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First published online August 16, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2956 -2963
doi:10.1634/stemcells.2007-0187; www.StemCells.com
© 2007 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Acceleration of Sensory Neural Regeneration and Wound Healing with Human Mesenchymal Stem Cells in Immunodeficient Rats

Toshifumi Imaizumia, Sadanori Akitaa, Kozo Akinob, Akiyoshi Hiranoa

Divisions of aPlastic and Reconstructive Surgery and
bAnatomy and Neurobiology, Department of Developmental and Reconstructive Medicine, Nagasaki University, Graduate School of Biomedical and Sciences, Nagasaki, Japan

Key Words. Human mesenchymal stem cell • Nude rat • Green fluorescent protein • Tracking • Differentiation Fibroblast growth factor • Vascularized epigastric flap • Lower extremity wound healing

Correspondence: Sadanori Akita, M.D., Ph.D., Nagasaki University, School of Medicine, 1-7-1 Sakamoto Machi, Nagasaki 8528501, Japan. Telephone: +81-95-849-7327; Fax: +81-95-849-7330; e-mail: akitas{at}hf.rim.or.jp

Received March 20, 2007; accepted for publication July 9, 2007.
First published online in STEM CELLS EXPRESS   August 16, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The sensory nerve is highly involved in lower extremity wound healing. In diabetic and vascular diseases, impaired nerve function and blood flow delay wound healing. Tissue regeneration using adult stem cells is a targeted therapeutic modality in disorders of nerve and blood supply. Effective delivery using an autologous vascularized fascial flap as a vehicle of stem cells leads to severed sensory nerve recovery, local tissue blood flow, and wound healing. Human MSCs (hMSCs) were transfected with green fluorescent protein (GFP) cDNA and tested for efficiency and proliferation in vitro. The nude rat model with femoral vessel and saphenous nerve severance and ligation was wrapped with a vascularized epigastric flap for GFP-hMSC, fibroblast growth factor-2 (FGF-2), or a combination of both after 2 weeks. Maximum nerve conduction velocity recovered to 70% of the presurgical level in the GFP-hMSC- and FGF-2-treated group at 2 weeks. Blood flow and nerve conduction velocity were positively correlated at 1 week. Wound healing in the ipsilateral paw had significantly improved by 1 week. Histologically, blood vessels and nerves are very organized, and regenerated neuron immunoreactivity of GAP-43 and a nerve regrowth marker of S-100 were remarkable in the human GFP (hGFP)-hMSC and FGF-2-treated group at 2 weeks; therefore, sensory nerve regeneration, blood flow, and wound healing were improved by the administration of stem cells and FGF-2 via a vascularized flap. This may be implicated in clinical denervated and reduced circulation tissue wound healing.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Denervation or the impairment of motor, sympathetic, or sensory nerves may result in delayed skin wound healing [1–3]. Nerve repair or regeneration has been attempted by various methods, including immediate end-on-end repair with brain-derived neurotrophic factor [4] and thin-walled nerve guide or autologous nerve graft [5]. Among these peripheral nerve regenerations, stem cell transplantation may be a future modality. Skin-derived stem cells are able to regenerate nerves with nerve guides [6], whereas bone marrow mesenchymal stem cells in vitro are able to express glial markers and induce nerve regeneration with glial growth factor [7]. Peripheral nerve involvement in healing problems was demonstrated in an experimental model of the rabbit medial collateral ligament [8]. Also, peripheral nerve impairment directs neuropathy and is worsened with ischemic physical conditions. Reduced sensation, as well as motor or autonomic deficits, may lead to diabetic neuropathy and ulcers [9].

In lower peripheral nerve impairment, clinical saphenous nerve injuries are sometimes accompanied with a saphenous vein stripping procedure and objectively assessed with clinical symptoms [10], causing sensory disturbances in the medical thigh, knee, calf, and sole, as well as instability of these inner lower extremity structures due to loss of motor nerve government. In this regard, investigation of the involvement of the saphenous nerve in sole wound healing may bring about insight into the role of the peripheral nerve in in vivo wound healing with clinical relevance.

Fibroblast growth factors (FGFs), especially FGF-2 or basic FGF, hasten nerve regeneration after sciatic nerve crush by proliferating Schwann cells, inducing axonal growth, and prohibiting remyelination in a transgenic mouse model [11]. Schwann cells overexpressing the FGF-2 isoform successfully improve peripheral nerve regeneration when cells are grafted with Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com) [12].

