MESENCHYMAL STEM CELL THERAPY TO PROMOTE LIMB

TRANSPLANT FUNCTIONAL RECOVERY

EMILIE B. FITZPATRICK, M.D.,1 MARY J. DEHART, B.S.,2 TOMMY A. BROWN, M.D.,1 and SHASHIKUMAR K. SALGAR, Ph.D.2*

Background: Limb transplantation is a viable option for reconstruction after traumatic limb loss; however, functional recovery can be sub-
optimal. The aim of this study was to determine whether mesenchymal stem cell (MSC) administration can improve limb transplant func-
tional recovery. Methods: Orthotopic syngeneic hindlimb transplants were performed in Lewis rats, followed by topical and intravenous
injections of syngeneic MSCs (5 3 106) or vehicle. Transplanted limb sensory and motor functions were tested by cutaneous pain reaction
and walking track analysis, respectively. Results: MSCs expanded ex vivo were CD291, CD312, CD342, CD441, CD45low, CD901, MHC
Class-I1, Class-II2, and pluripotent. Greater than 90% of limb transplants survived. At 4 weeks post-transplantation, the mean sensory
nerve (tibial, peroneal, or sural) function in MSC (n 5 9) and vehicle (n 5 9) groups was <0.3 on a scale of Grades 0–3 (0 5 No function;
3 5 Normal). By 8 weeks, the sensory scores for tibial, peroneal, and sural nerves were 2.2 6 0.7, 1.2 6 0.5, and 1.7 6 0.9 in the vehicle,
and 2.6 6 0.4, 1.0 6 0.9, and 1.7 6 0.9 in the MSC group, respectively (n 5 9/group). At 4, 8, 16, and 24 weeks, the overall sensory func-
tion was higher in MSC group (7/group). Sciatic Function Index (SFI), a measure of motor function, could not be calculated because of
poor foot prints; therefore, a novel grading system was developed. Bone fusion/vascularization as determined by X-ray films/laser Doppler
(2 week post-transplantation) were normal (n 5 3/group). Gastrocnemius muscle was atrophied (P < 0.05), and flexion contractures were
evident by 24 weeks. Conclusions: Bone marrow-derived MSC therapy appears to improve sensory function recovery in a rat limb trans-
plant model. V C 2016 Wiley Periodicals, Inc. Microsurgery 00:000–000, 2016.

Vascularized composite allotransplantation (VCA), or
composite tissue allotransplantation (CTA), is an expand-
ing practice among subspecialty transplant centers in the
reconstruction of complex tissue defects, to improve
functional as well as cosmetic outcomes. VCA involves
transfer of composite tissue grafts (bone, muscle, skin,
nerve, vessel, etc.) from one individual to another, as a
single functional unit. It is being increasingly utilized in
the reconstruction of traumatic injuries, such as amputa-
tions and severe burns. This emerging therapy has partic-
ular relevance in the military population: as of February
2013, there were 1,715 reported battle-injury amputations
sustained during the conflicts in Afghanistan and Iraq.
Most of these (1,493) were classified as major limb loss.1
The quality of life of a patient who has lost a limb can
be enhanced with VCA, which is an elective procedure.2
Since the first successful hand transplantation in 19983
and face transplantation in 2005,4 >100 upper limb and
30 face transplantations have been performed world-
wide.5,6 Outcomes of upper limb and face transplants have
1
Department of Surgery, Madigan Army Medical Center, Tacoma, WA
98431
2
Department of Clinical Investigation, Madigan Army Medical Center,
Tacoma, WA 98431
Disclosure: The authors declare no conflicts of interest, financial or otherwise.
The views expressed are those of the author(s) and do not reflect the official
policy of the Department of the Army, the Department of Defense or the U.S.
Government.
*Correspondence to: S. K. Salgar, Ph.D., Lead Research Physiologist, Chair,
Scientific Review Committee, Department of Clinical Investigation, Madigan
Army Medical Center, 9040 Fitzsimmons Drive, Tacoma, WA 98431-1100.
E-mail: Shashikumar.k.salgar.civ@mail.mil
Received 2 December 2015; Revision accepted 31 March 2016; Accepted
22 April 2016
Published online 00 Month 2016 in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/micr.30068
been promising in terms of graft (transplant) survival, func-
tional recovery, and psychosocial impact.6–8 Lower limb
transplantation is generally contraindicated due to assumed
risks that outweigh the benefits; nonetheless, there are suc-
cessful reports.7,9,10 Lower limb prostheses are not feasible
for all patients; therefore, limb transplantation may be the
only alternative to those patients.10
Unlike visceral solid organ transplants (e.g., liver), VCA
requires optimal nerve regeneration and reinnervation of
graft motor and sensory targets, to attain intrinsic muscle
function and sensory input.11–13 The mammalian nervous
system has a limited axonal regeneration capacity following
injury.14,15 Functional recovery after peripheral nerve injury
requires axonal outgrowth, myelination, and targeted rein-
nervation.16–18 Microsurgical techniques have advanced
peripheral nerve repair considerably, allowing precise epi-
neural or perineural suture approximation. Novel cellular
therapies are sought to enhance nerve regeneration and func-
tion, including stem cell transplantation.14,16,19–24 The bene-
ficial effect of exogenous stem cells on peripheral nerve
regeneration is not completely understood. However, several
reports suggest that cell replacement, trophic factor pro-
duction, axon guidance/sorting, remyelination, immune
modulatory effects etc., of stem cells may all contribute to
peripheral nerve regeneration.14,23–32
Mesenchymal stem cells (MSCs) are pluripotent progen-
itor cells found in many peripheral tissues, including bone
marrow. They can be readily expanded ex vivo, and have the
potential to self-renew and differentiate into several lineages,
including neuronal cell types such as Schwann cells.14,33,34
MSCs have been shown to have therapeutic benefit in animal
models of Parkinson’s disease, multiple sclerosis, stroke, spi-
nal cord injury and peripheral nerve damage.35–38

