Cell sheets of human dental pulp stem cells for future application in bone replacement.

OBJECTIVES: To analyze the potential of human dental pulp stem cells (hDPSCs) for maintaining their undifferentiated status and osteogenic differentiation capacity when arranged in cell sheets (CSs) for future application in bone replacement. MATERIALS AND METHODS: CSs were formed after being induced for 10-15 days by clonogenic medium containing additional vitamin C (20 µg/ml). The cell viability of hDPSC4s in the CSs was followed until 96 h using the Live/Dead® assay. The cells of the CSs were enzymatically dissociated and then compared with the original hDPSC4s. The two cell types were characterized immunophenotypically by flow cytometry using specific mesenchymal stem cell-associated markers (CD105, CD146, CD44, STRO-1, and OCT3/4) and non-associated markers (CD34, CD45, and CD14). Osteogenic differentiation was analyzed with the Alizarin red assay. RESULTS: Living cells were observed until 96 h in the CSs. Both cell types exhibited osteogenic differentiation and expressed the specific undifferentiated MSC-associated markers. Cells spontaneously detached from the CSs attached and proliferated at the bottom of the culture dishes. CONCLUSIONS: Cells in the hDPSC4s cell sheets survived for at least 96 h. Moreover, the cells in the cell sheets retained their stemness and their osteogenic differentiation potential. CLINICAL RELEVANCE: Cell sheets of hDPSCs could be employed as natural tri-dimensional structures for treating bone loss. This technique would be useful particularly for critical bone defects or any type of bone defects in patients carrying diseases that impair bone regeneration, such as diabetes mellitus, medication-related osteonecrosis of the jaw (MRONJ), and osteoporosis.

Tricalcium phosphate-loaded injectable hydrogel as a promising osteogenic and bactericidal teicoplanin-delivery system for osteomyelitis treatment: An in vitro and in vivo investigation.

Osteomyelitis is an inflammation of bone tissue usually caused by pyogenic bacteria. The most recurrent clinical approach consists of bone debridement followed by parenteral administration of antibiotics. However, systemic antibiotic treatment has limitations regarding absorption rate and bioavailability over time. The main challenge of osteomyelitis treatment consists of coupling the persistent infection treatment with the regeneration of the bone debrided. In this work, we developed an injectable drug delivery system based on poloxamer 407 hydrogel containing undoped Mg, Zn-doped tricalcium phosphate (ß-TCP), and teicoplanin, a broad-spectrum antibiotic. We evaluated how the addition of teicoplanin and ß-TCP affected the micellization, gelation, particle size, and surface charge of the hydrogel. Later, we studied the hydrogel degradation and drug delivery kinetics. Finally, the bactericidal, biocompatibility, and osteogenic properties were evaluated through in vitro studies and confirmed by in vivo Wistar rat models. Teicoplanin was found to be encapsulated in the corona portions of the hydrogel micelles, yielding a bigger hydrodynamics radius. The encapsulated teicoplanin showed a sustained release over the evaluated period, enough to trigger antibacterial properties against Gram-positive bacteria. Besides, the formulations were biocompatible and showed bone healing ability and osteogenic properties. Finally, in vivo studies confirmed that the proposed locally injected formulations yielded osteomyelitis treatment with superior outcomes than parenteral administration while promoting bone regeneration. In conclusion, the presented formulations are promising drug delivery systems for osteomyelitis treatment and deserve further technological improvements.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation.

Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-ßI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

Photobiomodulation Therapy on the Palliative Care of Temporomandibular Disorder and Orofacial/Cervical Skull Pain: Preliminary Results from a Randomized Controlled Clinical Trial.

The main symptoms of temporomandibular disorders (TMDs) are pain from musculoskeletal and/or joint-in the head and neck region-and complaints of difficulty in mandibular movements. The photobiomodulation therapy (PBMT) has been reported as a promising treatment in the management of these symptoms. The objective of this research was to assess the effect of PBMT immediately after irradiation on TMDs symptoms under a prospective clinical trial, randomized, triple-blinded, placebo-controlled, and with two parallel arms. According to the RDC/TMD, maximum mouth opening (MMO) and pain in the orofacial/cervical muscles and temporomandibular joint (TMJ) were recorded. One hundred forty-five participants (71 placebo and 74 PBMT experimental) were analyzed after irradiation protocols (sham-PBMT or PBMT) at the orofacial/cervical skull musculature and at the TMJ. The results showed a reduction in the total pain score (p = 0.026), a reduction in the number of painful points (p = 0.013), and an increase in the MMO (p = 0.016) in the PBMT protocol group when compared to the placebo protocol (sham-PBMT). The PBMT was shown to be effective in reducing orofacial/cervical skull pain immediately after the irradiation. It is clinically relevant and should be taken into consideration by professionals who are dedicated to treating this pathology because, in addition to bringing comfort to patients who need dental treatment, it also consists of a low-cost and low technical complexity clinical approach.

