yan,Liu Hongwei Molecular medicine reports Gingival-derived mesenchymal stem cells (GMSCs) have recently been harvested; however, the use of GMSCs in periodontal tissue engineering requires further study. The present study established an indirect co‑culture system between rat apical tooth germ‑conditioned medium (APTG‑CM) and GMSCs, in order to determine the effects on periodontal tissue differentiation in vitro and in vivo. Using the limiting dilution technique, single‑colony derived human GMSCs and periodontal ligament stem cells (PDLSCs) were isolated and expanded to obtain homogeneous populations. PDLSCs were used as a positive control group. Cell cycle distribution, alkaline phosphatase (ALP) activity, mineralization behavior, expression of genes associated with a cementoblast phenotype (osteocalcin, bone sialoprotein, ALP, type I collagen, cementum‑derived protein 23), and in vivo differentiation capacities of GMSCs/PDLSCs co‑cultured with APTG‑CM were evaluated. Flow cytometry indicated that GMSCs and PDLSCs were positive for STRO‑1 and CD105, whereas CD45 expression was negative. The cell types were capable of forming colonies, and of osteogenic and adipogenic differentiation in response to appropriate stimuli. The induced GMSCs and PDLSCs exhibited numerous characteristics associated with cementoblast lineages, as indicated by increased proliferation and ALP activity, and upregulated expression of cementum‑associated genes in vitro. In vivo, cementum/periodontal ligament‑like structures were shown to form along the dentin surface and ceramic bovine bone in GMSCs and PDLSCs induced by APTG‑CM group. Conversely, vertical fibers could not insert in the control group, which was not co‑cultured with APTG‑CM. In conclusion, GMSCs are likely to have a role in periodontal tissue regeneration. In addition, APTG‑CM was able to provide a cementogenic microenvironment and promote differentiation of GMSCs along the cementoblastic lineage. 10.3892/mmr.2016.5726
    Periodontal tissue regeneration using the cytokine cocktail mimicking secretomes in the conditioned media from human mesenchymal stem cells. Sakaguchi Kohei,Katagiri Wataru,Osugi Masashi,Kawai Takamasa,Sugimura-Wakayama Yukiko,Hibi Hideharu Biochemical and biophysical research communications Secretomes in the conditioned media from human mesenchymal stem cells (MSC-CM) were previously demonstrated to promote periodontal tissue regeneration. By mixing insulin-like growth factor-1, vascular endothelial growth factor-A, and transforming growth factor-β1 which were included in MSC-CM, we made the cytokine cocktail (CC) mimicking MSC-CM, and then evaluated its efficacy on periodontal tissue regeneration. In vitro, CC promoted the migration of dog bone marrow-derived stem cells and periodontal ligament cells, and the tube formation of human umbilical vein endothelial cells. In vivo, class II furcation defects were surgically created at premolars in dogs. After 4 weeks of vinylpolysiloxane-induced inflammation, defects were filled with or without CC mixed in hydroxypropyl cellulose, or enamel matrix derivative (EMD). After 8 weeks, periodontal tissues were evaluated histologically and immunohistochemically. CC showed promotional effects on angiogenesis and formation of new bone and cementum. Osteogenesis by CC was greater than that by EMD and cementogenesis by CC was as well as that by EMD. CC may be promising for periodontal tissue regeneration. 10.1016/j.bbrc.2017.01.065
    Periodontal Tissue Regeneration Using Syngeneic Adipose-Derived Stromal Cells in a Mouse Model. Lemaitre Mathieu,Monsarrat Paul,Blasco-Baque Vincent,Loubières Pascale,Burcelin Rémy,Casteilla Louis,Planat-Bénard Valérie,Kémoun Philippe Stem cells translational medicine Current treatment of periodontitis is still associated with a high degree of variability in clinical outcomes. Recent advances in regenerative medicine by mesenchymal cells, including adipose stromal cells (ASC) have paved the way to improved periodontal regeneration (PD) but little is known about the biological processes involved. Here, we aimed to use syngeneic ASCs for periodontal regeneration in a new, relevant, bacteria-induced periodontitis model in mice. Periodontal defects were induced in female C57BL6/J mice by oral gavage with periodontal pathogens. We grafted 2 × 10 syngeneic mouse ASCs expressing green fluorescent protein (GFP) (GFP+/ASC) within a collagen vehicle in the lingual part of the first lower molar periodontium (experimental) while carrier alone was implanted in the contralateral side (control). Animals were sacrificed 0, 1, 6, and 12 weeks after treatment by GFP+/ASC or vehicle graft, and microscopic examination, immunofluorescence, and innovative bio-informatics histomorphometry methods were used to reveal deep periodontium changes. From 1 to 6 weeks after surgery, GFP+ cells were identified in the periodontal ligament (PDL), in experimental sites only. After 12 weeks, cementum regeneration, the organization of PDL fibers, the number of PD vessels, and bone morphogenetic protein-2 and osteopontin expression were greater in experimental sites than in controls. Specific stromal cell subsets were recruited in the newly formed tissue in ASC-implanted periodontium only. These data suggest that ASC grafting in diseased deep periodontium, relevant to human pathology, induces a significant improvement of the PDL microenvironment, leading to a recovery of tooth-supporting tissue homeostasis. Stem Cells Translational Medicine 2017;6:656-665. 10.5966/sctm.2016-0028
    Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment. Gauthier Philippe,Yu Zongdong,Tran Quynh T,Bhatti Fazal-Ur-Rehman,Zhu Xiaofei,Huang George T-J Cell and tissue research Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1/CD146, STRO-1/CD146 and STRO-1/CD146 subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration. 10.1007/s00441-016-2513-8
    Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells. Kim Eun-Cheol,Park Jaesuh,Kwon Il Keun,Lee Suk-Won,Park Su-Jung,Ahn Su-Jin Journal of periodontal & implant science Purpose:Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Methods:Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. Results:The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated. Conclusions:SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease. 10.5051/jpis.2017.47.5.273
    Influence of Micropatterning on Human Periodontal Ligament Cells' Behavior. Zheng Lisha,Jiang Jingyi,Gui Jinpeng,Zhang Lingyu,Liu Xiaoyi,Sun Yan,Fan Yubo Biophysical journal The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-μm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells' peripheral regions. Moreover, proliferation was promoted by 10- and 20-μm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells' stem cell markers. β-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration. 10.1016/j.bpj.2018.02.041
    In Vivo Periodontium Formation Around Titanium Implants Using Periodontal Ligament Cell Sheet. Washio Kaoru,Tsutsumi Yusuke,Tsumanuma Yuka,Yano Kosei,Srithanyarat Supreda Suphanantachat,Takagi Ryo,Ichinose Shizuko,Meinzer Walter,Yamato Masayuki,Okano Teruo,Hanawa Takao,Ishikawa Isao Tissue engineering. Part A Osseointegrated implants have been recognized as being very reliable and having long-term predictability. However, host defense mechanisms against infection have been known to be impaired around a dental implant because of the lack of a periodontal ligament (PDL). The purpose of our experimental design was to produce cementum and PDL on the implant surface adopting cell sheet technology. To this aim we used PDL-derived cells, which contain multipotential stem cells, as the cell source and we cultured them on an implant material constituted of commercially pure titanium treated with acid etching, blasting, and a calcium phosphate (CaP) coating to improve cell attachment. Implants with adhered human PDL cell sheets were transplanted into bone defects in athymic rat femurs as a xenogeneic model. Implants with adhered canine PDL-derived cell sheets were transplanted into canine mandibular bone as an autologous model. We confirmed that PDL-derived cells cultured with osteoinductive medium had the ability to induce cementum formation. The attachment of PDL cells onto the titanium surface with three surface treatments was accelerated, compared with that onto the smooth titanium surface, at 40 min after starting incubation. Results in the rat model showed that cementum-like and PDL-like tissue was partly observed on the titanium surface with three surface treatments in combination with adherent PDL-derived cell sheets. On the other hand, osseointegration was observed on almost all areas of the smooth titanium surface that had PDL-derived cell