Ó 2016 Wiley Periodicals, Inc.

2 Fitzpatrick et al.
Furthermore, MSCs have anti-inflammatory and immuno-
suppressive properties.39,40 Due to the unique combination
of proregenerative and immunomodulatory properties, MSC
therapy in the setting of VCA could potentially enhance
limb transplant function as well as promote tolerance.
Our objectives in this study were: (1) to examine whe-
ther bone marrow derived MSC administration can improve
sensory and motor nerve function recovery in limb trans-
plants; and (2) to successfully establish a rat orthotopic hind
limb transplant model at our institution for the first time.
MATERIALS AND METHODS
Animals
Ten- to 12-week-old inbred male Lewis (RT1l) rats,
weighing 300 g, were purchased from Harlan Sprague Daw-
ley (Indianapolis, IN). All experiments were conducted follow-
ing animal protocol approval by the Madigan Army Medical
Center Animal Care and Use Committee, and as per institu-
tional guidelines. Animals involved in this study were main-
tained in accordance with the ‘Guide for the Care and Use of
Laboratory Animals’ published by the National Research
Council/Institute of Laboratory Animal Research (ILAR).
Mesenchymal Stem Cell Isolation and Expansion
Rats were euthanized by injecting sodium pentobarbitol
(40–80 mg/rat) intraperitoneally. Bone marrow cells
(BMCs) were isolated from femur and tibia, and suspended
at 5–10 3 107 cells/mL in MSC complete or growth
medium (Dulbecco’s Modified Eagle’s medium2low glu-
cose, glutamax, pyruvate, 10% fetal bovine serum, penicil-
lin [100 U/mL], and streptomycin [100 mg/mL], Gibco/
Life Technologies, NY), as described previously.41 BMCs
were then plated at a density of 5 3 105 cells/cm2 and cul-
tured at 378C in 5% CO2 for 72 h in complete medium.
After 72 h, nonadherent cells in the supernatant were
removed completely and the medium was replaced with
fresh medium. Adherent cells were propagated for approxi-
mately 2–4 weeks, subcultured, and frozen (passage 3) in
freezing medium. Approximately 1–2 weeks prior to injec-
tion, frozen cells were thawed and expanded.
Mesenchymal Stem Cell Characterization
and Differentiation
The monoclonal antibodies [mAbs (clones)] used for
MSC characterization were as follows: antirat CD34
(ICO115) (Santa Cruz Biotechnology, Santa Cruz, CA);
anti-rat CD11b/c (OX-42), CD29 (Ha2-5), CD31
(TLD3A12), CD44H (OX-49), CD45 (OX-1), CD90 (OX-
7), class I-RT1.A (OX-18), class II-RT1.B (OX6), and iso-
type control antibodies (BD Biosciences, San Jose CA). All
antibodies used were fluorochrome labeled mAbs. The cells
obtained from independent cultures (passage 8; n  4)
were sorted using forward and side scatter properties by
Microsurgery DOI 10.1002/micr
flow cytometry (Guaua Technologies, Millipore, CA,
USA). The representative MSC population of interest was
gated and analyzed for cell surface markers (Fig. 1A). Ex
vivo expanded MSCs (passage  8; n  4) were studied for
their in vitro differentiation potential in to multilineages
using osteogenesis (A10072-01), adipogenesis (A10070-
01), and chondrogenesis (A10071-01) differentiation kits
(GIBCO, Life Technologies, NY) as per manufacturer’s
