13 research outputs found
Investigation of The Cellular Response to Bone Fractures : Evidence for Flexoelectricity
The recent discovery of bone flexoelectricity (strain-gradient-induced electrical polarization) suggests that flexoelectricity could have physiological effects in bones, and specifically near bone fractures, where flexoelectricity is theoretically highest. Here, we report a cytological study of the interaction between crack stress and bone cells. We have cultured MC3T3-E1 mouse osteoblastic cells in biomimetic microcracked hydroxyapatite substrates, differentiated into osteocytes and applied a strain gradient to the samples. The results show a strong apoptotic cellular response, whereby mechanical stimulation causes those cells near the crack to die, as indicated by live-dead and caspase staining. In addition, analysis two weeks post-stimulation shows increased cell attachment and mineralization around microcracks and a higher expression of osteocalcin -an osteogenic protein known to be promoted by physical exercise. The results are consistent with flexoelectricity playing at least two different roles in bone remodelling: apoptotic trigger of the repair protocol, and electro-stimulant of the bone-building activity of osteoblasts
Osteogenic differentiation strategies of Dental Pulp Pluripotent-like Stem Cells (DPPSC) for their potential application in Bone Tissue Engineering
Actualment, els defectes ossis, ja sigui deguts a traumes o a malalties, afecten a milions de persones a nivell mundial, fet que suposa una demanda constant al sistema sanitari per al reemplaçament o la restitució d’os. En els últims anys, les tècniques de medicina regenerativa i d’enginyeria tissular d’ós que combinen l’ús de biomaterials, cèl·lules i factors de creixement, han esdevingut un recurs terapèutic molt prometedor per al tractament de defectes ossis. Les cèl·lules mare pluripotents poden ser de gran valor en enginyeria tissular degut al seu potencial d’auto-renovació, la seva alta capacitat de proliferació i el seu potencial de diferenciació a la majoria de llinatges cel·lulars del cos. A més, poden servir com a model per a testar l’eficiència dels nous biomaterials ja que necessiten ser guiades pròpiament pel material per a diferenciar-se. Tot i aixÃ, encara no s’ha identificat un tipus cel·lular amb caracterÃstiques pluripotents, controlat genèticament i sense problemes ètics per a ser utilitzat en tècniques d’enginyeria tissular d’os. En estudis previs, el nostre grup ha descrit una nova població de cèl·lules mare amb caracterÃstiques pluripotents de la polpa dental del tercer molar: les DPPSC (de l’anglès, Dental Pulp Pluripotent-like Stem Cells). Aquestes cèl·lules semblen estar presents fins a edat avançada, tenen una bona estabilitat genètica i mostren caracterÃstiques pluripotents tÃpiques de cèl·lules mare embrionà ries. Fins ara, s’ha demostrat la seva capacitat de diferenciar-se a cèl·lules de teixits de les tres capes embrionà ries. A més a més, semblen tenir una major capacitat de diferenciar-se a cèl·lules del teixit ossi que altres poblacions de cèl·lules mare de la polpa dental.
Aquesta tesis doctoral pretén doncs introduir l’ús de les DPPSC com a model cel·lular alternatiu per a tècniques de medicina regenerativa i d’enginyeria tissular d’os. La tesis s’ha dividit en tres parts principals:
Com a primer pas, el procés de diferenciació osteogènica de les DPPSC i les seves habilitats per créixer, adherir-se i diferenciar-se han estat avaluades a partir de diferents tipus de biomaterials tradicionalment utilitzats en estudis de regeneració òssia (metalls o materials d’origen natural). Els resultats mostren un alt potencial de diferenciació osteogènica i d’adhesió de les DPPSC als materials estudiats, aixà com una alta estabilitat genètica. Proposant en conseqüència l’ús de les DPPSC com a bon model cel·lular per avaluar la biocompatibilitat i la capacitat osteogènica de diferents tipus de biomaterials.
A continuació, diferents estratègies s’han utilitzat per millorar el procés d’osteogènesis a partir de les DPPSC per a la seva potencial aplicació en terà pies d’enginyeria tissular d’os.
