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HUMAN ADIPOSE STEM/STROMAL CELL SPHEROIDS AS A MODEL OF OSTEOGENESIS
New bone formation may be desirable in a variety of clinical settings, such as osteoporosis and non-union fractures. Alternatively, stem cells derived from adipose tissue represent a promising cellular source for the formation of this tissue through tissue engineering, because of its easy accessibility and abundance. In this context, a new approach in the field of tissue engineering corresponds to the use of spheroids as modular units for engineering biological tissues.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
In this way, the aim of this study is to induce and padronize the osteogenesis process using human adipose tissue derived stem cells (ASC) in a three-dimensional micromolded agarose hydrogel culture. Human lipoaspirate samples were obtained according to the local research ethics committee and human adipose tissue stem cells were isolated by mechanical dissociation. One group of spheroids were maintained for three weeks in an osteogenic induction medium composed of 10 mM β-glycerophosphate, 10-7 mM Dexamethasone and 200 mM hrBMP-7 (human recombinant bone morphogenic protein 7). Another group of spheroids were first maintained for two weeks in a chondrogenic medium composed of 10-8 mM Dexamethasone and 10 ng/mL TGFβ-3. Then, the medium was exchanged for an osteogenic one composed of 10 mM β-glycerophosphate and 10-7 mM Dexamethasone for three weeks. Initially, spheroids diameter measurements were performed. In control spheroids, the range of diameter was 300-350 µM, in hrBMP-7 induced spheroids the range of diameter was between 350-400 µM and in TGFβ-3 induced spheroids the range of diameter was 400-450 µM. Regarding cell viability, this was shown to be high for the control and induced spheroids, once 85% of cells were negative for 7-AAD DNA intercalant. Hematoxylin and Eosin histological analyzes were performed to evaluate cell morphology and Alizarin Red O staining to identify extracellular mineralization, where we observed the presence of calcium deposits only in both induced conditions. The molecular analyses by reversal transcriptional polymerase chain reaction (rtPCR) showed a high relative expression of alkaline phosphatase (ALP) in hrBMP-7 induced spheroids when compared with control spheroids and a low relative expression of chondrogenesis master gene Sox9 in hrBMP-7 induced spheroids. Simultaneously, immunohistochemistry was performed using antibodies to components of bone extracellular matrix, as type I collagen, osteocalcin, osteopontin, biglycan and tenascin C, where the TGFβ-3 induced spheroids showed a better distribution of those molecules in the last week of culture when compared to hrBMP-7 induced and control spheroids. The surface of spheroids was visualized by scanning electron microscopy, in addition to an elemental EDS analysis for the calcium and phosphate elements, present only in induced spheroids of both conditions. At the end, mechanical resistance to compression were evaluated utilizing Microsquisher (Cell Scale), in which TGFβ-3 induced spheroids presented a better performance in the third week of culture, reaching values of 400 µN of force, while control and hrBMP-7 induced spheroids presented values of 20-30 µN of force.
Considerações Finais/Final considerations/Consideraciones finales
We were able to compare two different protocols for osteogenic induction in ASC spheroids and realize that TGFβ-3 induced spheroids showed a better response in analyses of osteogenic differentiation efficiency.
Palavras-chave/Key words/Palabras clave
Spheroids, Osteogenesis, Tissue Engineering
GABRIELA SOARES KRONEMBERGER, ANDERSON BEATRICI, KARINA RIBEIRO SILVA, JOSÉ MAURO GRANJEIRO, LEANDRA SANTOS BAPTISTA