instructions. Osteocyte (red stained calcium deposits), adi-
pocyte (red stained lipid granules), and chondrocyte (blue
stain indicating synthesis of proteoglycans by chondrocytes
and developing chondrogenic pellets) differentiation are
shown in Figure 1B.
MSC Transplantation
Briefly, ex vivo expanded MSCs (passage 8) were
suspended in cold phosphate buffered saline (calcium and
magnesium free), filtered (40–70 mm pore size) to create
a homogeneous solution, and kept on ice until injected
(<2 h). These measures were to prevent cell aggregation
and decrease the risk of animal death due to pulmonary
embolism. Topical MSC administration: following limb
transplantation, MSCs (5 3 106 cells per animal in
0.5 mL saline) or vehicle were infused locally at the
sites of bone fusion, vascular anastomosis and nerve
repair prior to muscle approximation and skin closure.
This was done only once on the day of surgery. Intrave-
nous administration: immediately after surgery and clos-
ing the surgical wound, MSCs (5 3 106 cells per animal
in 1–1.5 mL of saline) or vehicle were injected (Injection
1) intravenously (IV) over 2 min via the dorsal penile
vein. Three more intravenous MSC (5 3 106 cells) or
vehicle injections (Injections 2, 3, and 4) were repeated
at weekly intervals.
Surgical Procedure
The general surgical techniques used for orthotopic
limb transplantation are as previously described.42,43
Briefly, the animal was anesthetized with intraperitoneal
(IP) injection of ketamine (40–80 mg/kg) and xylazine
(5–10 mg/kg), and anesthesia was maintained with inhal-
ant 1–2% isoflurane. The surgical site was prepared and
a skin incision was made around the circumference of
the right hind limb at the level of the inguinal ligament.
Beginning laterally, the skin of the upper thigh was
mobilized to expose the biceps femoris. The biceps fem-
oris was then divided near the distal attachments to the
stifle and tibia, leaving a sufficient edge of residual tissue
for later repair. The biceps was reflected to expose the
sciatic nerve within the stifle fossa. The sciatic nerve
was dissected out proximally to the point of emergence
from below the gluteus muscle, preserving mesoneurial
tissue. The sciatic nerve was transected proximally after
a tag suture of 10-0 nylon was placed. The femoral
 Stem Cells and Limb Transplantation 3

Figure 1. Mesenchymal stem cell (MSC): A. Characterization—MSC (passage 8; n  4) were stained with fluorescently labeled antigen
specific monoclonal antibodies and analyzed by FACS (fluorescence activated cell sorter) using flow cytometer. The MSCs were highly
positive for CD29, CD90, CD44 markers, moderately positive for RT1.A (Class I), and negative for CD31 (data not shown), CD34, CD45,
and RT1.B (Class II); representative histograms are shown in the figure. B. MSC differentiation—MSC (passage 8; n  4) were cultured
with osteogenesis, adipogenesis and chondrogenesis differentiation medium and stained with 2% Alizarin red, 0.5% Oil O Red, and 1%
Alcian Blue, respectively. The red calcium deposits are indicative of osteocyte, red lipid droplets are indicative of adipocyte, and blue carti-
laginous pellets are indicative of chondrocyte differentiation.

