384773 Transdifferentiated Mesenchymal Stem Cells in Micropatterned Nerve Guidance Conduits for Peripheral Nerve Regeneration
Peripheral nerves are responsible for communication between the central nervous system (CNS) and other parts of the body such as muscles and skin. Accidental trauma can damage peripheral nerves and can result in sensory and motor dysfunctions. The peripheral nervous system is capable of repair, recovery and regeneration after a mild crush injury but in cases of severe injury resulting in nerve transection, exogenous treatment is required. A common treatment for such severe injuries is an autologous nerve graft (ANG) where a segment of healthy nerve is used for connecting the severed ends of the nerve at the site of injury. ANGs are considered as the “gold standard” treatment for such injuries. However, there are a number of limitations associated with ANGs including donor site morbidity and the limited length of available graft material. Due to these limitations alternative strategies are under investigation for treating peripheral nerve injuries (PNIs). One such strategy is to engineer a nerve guidance conduit (NGCs) that acts as a topographical cue in facilitating the growth and alignment of regenerating axons. Another strategy is to transplant Schwann cells (SCs) as a cellular replacement of native SCs lost during injury. SCs are glial cells of the peripheral nervous system and are responsible for providing trophic support to the regenerating axons. Though SC transplantation enhances nerve regeneration, there is no easy source for obtaining SCs except from a healthy nerve. As an alternative, mesenchymal stem cells (MSCs) are being investigated as cellular replacement to SCs. MSCs are multipotent cells capable of differentiating into adipocytes, osteoblasts and chondrocytes and can easily be isolated from bone marrow via routine surgical procedure. Furthermore it has been shown that under appropriate induction procedures, MSCs can transdifferentiate into SC-like cells. Transdifferentiated MSCs (tMSCs) differ from undifferentiated MSCs (uMSCs) in terms of expression of various SC specific proteins such as S100β and p75NTR.
Previously our group has shown that micropatterned conduits seeded with SCs enhanced nerve regeneration in a rat sciatic nerve injury model (Rutkowski et. al, 2004). But because of the limitations associated with SC grafts we are investigating the efficacy of tMSCs as SC replacements. Our hypothesis is to synergistically combine topographical cues in the form of micropatterned conduits and biological cues in the form of tMSCs to enhance nerve regeneration. In the future this strategy may translate into using patient specific autologous tMSCs for transplantation purposes.
Recently we have demonstrated that the topography of a biodegradable poly (lactic acid) (PLA) micropatterned substrate does not affect transdifferentiation of MSCs (Sharma et. al. 2014, submitted). In addition, a BrdU incorporation assay demonstrated that MSCs proliferation was similar on micropatterned and smooth films. No difference in the percentage of S100β and p75NTR immunostained tMSCs was observed between micropatterned and smooth PLA films but was found to be significantly higher when compared to uMSCs growing on similar substrates under control conditions. However, tMSCs growing on the patterned films were found to be significantly oriented and elongated in the direction of the micropatterns as compared to the tMSCs growing on smooth films. These results demonstrate that micropatterning can be used as an effective tool in controlling the morphology and alignment of tMSCs without causing any detrimental effect on transdifferentiation and proliferation of MSCs.
Currently we have initiated in vivo studies using micropatterned PLA conduits in a sciatic nerve transection (10 mm gap) model in young adult Brown Norway rats. We have grafted conduits into three different groups of animals, (I) Micropatterned PLA conduits seeded with tMSCs, (II) Micropatterned PLA conduits seeded with uMSCs and (III) Empty micropatterned PLA conduits. Behavioral studies designed to assess possible recovery of motor and sensory function are underway to test for differences between the three groups. At the end of the experiment the animals will be euthanized and an immunohistochemical and morphometric analysis will be conducted to assess the extent of possible nerve regeneration and growth.
To provide additional trophic support to the regenerating axons, we have developed nerve growth factor (NGF) encapsulated biodegradable polymeric nanoparticles (NPs) using polyanhydrides. These NPs are capable of releasing bioactive NGF for up to a month.
Our future studies will combine regenerative cues such as micropattern conduits, tMSCs and NGF encapsulated nanoparticles to enhance nerve regeneration in the rat sciatic nerve injury model.
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