Human mesenchymal stem cells proliferate in culture with attached well-spread fibroblast-like cell morphology [13]. The growth kinetics and differentiation were studied with human mesenchymal stem cells in subcultivation, followed by cryopreservation [14]. The early phase of cell profiles and the expression pattern by cytokines of the human mesenchymal stem cells were investigated in vitro [15]. Bone marrow derived human MSCs (hMSCs) are stringently sorted by cell surface antigen markers to exclude hematopoietic markers such as CD14, CD45, and CD34 and include mesenchymal markers such as CD105, CD166, CD29, and CD44 [13, 14]. Mesenchymal stem cells have the capacity to differentiate and regenerate nerves under certain conditions [16] and are available for supporting nerve regeneration and myelination in rats when transdifferentiated mesenchymal stem cells are grafted [17].

The hMSCs are able to differentiate into bone in cranial bone defects and ectopic bone formation with an appropriate cell carrier and cytokines [18, 19]. The superficial epigastric fascial flap used in the ectopic bone formation model is an ideal tool for transferring hMSCs and cytokines. As hMSCs and FGF-2 separately induce peripheral nerve regeneration, combined treatment may be more efficient for nerve regeneration. The saphenous nerve severing and ligating model was tested for nerve regeneration by hMSC and FGF-2 administration via a vascularized epigastric flap, and sole wound healing was also investigated.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human Mesenchymal Stem Cells
Human mesenchymal stem cells from a single human bone-marrow donor were isolated by density gradient centrifugation and were strictly sorted as positive for markers such as CD105, CD166, CD29, and CD44 and negative for cell surface markers such as CD14, CD34, and CD45. These hMSCs were purchased from Lonza Walkerville, Inc. (Walkersville, MD, http://www.lonzabioscience.com), and cryopreserved cells were thawed immediately according to the manufacturer's instructions. Two independent donor-derived hMSCs (white female; lots 1F0658 and 1F1061) were used in the experiments. The cells were cultured in "basic medium" of Dulbecco's modified Eagle's medium (DMEM) containing low glucose supplemented with 10% fetal bovine serum (FBS) (heat-inactivated; Gibco, Tokyo, http://www.invitrogen.com), 200 mM L-glutamine, and penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in 95% humidified air and 5% CO2. The medium was changed every 3 days until the cells reached approximately 80%–90% of total confluence, and then the cells were passaged up to three times. Growth characteristics during the four passages in FBS were indistinguishable. The cells were washed using 10 ml of phosphate-buffered saline (PBS) and then liberated by exposure to 0.25% trypsin/1 mM EDTA (Gibco) for 3 minutes at 37°C, followed by tapping the dishes and the addition of 5 ml of culture medium. The cells were centrifuged at 400g and then resuspended in basic medium for the following in vitro examinations. The other cells were stored at –70°C until used in a solution containing 5% human serum albumin (IS Japan, Co., Ltd., Saitama, Japan, http://www.isjpn.co.jp) and 10% dimethyl sulfoxide (Sigma-Aldrich, Tokyo, http://www.sigmaaldrich.com) according to the manufacturers' instructions. Cells were counted three times using a Beckman Coulter Cell and Particle Counter (Beckman Coulter, Tokyo, http://www.beckmancoulter.com).

GFP-hMSC Cell Preparation
hMSCs (5 x 104) were seeded into 24-well plates with 500 µl of DMEM containing low glucose supplemented with 10% FBS, without antibiotics, at approximately 90% confluence overnight. The next day, 1 µl of gently mixed Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) was diluted in 50 µl of Opti-MEM-1 Reduced Serum Medium (Invitrogen). One µg of green fluorescent protein (GFP) DNA plasmid (pIRES-EGFP) (Clontech, Mountain View, CA, http://www.clontech.com), which contains human cytomegalovirus promoter, enhanced green fluorescent protein gene, and the bovine growth hormone poly(A) signal, was mixed with 50 µl of Opti-MEM-1 Reduced Serum Medium. Immediately, another 50 µl of Opti-MEM-1 Reduced Serum Medium was added to each well containing 50 µl of Opti-MEM-1 Reduced Serum Medium with Lipofectamine 2000. After a 5-minute incubation, the complex of plasmid and Lipofectamine 2000 was left at room temperature for 20 minutes and then added to each well of cells with gentle rocking. The complex-added wells were incubated for 3, 6, 18, or 24 hours at 37°C. The medium was changed to basic medium supplemented with 50 mg/ml Geneticin (G-418 sulfate; Gibco) instead of penicillin (100 U/ml) and streptomycin (100 µg/ml) for cell selection. The cell culture continued for 4 days before the first passage (P1), and G-418-added medium was continued for 3 weeks for complete selection of cells. The passage was repeated when cells reached 80%–90% confluence in each culture plate. Transfection efficacy was confirmed by fluorescent microscopy, and fluorescent cells were counted five times under each condition. DNA experiments followed the Nagasaki University Safety Committee guidelines for handling recombinant DNA, which were approved on July 4, 2002 (no. 0207040655).