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4 Fitzpatrick et al.
vessels were isolated from the inguinal ligament to the
level of the epigastric takeoff. Tag sutures of 10-0 nylon
were placed proximally on the femoral vessels. All mus-
cle groups were then sharply divided slightly proximal to
the level of the mid-femur. Heparin (300 mL [50 IU])
was administered via the tail vein for anticoagulation.
The femoral vessels were then clamped above the previ-
ously placed suture tags and transected. Using a 22-
gauge angiocatheter, approximately 5–10 mL of ice-cold
TM
Plegisol solution (Hospira, Lake Forest, IL) was per-
fused through the femoral artery, until the venous efflu-
ent was clear. The osteotomy was performed at the mid
R
femur using a rotary saw (DREMELV 7300-N/8 Mini-
Mite 4.8-V, Robert Bosch Tool Corporation, Racine, WI)
with a stainless steel saw blade. Once detached, the
donor limb (graft) was wrapped in moist gauze and
placed on ice, until the recipient animal was prepared
and ready for transplantation. The donor animal was
euthanized.
The recipient animal received a preoperative antibi-
otic, cefazolin (25 mg/kg body weight) subcutaneously
(SQ), and was anesthetized and prepared for surgery. The
right hind limb was removed in the same fashion as for
the donor, with division of the femoral vessels, sciatic
nerve, and muscle groups occurring further distally to
preserve adequate tissue for approximation with the
donor limb. The donor limb was transplanted by per-
forming osteosynthesis of the femur using an intramedul-
lary pin, as described previously43 and orthopedic bone
cement (Simplex P, Stryker, Mahway, NJ), to achieve a
rigid fixation. The vascular anastomoses were performed
using the vascular cuff technique, as described previ-
ously.42,44 Briefly, thin-walled polyimide tubing (River-
tech Medical LLC, Chattanooga, TN) was used to
fashion arterial (0.724 mm inside diameter, 0.025 mm
wall thickness) and venous (1.151 mm inside diameter,
0.025 mm wall thickness) cuffs approximately 2.5 mm in
length. The sciatic nerve was repaired using 2–4 simple
interrupted epineurial sutures of 10-0 nylon (Figs. 2A
and 2B). MSCs or vehicle was administered topically at
the sites of the nerve repair and vascular anastomoses,
and injected at the site of muscle repair and bone fusion.
All muscle groups were approximated with 6-0 prolene.
The skin was closed with interrupted 4-0 nylon sutures
and stainless steel clips.
Postoperative Management
Lactated ringers solution (5 mL, SQ), buprenorphine
(0.02–0.05 mg/kg, SQ every 12 h as needed) and cefazo-
lin (20 mg/kg, SQ every 12 h for 3 days) was adminis-
tered postoperatively. Daily/weekly body weights were
monitored and animals were closely observed for signs
of pain or distress. Physiotherapy was provided 1–2 times
per week, beginning 1–2 weeks after surgery. The animal
Microsurgery DOI 10.1002/micr
was manually restrained and the transplanted limb was
gently and repeatedly manipulated through the normal
range of motion.43 Each session lasted up to 5 min, as
long as the animal tolerated it well.
Experimental Design
Group A (n 5 9 rats) received syngeneic MSCs, and
Group B (n 5 9 rats) received vehicle (saline control).
Both groups underwent right hind limb transplantation,
followed by MSCs or vehicle administration. Starting 1
week post-transplantation, all animals received manual
physiotherapy for the transplanted limb (5 min, 1–2
times per week) as described previously.43 The following
parameters were studied: cutaneous pain reaction test for
sensory nerve function was done on a weekly basis and
walking track analysis for motor function every two
weeks. Randomly selected animals (2 week post-
transplantation; n 5 3 from each group) underwent laser
Doppler imaging to assess vascularization of the trans-
planted limb, and radiologic analysis to assess fusion of
the donor and native femoral ends. Animals were eutha-
nized at 26–32 weeks post-transplantation and gastrocne-
mius muscle weights (transplanted limb and contralateral
native limb) were recorded. In addition, donor and recipi-
ent animal weights at the time of transplantation, recipi-
ent weight at euthanasia, and transplant limb ischemic
time were recorded.
ASSESSMENT OF LIMB FUNCTIONAL RECOVERY
Cutaneous Pain Reaction (the Flexor
“Withdrawal” Spinal Reflex)
The somatosensory reinnervation of the transplanted
limb was assessed by cutaneous pain reaction in response
to a painful stimulus applied to the distal limb, as described
previously.43 Testing began 2 weeks post-transplantation
and continued at weekly intervals until the end of the study
period. Animals were not sedated or anesthetized for this
analysis. Briefly, animals were restrained by a gentle hand-
hold with the hindlimbs in suspension, and testing began
once the animal was relaxed. Atraumatic forceps were used
to apply a brief painful stimulus in selected areas of the
foot, innervated by the tibial, peroneal, sural and saphenous
nerves (Fig. 3A), as described previously.43,45 The stimulus
was first applied to the native (left) hind limb, and the
response was observed. The stimulus was then applied in
the same area on the transplanted hindlimb (right), and the
response was graded in comparison to the native limb. The
withdrawal reflex was graded using a subjective scale as
described previously46: 0, No response; 1, Mild response;
2, Moderate response; 3, Strong response (normal). Each of
the four nerve territories was tested three times per session
and each response graded separately (Fig. 3). It should be
noted that sensory testing used in this study is a standard
 Stem Cells and Limb Transplantation 5

