ADSCs promote peripheral nerve repair can be harvested easily by a safe and conventional in a humidified atmosphere with 5% CO2 at 37°C liposuction procedure from subcutaneous fat tissue; for 48 h after which they were quickly used for in the frequency of ADSCs in adipose tissue is much vivo experiments. higher than that of MSCs in bone marrow [11]; and ADSCs proliferate significantly faster than BMMSCs Grafting procedure [12, 13]. The apparent advantages of ADSCs led us Twenty adult female Wistar rats (200-250 g), to investigate whether they may be ideal trans- divided into ADSC-treated (n = 10) and control plantable cells for peripheral nerve repair. In this (n = 10) groups, were anesthetized with chloral study we employed a rat model of peripheral nerve hydrate (350 mg/kg) during all surgical procedures. injury and demonstrated that implantation of After skin incision, the sciatic nerve was exposed ADSCs promotes peripheral nerve repair. using a muscle splitting incision. With the aid of an operation microscope, the right sciatic nerve Material and methods (15-mm) was severed and removed near the Preparation and characterization of adipose- obturator tendon in the mid-thigh. A 15-mm nerve derived stem cells graft was interposed into this nerve gap. The graft was coapted to the host nerve stumps by Rat ADSCs were isolated as described previously epineurial neurorrhaphy using one 9-0 Ethilon [14]. Briefly, the inguinal fat pad was harvested from suture at each end. The ADSC-treated group a 4-week old male Wistar rat and the adipose tissue received ADSC-laden grafts and the control group was carefully dissected. The adipose tissue was received non-ADSC-laden grafts. Both proximal and digested using collagenase type I (Gibco, USA) and distal nerve stumps were anchored into the graft then dissociated mechanically. The suspension was with 9-0 nylon microsutures, and the skin was centrifuged to separate the floating adipocytes from closed with wound clips. the stromal vascular fraction. The cells in the stromal vascular fraction were cultured in DMEM Walking-track analysis (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and the culture medium was changed Functional recovery was assessed by calculating 24 h later to eliminate the non-adherent cells. The the sciatic functional index (SFI) value [15]. SFI = cells were passaged 3-5 times before being used –38.3[(EPL-NPL)/NPL] + 109.5[(ETS-NTS)/NTS] + for subsequent experiments. To characterize the + 13.3[(EIT-NIT)/NIT] – 8.8 based on analysis of multi-potential differentiation capacity of ADSCs, walking tracks [16, 17]. Postoperatively, animals were the cells grown to at least 80% confluence were assessed every 4 weeks to week 12. The investi- treated for 3 weeks with either osteogenic induction gators were blinded to the animal groups during medium or adipogenic induction medium. Osteo- walking-track analysis. genic induction medium was DMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM Electrophysiological analysis ascorbate-2-phosphate, and 10 mM β-glycero- The rats were anesthetized with chloral hydrate phosphate. Adipogenic induction medium was (350 mg/kg) and the right sciatic nerve was DMEM supplemented with 10% FBS, 0.5 mM exposed thoroughly. A crook-shaped silver needle isobutyl-methylxanthine (IBMX), 1 μM dexametha- electrode was placed on the proximal end and distal sone, 10 μM insulin, and 200 μM indomethacin. Rat end of grafts. The proximal end was stimulated, and ADSCs were then stained with Alizarin Red S or Oil- was record at the distal end. The distance between Red O to confirm the osteogenic and adipogenic the two electrodes was measured with a sliding differentiation, respectively. caliper of 0.2 mm precision. Nerve conduction velocity, latent period, and peak amplitude were In vitro construction of nerve graft recorded. A total of 2 × 106 ADSCs in 100 μl DMEM were injected into a 1.5-cm long acellular nerve allograft Weight of the gastrocnemius and soleus using a microinjector under an SXP-10 microscope muscles at 10× magnification. To perform the injection, the microinjector was inserted through the full length The rats were anaesthetized with chloral hydrate of the nerve section, and cells were injected in (350 mg/kg) 12 weeks after operation. The gastrocne- equal volumes at four evenly spaced points as the mius and soleus muscles were harvested from the injector was withdrawn. Another set of acellular experimental and control sides, tendons were nerve was used as the control group with no ADSC- trimmed and muscles were weighed. A conservation laden grafts, in which 100 μl DMEM was injected muscle-mass ratio was recorded for each animal by into the nerve grafts. The nerve grafts were then dividing the experimental-side muscle mass by the incubated in DMEM supplemented with 10% FBS control-side muscle mass [18, 19]. Arch Med Sci 4, August / 2011 593