RESUMO
The decellularized extracellular matrix (dECM) is capable of promoting stem cell proliferation, migration, adhesion, and differentiation. It is a promising biomaterial for application and clinical translation in the field of periodontal tissue engineering as it most effectively preserves the complex array of ECM components as they are in native tissue, providing ideal cues for regeneration and repair of damaged periodontal tissue. dECMs of different origins have different advantages and characteristics in promoting the regeneration of periodontal tissue. dECM can be used directly or dissolved in liquid for better flowability. Multiple ways were developed to improve the mechanical strength of dECM, such as functionalized scaffolds with cells that harvest scaffold-supported dECM through decellularization or crosslinked soluble dECM that can form injectable hydrogels for periodontal tissue repair. dECM has found recent success in many periodontal regeneration and repair therapies. This review focuses on the repairing effect of dECM in periodontal tissue engineering, with variations in cell/tissue sources, and specifically discusses the future trend of periodontal regeneration and the future role of soluble dECM in entire periodontal tissue regeneration.
Assuntos
Matriz Extracelular Descelularizada , Matriz Extracelular , Periodonto , Regeneração , Matriz Extracelular/metabolismo , Hidrogéis/farmacologia , Engenharia Tecidual , Periodonto/citologia , Periodonto/fisiologiaRESUMO
Teeth and the surrounding periodontal tissues are affected by many pathologies that compromise their integrity and significantly affect life quality. The study of the main dental tissues, the dental pulp and periodontium, is made arduous by their close association with highly mineralized tissues (dentin, cementum, and alveolar bone). Here we describe a protocol to isolate all cells composing human dental pulp and periodontium for single-cell RNA sequencing analysis. For complete details on the use and execution of this protocol, please refer to Pagella et al. (2021).
Assuntos
Separação Celular/métodos , Polpa Dentária/citologia , Periodonto/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Dente/citologiaRESUMO
Periodontitis is a chronic inflammatory disease characterized by loss of attachment and destruction of the periodontium. Decellularized sheet, as an advanced tissue regeneration engineering biomaterial, has been researched and applied in many fields, but its effects on periodontal regeneration remain unclear. In this study, the biological properties of decellularized human periodontal ligament cell (dHPDLC) sheets were evaluated in vitro. Polycaprolactone/gelatin (PCL/GE) nanofibers were fabricated as a carrier to enhance the mechanical strength of the dHPDLC sheet. 15-deoxy-[Formula: see text]-prostaglandin J2 (15d-PGJ2) nanoparticles were added for anti-inflammation and regeneration improvement. For in vivo analysis, dHPDLC sheets combined with 15d-PGJ2 nanoparticles, with or without PCL/GE, were implanted into rat periodontal defects. The periodontal regeneration effects were identified by microcomputed tomography (micro-CT) and histological staining, and immunohistochemistry. The results revealed that DNA content was reduced by 96.6%. The hepatocyte growth factor, vascular endothelial growth factor, and basic fibroblast growth factor were preserved but reduced. The expressions or distribution of collagen I and fibronectin were similar in dHPDLC and nondecellularized cell sheets. The dHPDLC sheets maintained the intact structure of the extracellular matrix. It could be recellularized by allogeneic human periodontal stem ligament cells and retain osteoinductive potential. Newly formed bone, cementum, and PDL were observed in dHPDLC sheets combined with 15d-PGJ2 groups, with or without PCL/GE nanofibers, for four weeks post-operation in vivo. Bringing together all these points, this new construct of dHPDLC sheets can be a potential candidate for periodontal regeneration in an inflammatory environment of the oral cavity.