sheets, but did not have the three surface treatments. In the canine model, histological observation indicated that formation of cementum-like and PDL-like tissue was induced on the titanium surface with surface treatments and that the PDL-like tissue was perpendicularly oriented between the titanium surface with cementum-like tissue and the bone. Results demonstrate that a periodontal-like structure was formed around a titanium implant, which is similar to the environment existing around a natural tooth. The clinical application of dental implants combined with a cell sheet technique may be feasible as an alternative implant therapy. Furthermore, application of this methodology may play an innovative role in the periodontal, prosthetic, and orthodontic fields in dentistry. 10.1089/ten.TEA.2017.0405
    The Fate of Transplanted Periodontal Ligament Stem Cells in Surgically Created Periodontal Defects in Rats. Iwasaki Kengo,Akazawa Keiko,Nagata Mizuki,Komaki Motohiro,Honda Izumi,Morioka Chikako,Yokoyama Naoki,Ayame Hirohito,Yamaki Kazumasa,Tanaka Yuichi,Kimura Tsuyoshi,Kishida Akio,Watabe Tetsuro,Morita Ikuo International journal of molecular sciences Periodontal disease is chronic inflammation that leads to the destruction of tooth-supporting periodontal tissues. We devised a novel method ("cell transfer technology") to transfer cells onto a scaffold surface and reported the potential of the technique for regenerative medicine. The aim of this study is to examine the efficacy of this technique in periodontal regeneration and the fate of transplanted cells. Human periodontal ligament stem cells (PDLSCs) were transferred to decellularized amniotic membrane and transplanted into periodontal defects in rats. Regeneration of tissues was examined by microcomputed tomography and histological observation. The fate of transplanted PDLSCs was traced using PKH26 and human Alu sequence detection by PCR. Imaging showed more bone in PDLSC-transplanted defects than those in control (amnion only). Histological examination confirmed the enhanced periodontal tissue formation in PDLSC defects. New formation of cementum, periodontal ligament, and bone were prominently observed in PDLSC defects. PKH26-labeled PDLSCs were found at limited areas in regenerated periodontal tissues. Human Alu sequence detection revealed that the level of Alu sequence was not increased, but rather decreased. This study describes a novel stem cell transplantation strategy for periodontal disease using the cell transfer technology and offers new insight for cell-based periodontal regeneration. 10.3390/ijms20010192
    Periodontal Tissue Engineering with a Multiphasic Construct and Cell Sheets. Vaquette C,Saifzadeh S,Farag A,Hutmacher D W,Ivanovski S Journal of dental research This study reports on scaffold-based periodontal tissue engineering in a large preclinical animal model. A biphasic scaffold consisting of bone and periodontal ligament compartments manufactured by melt and solution electrospinning, respectively, was used for the delivery of in vitro matured cell sheets from 3 sources: gingival cells (GCs), bone marrow-derived mesenchymal stromal cells (Bm-MSCs), and periodontal ligament cells (PDLCs). The construct featured a 3-dimensional fibrous bone compartment with macroscopic pore size, while the periodontal compartment consisted of a flexible porous membrane for cell sheet delivery. The regenerative performance of the constructs was radiographically and histologically assessed in surgically created periodontal defects in sheep following 5 and 10 wk of healing. Histologic observation demonstrated that the constructs maintained their shape and volume throughout the entirety of the in vivo study and were well integrated with the surrounding tissue. There was also excellent tissue integration between the bone and periodontal ligament compartments as well as the tooth root interface, enabling the attachment of periodontal ligament fibers into newly formed cementum and bone. Bone coverage along the root surface increased between weeks 5 and 10 in the Bm-MSC and PDLC groups. At week 10, the micro-computed tomography results showed that the PDLC group had greater bone fill as compared with the empty scaffold, while the GC group had less bone than the 3 other groups (control, Bm-MSC, and PDLC). Periodontal regeneration, as measured by histologically verified new bone and cementum formation with obliquely inserted periodontal ligament fibers, increased between 5 and 10 wk for the empty, Bm-MSC, and PDLC groups, while the GC group was inferior to the Bm-MSC and PDLC groups at 10 wk. This study demonstrates that periodontal regeneration can be achieved via the utilization of a multiphasic construct, with Bm-MSCs and PDLCs obtaining superior results as compared with GC-derived cell sheets. 10.1177/0022034519837967