Figure 2. Rat hindlimb transplantation: A. Schematic of donor and recipient surgeries; B. Transplant surgery—under general anesthesia
(B1) medial (B2) and lateral (B3) aspects of the thigh exposed show femoral vessels and sciatic nerve, respectively. The limb was
removed at the mid femur from the donor animal (B4). The recipient animal was prepared by removing the right hind limb (B4) and the
donor limb was attached to the recipient stabilizing with intramedullary pin and bone cement (B4–5). Medially, femoral vessels were anas-
tomosed using vascular cuff technique (B6–8). Laterally, the donor and recipient sciatic nerve ends were approximated surgically with
10.0 silk sutures (B9–10). The opposing respective muscles/tissues of the donor and recipient were aligned and sutured. Skin was
approximated using clips (B11). C. Laser Doppler analysis—two or more weeks following limb transplantation laser Doppler analysis
revealed normal vascularization. D. Radiograph analysis—six to eight weeks following transplantation, radiographs confirmed significant
bone fusion and intramedullary pin (rod) in proper position. a, Artery; v, vein; D, donor; R, recipient; DSN, donor sciatic nerve; RSN, recip-
ient sciatic nerve; SNR, sciatic nerve repair; Tx, transplanted limb; N, native limb; IMP, intramedullary pin.

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6 Fitzpatrick et al.

Figure 3. Cutaneous pain reaction assessment: A. Sensory nerve function—was assessed by pinch technique in the territories of the tibial
(T), peroneal (P), sural (Sur), and saphenous (S) nerves as described, previously.42 Withdrawal/vocal response was scored in comparison
to the normal limb (0 5 no response, 1 5 slight, 2 5 moderate, 3 5 normal). L, lateral; M, medial. B–E. Mean sensory score of individual
nerves in vehicle and mesenchymal stem cell (MSC) treated groups up to 26 weeks post-transplantation. Tibial nerve function was first
recovered followed by sural, peroneal, and saphenous nerves. F. Overall sensory nerve function—there was no significant (P > 0.05) dif-
ference between vehicle and MSC treated groups. However, sensory function recovery was slightly better in MSC group than in vehicle
group.

technique used by several investigators in the past, and is
subjective in nature; investigator in this study was very
well trained to perform such a testing.
Walking Track Analysis
Motor function of the transplanted limb was assessed by
walking track analysis, as described previously.47–50 Briefly,
animals were tested in a confined walkway (approximately
10 cm wide 3 10 cm high 3 70 cm long), which was lined
with white paper and led into a dark shelter. Water-soluble
black ink was applied directly to the plantar surfaces of the
hind feet, and the animal was placed at the end of the walk-
way and allowed to walk down the corridor into the shelter;
this was repeated three times, with fresh ink applied each
time. Animals underwent conditioning trials 3–5 days prior to
transplantation. Testing began 2 weeks post-transplantation
and continued at 2-week intervals until the end of study
period. The sciatic function index (SFI), a conventional mea-
sure used to assess hindlimb motor function, 47requires mea-
surement of three print characteristics: print length [distance
between the heel and 3rd toe], toe spread [distance between
the 1st and 5th toe] and intermediary toe spread [distance
between the 2nd and 4th toe]. Due to the poor quality of foot
prints obtained in this study, the SFI could not be calculated.
Therefore, a novel grading scale was developed based on the
presence of a heel print and the number of individual toe
prints. The prints were graded from 0 (no print) to 4 (complete
print) as shown in Figure 4.

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 Stem Cells and Limb Transplantation 7

Figure 4. Walking track analysis: (A) Progression of walking track (foot prints)—normal and transplanted limb foot prints were obtained as
described previously.47,50 In both vehicle and MSC treated animals, the transplant limb foot prints became less distinct with time due to
progressive foot flexion contractures in the transplanted limb (4D). (B) Grading system for foot print analysis—we developed a novel grad-
ing system (grades 0–4) based on the quality of foot prints available to us. (C) Walking track analysis—there was no significant (P > 0.05)
difference between vehicle and MSC treated groups until 26 weeks post-transplantation. (D) Foot flexion contracture—all animals devel-
oped some degree of foot contracture by 20 weeks post-transplantation despite manual physiotherapy.