Assuntos
Matriz Extracelular Descelularizada , Nanopartículas/química , Ligamento Periodontal/citologia , Periodonto , Prostaglandina D2/análogos & derivados , Animais , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Regeneração Tecidual Guiada Periodontal , Masculino , Periodonto/citologia , Periodonto/efeitos dos fármacos , Prostaglandina D2/química , Prostaglandina D2/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Thermosensitive hydrogels could function as scaffolds and delivery vehicle of natural flavonoids. The current study aimed to investigate effects of chitosan/collagen ratios on properties of thermosensitive beta-glycerophosphate (bGP) chitosan/collagen hydrogels as delivery vehicle of quercetin and then examined effects of quercetin-hydrogels on growth and cell viability of human periodontal ligament stem cells (hPDLSCs). Microstructure and physical, mechanical and antioxidant properties and quercetin release profiles of the hydrogels were investigated. Fourier transform infrared spectroscopy and X-ray powder diffraction analyses were performed to examine gelation process of the hydrogels. Antioxidant assays were conducted to measure antioxidant capacity of quercetin-hydrogels. It was found that bGP-chitosan/collagen hydrogels exhibited porous structures with interconnected pore architecture and could sustain quercetin release. Chitosan content improved well defined porous structure, increased porosity of the hydrogels and decreased releasing rate of quercetin from the hydrogels. The quercetin-bGP-2:1 (wt/wt) chitosan/collagen hydrogels exhibited antioxidant capacity and were able to promote growth of hPDLSCs in a dose dependent manner. In conclusion, the thermosensitive quercetin-bGP-2:1 (wt/wt) chitosan/collagen hydrogel demonstrated optimal properties of scaffolds for bone tissue engineering and sustained release of natural flavonoids. Incorporating quercetin in the chitosan/collagen hydrogel enhanced bioactive microenvironment that supported stem cell encapsulation.
Assuntos
Quitosana/farmacologia , Colágeno/farmacologia , Hidrogéis/farmacologia , Ligamento Periodontal/citologia , Periodonto/citologia , Quercetina/farmacologia , Células-Tronco/efeitos dos fármacos , Antioxidantes/química , Materiais Biocompatíveis , Regeneração Óssea , Sobrevivência Celular , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres , Humanos , Ligamento Periodontal/efeitos dos fármacos , Periodonto/efeitos dos fármacos , Porosidade , Engenharia TecidualRESUMO
Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.
Assuntos
Materiais Biocompatíveis , Meios de Cultura/farmacologia , Osteoclastos/citologia , Periodonto/citologia , Engenharia Tecidual/métodos , Adulto , Animais , Transplante de Medula Óssea , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Teste de Materiais , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Adulto JovemRESUMO
The aim of the study was to clarify whether orthodontic forces and periodontitis interact with respect to the anti-apoptotic molecules superoxide dismutase 2 (SOD2) and baculoviral IAP repeat-containing protein 3 (BIRC3). SOD2, BIRC3, and the apoptotic markers caspases 3 (CASP3) and 9 (CASP9) were analyzed in gingiva from periodontally healthy and periodontitis subjects by real-time PCR and immunohistochemistry. SOD2 and BIRC3 were also studied in gingiva from rats with experimental periodontitis and/or orthodontic tooth movement. Additionally, SOD2 and BIRC3 levels were examined in human periodontal fibroblasts incubated with Fusobacterium nucleatum and/or subjected to mechanical forces. Gingiva from periodontitis patients showed significantly higher SOD2, BIRC3, CASP3, and CASP9 levels than periodontally healthy gingiva. SOD2 and BIRC3 expressions were also significantly increased in the gingiva from rats with experimental periodontitis, but the upregulation of both molecules was significantly diminished in the concomitant presence of orthodontic tooth movement. In vitro, SOD2 and BIRC3 levels were significantly increased by F. nucleatum, but this stimulatory effect was also significantly inhibited by mechanical forces. Our study suggests that SOD2 and BIRC3 are produced in periodontal infection as a protective mechanism against exaggerated apoptosis. In the concomitant presence of orthodontic forces, this protective anti-apoptotic mechanism may get lost.
Assuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Regulação da Expressão Gênica , Ligamento Periodontal/metabolismo , Periodonto/metabolismo , Superóxido Dismutase/genética , Animais , Apoptose/genética , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fusobacterium nucleatum/fisiologia , Gengiva/citologia , Gengiva/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Ligamento Periodontal/citologia , Ligamento Periodontal/microbiologia , Periodonto/citologia , Periodonto/microbiologia , Ratos , Superóxido Dismutase/metabolismoRESUMO
Human periodontal ligament stem cells (hPDLSCs) sheets play an important role in periodontal tissue engineering. Low-intensity pulsed ultrasound (LIPUS) has been reported as an effective stimulus to regulate cell biological behavior. The present study aims to explore the potential of LIPUS to promote the formation and function of hPDLSC sheets (hPDLSCSs). Hematoxylin-eosin (H&E) staining, western blot, real-time PCR, alkaline phosphatase (ALP), and alizarin red staining were used to evaluate the formation and osteogenic effect of LIPUS on hPDLSCSs in vitro. Hydroxyapatite with or without hPDLSCSs was transplanted in the subcutaneous pockets on the back of nude mice and histological analysis was performed. H&E staining showed increased synthesis of extracellular matrix (ECM) and real-time PCR detected a significant increase in ECM-related genes after LIPUS treatment. In addition, LIPUS could promote the expression of osteogenic differentiation-related genes and proteins. ALP and alizarin red staining also found LIPUS enhanced the osteogenesis of hPDLSCSs. After transplantation in vivo, more dense collagen fibers similar to periodontal ligament were regenerated. Collectively, these results indicate that LIPUS not only promotes the formation and osteogenic differentiation of hPDLSCSs but also is a potential treatment strategy for periodontal tissue engineering.
