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1.
Tissue Eng Part A ; 21(21-22): 2639-48, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26414682

RESUMO

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.


Assuntos
Células-Tronco Adultas/fisiologia , Sangue/metabolismo , Meios de Cultura/química , Polpa Dentária/citologia , Engenharia Tecidual/métodos , Dente/citologia , Adolescente , Células-Tronco Adultas/citologia , Técnicas de Cultura Celular por Lotes , Proliferação de Células , Criança , Polpa Dentária/fisiologia , Feminino , Humanos , Masculino , Técnicas de Cultura de Órgãos/métodos , Técnicas de Cultura de Tecidos/métodos , Dente/crescimento & desenvolvimento
2.
Tissue Eng Part A ; 19(23-24): 2537-43, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23863195

RESUMO

New techniques for tissue engineering (TE) are rapidly emerging. The basic concept of autologous TE is to isolate cells from small biopsy specimens, and to expand these cells in culture for subsequent seeding onto biodegradable scaffolds. Nanocrystalline diamond films have attracted the attention of researchers from a variety of different areas in recent years, due to their unique and exceptional properties. In this approach, human dental stem cells (hDSCs) were characterized by flow cytometry and grown on diamond films with hydrogen (H)-terminated and oxygen (O)-terminated surfaces for 28 days, and then removed by lysis and washing with distilled water. Energy dispersive spectroscopy analysis was performed, showing that the regions with O-terminated surfaces contained much higher levels of deposited calcium, oxygen, and phosphorus. These results suggest that the extracellular matrix was considerably more developed in the O-terminated regions, as compared with the H-terminated regions. In addition, optical microscopy of hDSCs cultured on the diamond substrate with H- and O-terminated surfaces, before washing with distilled water, showed preferential directions of the cells arrangement, where orthogonal lines suggest that the cells appeared to be following the O-terminated regions or hydrophilic surface. These findings suggest that O-terminated diamond surfaces prepared on biodegradable scaffolds can be useful for mineralized dental tissue formation.


Assuntos
Nanodiamantes/química , Células-Tronco/citologia , Engenharia Tecidual , Alicerces Teciduais/química , Dente/citologia , Células Cultivadas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células-Tronco/metabolismo , Dente/metabolismo
3.
J Mol Histol ; 43(1): 89-94, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22109772

RESUMO

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.


Assuntos
Células-Tronco Adultas/citologia , Aberrações Cromossômicas , Polpa Dentária/ultraestrutura , Instabilidade Genômica , Linhagem Celular , Bandeamento Cromossômico , Humanos , Hibridização in Situ Fluorescente , Cariotipagem
4.
Artif Organs ; 35(7): E129-35, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21702761

RESUMO

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.


Assuntos
Dente Serotino/citologia , Dente Serotino/crescimento & desenvolvimento , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adolescente , Adulto , Células Cultivadas , Criança , Feminino , Humanos , Masculino , Dente Serotino/diagnóstico por imagem , Odontogênese , Radiografia , Adulto Jovem
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