Laser Doppler and Radiologic Analyses
Animals randomly selected 2 weeks post-transplantation
underwent laser Doppler analysis to assess vascularization
of the transplanted limb. Under isoflurane anesthesia, the
hind limbs were shaved and the animal was placed in dorsal
recumbency in the laser Doppler imager, which was pro-
grammed to scan both hind limbs. The images were ana-
lyzed using the laser Doppler software, and the native limb
served as the control. For radiologic analysis, animals were
anesthetized and placed in the Kodak in vivo Imaging
System-FX (Carestream Health Inc., CA) in dorsal or ven-
tral recumbency. Both hindlimbs were included in the

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8 Fitzpatrick et al.

Table 1. Animal Weight, Ischemic Time, and Gastrocnemius Muscle Weight (Mean 6 SD) in Limb Transplanted Animals
P value between
columns (vehicle vs. P value between P value between
Group Vehicle MSC MSC) rows (vehicle) rows (MSC)
Donor weight (g) 288.5 6 19.7 297.4 6 38.9 P > 0.05 Donor vs. recipient Donor vs. recipient
(n 5 9)a (n 5 9)a weight P > 0.05b P > 0.05b
Recipient weight (g) 300.5 6 19.7 312.6 6 48.4 P > 0.05
(n 5 9)a (n 5 9)a
Ischemic time (min) 167.7 6 14.3 170.8 6 31.4 P > 0.05
(n 5 9)a (n 5 9)a
Weight at euthanasia (g) 441.6 6 24.3 460.4 6 63.1 P > 0.05
(n 5 7)a (n 5 8)a
Normal limb gastrocnemius 2.3 6 0.2 2.4 6 0.15 P > 0.05 Normal vs. transplant Normal vs. transplant
muscle weight (g) (n 5 7)a (n 5 8)a limb weight P < 0.05c limb weight
Transplanted limb gastrocnemius 1.52 6 0.3 1.37 6 0.2 P > 0.05 P < 0.05c
muscle weight (g) (n 5 7)a (n 5 8)a
MSC, mesenchymal stem cells.
a
Common superscripts (‘a’) between columns (vehicle and MSC) indicate no significant (P > 0.05) difference.
b
Body weight between donor and recipient animals at the time of transplantation in vehicle or MSC group did not vary significantly (P > 0.05).
c
Transplant gastrocnemius muscle weight was significantly (P < 0.05) reduced in both vehicle and MSC treated groups compared to their contralateral normal
limbs.

image field. The images were analyzed to assess fusion of
the donor and native femoral ends.
Gastrocnemius Muscle Mass
After euthanasia, the gastrocnemius muscles of the
transplanted and native hind limbs were carefully dis-
sected, harvested, and weighed; the mean gastrocnemius
muscle weights were compared between the groups.
Statistical Analysis
Statistical analysis was performed with SPSS software
version PASW Statistics18 (SPSS Inc., Chicago, IL). The
data between two groups were compared by Student t
test or ANOVA with Bonferroni correction. All P values
were two-tailed and values <0.05 were considered to
indicate statistical significance.
RESULTS
CD45 (hematopoietic stem cell-specific markers). Ex vivo
cultured MSCs (passage 8) were CD291, CD901,
CD441, CD31–, CD34–, CD45low, CD11b/clow, MHC
class I (RT1.A)1, and MHC class II (RT1.B) (Fig. 1A).
The MSC markers CD29 and CD90 were abundantly
expressed (>90%), and hematopoietic stem cell markers
(CD31, CD34, CD45) were least expressed (<1%). The
class I (RT1.A) was moderately expressed (<50%), and
Class II (RT1.B) was least expressed (<1%).
MSC differentiation
MSCs cultured in osteogenesis, adipogenesis, and
chondrogenesis differentiation media formed calcium
deposits (stained red) produced from osteocytes, lipids
(stained red) produced in adipocytes, and chondrogenic
pellets (stained blue) containing proteoglycans produced
from chondrocytes, respectively, as shown in Figure 1B.