Assuntos
Ligamento Periodontal/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Células Cultivadas , Humanos , Camundongos , Camundongos Nus , Osteogênese/efeitos da radiação , Periodonto/citologia , Transplante de Células-Tronco , Células-Tronco/efeitos da radiação , Engenharia Tecidual , Ondas UltrassônicasRESUMO
Destruction of the alveolar bone in the jaws can occur due to periodontitis, trauma or following tumor resection. Common reconstructive therapy can include the use of bone grafts with limited predictability and efficacy. Romosozumab, approved by the FDA in 2019, is a humanized sclerostin-neutralizing antibody (Scl-Ab) indicated in postmenopausal women with osteoporosis at high risk for fracture. Preclinical models show that Scl-Ab administration preserves bone volume during periodontal disease, repairs bone defects surrounding dental implants, and reverses alveolar bone loss following extraction socket remodeling. To date, there are no studies evaluating Scl-Ab to repair osseous defects around teeth or to identify the efficacy of locally-delivered Scl-Ab for targeted drug delivery. In this investigation, the use of systemically-delivered versus low dose locally-delivered Scl-Ab via poly(lactic-co-glycolic) acid (PLGA) microspheres (MSs) was compared at experimentally-created alveolar bone defects in rats. Systemic Scl-Ab administration improved bone regeneration and tended to increase cementogenesis measured by histology and microcomputed tomography, while Scl-Ab delivered by MSs did not result in enhancements in bone or cemental repair compared to MSs alone or control. In conclusion, systemic administration of Scl-Ab promotes bone and cemental regeneration while local, low dose delivery did not heal periodontal osseous defects in this study.
Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Anticorpos Monoclonais/administração & dosagem , Proteínas Morfogenéticas Ósseas/imunologia , Marcadores Genéticos/imunologia , Microesferas , Periodonto/citologia , Regeneração , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/patologia , Animais , Masculino , Periodonto/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
OBJECTIVE: To elucidate the role of bone marrow-mesenchymal stem cells (BM-MSCs) on the structure of periodontal tissues in carbimazole (antithyroid drug) treated rats at different durations. DESIGN: 28 albino rats were divided into: Group I: received distilled water. Group II: received therapeutic dose of carbimazole. Group III: received carbimazole then single injection of BM-MSCs by the end of 3rd week. Group IV: received carbimazole and single injection of BM-MSCs at the beginning of the experiment. Specimens were examined by light microscope. New collagen and ß-catenin-immunoreactivity area% were assessed histomorphometrically, and statistically using ANOVA test. RESULTS: Histological examination revealed normal periodontal tissues structure in Groups I & IV. Group II showed disorganized periodontal ligament fibers and different stainability of cementum and alveolar bone. Group III illustrated dense periodontal ligament fibers, normal stainability of cementum and most of alveolar bone. Masson's trichrome results of Groups I & IV illustrated large areas of new collagen in periodontal ligament, old collagen in cementum and intermingled old and new collagen in alveolar bone. Group II showed old collagen. Group III revealed only new collagen. ß-catenin-immunoreactivity was strong in Groups I & IV, negative in Group II and moderate in Group III. Statistically, Group III showed highest mean of new collagen area% followed by Groups I, IV and II respectively. Highest mean of ß-catenin-immunoreactivity area% was for Group I followed by Groups IV, III and II respectively. CONCLUSIONS: Carbimazole has damaging effects and BM-MSCs are capable to mend these destructive outcomes in time dependent manner.