Dental tissue-derived stem cell sheet biotechnology for periodontal tissue regeneration: A systematic review.

OBJECTIVE: This study aimed to conduct a systematic review of the use of a cell sheet formed by mesenchymal stem cells derived from dental tissues (ddMSCs) for periodontal tissue regeneration in animal models in comparison with any other type of regenerative treatment. DESIGN: PubMed and Scopus databases were searched for relevant studies up to December 2020. The review was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. RESULTS: Of the 1542 potentially relevant articles initially identified, 33 fulfilled the eligibility criteria and were considered for this review. Even with a wide variety of selected study methods, the periodontal tissue was always regenerated; this indicates the potential for the use of these cell sheets in the future of periodontics. However, this regeneration process is not always complete. CONCLUSION: Despite the implantation, ddMSCs sheets have a great potential to be used in the regeneration of periodontal tissue. More in vivo studies should be conducted using standardized techniques for cell sheet implantation to obtain more robust evidence of the relevance of using this modality of cell therapy for periodontal tissue regeneration.

Effect of human dental pulp stem cell conditioned medium in the dentin-pulp complex regeneration: A pilot in vivo study.

BACKGROUND: Dental trauma, restorative operative procedures and/or caries lesions can expose the dental pulp. Facing this clinical condition, where the maintenance of the dentin-pulp complex vitality is imperative, is challenging in Dentistry. Dental pulp stem cells conditioned medium contains trophic factors that could help in this task. This in vivo pilot study aimed to evaluate the effects of the human dental pulp stem cells conditioned medium on the dental pulp tissue response to vital pulp therapy. MATERIAL AND METHODS: Concentrated conditioned medium was obtained by incubating characterized human dental pulp stem cells with fresh culture medium. Pulp exposures performed at the first upper molars (n = 20) of Wistar rats were directly capped with: MTA or MTA + Conditioned Medium. Four and 8 weeks later, the samples were qualitatively analyzed in histological sections (H&E). RESULTS: When the conditioned medium was associated with MTA, there were a high percentage of samples presenting formation of dentin bridges and small percentage of pulp tissue with inflammatory signs in both experimental times. The conditioned medium improved the organization of the newly formed hard tissue. CONCLUSIONS: The association of dental pulp stem cell conditioned medium with MTA showed beneficial effects on dentin-pulp complex regeneration and has promising potential for studies in regenerative dentistry.

Physical and Biological Properties of a Chitosan Hydrogel Scaffold Associated to Photobiomodulation Therapy for Dental Pulp Regeneration: An In Vitro and In Vivo Study.

BACKGROUND: The regeneration of dental pulp, especially in cases of pulp death of immature teeth, is the goal of the regenerative endodontic procedures (REPs) that are based on tissue engineering principles, consisting of stem cells, growth factors, and scaffolds. Photobiomodulation therapy (PBMT) showed to improve dental pulp regeneration through cell homing approaches in preclinical studies and has been proposed as the fourth element of tissue engineering. However, when a blood clot was used as a scaffold in one of these previous studies, only 30% of success was achieved. The authors pointed out the instability of the blood clot as the regeneration shortcoming. Then, to circumvent this problem, a new scaffold was developed to be applied with the blood clot. The hypothesis of the present study was that an experimental injectable chitosan hydrogel would facilitate the three-dimensional spatial organization of endogenous stem cells in dental pulp regeneration with no interference on the positive influence of PBMT. METHODS: For the in vitro analysis, stem cells from the apical papilla (SCAPs) were characterized by flow cytometry and applied in the chitosan scaffold for evaluating adhesion, migration, and proliferation. For the in vivo analysis, the chitosan scaffold was applied in a rodent orthotopic dental pulp regeneration model under the influence of PBMT (660 nm; power output of 20 mW, beam area of 0.028 cm2, and energy density of 5 J/cm2). RESULTS: The scaffold tested in this study allowed significantly higher viability, proliferation, and migration of SCAPs in vitro when PBMT was applied, especially with the energy density of 5 J/cm2. These results were in consonance to those of the in vivo data, where pulp-like tissue formation was observed inside the root canal. CONCLUSION: Chitosan hydrogel when applied with a blood clot and PBMT could in the future improve previous results of dental pulp regeneration through cell homing approaches.