Animal Characteristics Limb Transplant Surgery

The donor rat body weight was 288.5 6 19.7 and
297.4 6 38.9 g in vehicle and MSC treated groups,
respectively; the recipient rat weight was 300.5 6 19.7
and 312.6 6 48.4 g in vehicle and MSC treated groups,
respectively. There was no significant difference
(P > 0.05) between donor and recipient weights in either
vehicle or MSC treated groups (Table 1).
Mesenchymal Stem Cell Characteristics and
Differentiation
MSCs were confirmed on the basis of previously
accepted cell surface markers,51 including the presence
of CD29 and CD90 (stromal cell-specific markers), and
the absence or low expression of CD34, CD31, and
Overall, the limb transplant surgery was highly suc-
cessful, with >90% limb transplant survival at 2 weeks
post-transplantation. The operative time for donor surgery
was 75 min, and for recipient surgery (limb removal
and transplantation) was 120 min. The ischemic time
(time from donor limb vascular clamping to reperfusion)
was 167.7 6 14.3 and 170.8 6 31.4 min in vehicle and
MSC treated groups, respectively, and did not differ sig-
nificantly (P > 0.05) (Table 1).
Postoperatively, all animals developed edema in their
transplanted limbs that persisted for approximately 1
week. Animals bore weight (partially) on the transplanted
limb by 1–2 weeks, and started moving around actively
by 3–4 weeks.

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 Stem Cells and Limb Transplantation 9

0.16 6 0.25
1.52 6 0.47
1.91 6 0.57
2.25 6 0.34
2.39 6 0.33
2.68 6 0.21
MSC, mesenchymal stem cell injected group; Tx, transplantation. Sensory nerve function was graded from 0 to 3; 0, no response; 1, mild; 2, moderate; and 3, normal (see “ Materials and Methods” ). Sen-
sory response to individual nerve (tibial, saphenous, peroneal, sural) stimuli significantly (P < 0.05) improved over time in both vehicle and MSC groups. However, there was no significant (P > 0.05) differ-
ence between vehicle and MSC treated groups at any time point post-Tx tested. Interestingly, the total sensory score (determined by averaging individual nerve scores) in MSC treated group was better
Osteosynthesis of the femur using an intramedullary

MSC
rod and bone cement provided adequate fixation (Fig.
2B; 4, 5). Furthermore, radiographs demonstrated proper
Total bone fusion (Fig. 2D). The “cuff” technique employed
for vascular anastomosis (Fig. 2B; 7, 8) was highly effi-
cient; none of the transplants showed signs of vascular
0.21 6 0.20
1.35 6 0.33
1.73 6 0.36
2.16 6 0.27
2.47 6 0.24
2.36 6 0.36
Table 2. Sensory Nerve Function Score (Mean 6 SD) as Determined by Cutaneous Pain Reaction (Pinch Reflex) Test in the Transplanted Foot

Vehicle

leak or cuff failure during the entire post-transplantation

period. The sciatic nerve repair is shown in Figure 2B;
9, 10. The surgical wounds healed well, and sutures were
removed by 10–12 days. Several animals partially
removed the skin clips, which required replacements.
1.70 6 0.89
1.63 6 0.77
2.24 6 0.74
2.62 6 0.49
2.67 6 0.30
0.0401 6 1

One animal was euthanized due to self-mutilation of the

MSC

transplanted limb. The laser Doppler imaging of trans-

planted limb confirmed adequate vascularization (Fig.
Sural

2C), in addition to the visual assessment of pink toes for

blood supply.
0.33 6 0.71
1.67 6 0.94
1.78 6 0.80
2.00 6 0.37
2.54 6 0.56
2.46 6 0.40
Vehicle

ASSESSMENT OF LIMB FUNCTIONAL RECOVERY

Cutaneous Pain Reaction Test

Normal innervation results in an immediate with-
0.07 6 0.22
1.00 6 0.91
1.33 6 0.73
1.52 6 0.54
1.81 6 0.57
2.33 6 0.56

drawal response, with or without vocalization which was

MSC

graded 0–3 (see “Materials and Methods”). The mean

sensory function scores in the territories of individual
Peroneal

nerves (Fig. 3A) are presented in Table 2 and Figures

3B–3F. Sensory recovery was earliest in the distribu-
0.30 6 0.51
1.17 6 0.47
1.19 6 0.90
1.67 6 0.96
2.00 6 0.56
2.33 6 0.62

tion of the tibial nerve, followed by the sural, peroneal,

Vehicle

and saphenous nerve territories. At 4 weeks, post-

transplantation, the overall sensory nerve function was
grade 0.21 6 0.20 and 0.16 6 0.25 in vehicle and MSC
treated animals, respectively. However, by 6–8 weeks it
0.00 6 0.00
0.81 6 0.94
1.75 6 1.28
2.29 6 0.91
2.190 6 .79
2.780 6 .40

was significantly (P < 0.05) higher and clearly recogniz-

MSC

able, grade 1.0 (Fig. 3F), in both vehicle and MSC

treated groups. The overall sensory functional recovery
Saphenous

improved over time, reaching a maximum of grade 2.5.