Una nova famÃlia de polÃmers ha estat utilitzada com a terà pia gènica no viral per a la millora de la diferenciació osteogènica en DPPSC a partir del silenciament de gens de pluripotència i la sobre-expressió de gens de osteogènesis. Concretament, s’ha utilitzat com a vector la combinació de poli(β-amino ester)s (pBAEs) amb oligopèptids d’origen natural per tal de millorar la seva biocompatibilitat amb les DPPSC i alliberar-ne simultà niament anti-OCT3/4 siRNA, anti-NANOG siRNA i plà smid RUNX2. Els resultats mostren que les DPPSC poden ser perfectament transfectades amb aquests polÃmers mantenint la viabilitat cel·lular i l’estabilitat genètica. A més a més, la combinació de silenciar OCT3/4 i potenciar RUNX2 n’accelera la diferenciació osteogènica.
Seguidament, diferents estratègies han estat provades per tal d’induir vascularització en els constructes d’enginyeria tissular a partir de les DPPSC, fet important per a garantir la supervivència dels constructes un cop implantats in vivo i que a l’hora permet millorar la osteogènesis. Resultats previs mostren que les DPPSC tenen un alt potencial endotelial. Per tant, en aquest estudi, s’ha suggerit la combinació de DPPSC pre-diferenciades a teixit ossi amb DPPSC pre-diferenciades a teixit endotelial com a una bona estratègia per induir os vascularitzat a partir d’una única població de cèl·lules mare. AixÃ, diferents sistemes de co-cultius amb DPPSC han estat analitzats. A més a més, per tal d’apropar els resultats a la terà pia clÃnica, tant les diferenciacions en monocultiu com en co-cultiu de les DPPSC han estat testades en medis lliures de components animals, reemplaçant el sèrum fetal bovà per sèrum humà . Per altra banda, l’efecte de vidres bioactius (BaG, de l’anglès Bioactive Glasses), caracteritzats per les seves propietats osteogèniques, també ha estat investigat en monocultius i co-cultius de DPPSC. Els resultats mostren que el medi endotelial condicionat amb extractes de BaG pot promoure tant el potencial osteogènic com l’endotelial de les DPPSC, promovent la formació d’estructures vasculars en els sistemes de co-cultiu. AixÃ, el co-cultiu de DPPSC en combinació amb BaG i en medis lliures d’origen animal proporciona un nou sistema per a la vascularització in vitro de constructes ossis d’enginyeria tissular.
En conclusió, els resultats dels estudis realitzats en aquesta tesis doctoral proposen l’ús de les DPPSC per diferents tècniques d’enginyeria tissular d’os i medicina regenerativa
Investigation of The Cellular Response to Bone Fractures : Evidence for Flexoelectricity
The recent discovery of bone flexoelectricity (strain-gradient-induced electrical polarization) suggests that flexoelectricity could have physiological effects in bones, and specifically near bone fractures, where flexoelectricity is theoretically highest. Here, we report a cytological study of the interaction between crack stress and bone cells. We have cultured MC3T3-E1 mouse osteoblastic cells in biomimetic microcracked hydroxyapatite substrates, differentiated into osteocytes and applied a strain gradient to the samples. The results show a strong apoptotic cellular response, whereby mechanical stimulation causes those cells near the crack to die, as indicated by live-dead and caspase staining. In addition, analysis two weeks post-stimulation shows increased cell attachment and mineralization around microcracks and a higher expression of osteocalcin -an osteogenic protein known to be promoted by physical exercise. The results are consistent with flexoelectricity playing at least two different roles in bone remodelling: apoptotic trigger of the repair protocol, and electro-stimulant of the bone-building activity of osteoblasts
S53P4 Bioactive glass inorganic ions for vascularized bone tissue engineering by dental pulp pluripotent-like stem cell cocultures
Vascularization of large bone grafts is one of the main challenges that limit the clinical application of bone tissue engineering (BTE). In this way, cell cocultures, which involve the cross-talk between endothelial and osteogenic cells, have shown to be an effective strategy for in vitro prevascularization. Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSCs), shows high plasticity and great capacity to differentiate into different tissues. Here, we suggested a combination of bone-like DPPSC and endothelial-like DPPSC to induce vascularized bone formation from a unique stem cell population. In addition, we evaluated the use of inorganic ions dissolved from S53P4 bioactive glass (BaG) in different medium compositions. Results show that endothelial medium with BaG extracts provides an effective way to enhance both endothelial and osteogenic processes, supporting the formation of vascular-like structures and mineralization simultaneously. Furthermore, 3D DPPSC cocultures in the same medium conditions demonstrated the formation of vessel-like structures that appear to be functional as indicated by the presence of an internal lumen. Overall, these results would provide a new promising system for the prevascularization of BTE constructs.This work was partially funded by the Universitat International de Catalunya (UIC Barcelona) and the University of Tampere (UTA). UIC acknowledges Agencia de Gestio d’ajuts Universitaris i de Recerca, Generalitat de Catalunya (SGR 2014) n 1060. The authors R. Nuñez-Toldra and B.
