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Presenter: Maria, Madajka, Cleveland, United States
Authors: Madajka M., Mendiola A., Przybyla B., Siemionow M.
MESENCHYMAL STEM CELLS AND CELLULAR TRANSPLANTATION
M. Madajka1, A. Mendiola2, B. Przybyla2, M. Siemionow2
1Plastic Surgery, Cleveland Clinic Foundation, Cleveland/OH/UNITED STATES OF AMERICA, 2Plastic Surgery, Cleveland Clinic Foundation, Cleveland/UNITED STATES OF AMERICA
Body: Introduction: Nerve regeneration is one of the critical steps following transplantation, and is absolutely necessary for successful recovery. In the USA there are over 50,000 peripheral nerve procedures per year with overall cost exceeding 7 billion dollars. Current techniques such as cable nerve grafts or conduits from biodegradable polymers have limitations due to postoperative complications such as sensory loss, scarring, painful neuroma and the restoration of the damage exceeding 4cm is still complicated. Limited efficacy requires novel approaches and in our research we investigate two main objectives:1) To developed a new surgical protocol with the use of epineural sheath filled with BMSC to regenerate peripheral nerve gaps; 2) To characterize the neurotrophic capabilities of new conduit together with the multipotential capabilities of BMSC. Methods for objective 1: Epineural sheaths were collected and filled with 3x106 BMSC stained with PHK and incubated in complete α-MEM medium for 14 days. After that time the constructs were used for restoration of 2cm peripheral nerve gaps in total of 30 Lewis rats. Animals were neurologically evaluated after 6, 12 and 24 weeks. Nerve samples were harvested and the progression of axon growth was examined by toluidine blue staining. Additional evaluation of nerve regeneration was completed by fluorescent staining of H-neurofilaments, CD31, S-100, laminin B, VEGF and GFAP. Methods for objective 2: Neurotrophic potential of epineurial sheath alone and with the content of BMSC was tested in vitro for 3 weeks. Four combinations of epineural sheaths/BMSC were tested: LEW/LEW, ACI/ACI, ACI/LEW, LEW/ACI. Media samples were collected every 3 days, including collection from BMSC cultured alone and epineural sheaths only. ELISA test was utilized to measure the level of released NGF in collected medium to determine its maximum concentration and optimum activity time for the construct. Additionally, 12 epineural sheaths filled with LEW BMSC were created and incubated 2 weeks in complete α- MEM medium enriched with NGF (100ng/mL). All constructs were than implanted into LEW rats and collected after 6 or 12 weeks. Nerve samples were harvested and immunostained. Results: NGF release was detected in vitro after 4 days in culture in case of epineurial sheats filled with BMSC and reached the maximum level of 1000pg/mL by day 10. Cultures of only BMSC or epineural sheaths had 2 weeks delay in the release of NGF, which was detected at the concentration range of 30-200pg/mL. Immunohistochemistry revealed that expression of H-neurofilaments was detectable 6 weeks after implantation. CD31 positive cells were present mostly inside of epineural tube and co-localized with dividing cells. The presence of VEGF, GFAP and S-100 was detected at low levels. Conclusions: The construct made of epineural sheath filled with BMSC has a high capacity to maintain stable secretion of NGF, which could be the most critical neutrotrophic factor. The growth of neurofilaments in the lumen of the tube indicates successful nerve growth. The presence of endothelial markers on the surface of dividing cells inside the construct reflects self-maintenance of their pro-regenerative activity.
Disclosure: All authors have declared no conflicts of interest.
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