    Periodontal healing using a collagen matrix with periodontal ligament progenitor cells in a dehiscence defect model in beagle dogs. Yoo Seung-Yoon,Lee Jung-Seok,Cha Jae-Kook,Kim Seul-Ki,Kim Chang-Sung Journal of periodontal & implant science Purpose:To histologically characterize periodontal healing at 8 weeks in surgically created dehiscence defects in beagle dogs that received a collagen matrix with periodontal ligament (PDL) progenitor cells. Methods:The bilateral maxillary premolars and first molars in 6 animals were used. Standardized experimental dehiscence defects were made on the buccal side of 3 premolars, and primary culturing of PDL progenitor cells was performed on the molars. Collagen matrix was used as a scaffold and a delivery system for PDL progenitor cells. The experimental sites were grafted with collagen matrix (COL), PDL progenitor cells with collagen matrix (COL/CELL), or left without any material (CTL). Histologic and histomorphometric analyses were performed after 8 weeks. Results:The defect height from the cementoenamel junction to the most apical point of cementum removal did not significantly differ across the CTL, COL, and COL/CELL groups, at 4.57±0.28, 4.56±0.41, and 4.64±0.27 mm (mean ± standard deviation), respectively; the corresponding values for epithelial adhesion were 1.41±0.51, 0.85±0.29, and 0.30±0.41 mm (<0.05), the heights of new bone regeneration were 1.32±0.44, 1.65±0.52, and 1.93±0.61 mm (<0.05), and the cementum regeneration values were 1.15±0.42, 1.81±0.46, and 2.57±0.56 mm (<0.05). There was significantly more new bone formation in the COL/CELL group than in the CTL group, and new cementum length was also significantly higher in the COL/CELL group. However, there were no significant differences in the width of new cementum among the groups. Conclusions:PDL progenitor cells carried by a synthetic collagen matrix may enhance periodontal regeneration, including cementum and new bone formation. 10.5051/jpis.2019.49.4.215
    Split-mouth evaluation of connective tissue graft with or without enamel matrix derivative for the treatment of isolated gingival recession defects in dogs. Shirakata Y,Nakamura T,Shinohara Y,Nakamura-Hasegawa K,Hashiguchi C,Takeuchi N,Imafuji T,Sculean A,Noguchi K Clinical oral investigations OBJECTIVES:The potential additive effect of an enamel matrix derivative (EMD) to a subepithelial connective tissue graft (CTG) for recession coverage is still controversially discussed. Therefore, the aim of this study was to histologically evaluate the healing of gingival recessions treated with coronally advanced flap (CAF) and CTG with or without EMD in dogs. MATERIALS AND METHODS:Gingival recession defects (5 mm wide and 7 mm deep) were surgically created on the labial side of bilateral maxillary canines in 7 dogs. After 8 weeks of plaque accumulation and subsequent 2 weeks of chemical plaque control, the 14 chronic defects were randomized to receive either CAF with CTG (CAF/CTG) or CAF with CTG and EMD (CAF/CTG/EMD). The animals were sacrificed 10 weeks after reconstructive surgery for histologic evaluation. RESULTS:Treatment with CAF/CTG/EMD demonstrated statistically significantly better results in terms of probing pocket depth reduction (P < 0.05) and clinical attachment level gain (P < 0.001). The length of the epithelium was statistically significantly shorter in the CAF/CTG/EMD group than in the CAF/CTG group (1.00 ± 0.75 mm vs. 2.38 ± 1.48 mm, respectively, P < 0.01). Cementum formation was statistically significantly greater in the CAF/CTG/EMD group than following treatment with the CAF/CTG group (3.20 ± 0.89 mm vs. 1.88 ± 1.58 mm, respectively, P < 0.01). The CAF/CTG/EMD group showed statistically significantly greater complete periodontal regeneration (i.e., new cementum, new periodontal ligament, and new bone) than treatment with CAF/CTG (0.54 ± 0.73 mm vs. 0.07 ± 0.27 mm, respectively, P < 0.05). CONCLUSION:Within their limits, the present findings indicate that the additional use of EMD in conjunction with CAF + CTG favors periodontal regeneration in gingival recession defects. CLINICAL RELEVANCE:The present findings support the use of EMD combined with CTG and CAF for promoting periodontal regeneration in isolated gingival recession defects. 10.1007/s00784-018-2750-1