Bosch were funded by the predoctoral grant Junior Faculty awarded by the Obra Social La Caixa and the UIC. The author R. Nuñez Toldra was also funded by a travel grant awarded by Boehringer Ingelheim Fonds. The work at UTA was funded by Tekes, the Finnish Funding Agency for Innovation, the Academy of Finland, and the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital.Peer reviewe
S53P4 Bioactive Glass Inorganic Ions for Vascularized Bone Tissue Engineering by Dental Pulp Pluripotent-Like Stem Cell Cocultures
The Effect of Commercially Available Endodontic Cements and Biomaterials on Osteogenic Differentiation of Dental Pulp Pluripotent-Like Stem Cells
The aim of this study is to compare the osteogenic differentiation capacity of the dental pulp pluripotent-like stem cells (DPPSCs) using conditional media pretreated with ProRoot-MTA, Biodentine (BD) or the newly manufactured pure Portland cement Med-PZ (MZ). DPPSCs, isolated from human third molars, are the most relevant cell model to draw conclusions about the role of biomaterials on dental tissue regeneration. Cytotoxicity, alkaline phosphatase (ALP) activity, and calcium deposition analysis were evaluated at different differentiation time points. Gene expression of key osteogenic markers (RUNX2, Collagen I and Osteocalcin) was determined by qRT-PCR analysis. The osteogenic capacity of cells cultured in conditioned media prepared from MZ or MTA cements was comparable. BD conditioned media supported cell proliferation but failed to induce osteogenesis. Relative to controls and other cements, high osteogenic gene expression was observed in cultures pre-treated with the novel endodontic cement MZ. In conclusion, the in vitro behavior of a MZ- endodontic cement was evaluated, showing similar enhanced cell proliferation compared to other commercially available cements but with an enhanced osteogenic capacity with prospective potential as a novel cement for endodontic treatments
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Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept
Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct
in vitro
the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques
Additional file 1: Figure S1. of Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation
Characterization of undifferentiated DPPSC. a Cell morphology of DPPSC from passage 10 observed with optic microscopy. DPPSC are characterized as small-sized cells with large nuclei and low cytoplasm content. b Immunofluorescence analysis of OCT3/4-FITC, SSEA4-PE, and Merge. Hoechst (HT) as a nucleus control. DPPSC were positive for these embryonic markers, and both were located in the nucleus. c FACS analysis of DPPSC. c1 FACS analysis of membrane markers: CD105 (92,15%), CD29 (99,63%), CD146 (15,54%) and CD45 (0.04%). c2 FACS analysis of pluripotency nuclear markers: OCT3/4 (76,72%) and NANOG (30,18%). d RT-PCR of OCT3/4, NANOG and SOX2 expresions in DPPSC and DPMSC. e Western Blot analysis of OCT3/4 in DPPSC and DPMSC at different time points (5, 10 and 15 passages). GAPDH as a housekeeping. (TIF 1031Â kb
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Additional file 1: Table S1. of Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration
List of primers used for cDNA amplification in qRT-PCR analyses. (DOCX 11 kb