Successful application of antimicrobial photodynamic and photobiomodulation therapies for controlling osteoradionecrosis and xerostomia after laryngeal carcinoma treatment: A case report of full oral rehabilitation.

We present a clinical case where a conservative treatment based on photonics [antimicrobial Photodynamic Therapy (aPDT) associated to Photobiomodulation therapy (PBMT)] of a patient with osteoradionecrosis (ORN) due to radiotherapy treatment of a laryngeal cancer. As a result of this combined treatment the ORN was controlled (e.g. the necrosis, infection and suppuration disappeared). Moreover, the symptoms reported by the patient (pain and xerostomia) also diminished along with the repair of oral mucosa. In future cases this combined therapy (e.g. PBM therapy and the aPDT) will be further examined.

The effect of three different pulp capping cements on mineralization of dental pulp stem cells.

This study evaluated the osteogenic differentiation of human dental pulp stem cells in response to substances released by the pulp capping agents, Biodentine (BD), mineral trioxide aggregate (MTA) and two-paste calcium hydroxide cement (CHC), along with their physicochemical characteristics. The dimensional stability test showed that of the materials studied, only BD met the standards recommended by the International Organization for Standardization (ISO) for pulp capping materials and thus can be used safely. In the chemical tests, BD was the most stable material. In the Alizarin red S test, BD formed the higher amount of mineralized nodules in the mineralizing medium and also formed mineralized nodules in a non-mineralizing medium. BD releases substances that can significantly induce formation of the human dental pulp stem cell-mineralized extracellular matrix, with physicochemical characteristics that are more conducive to pulp repair than those of MTA and CHC.

Effect of titania addition and sintering temperature on the microstructure, optical, mechanical and biological properties of the Y-TZP/TiO2 composite.

OBJECTIVE: The aims of this study were: 1) to evaluate the effect of sintering temperature on microstructure, density and flexural strength of a 3Y-TZP/TiO2 composite containing 12.5 wt% of TiO2 compared to 3Y-TZP specimens (control); 2) to compare 3Y-TZP with the experimental 3Y-TZP/TiO2 composite, both sintered at 1400 °C, with respect to the following parameters: optical properties, characteristic strength, Weibull modulus, fatigue behavior, induction of osteoblasts proliferation and differentiation (mineralization nodules formation). METHODS: The 3Y-TZP and 3Y-TZP/TiO2 powders were uniaxially pressed and sintered at 1200 °C, 1300 °C, 1400 °C or 1500 °C for one hour in a furnace. The microstructural analysis consisted of X-ray diffraction and scanning electron microscopy. The density was measured by the Archimedes' principle and the flexural strength was obtained by the biaxial flexure test. The optical properties were measured using a spectrophotometer operating in the visible light wavelength range. The step-stress accelerated life testing was performed by the pneumatic mechanical cycler and the biological behavior achieved by using osteoblast-like cells (Osteo-1 cell line). RESULTS: Tetragonal zirconia was identified in all groups and cubic zirconia was identified only at 3Y-TZP group. The addition of TiO2 decreased the values of density and flexural strength of the composite 3Y-TZP/TiO2 in relation to 3Y-TZP regardless of the sintering temperature. The color difference between the two materials was not significant regarding L*a*b* parameters. The composite showed higher probability of failure, and induced higher proliferation and differentiation than control. SIGNIFICANCE: The composite developed have good aesthetic and biologics properties. However, its microstructure and mechanical properties need to be improved for future dental implant applications.

Photobiomodulation as adjunctive therapy for guided bone regeneration. A microCT study in osteoporotic rat model.