Assuntos
Carbimazol/efeitos adversos , Células-Tronco Mesenquimais/citologia , Periodonto/citologia , Animais , Células da Medula Óssea , Transplante de Células-Tronco Mesenquimais , Ligamento Periodontal , RatosRESUMO
Bone defects due to trauma or diseases still pose a clinical challenge to be resolved in the current tissue engineering approaches. As an alternative to traditional methods to restore bone defects, such as autografts, bone tissue engineering aims to achieve new bone formation via novel biomaterials used in combination with multipotent stem cells and bioactive molecules. Mesenchymal stem cells (MSCs) can be successfully isolated from various dental tissues at different stages of development including dental pulp, apical papilla, dental follicle, tooth germ, deciduous teeth, periodontal ligament and gingiva. A wide range of biomaterials including polymers, ceramics and composites have been investigated for their potential as an ideal bone scaffold material. This article reviews the properties and the manufacturing methods of biomaterials used in bone tissue engineering, and provides an overview of bone tissue regeneration approaches of scaffold and dental stem cell combinations as well as their limitations.
Assuntos
Regeneração Óssea , Polpa Dentária/citologia , Periodonto/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Germe de Dente/citologia , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.
Assuntos
Periodonto/fisiologia , Regeneração/fisiologia , Células 3T3 , Animais , Células Cultivadas , Cemento Dentário/citologia , Cemento Dentário/fisiologia , Cemento Dentário/transplante , Feminino , Regeneração Tecidual Guiada Periodontal/métodos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteoblastos/transplante , Ligamento Periodontal/citologia , Ligamento Periodontal/fisiologia , Ligamento Periodontal/transplante , Periodonto/anatomia & histologia , Periodonto/citologia , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodos , Microtomografia por Raio-XRESUMO
Healing and regeneration of periodontium are considered as a complex physiological process. Therefore, treatments need to be addressed with highly effective components modulating the multiple pathways. In this study, exopolysaccharide (EPS) produced by Weissella cibaria EIR/P2, was partially purified from the culture supernatant and subjected to characterization within the aim of evaluating its potential for periodontal regeneration. High-Performance Liquid Chromatography analysis revealed a single-peak corresponding to the glucose which identified the EPS as dextran. Fourier transform-infrared spectra were also displayed characteristic peaks for polysaccharides. According to the results of gel permeation/size exclusion-chromatography, the molecular mass was determined to be 8 × 106 Da. To clarify its anti-bacterial activity on Streptococcus mutans, effects on viability and biofilm formation was evaluated. At 50 mg/mL, dextran exhibited a bactericidal effect with 70% inhibition on biofilm formation. Besides, dose-dependent antioxidant effects were also detected. The efficacy of dextran in enhancing the viability of human periodontal ligament fibroblast cells (hPDLFCs) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay, and an increase was observed in the viability of hPDLFCs. In conclusion, dextran derived from W. cibaria can be potentially used as a multi-functional bioactive polymer in the design of new therapeutic strategies to promote healing and regeneration of periodontium.
Assuntos
Materiais Biocompatíveis/farmacologia , Periodonto/citologia , Polissacarídeos Bacterianos/farmacologia , Weissella/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Materiais Biocompatíveis/isolamento & purificação , Biofilmes/efeitos dos fármacos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Dextranos/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Periodonto/efeitos dos fármacos , Polissacarídeos Bacterianos/isolamento & purificação , Regeneração/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/crescimento & desenvolvimentoRESUMO
Ultra-fine grained biodegradable Mg-based Mg1Zn1Mn0.3â¯Zr - HA and Mg4Y5.5Dy0.5â¯Zr - 45S5 Bioglass composites have shown great medical potential. Two types of these Mg-based biomaterials subjected to different treatments were tested and as shown earlier they are biocompatible. The aim of the study is to determine how much culture media incubated with these ultra-fine trained Mg-based composites can cause inflammatory reactions and /or periodontal cell death. The incubation of composites in the medium releases metal ions into the solution. It can be assumed that this process is permanent and also occurs in the human body. The results have shown that the effect of proinflammatory IL-6 and TNF- cytokines results in the strongest production of the acute phase proteins in the first day on the Mg1Zn1Mn0.3â¯Zr-5â¯wt.% HA-1â¯wt. % Ag HF-treated biocomposite after immersion for 2â¯h in 40 % HF and then the fastest decrease in these processes on the third day. In turn, the inflammatory process induced on the Mg1Zn1Mn0.3â¯Zr-5â¯wt.% HA-1â¯wt. % Ag biomaterial, in BAX / BCL ratio assessment, is the strongest on the third day and maintains a significantly high level on the following day, which, at the same time, confirms its persistence and development. In addition, these results confirm the successively generated necrotic processes. Ions can induce inflammatory reactions, which in the case of the implant may take a long time, which results in the loss of the implant. Even if the material is biocompatible in rapid in-vitro tests, it can induce inflammation in the body after some time due to the release of ions. Not every treatment improves the material's properties in terms of subsequent safety.
