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Paper Information

Journal:   CELL JOURNAL (YAKHTEH)   SUMMER 2011 , Volume 13 , Number SUPPLEMENT 3 (7TH CONGRESS ON STEM CELL BIOLOGY AND TECHNOLOGY); Page(s) 47 To 48.
 
Paper: 

GROWING VASCULARISED THICK TISSUE IN A NOVEL DESIGNED BIOREACTOR

 
 
Author(s):  SHAHHOSSEIN DASTJERDI S.*, CROLL T., MANTON K., UPTON Z.
 
* INSTITUTE OF HEALTH AND BIOMEDICAL INNOVATION, QUEENSLAND UNIVERSITY OF TECHNOLOGY, BRISBANE, AUSTRALIA
 
Abstract: 
Objective: Organ transplantation as a medical treatment is a last resort, life saving option for some injuries and diseases. A major problem, however, is the scarcity of donor tissue and organs. Current use of engineered human tissue is limited to thin tissues (100-200 mm). For other thick tissues, fully functional blood vessels must be created. Human mesenchymal stem cells (hMSCs) are known for their ability to self renew, undergo cloning and differentiate into other cells.
Moreover, the ability of MSCs to mediate immunosuppression gives them an important role in limiting the rejection of foreign tissue in regenerative medicine.
Materials and Methods: In this project, 5% scaffolds constructed from Poly lactic acid (PLA) by a thermally induced phase separation (TIPS) technique have been utilized as a biodegradable 3D structure. PLA was dissolved in ethylene carbonate (EC) at 70°C. The polymer solution was then cast in a cylindrical mould. Next, crystallisation was induced manually at room temperature. The solidified polymer solution was then kept at 4°C cold room overnight before leaching. Next, the solidified PLA/EC was leached in pre-cooled de-ionized (DI) water at 4cC for 2-3 days to remove EC. Finally, the scaffold samples were recovered from the DI water and transferred into a vacuum container overnight. These fabricated scaffolds were then cultivated with hMSCs. Initially, static culture conditions were tested and then hMSCs were seeded within the bioreactor to compare the results of static and dynamic culture conditions.
Results: Thick scaffolds were seeded with hMSCs in 3D static conditions. The media was changed every 2-3 days over a period of 2 weeks. MTT and confocal were indicated that the number of viable seeded cells with metabolic activity were higher on the top and sides of the scaffold in comparison with the bottom in static culture conditions.
Then, thick scaffolds were seeded in 3D dynamic conditions within the bioreactor, and were cultured for 2 weeks. MTT and confocal staining revealed uniform seeding of the hMSCs; this presumably resulting from the continuous drip feeding loop within the bioreactor in dynamic culture conditions. Thin scaffolds (were also examined when seeding the hMSCs in 3D static conditions and were cultured for 8 weeks. Differentiated osteogenic cells were stained with Alizarin Red-S. These cells actively produced extra cellular matrix (ECM), albeit the lower number of visible nuclei suggests that the cell density is still quite low. Finally, quantitation of hMSC differentiation to osteogenic cells was confirmed by quantitative RT-PCR. RNA concentrations from 64 to 200 ng/
ml were extracted from the hMSC cells in the scaffold using Trizol. RT-PCR subsequently revealed the presence of osteogenic markers; Osteocalcin, Collagen 1A and RUNX2.
Conclusion: The goal of this research is to facilitate culture of hMSCs seeded throughout a 3D scaffold that can be fed with nutrients using the media with the established growth factor and hormone cocktails. At the same time, a suitable environment can be provided to allow the sprouting and growth of a blood vessel network which may lead to the production of vascularised 3D hMSCs seeded scaffolds with enhanced thickness (1 cm).
 
Keyword(s): ORGAN TRANSPLANTATION, HUMAN MESENCHYMAL STEM CELLS, TISSUE ENGINEERING, VASCULARISATION, BIOREACTOR
 
References: 
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