Regeneration of diseased bone is challenging. Guided bone regeneration (GBR) has been applied to favor the bone repair. Photobiomodulation (PBM) is also a recognized therapy able to improve bone repair in healthy and diseased individuals. Thus, with the hypothesis that PBM therapy could improve the GBR of diseased bone, this study evaluated the effect of PBM as adjunctive therapy to GBR in osteoporotic rats. Osteoporosis was induced in rats using the oophorectomy model. Then, 5-mm calvaria bone defects were created and treated according to the experimental groups, as follows: with no further treatment (Control); conventional GBR (Membrane), GBR and PBM applied with 3 s, 4 J/cm2 and 0.12 J per point (PBM-1) and GBR and PBM applied with 10s, 14 J/cm2, 0.4 J per point (PBM-2). PBM therapy (808 nm, 40 mW, 1.42 W/cm2) was applied immediately, 48 and 96 h postoperatively. Four and eight weeks later, the samples were harvested and processed for micro-computerized tomography (Micro CT). Data were statistically compared (p < 0.05). From 4 to 8 weeks mostly significant changes were observed in the PBM groups. The bone volume fraction and number of trabeculae of the PBM groups, especially the PBM-1, were significantly higher than those of Control (p < 0.0001). The values of thickness and separation of the trabeculae and structural model index of the PBM groups were significantly smaller than Control (p < 0.0001). The connectivity density was significantly higher on Membrane and PBM groups than Control (p < 0.0004). The application of PBM as adjunctive therapy to GBR results in enhanced bone formation and maturation in comparison to the conventional GBR in the regeneration of lesions of osteoporotic bone in rats. Overviewing the challenges that face bone regeneration in patients with osteoporosis, our findings open new perspectives on the treatment of bone defects under osteoporotic conditions.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

Abstract Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-βI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

Angiogenic properties of dental pulp stem cells conditioned medium on endothelial cells in vitro and in rodent orthotopic dental pulp regeneration.

OBJECTIVES: To evaluate the effect of SHED-CM on the proliferation, differentiation, migration ability, cell death, gene expression and production of VEGF of HUVEC in vitro and in a rodent orthotopic dental pulp regeneration. METHODS: Three culture media [M199, DMEM/Ham's F12 and DMEM/Ham's F12 conditioned by SHEDs] were used as experimental groups. SHED-CM was prepared maintaining confluent cells in culture without serum for 3 days. The proliferation and cell death marker of HUVECs were assessed using flow cytometry. The capacity of formation of vascular-like structures was analyzed in cells grown over Matrigel® in hypoxic condition. HUVECs migration was followed using the scratch test. VEGF-A expression in HUVECs was assessed using real time RT-qPCR; and VEGF synthesis with ELISA test. SHED-CM was also applied in rodent ortotopic model of dental pulp regeneration in rats. The formed tissue was submitted to histological and immunohistochemical analyses. RESULTS: SHED-CM promoted significantly lower expression of 7AAD in HUVECs; whereas the expression of the Ki67 was similar in all groups. The vascular-like structures were observed in all groups. Migration of SHED-CM group was faster than DMEM/Ham's F12. SHED-CM induced similar expression of VEGF-A than M199, and higher than DMEM/Ham's F12. SHED-CM induced significantly higher VEGF synthesis than other media. SHED-CM induced formation of a vascularized connective tissue inside the root canal. CONCLUSION: The study showed that SHEDs release angiogenic and cytoprotective factors, which are of great importance for tissue engineering. CLINICAL SIGNIFICANCE: SHED-CM could be an option to the use of stem cells in tissue engineering.

Hemolasertherapy: A Novel Procedure for Gingival Papilla Regeneration-Case Report.

BACKGROUND: Interdental papilla is of major importance to patients' orofacial aesthetics, especially regarding anterior teeth as part of the smile's harmony. Loss of gingival tissue, which constitutes interdental papilla, forms what in odontology is called black spaces. This loss, besides affecting the smile's aesthetics, also provokes phonetic and functional damage. OBJECTIVE: The objective of the authors is to present the result of three clinical cases treated with an innovative technique called hemolasertherapy, which stimulates growth of gingival papilla and thus permanently fills in the black spaces. METHODS: The photobiomodulation therapy (PBMT) used a 660 nm diode laser (Laser Duo, MMO-São Carlos, SP, Brazil), punctual, contact mode in two steps: before the bleeding (first PBMT) and immediately after bleeding (second PBMT). Parameters used were power output: 100 mW, CW; diameter tip: 5 mm; spot area: 0.19 cm2; irradiation exposure time per point: 20 sec; 14 points per daily session; total of 2 sessions, with a 1-week interval; E: 2 J per point; E: per daily session, 28 J; irradiance per point: 0.52 W/cm2; fluence per point: 10.4 J/cm2. Total in two daily sessions: total energy: 56 J; total fluence: 294.75 J/cm, 560 sec total time. An in vitro preliminary study was simultaneously carried out to demonstrate what could happen at cellular level in hemotherapy clinical cases associated with PBMT laser application. RESULTS: This initial study demonstrated that the blood clot originated from the bleeding provoked in the gingival area is rich in mesenchymal stem cells. PBMT enables preservation, viability, and further differentiation, stimulating the return of gingival stem cells, which would support their survival and differentiation in the blood clot, thus favoring interdental papilla regeneration. CONCLUSIONS: Follow-up was done for a time span of 4-5 years and considered excellent with regard to papilla preservation.

