*Purpose: Although, over 200 clinical Vascularized Composite Allotransplantation (VCA) such as hands, legs, and face have been performed worldwide, still there are many challenges such as immune rejection and incomplete recovery of motor and sensory function. The objective of this study was to investigate whether adult Mesenchymal Stem Cells (MSCs) and Granulocyte-Colony Stimulating Factor (G-CSF) can improve limb functional outcomes in a rat sciatic nerve transection repair model.

*Methods: Under general anesthesia the right hind limb (femoral region) of the Lewis rat was exposed, the sciatic nerve was identified and transected; after an hour the epineurium of the proximal and distal ends of transected nerve were approximated using 10-O sutures. Bone marrow derived syngeneic MSCs (2 million/rat), G-CSF (50-100 µg/kg), and/or Vehicle were administered locally via hydrogel at the site of nerve repair, i.v./i.p., and to gastrocnemius muscle. The motor function was assessed by walking track analysis and sensory function by cutaneous pain reaction test. All animals received manual physiotherpay once a week 5-10 minutes and were group housed on wire mesh floor for 6-8 hours per day.

*Results: MSCs expanded ex vivo were CD29+, CD90+, CD34-, CD31-, MHC Class I+, Class II- and multipotent as determined by ex vivo differentiation in to adipocytes, osteocytes and chondrocytes. At two weeks post-nerve repair, total sensory function (peroneal, tibial, saphenous and sural) in all groups was ~1.8 on a scale of Grade 0-3 (0=No function; 3= Normal function); however, peroneal nerve function was ~ 2.8 (n=6/group). By 4 weeks total sensory function was 2.2±1.0 and 2.0±1.4 in MSC+G-CSF and MSC+vehicle groups, respectively. At 8 weeks, sensory function was restored to nearly normal in MSC+G-CSF (~2.8) but not in MSC+vehicle control (~2.3). At 8 weeks, motor function as determined by walking foot print grades 0-4 (0=no print; 4=heel plus 4-5 toe prints) was 3.2±1 and 1.8±1.2; and at 16 weeks 3.0±0.9 and 2.0±0.6 in MSC+G-CSF and MSC+vehicle groups, respectively. Motor function was significantly (P<0.05) improved in MSC+G-CSF treated group. Also, G-CSF administration to gastrocnemius muscle mitigated muscle atrophy markedly. Flexion contractures graded 0-4 (0= no contracture; 4= >90 degrees contracture) were significantly (P<0.01) reduced in G-CSF+MSC (1.5±0.8) treated animals compared to MSC+vehicle control (3.2±0.75). In a parallel study, we observed significant improvement in nerve regeneration by histology and functional outcomes with MSC therapy (n≥6) compared to vehicle alone controls.

*Conclusions: We believe MSC with G-CSF combination therapy can be successfully used to improve functional outcomes in limb transplantation and sciatic nerve injury.

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