    The effect of CPNE7 on periodontal regeneration. Choung Han-Wool,Lee Dong-Seol,Park Yeoung-Hyun,Lee Yoon Seon,Bai Shengfeng,Yoo Su-Hyang,Lee Jong-Ho,You Hyung-Keun,Park Joo-Cheol Connective tissue research : Preameloblast-conditioned medium (PA-CM), as a mixture of dental epithelium-derived factors, has been reported to regenerate dentin and periodontal tissues and . The aim of this study was to investigate the biological effect of Cpne7 on the proliferation, migration, and cementoblast differentiation of periodontal cells , and on the regeneration of periodontal tissue using periodontal defect model with canine . : The effect of Cpne7 on cell proliferation, migration, and cementoblast differentiation of periodontal cells were evaluated . A periodontal defect canine model was designed and the defects were divided into five groups: Group 1: No treatment (negative control), Group 2: Collagen carrier only, Group 3: PA-CM with collagen carrier (positive control), Group 4: PA-CM + CPNE7 Antibody (Ab) with collagen carrier, and Group 5: recombinant CPNE7 (rCPNE7) protein with collagen carrier. : Cpne7 was expressed in HERS cells and periodontal ligament (PDL) fibers. By real-time PCR, Cpne7 increased expression of Cap compared to the control. In the periodontal defect canine model, rCPNE7 or PA-CM regenerated periodontal complex, and the arrangement of the newly formed PDL-like fibers were perpendicular to the newly formed cementum and alveolar bone like Sharpey's fibers in natural teeth, while PA-CM + CPNE7 Ab showed irregular arrangement of the newly formed PDL-like fibers compared to the rCPNE7 or PA-CM group. : These findings suggest that Cpne7 may have a functional role in periodontal regeneration by supporting periodontal cell attachment to cementum and facilitating physiological arrangement of PDL fibers. 10.1080/03008207.2019.1574776
    Self-assembling polymeric dendritic peptide as functional osteogenic matrix for periodontal regeneration scaffolds-an in vitro study. Das Eva C,Dhawan Sameer,Babu Jisha,Anil Kumar P R,Kumary Thrikkovil Variathu,Haridas V,Komath Manoj Journal of periodontal research OBJECTIVE:Regeneration of periodontal defects is challenging as it necessitates the formation of complex tissue structure with cementum, periodontal ligament, and alveolar bone. Rather than the conventional barrier membranes, scaffolds mimicking extracellular matrix (ECM) can achieve faster healing as they promote migration, adhesion, and differentiation of native progenitor cells. This work explores the possibility of a functional osteogenic matrix based on self-assembling peptide appended dendritic polydiacetylene in regenerating diseased periodontia. METHOD:The amino acid lysine was appended onto a diacetylene core, which was converted to a polymeric dendritic lysine matrix (Lys-PDA) through photopolymerization. This bioactive matrix was evaluated in vitro for the viability, adhesion, spreading, and differentiation of cultured human periodontal ligament (hPDL) progenitor cells. Its osteogenic differentiation was analysed by histologic staining and expression of osteogenic markers (alkaline phosphatase and Osteonectin). Electrospun polycaprolactone (PCL) mat, a candidate barrier material, was fabricated and functionalized with Lys-PDA matrix, and the cell viability, adhesion, and spreading of hPDL cells were evaluated. RESULTS:The dendritic Lys-PDA matrix well supported the hPDL cell growth and differentiation. The cells were viable and showed good cytoskeletal organization. Early expression of osteogenic markers and mineralization was noted in vitro in the presence of Lys-PDA matrix. The electrospun PCL mat functionalized with Lys-PDA maintained the viability, morphology, and spreading of the hPDL cells. SIGNIFICANCE:The ECM mimetic dendritic peptide matrices are capable of hosting and differentiating cells which can lead to the regeneration of periodontal tissue architecture. They could be used in conjunction with barrier membranes for better results. 10.1111/jre.12647