Photobiomodulation of Dental Derived Mesenchymal Stem Cells: A Systematic Review.

OBJECTIVE: This study aimed to conduct a systematic review of the literature published from 2000 to August 2015, to investigate the effect of photobiomodulation (PBM) therapy on dentoalveolar-derived mesenchymal stem cells (ddMSCs), assessing whether a clear conclusion can be reached from the data presented. BACKGROUND DATA: Systematic reviews provide the best evidence on the effectiveness of a procedure and permit investigation of factors that may influence the performance of a method. To the best of our knowledge, no previous systematic review has evaluated the effects of PBM only on ddMSCs. METHODS: The search was conducted in PubMed /MEDLINE®, Scopus and Web of Science databases, and reported according to the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA Statement). Original research articles investigating the effects of PBM therapy on ddMSCs, published from 2000 to August 2015, were retrieved and used for this review according to the following eligibility criteria: evaluating PBM therapy, assessing stem cells of dentoalveolar origin, published in English, dealing with cells characterized as stem cells, and using light that did not need external chromophores. RESULTS: From the initial 3467 potentially relevant articles identified, 6 were excluded because they were duplicates, and 3453 were considered ineligible based on the inclusion criteria. Therefore, eight articles remained, and these were fully analyzed in order to closely check exclusion criteria items. Only one of them was excluded because the cultured cells studied were not characterized as stem cells. Finally, seven articles served as the basis for this systematic review. CONCLUSIONS: PBM therapy has no deleterious effects on ddMSCs. Although no other clear conclusion was obtained because of the scarce number of publications, the results of these studies are pointing to an important tendency of PBM therapy to improve ddMSCs' viability and proliferation.

Efeito da Laserfototerapia associada ou não à Vitamina C na indução de membranas celulares (cell sheets) de células-tronco da polpa dentária humana / Effect of laserphototherapy associated or not to Vitamin C in the induction of cell sheets of human dental pulp stem cells

Membranas celulares (MCs; Cell Sheets), constituídas por células-tronco (CTs), são autodestacáveis da placa de cultivo, e sem subcultivos geram grande quantidade de células que podem ser transplantadas de maneira mais próxima da fisiologia celular, mantendo-se as ligaçُões celulares e a matriz extracelular produzidas em cultura. O Ácido ascórbico ou vitamina C (VC) tem efeito indutor da formação destas MCs, aumentando a longevidade e tempo de indiferenciação das CTs. A similaridade observada entre respostas biológicas da VC em MCs e aquelas da Laserfototerapia (LFT) sobre células e tecidos, nos levou à hipótese de que estas terapias poderiam se complementar melhorando o prognóstico de futura aplicação clínica dessas MCs em regenerações tecidos de interesse odontológico. Para testar essa hipótese, LFT e VC foram aplicadas associadas ou não na indução de MCs de células-tronco da polpa dentária humana (hDPSCs). Para tanto, hDPSCs descongeladas, que expressaram níveis típicos de marcadores de superfície de células-tronco mesenquimais, foram plaqueadas em placas de 6 poços (5x104 células por poço). Vinte e quatro horas depois do plaqueamento as culturas foram submetidas aos tratamentos dos grupos experimentais: Controle: hDPSCs em P3 cultivadas com meio clonogênico; Senescente: hDPSCs em P27 cultivadas com meio clonogênico; VC: P3 cultivadas com meio clonogênico acrescido de VC (20µg/ml); Laser: P3 cultivadas com meio clonogênico e submetido à LFT (contato e pontual - 5 pontos / poço, 660 nm, 20 mW, 0,028 cm², 0,71 W/cm², 7 segundos, 5 J/cm², 0,14 J por ponto, 48 horas de intervalo) e Laser+VC: P3 cultivadas com meio clonogênico acrescido de VC e submetido