GFP-Transfected hMSC Cell Growth Curve in Passage
After cells were successfully transfected with GFP DNA plasmid, the growth curve was tested from day 0 at the time of cell culture to day 3 to determine the best efficiency of cell transfection and proliferation.

FGF-2
Genetically recombinant human FGF-2 (Trafermin) was purchased from Kaken Pharmaceutical Co. Ltd (Tokyo, http://www.kaken.co.jp/english). Freeze-dried samples were dissolved in PBS and dissolved in culture medium 30 minutes before in vitro use. The concentration of basic FGF (bFGF) was within the physiological range, according to the manufacturer's instructions.

Animal Protocol
Fifty male F344/NJCl-rnu nude rats, ages 8–9 weeks and weighing 250–300 g, that were deficient in T-cell function, were used in this experiment. Animals were obtained from CLEA Japan (Tokyo, http://www.clea-japan.com) and housed in the laboratory animal center for biomedical research (Nagasaki University School of Medicine, Nagasaki, Japan), and the protocol of the animal experiment was approved by the Institutional Animal Care and Use Committee of Nagasaki University (0308150310). They were handled according to the guidelines established for animal care at the center. Each rat had free access to both sterile water and standard rodent soft chow ad libitum.

Surgical Procedure
Rats were anesthetized with 40 mg/kg body weight intraperitoneal injection of pentobarbital sodium, United States Pharmacopeial Convention (Nembutal) (Abbott, Abbott Park, IL, http://www.abbott.com). The animals were then placed in the supine position, and initially, a 4 x 4 cm2 axial-pattern fasciocutaneous flap was made. All of the branches from the flap pedicle (superficial epigastric artery and veins) were kept intact. After flap elevation, the cutaneous portion was removed, and the remaining pedicled fascial flap was then used to wrap the transected saphenous nerves and femoral vessels and for subsequent cell and cytokine delivery [20]. The actual size of the flap used for wrapping was 1.5 x 1.5 cm2, and both vessels and nerves were totally wrapped with the fascial (connective tissue) portion. All animals received 0.1 ml of DMEM containing either 5 x 106 GFP-transfected hMSCs, bFGF (10 µg), combined administration of the two, or vehicle of DMEM alone within 30 minutes prior to injection. Injection of the above solutions was into the proximal site of the femoral artery and veins, where the superficial epigastric vessels originate, after the distal site of the bifurcated femoral artery to the epigastric artery and veins was clamped, where the severed femoral artery and veins were also wrapped with a vascularized flap. All clamps were removed after a 10-minute incubation of the injection, as described previously [20]. Severance and ligation of the femoral vessels and saphenous nerve were confirmed by using 3-0 nylon on both proximal and distal stumps.

Rats were divided into the following groups:

  1. No-reconstruction group (n = 10): no reconstruction after severing and ligating both saphenous nerve and femoral vessels.
  2. Control group (vascularized epigastric flap alone) (n = 10): 5 µl of DMEM was added through the superficial epigastic artery.
  3. hMSC treatment group (n = 10): 5 µl of DMEM solution containing 5 x 106 GFP-transfected hMSCs was prepared 30 minutes before transplantation for each epigastric flap.
  4. FGF-2 group (n = 10): 5 µl of DMEM solution containing 10 µg of FGF-2 was prepared 30 minutes before transplantation for each epigastric flap.
  5. Combined group (n = 10): both 5 x 106 GFP-transfected hMSCs and 10 µg of FGF-2 were prepared 30 minutes before transplantation for each epigastric flap.