Assuntos
Materiais Biocompatíveis/farmacologia , Compostos de Magnésio/farmacologia , Magnésio/farmacologia , Teste de Materiais/métodos , Periodonto/efeitos dos fármacos , Células Cultivadas , Cerâmica/farmacologia , Vidro , Humanos , Inflamação/induzido quimicamente , Interleucina-6/biossíntese , Osteoblastos/efeitos dos fármacos , Periodonto/citologia , Próteses e Implantes , Propriedades de Superfície , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Ferritin, an iron-binding protein, is composed of two subunits, a heavy chain and a light chain. It regulates many biological functions, such as proliferation, angiogenesis, and immunosuppression. The objective of this study was to determine the expression and distribution of ferritin in the periodontal tissuesof primates.First, we assessed the expression of ferritin in primary cultured cells isolated from human periodontal tissues using the polymerase chain reaction and immunofluorescent staining. Second, we investigated the expression and distribution of ferritin in the periodontal tissues of Macaca fascicularis, human gingival tissues, and human gingival carcinoma tissues using immunohistochemistry.Both protein and mRNA of ferritin were constitutively present in human primary cultured cells, including those from the dental apical papilla, periodontal ligament, dental pulp, and gingival epithelium, as well as gingival fibroblasts. In M. fascicularistissues, the immunohistochemical staining was particularly strong in blood vessel and mineralizing areas of the dental pulp and periodontal ligament. Ferritin heavy chain exhibited specific immunopositivity in in the stratum basale of the epithelium in human gingival tissue and strong immunostaining was found in peripheral regions of gingival carcinoma sites. Ferritin is constitutivelypresent andwidelydistributed in the periodontal tissues of primates. Ferritin may play roles in epithelial proliferation, vascular angiogenesis, and mineralization in these tissues.
Assuntos
Ferritinas/metabolismo , Periodonto/metabolismo , Animais , Células Cultivadas , Gengiva/metabolismo , Neoplasias Gengivais/metabolismo , Humanos , Macaca fascicularis , Masculino , Periodonto/citologiaRESUMO
Periodontal ligament stem cells (PDLSCs) can repair alveolar bone defects in periodontitis in a microenvironment context-dependent manner. This study aimed to determine whether different extracellular matrices (ECMs) exert diverse effects on osteogenic differentiation of PDLSCs and accurately control alveolar bone defect repair. Methods: The characteristics of PDLSCs and bone marrow mesenchymal stem cells (BMSCs) with respect to surface markers and multi-differentiation ability were determined. Then, we prepared periodontal ligament cells (PDLCs)-derived and bone marrow cells (BMCs)-derived ECMs (P-ECM and B-ECM) and the related decellularized ECMs (dECMs). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and protein mass spectrometry were used to distinguish the ECMs. The expression of Type IV collagen A2 (COL4A2) in the ECMs was inhibited by siRNA or activated by lentiviral transduction of relevant cells. The stemness, proliferation, and differentiation of PDLSCs were determined in vitro in different dECMs. For the in vivo analysis, different dECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted into immunocompromised mice or in defects in rat alveolar bone. The repair effects were identified by histological or immunohistochemical staining and micro-CT. Results: B-dECM exhibited more compact fibers than P-dECM, as revealed by TEM, SEM, and AFM. Protein mass spectrometry showed that COL4A2 was significantly increased in B-dECM compared with P-dECM. PDLSCs displayed stronger proliferation, stemness, and osteogenic differentiation ability when cultured on B-dECM than P-dECM. Interestingly, B-dECM enhanced the osteogenic differentiation of PDLSCs to a greater extent than P-dECM both in vitro and in vivo, whereas downregulation of COL4A2 in B-dECM showed the opposite results. Furthermore, the classical Wnt/ß-catenin pathway was found to play an important role in the negative regulation of osteogenesis through COL4A2, confirmed by experiments with the Wnt inhibitor DKK-1 and the Wnt activator Wnt3a. Conclusion: These findings indicate that COL4A2 in the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of the Wnt/ß-catenin pathway, which can be used as a potential therapeutic strategy to repair bone defects.
