Induction of Hyaluronic Acid Production from Amniotic Epithelia and Irradiated Pasteurella Multocida Against bone Marrow Cells Aging in Vitro

Document Type : Original Article

Author

The Department of Radiation Microbiology*, National Center for Radiation Research & Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt and Department of Animal Biotechnology**, Genetic Engineering & Biotechnology Research Institute (GEBRI-USC), University of Sadat City, Egypt

Abstract

Background: Hyaluronic acid (HA) is a biopolymer mol-ecule that stimulates cell growth by supporting the building of the extracellular matrix (ECM). Therefore, it has been applied in many biomedical researches. The common biological re-sources for producing HA are animal tissues and bacteria. Cell aging is attributed to the reduction of cell division capacity and ECM quality. Aim of Study: The current study was aimed to evaluate the effect of irradiation on the productivity of Pasteurella multocida to HA and compare its biological effect with a hu-man-sourced HA. Material and Methods: Human amniotic epithelial cells (HAECs) were isolated from the amniotic membrane and seed-ed in vitro in to cell culture dishes with optimized different ex-perimental conditions to produce HA. This was verified by gene expression profiling of three enzymes (HAS1, HAS2, HAS3 for HA synthesis using quantitative PCR analysis. P. multocida was induced to produce large quantities of HA by gamma irradia-tion. Then, the produced HA from HAECs (HAECs-HA) and P.multocida (Pm-HA) were extracted by ethanol-precipitation methodology. Then the extracted HAs were analysed and com-pared against a standard HA (sHA) by Fourier transform infra-red (FTIR) assay. Aged murine bone marrow cells (m BMCs) were isolated from a femur of a 2-year-old rat, and cultivated in replicates into a 96-well cell culture plate. Each triplicate of m BMCs cultures was treated with one of the extracted HA (HAECs-HA or Pm-HA). Non-treated cultures were assigned as control. Cellular migration and proliferation were evaluat-ed by microscopic examination and MTT(3-[4,5-dimethylthi-azol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Results: The optimized HAECs cultures for producing HA were determined inthe multi-layering-conditioned cells and those in the agitated culture. The principalsyn thesis enzymes that express HA in HAECs were HAS2 followed by HAS3. The Pm-HA was increased in the P. multocida culture after the induction by a dose of 2 KGy gamma irradiation more than oth-er irradiation doses. FTIR assay showed chemical similarities between both synthetic HA products with sHA. The microscop-ic evaluation revealed the superior effect of HAECs-HA in the improvement of BMCs growth (until confluence) and migration than Pm-HA and saline (couldn’t reach confluence). This was also confirmed by the results of the MTT assay. Conclusion: This observatory study consider as the first approach for producing HA from HAECs culture in vitro that has a biological effect to improve the migration and the growth of aged mBMCs more than the bacterial HA. Therefore, these outcomes encourage further studies to evaluate the clinical fea-sibility of using HAECs-HA as an anti-aging ingredient.

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