Blood flow was measured using a noncontact laser blood flowmeter (ALF 21N; Advance Co. Ltd, Tokyo, http://www.advance.jp/english). The maximum sensory nerve conduction velocity was determined with a digital stimulator (model PG4000A; Cygnus Technology, Inc., Delaware Water Gap, PA, http://www.cygnustech.com) and a gated constant current source stimulus isolator (model SIU90; Cygnus Technology) and amplified with a differential extracellular amplifier (model ER-1; Cygnus Technology). Needle electrodes were set with a 5-mm gap at the saphenous nerve after meticulous dissection from other tissues under a microscope, and all nerve experiments were shielded by a magnetic shield cage (Astec Co., Ltd., Fukuoka, Japan, http://www.astec.com). The "earth" of the needles was inserted into the distant lower calf, and a pair of excitation needles and a pair of collection needles were placed with a 5-mm gap for velocity measurement. All blood flow data and nerve conduction velocity data were immediately transferred to an Analogue-Digital converter of PowerLab/4ST (ML 750; ADInstruments Japan Inc., Nagoya, Japan, http://www.adinstruments.com), and data were analyzed with the accompanying software of Scope v3.7 for nerve conduction and Chart v4.2.3 for blood flow.

Data samplings were repeated five times, and the mean value for each animal was adopted for further group analyses. Measurements were performed at the distal periphery of the severed nerves and vessels preligation (10 animals per group), immediately after ligation (10 animals per group), at 1 week (5 animals per group), and at 2 weeks (5 animals per group).

After terminating blood flow and maximum nerve conduction velocity experiments, inguinal wounds were sutured with 5-0 nylon, and an ipsilateral foot paw wound was created with 4-mm punch biopsy instruments. The wounds were covered with transparent semipermeable membranes (Cathereep Flexible, Soft & Smooth roll, number 1510; Nichiban Co., Ltd., Tokyo, http://www.nichiban.co.jp/medical). The experimental diagram of nerve severance, vessel ligation, vascularized flap wrapping, blood flow, and nerve velocity measurement is shown in Figure 1.


Figure 1
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  		Figure 1. Diagram of the experiment. (A): Vascularized fasciocutaneous flap and severance of the nerve and artery. A 4 x 4 cm2 fasciocutaneous flap, which is a vascularized superficial epigastric flap bifurcated from the proximal femoral artery, was elevated, and the femoral artery peripheral to the superficial epigastric artery and saphenous nerve was severed and ligated. (B): Wrapping the severed nerve and artery with vascularized epigastric flap and creation of 4-mm diameter punch hole in the paw. After removal of the circumferential excessive tissue and skin portion of the flap, the severed nerve and artery were wrapped with a reduced-size 1.5 x 1.5 cm2 vascularized flap. Nerve conduction velocity was measured just proximal to the severance, with a pair of stimulus electrodes distally and a pair of action potential electrodes proximally. A 4-mm diameter punch hole was created in the ipsilateral foot. Abbreviation: A&V, artery and veins.

 
Wound sizes were photographed in the supine position using a 600 million dots-per-inch digital camera (Cyber-shot; Sony, Tokyo, http://www.sony.com) and an image analyzer (NIH Image, version 1.62) on a Macintosh computer. The macroscopic size of the remaining wound was calculated three times for each group of five animals at 1, 3, 7, and 14 days following the previous wound healing experiment [21].

Animals were kept at a constant room temperature of 25°C with 55% humidity, and ankle body temperature was measured using a noncontact thermometer (UNIVLOT, UT-2) at the beginning and end of experiments. Animals were euthanized at 1 (five animals per each group) and 2 (five animals per each group) weeks postoperatively. Sections including the possible reconstructed nerve and vessels were fixed in 4% paraformaldehyde solution and processed for H&E for histology and immunohistochemistry.

Histology
Specimens were fixed in cold 4% paraformaldehyde solution for 2 weeks, decalcified in EDTA, embedded in paraffin, and cut into 5-µm-thick sections. Slides were stained with H&E.

Immunohistochemistry
For axonal regeneration marker analysis, immunostaining for growth-associated protein (GAP)-43, the mouse monoclonal GAP-43 antibody (catalog no. sc-33705; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) was used. This GAP-43 antibody was raised against full-length GAP-43 of rat origin and was able to detect axonal membranous protein of rats, mice, and humans. The primary antibody was incubated overnight at a dilution of 1:50 by PBS per the manufacturer's recommendations, at 4°C, followed by a rabbit anti-mouse IgG rhodamine-conjugated secondary antibody (catalog no. AP160R; Chemicon, Temecula, CA, http://www.chemicon.com) at 1:500 for 1 hour at room temperature and 4',6-diamidinophenylindole dihydrochloride (DAPI) for nuclear DNA counterstaining DAPI (catalog no. 340-07971; Dojindo Molecular Technologies Inc., Kumamoto, Japan, http://www.dojindo.com) at 1:100 for 1 hour at room temperature.