à LFT. Em 24 horas, 7 e 13 dias as hDPSCs dos diferentes grupos experimentais foram observadas macro e microscopicamente, e atividade da enzima telomerase foi avaliada por PCR-TRAP, complementado por ELISA. Para a avaliação da expressão de genes relacionados à natureza e indiferenciação (Mitofilina e Oct 4) e à longevidade (fase catalíca da enzima telomerase - hTERT); bem como à senescência das células do grupo senescente (­­ß-galactosidase), as hDPSCs de todos os grupos experimentais foram submetidas ao RT-qPCR As hDPSCs foram capazes de formar MCs somente nos grupos VC e Laser+VC (100%), entre 10 e 13 dias. As MCs do grupo Laser+VC apresentaram maior facilidade na manipulação. Atividade de Telomerase nas hDPSCs foi observada somente em 24 horas (Controle e LFT) e em 7 dias (VC e Laser+VC). Os marcadores de indiferenciação (Oct 4) e mesenquimal (mitofilina), bem como a hTERT foram expressos nas hDPSCs de todos os grupos experimentais. O Oct4 e o hTERT, em 7 dias, apresentaram expressões significativamente maiores nos grupos VC e Laser+VC em comparação com os demais (p < 0,0001, p = 0,0009, respectivamente). A expressão da mitofilina foi significativamente maior no grupo Laser+VC, em 7 dias (p =0,033). A técnica de obtenção de MCs de hDPSCs por essa metodologia foi considerada adequada para ser testada em procedimentos regenerativos. A LFT quando associada à VC não interferiu na formação das MCs, nem na manutenção da longevidade e indiferenciação das hDPSCs. Adicionalmente, a LFT melhorou a manipulação das MCs. Assim sendo, a associação de VC e LFT na indução de MCs parece promissora para futura utilização de MCs na odontologia regenerativa.

Cell Sheets, consisting of stem cells (SCs) are self detachable from the cultivation plate, and with no subcultivation can generate large amount of cells. The cell sheets can be transplanted closer to cell physiology environment by keeping the cell connections and the extracellular matrix produced in culture. Ascorbic acid or Vitamin C (VC) has inductive effect on cell sheet formation, increasing the longevity and the stemness of the cell for long period of time. The similarity between biological responses of VC in cell sheets and those of Laserphototherapy (LPT, Laser) on cells and tissues led us to hypothesize that these therapies could improve the prognosis of future clinical application of these cell sheets in regeneration of dental tissues. To test this hypothesis, LPT and VC were applied, associated or not, to induce human dental pulp stem cells (hDPSCs). Therefore, hDPSCs, which expressed typical levels of mesenchymal stem cell surface markers, were plated in 6-well plates (5x104 cells per well). Twenty-four hours later they were subjected to the treatment of experimental groups: Control: hDPSCs in P3 cultured with regular medium; Senescent: hDPSCs in P27 cultured with regular medium; VC: P3 cultured with regular medium supplemented with VC (20 ?g/ml); Laser: P3 cultures with regular medium and submitted to LPT (punctual and contact mode-5 points / well, 660 nm, 20 mW, 0.028 cm², 0.71 W/cm²,

7 sec, 5 J/cm², 0.14 J per point, 48 hours-intervals) and Laser+VC: P3 cultured with regular medium supplemented with VC and submitted to LPT Within 24 hours, 7 and 13 days the hDPSCs of the different experimental groups were observed macroscopically and microscopically, and the telomerase enzyme activity was assessed by PCR-TRAP, complemented by ELISA. To evaluate the expression of genes related to the nature and differentiation (Mitofilina and Oct 4), longevity (catalytic phase of telomerase-hTERT enzyme), and the senescence of the senescent group cells (?-galactosidase), the hDPSCs of all experimental groups were subjected to RT-qPCR. The RT-qPCR data were compared by ANOVA complemented by the Tukey's test (p <= 0.05). The hDPSCs were able to form cell sheets only in the VC and Laser+VC groups (100%). Additionally, the cell sheets of the Laser+VC group presented easier handling...