Assuntos
Colágeno Tipo IV/metabolismo , Osteogênese , Periodontite/metabolismo , Animais , Colágeno Tipo IV/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais , Camundongos , Periodontite/genética , Periodontite/fisiopatologia , Periodonto/citologia , Periodonto/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Caroteno/genética , beta Caroteno/metabolismoRESUMO
Periodontium is consisted of root cementum, bone lining the tooth socket, gingiva facing the tooth, and periodontal ligament (PDL). Its primary functions are support of the tooth and protection of tooth, nerve, and blood vessels from injury by mechanical loading. Severe periodontitis induces the destruction of periodontium and results in a significant cause of tooth loss among adults. Unfortunately, conventional therapies such as scaling and root planning are often only palliative. Therefore, the ultimate goal of the treatment for periodontitis is to restore disrupted periodontium to its original shape and function. Tissue engineering refers to the process of combining cells, scaffolds, and signaling molecules for the production of functional tissues to restore, maintain, and improve damaged organs. The discovery of periodontal ligament stem cells (PDLSCs) highlighted the possibility for development of tissue engineering technology-based therapeutics for disrupted periodontium. PDLSCs are a kind of somatic stem cells that show potential to differentiate into multiple cell types and undergo robust clonal self-renewal. Therefore, PDLSCs are considered a highly promising stem cell population for regenerative therapy in periodontium; however, their rarity prevents the progression of basic and clinical researches. In this review, we summarize recent research advancement and accumulated information regarding the self-renewal capacity, multipotency, and immunomodulatory effect of PDLSCs, as well as their contribution to repair and regeneration of periodontium and other tissues. We also discuss the possibility of PDLSCs for clinical application of regenerative medicine and provide an outline of the genetic approaches to overcome the issue about the rarity of PDLSCs.
Assuntos
Regeneração Tecidual Guiada Periodontal/métodos , Ligamento Periodontal/citologia , Periodonto/fisiologia , Regeneração/fisiologia , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Adulto , Humanos , Ligamento Periodontal/transplante , Periodonto/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/citologiaRESUMO
Several therapeutic strategies are currently in development for severe periodontitis and other associated chronic inflammatory diseases. Guided tissue regeneration of the periodontium is based on surgical implantation of natural or synthetic polymers conditioned as membranes, injectable biomaterials (hydrogels), or three-dimensional (3D) matrices. Combinations of biomaterials with bioactive factors represent the next generation of regenerative strategy. Cell delivery strategy based on scaffold-cell constructs showed potential in periodontitis treatment. Bioengineering of periodontal tissues using cell sheets and genetically modified stem cells is currently proposed to complete existing (pre)clinical procedures for periodontal regeneration. 3D structures can be built using computer-assisted manufacturing technologies to improve the implant architecture effect on new tissue formation. The aim of this review was to summarize the advantages and drawbacks of biomimetic composite matrices used as biomaterials for periodontal tissue engineering. Their conditioning as two-dimensional or 3D scaffolds using conventional or emerging technologies was also discussed. Further biotechnologies are required for developing novel products tailored to stimulate periodontal regeneration. Additional preclinical studies will be useful to closely investigate the mechanisms and identify specific markers involved in cell-implant interactions, envisaging further clinical tests. Future therapeutic protocols will be developed based on these novel procedures and techniques.