For detection of the regenerated nerve fiber, S-100 immunohistochemistry was performed. Rabbit polyclonal anti-S-100 antibody (catalog no. N1573, ready-to-use; Dako Japan Inc., Kyoto, Japan, http://www.dako.com) was incubated overnight at room temperature, and then the anti-rabbit Histofine simple stain MAX PO kit (code 414151F; Nichirei, Tokyo, http://www.nichirei.co.jp/english/index.html) followed as a universal immunoenzyme polymer method according to the manufacturer's instructions with diaminobenzamine chromogen, and finally hematoxylin staining was used for counterstaining. All fluorescent analyses were performed using the Lumina Vision (Mitani Corporation, Fukui, Japan, http://www.mitani-corp.co.jp) bioimaging analysis system for GFP (green), DAPI (blue), and GAP-43 (red).

Statistical Analysis
The results of the percentage of hGFP-positive hMSCs and GFP-hMSC proliferation analyses are expressed as the means ± SD. Data were statistically compared with unpaired t test, and p values less than 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
DNA Transfection Efficiency
The percentage of transfection efficiency of GFP to hMSCs was investigated with 3-, 6-, 18-, and 24-hour incubations before cell selection and each passage from P1 to P4. The 3- and 6-hour incubations demonstrated statistical significance from P2 to P3 compared with 18- and 24-hour incubations (90.3% ± 5.3%, 87.1% ± 5.0%, 86.8% ± 5.8%, 88.1% ± 4.6%, 85.1% ± 3.4%, 81.5% ± 5.4%, 74.9% ± 4.8%, 69.3% ± 2.2%, 83.2% ± 5.7%, 85.7% ± 3.4%, 17.8% ± 2.8%, and 14.1% ± 2.7% for the P1-3 hour, P1-6 hour, P1-18 hour, P1-24 hour, P2-3 hour, P2-6 hour, P2-18 hour, P2-24 hour, P3-3 hour, P3-6 hour, P3-18 hour, and P3-24 hour incubations, respectively; p < .01 between 3- or 6-hour incubation vs. 18- or 24-hour incubation at each passage). The 3- and 6-hour incubations of P4 were significantly less than those of P1 to P3 (Fig. 2A). Thus, cell growth and in vivo transplantation of GFP-transfected hMSCs were determined at 3 or 6 hours of P1 to P3.


Figure 2
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  		Figure 2. Efficiency of the GFP gene in hMSCs and proliferation. (A): hMSCs were transfected with the GFP gene at various incubation times. The highest transfection efficiency was with either 3- or 6-h incubation and up to three Ps. (B): The cell proliferation curve after GFP transfection was followed up to day 3. There was a significance between P1 cells and P2 or P3 cells from day 1 to day 3 (p < .01). There was no significant cell proliferation between control (no human GFP transfection) and P1 cells at any time point. Abbreviations: GFP, green fluorescent protein; h, hour; hMSC, human MSC; P, passage.

 
Cell Growth of GFP-Transfected hMSCs
Initially, 2.5 x 104 of both 3-hour and 6-hour cells were counted at day 0 of the control (no transfected hMSCs) and first-, second-, and third-passage cells. Both control and first-passage cells proliferated more than three times at day 3, whereas second- and third-passage cells proliferated fewer than two times at day 3. There were significant differences between control or first-passage cells and second- or third-passage cells from day 1 to day 3 (Fig. 2B). We therefore decided to use 3- or 6-hour-incubated first-passage GFP-transfected hMSCs in vivo. All cell counting was measured in triplicate, and the average value was calculated for each plate. Cell death was minimal, and trypan blue cell viability assay revealed fewer than 3% nonviable cells up to day 3 in FBS.

Blood Flow
Tissue blood flow varied among animals. Initial blood flow at just before severance and ligation was 10–30 ml/minute per 100 g (tissue) and dropped to almost 0 just after ligation. Blood flow of each animal either 1 week or 2 weeks later was normalized to each animal's blood flow at preligation. Relative blood flow in the no-reconstruction group was 0.62 and 1.00 at 1 week and 2 weeks after experiment, respectively. There was a significant difference between 1 week and 2 weeks in the no-reconstruction group. The value in the control group with the vascularized epigastric flap for the defects demonstrated significantly increased blood flow at 1 week with a relative value of 1.71 compared with the no-reconstruction group, but there was no significant difference between 1 week and 2 weeks. Similarly, there was a significant increase in the FGF-2-treated group at 1 week with a relative value of 1.83 compared with the no-reconstruction group. The value at 2 weeks of the FGF-2-treated group was even lower, 1.78. The GFP-hMSC-treated group and GFP-hMSC- plus FGF-2-treated group were 1.59 and 1.51 at 1 week, respectively, and there was a significant increase by 2 weeks in both groups, to 2.15 and 2.29, respectively. At 2 weeks, combined GFP-hMSC and FGF-2 treatment demonstrated the most significant blood flow increase. Except for the GFP-hMSC group, there was a significant difference (1.00, 1.71, and 1.78 for no-reconstruction, control, and FGF-2 groups, respectively; p < .01) (Fig. 3).


Figure 3
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  		Figure 3. Tissue blood flow relative to the presurgical value. A noncontact laser blood flowmeter was used. Data samplings were repeated five times, and the mean value for each animal was adopted for further group analyses. Measurements were performed at the distal periphery of the severed and ligated femoral vessels preligation (10 animals per group), immediately after ligation (10 animals per group), at 1 w (5 animals per group), and at 2 w (5 animals per group). There was a significant blood flow increase in all group using the vascularized epigastric flap compared with the no-reconstruction group at 1 and 2 w (p < .01). The green fluorescent protein-hMSC and FGF-2 group demonstrated a significant time-dependent increase compared with control (flap-alone) and FGF-2-alone groups at 2 w (p < .01). Abbreviations: FGF, fibroblast growth factor; hMSC, human MSC; w, week(s).

 
Nerve Conduction Velocity
Baseline sensory maximum nerve conduction velocity (sMNCV) was obtained just before nerve severance and ligation for each animal. Presurgical sMNCV varied from 3.23 to 33.33 m/second. Once the nerve was severed, the sMNCV immediately after surgery was 0 m/second for all examined nerves. The sMNCV of each animal either 1 week or 2 weeks later was normalized to each animal's sMNCV presurgery.

The relative value of no reconstruction was 0.05 and 0.08 at 1 and 2 weeks, respectively. All groups except the control at 2 weeks demonstrated significantly improved sMNCV compared with no reconstruction (0.13, 0.14, 0.14, 0.30, 0.15, 0.21, 0.44, and 0.68 for control at 1 week, control at 2 weeks, GFP-hMSC at 1 week, GFP-hMSC at 2 weeks, FGF-2 at 1 week, FGF-2 at 2 weeks, GFP-hMSC and FGF-2 at 1 week, and GFP-hMSC and FGF-2 at 2 weeks, respectively; p < .01 compared with each week of no reconstruction except the 2-week control). The value of sMNCV in the GFP-hMSC group and the GFP-hMSC with FGF-2 group demonstrated a significant increase between 1 and 2 weeks after surgery (0.14 and 0.44 vs. 0.30 and 0.68 for the GFP-hMSC and GFP-hMSC with FGF-2 group at 1 week vs. the GFP-hMSC and GFP-hMSC with FGF-2 group at 2 weeks, respectively; p < .01) (Fig. 4).


Figure 4
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  		Figure 4. Sensory nerve maximum conduction velocity relative to the presurgical value. Needle electrodes were set with a 5-mm gap at the saphenous nerve after meticulous dissection from other tissues under a microscope, and all nerve experiments were shielded by a magnetic shield cage. All nerve conduction velocity data were immediately transferred to an AD converter. Data samplings were repeated five times, and the mean value for each animal was adopted for further group analyses. Measurements were performed at the distal periphery of the severed nerves and vessels preligation (10 animals per group), immediately after ligation (10 animals per group), at 1 w (5 animals per group), and at 2 w (5 animals per group). There was a significant nerve conduction velocity increase in the green fluorescent protein (GFP)-hMSC and FGF-2 group compared with other groups at 1 and 2 w (p < .01). The GFP-hMSC-alone and GFP-hMSC and FGF-2 groups demonstrated a significant time-dependent increase (p < .01). The GFP-hMSC and FGF-2 group demonstrated the most significant increase at 2 w (p < .01). Abbreviations: FGF, fibroblast growth factor; hMSC, human MSC; w, week(s).