Assuntos
Materiais Biocompatíveis/uso terapêutico , Regeneração Tecidual Guiada Periodontal/métodos , Periodonto/fisiologia , Engenharia Tecidual/métodos , Materiais Biocompatíveis/química , Humanos , Hidrogéis/química , Hidrogéis/uso terapêutico , Ligamento Periodontal/citologia , Ligamento Periodontal/fisiologia , Ligamento Periodontal/transplante , Periodontite/terapia , Periodonto/citologia , Alicerces Teciduais/químicaRESUMO
Periodontitis is a common chronic inflammatory disease that affects tooth-supporting tissues. We engineer a multifunctional periodontal membrane for the guided tissue regeneration of lost periodontal tissues. The major drawback of current periodontal membranes is the lack of tissue regeneration properties. Here, a series of nanofibrous membranes based on poly(ε-caprolactone) with tunable biochemical and biophysical properties were developed for periodontal tissue regeneration. The engineered membranes were surface coated using biomimetic polydopamine to promote the adhesion of therapeutic proteins and cells. We demonstrate successful cellular localization on the surface of the engineered membrane by morphological patterning. Polydopamine accelerates osteogenic differentiation of dental-derived stem cells by promoting hydroxyapatite mineralization. Such multiscale designs can mimic the complex extracellular environment of periodontal tissue and serve as functional tissue constructs for periodontal regeneration. In a periodontal defect model in rats, our engineered periodontal membrane successfully promoted the regeneration of periodontal tissue and bone repair. Altogether, our data demonstrate that our biomimetic membranes have potential as protein/cell delivery platforms for periodontal tissue engineering.
Assuntos
Indóis/química , Células-Tronco Mesenquimais/citologia , Nanofibras/química , Periodonto/citologia , Polímeros/química , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Regeneração Óssea , Células Cultivadas , Humanos , Masculino , Membranas Artificiais , Nanofibras/ultraestrutura , Osteogênese , Poliésteres/química , Ratos , Alicerces Teciduais/químicaRESUMO
Thixotropic clays have favorable properties for tissue regeneration. Hypoxia mimetic agents showed promising results in pre-clinical models for hard and soft tissue regeneration. It is unclear if clays can be used as carrier for hypoxia mimetic agent in a periodontal regenerative setting. Here, we tested the response of human fibroblasts of the periodontal soft tissue to synthetic clay hydrogels and assessed hypoxia mimetic agent release. Cells were cultured on synthetic clay hydrogels (5.00%-0.15%). We assessed viability and differentiation capacity with resazurin-based toxicity assays, MTT staining, Live-Dead staining, and alkaline phosphatase staining. To reveal the response of fibroblasts to hypoxia mimetic agent-loaded clay hydrogels, cells were exposed to clay supplemented with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2. Supernatants from hypoxia mimetic agent-loaded clay hydrogels were harvested and replaced with medium at hour 1, 3, 6, 24, 48, and 72. To reveal the hypoxia mimetic capacity of supernatants, vascular endothelial growth factor production in the fibroblasts was assessed in the culture medium. Our data show that clay did not induce relevant toxic effects in the fibroblasts which remained capable to differentiate into alkaline phosphatase-positive cells at the relevant concentrations. Fibroblasts cultured on clay hydrogel loaded with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2 remained vital, however, no significant increase in vascular endothelial growth factor levels was found in the culture medium. Only dimethyloxalylglycine-loaded clay supernatants taken in the first hours stimulated vascular endothelial growth factor production in fibroblasts. In conclusion no pronounced toxic effects of synthetic clay were observed. Supplementation with dimethyloxalylglycine leads to hypoxia mimetic activity. This pilot study provides first insights into the impact of synthetic clay on periodontal tissue.
Assuntos
Hipóxia Celular/efeitos dos fármacos , Argila/química , Fibroblastos/efeitos dos fármacos , Hidrogéis/química , Periodonto/citologia , Aminoácidos Dicarboxílicos/administração & dosagem , Aminoácidos Dicarboxílicos/farmacologia , Materiais Biocompatíveis/química , Células Cultivadas , Cobalto/administração & dosagem , Cobalto/farmacologia , Desferroxamina/administração & dosagem , Desferroxamina/farmacologia , Sistemas de Liberação de Medicamentos , Fibroblastos/citologia , Humanos , Mimosina/administração & dosagem , Mimosina/farmacologia , Periodonto/efeitos dos fármacos , Alicerces Teciduais/químicaRESUMO
Introduction: Preameloblast-conditioned medium (PA-CM), as a mixture of dental epithelium-derived factors, has been reported to regenerate dentin and periodontal tissues in vitro and in vivo. The aim of this study was to investigate the biological effect of Cpne7 on the proliferation, migration, and cementoblast differentiation of periodontal cells in vitro, and on the regeneration of periodontal tissue using periodontal defect model with canine in vivo. Materials and methods: The effect of Cpne7 on cell proliferation, migration, and cementoblast differentiation of periodontal cells were evaluated in vitro. 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. Results: 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. Conclusion: 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.