 
Correlation Between Maximum Sensory Nerve Conduction Velocity and Tissue Blood Flow
The correlation between maximum sensory conduction velocity and tissue blood flow was analyzed with a simple regression curve of the independent value of all blood flow and of a dependent value of maximum nerve conduction velocity. A significant positive correlation was observed between the two with the following formula: y = 0.22x – 0.12; r = 0.58; p < .01.

Wound Healing of the Paw
The wound size was initially 12.56 mm2 at day 0. At day 1, the mean wound size with no reconstruction, the control, GFP-hMSC, FGF-2, and GFP-hMSC and FGF-2 was 12.46, 12.95, 12.64, 12.42, and 13.00 mm2, respectively. There was no significant difference from day 1 to day 3. The wound size was significantly smaller in the GFP-hMSC-treated wound group than any other group at day 7. Wounds in all groups but the no-reconstruction group were healed by day 14. The no-reconstruction group demonstrated wounds of 1.76 mm2 (Fig. 5).


Figure 5
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  		Figure 5. Wound healing curve of the ipsilateral paw. The initial wound size was 12.56 mm2 and was measured at days 1, 3, 7, and 14. The wound size in the supine position was photographed using a 600 million dots-per-inch digital camera and an image analyzer (NIH Image, version 1.62) on a Macintosh computer. The macroscopic size of the remaining wounds was calculated three times. Significantly accelerated wound healing was observed in the green fluorescent protein-hMSC and FGF-2 group compared with any other group at day 7 (p < .01). All wounds except the no-reconstruction group demonstrated complete healing by day 14. Abbreviations: FGF, fibroblast growth factor; hMSC, human MSC.

 
Histology
The distal region from the severed and ligated saphenous nerve and femoral artery was histologically examined at 2 weeks postoperatively. The inner layer of the control artery was hypertrophic, and the nerve was atrophic. In contrast, the FGF-2-treated artery and nerve were more vascularized around the neuron-vascular bundles. The GFP-hMSC-treated artery was more elastic, and the nerve demonstrated denser and tight organization; the GFP-hMSC-treated artery, demonstrating well-vascularized surroundings, and the saphenous nerve were located adjacent to the artery, with a tight connection. Nerves in the vascularized epigastric fascial flap were larger than those of severed and regenerated nerves in the distal region of the severance (Fig. 6A, 6B, 6H, 6I).


Figure 6
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  		Figure 6. Histology and immunohistochemistry of the artery and nerve at 2 weeks (x200). For histology, the inner layer of the control artery was hypertrophic, and the nerve was atrophic. The control nerve was fragile and isolated from the surrounding connective and capillary tissues. Combined GFP-human MSC (hMSC)- and fibroblast growth factor-2 (FGF-2)-treated artery was more elastic, and the nerve demonstrated more dense and tight organization (A, B, H, I). In the control, there was no immunoreactivity of GAP-43 or traceable GFP-positive cells except the internal elastic lamina, which is considered a nonspecific immunoreaction (C–F). The greatest immunoreactivity of GAP-43 was seen in the GFP-hMSC- and FGF-2-treated group. More intense immunoreactivity was observed near the artery and the GFP-hMSC and FGF-2 group mainly between the artery and the nerve (J–M). The arrows indicate triple coexpressions of GFP, GAP-43, and DAPI. S-100 protein was diffusely expressed in the combined GFP-hMSC and FGF-2 treatment group as brown chromogen (G, N). Abbreviations: DAPI, 4',6-diamidinophenylindole dihydrochloride; GFP, green fluorescent protein.

 
Tracking of GFP-Transfected Cells and Immunofluorescent GAP-43 Expressions
Grafted GFP-transfected hMSCs were traced 2 weeks postoperatively. In the control, there was no immunoreactivity of GAP-43 or traceable GFP-positive cells except the internal elastic lamina, which is considered a nonspecific immunoreaction. There was very faint GAP-43 immunoreactivity around the nerve of the FGF-2-treated group. The intensity of both GAP-43- and GFP-positive cells increased in the GFP-hMSC-treated group. Only the outer region of the nerve adjacent to the branch of the femoral artery demonstrated both GFP and GAP-43 with DAPI expression.

The greatest immunoreactivity of GAP-43 was in the GFP-hMSC- and FGF-2-treated group. More intense immunoreactivity was observed near the artery, and GFP-hMSCs were mainly between the artery and the nerve (Fig. 6C–6F, 6J–6M). There was coexpression in the envelope area of the nerve in the fascia of the vascularized epigastric fascial flap of the GFP-hMSC and FGF-2 group (Fig. 7).


Figure 7
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  		Figure 7. Immunoreactivity of GAP-43, GFP, and DAPI in control and GFP-hMSC groups at 2 weeks (x200). The outer areas of the nerve in the GFP-hMSC and FGF-2 treatment group demonstrated the coexpression of Gap-43, GFP, and DAPI. The arrows indicate triple coexpressions of GFP, GAP-43, and DAPI. Abbreviations: DAPI, 4',6-diamidinophenylindole dihydrochloride; FGF, fibroblast growth factor; GFP, green fluorescent protein; hGFP, human green fluorescent protein; hMSC, human MSC.

 
S-100 Immunohistochemistry
Combined GFP-transfected hMSCs and FGF-2 demonstrated clear and diffuse S-100 immunoreactivity in the saphenous nerve. In the control, there was no S-100 immunoreactivity in control group immunohistochemistry (Fig. 6G, 6N).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The human green fluorescent protein plasmid was successfully transfected to bone marrow-derived human mesenchymal stem cells. Transfection efficiency was conserved up to the third passage with 3- or 6-hour incubation. With 18- and 24-hour incubation, transfection efficiency significantly dropped at second and third passages compared with those with 3- and 6-hour incubation. In terms of cell proliferation, only first-passage cells demonstrated proliferation similar to that of the nontransfected control. In the next in vivo experiment, 3- or 6-hour-incubated first-passage cells were used. In the severed and ligated saphenous nerve and femoral artery, which were wrapped by a superficial epigastric vessel vascularized fascial flap as a carrier of FGF-2 and GFP-hMSC, relative blood flow was significantly increased time-dependently in the no-reconstruction group, GFP-hMSC, and GFP-hMSC and FGF-2 groups between 1 week and 2 weeks. Efficient blood flow increase was observed when the experiment was performed with GFP-hMSCs through the vascularized epigastric artery. Maximum conduction velocity of the saphenous nerve was most significantly well-recovered in GFP-hMSC and FGF-2 treatment at 1 and 2 weeks compared with any other treatment. There was almost 70% recovery to presurgical levels at 2 weeks in GFP-hMSC and FGF-2 treatment. The regenerated nerve was found at the distal site of the severed nerve. GAP-43 immunoreactivity was most clearly observed when reconstruction was performed with GFP-hMSC and FGF-2 treatment. The location of GAP-43 was mainly perineural tissues between the nerve and artery. This suggests that the bloodstream induced nerve regeneration. Nerve conduction velocity and blood flow are important, as a significant decrease of sciatic nerve blood flow and saphenous nerve conduction velocity was observed in streptozotocin-induced diabetic rats [22]. Basic fibroblast growth factor alone directly promotes the extension of regeneration of the frozen saphenous nerve in vivo [23] at 2 and 5 days. The nerve fiber regrowth demonstrated by S-100 immunohistochemistry was observed in the combined GFP-hMSC- and FGF-2-treated group, which is further evidence of this treatment efficacy in nerve regeneration [6]. In our experiments, FGF-2 enhanced tissue blood flow compared with no reconstruction at 1 and 2 weeks. The correlation between blood flow and nerve conduction velocity demonstrated a significant positive correction. The greater blood flow in the severed nerve in situ promotes better recovery of sensory nerve conduction. Blood flow is synergistically increased in the presence of GFP-hMSC at 2 weeks. This may be because FGF-2 induces blood flow not only via the arterial pedicle but by direct proliferation and differentiation effects to hMSCs, which was also observed in another wound healing model using a bilayered collagen sponge [21]. Wound healing was significantly improved with GFP-hMSC and FGF-2 treatment by 1 week. All wounds with a vascularized epigastric flap were completely healed by 2 weeks. Wounds with no reconstruction by a flap showed delayed healing at 1 and 2 weeks, and the wound was not healed by 2 weeks, whereas wounds of other groups with vascularized flap augmentation demonstrated complete healing. This investigation may contribute to human mesenchymal stem cell grafting by a vascularized epigastric flap model with cytokines leading to more local blood flow and nerve recovery, which is a fundamental factor in traumatized, diabetic, and vascular clinical settings.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This study was supported by Grants 16390511, 16591795, 16659487, 16791091, 17659562, 17659563, 18390478, 18591967, and 18659526 from the Ministry of Education, Sports and Culture of Japan.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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