There was no significant difference (P > 0.05) in overall
sensory nerve function recovery between the vehicle and
0.00 6 0.00
0.42 6 0.58
1.22 6 0.97
2.00 6 0.87
2.42 6 0.43
1.67 6 0.93
Vehicle

MSC treated groups. However, MSC administration

enhanced sensory functional recovery marginally (Fig.
3F; Table 2).
than vehicle group but was not significant (P > 0.05).

Walking Track Analysis

0.52 6 0.78
2.56 6 0.41
2.92 6 0.15
2.95 6 0.13
2.95 6 0.13
2.94 6 0.14

Using the grading system developed in this study (see

MSC

“Materials and Methods”), unexpectedly, the walking

track analysis revealed progressively poorer foot prints
Tibial

with time (up to 26 weeks) in both vehicle and MSC

treated groups (Fig. 4A). The grading system (grades 0–
0.22 6 0.44
2.17 6 0.69
2.85 6 0.34
2.96 6 0.11
2.92 6 0.15
3.00 6 0.00

4) is presented in Figure 4B. There was no significant

Vehicle

(P > 0.05) difference in mean print grades between the

vehicle and MSC treated groups (Fig. 4C). The cause for
poor prints was progressive flexion–contractures of the
foot (Fig. 4D). The severity of flexion contracture
post-Tx
Week

increased with the time post-transplantation; ultimately,

12
16
20
24

the motor function could not be assessed effectively.

4
8

Microsurgery DOI 10.1002/micr

10 Fitzpatrick et al.
Gastrocnemius Muscle Mass
The gastrocnemius muscle mass was significantly
(P < 0.05) reduced in the transplanted limb (1.37 6 0.2 g)
compared to normal limb (2.4 6 0.15 g) in MSC treated
group; and a similar finding was observed in vehicle
treated group (Table 1). There was no difference
(P > 0.05) in gastrocnemius muscle mass between normal
limbs (left) of vehicle and MSC treated animals, and
between transplanted limbs (right) of vehicle and MSC
treated animals.
DISCUSSION
We examined whether bone marrow derived MSC
administration can improve sensory and motor function
recovery in an orthotopic rat hind limb transplant model.
Our limb transplant surgical procedure included utilization
of the vascular cuff technique to anastomose donor and
recipient vessels, intramedullary pin and bone cement for
stable bone fixation, and sciatic nerve repair with epineurial
sutures. Our findings include restoration of limb transplant
sensory function (>75%), which was slightly improved
with MSC therapy, incomplete motor function recovery
secondary to flexion–contractures, and highly successful
surgical technique with >90% limb transplant survival and
<3 h of ischemia time. The follow-up period post-
transplantation in this study was about 26 weeks.
MSC therapy appeared to improve sciatic nerve func-
tion recovery, but was not significant. In our model,
MSCs were applied to the nerve repair after the donor
and recipient sciatic nerves were approximated with sur-
gical sutures. Goel and co-workers14 transplanted bone-
marrow derived mononuclear cells between the proximal
and distal ends of transected sciatic nerve in a nerve
transection model and found accelerated and enhanced
nerve regeneration. This effect may be due to stem cell
trophic factors causing axonal growth and stem cell dif-
ferentiation into Schwann-like cells, leading to myelin
reformation. In another study, transplanted Schwann cells
(derived from bone marrow MSCs) into transected sciatic
nerve resulted in vigorous nerve regeneration with myelin
synthesis in rats.35 There are other reports that demon-
strated the ability of bone marrow MSCs in differentiat-
ing into Schwann cell-like cells both in vivo and in vitro
and induce myelination of regenerated nerve fibers after
sciatic nerve injury.28,52,53 It should be noted that none
of the above studies described were in a limb transplant
model. We believe a similar mechanism should be
expected in a limb transplant model. To our knowledge,
there are no reports on the efficacy of bone marrow-
derived MSCs on limb nerve function recovery in an
orthotopic rat limb transplant model. Ours was the first
attempt in which MSC administration appeared to have
some benefit (sensory nerve function recovery).
Microsurgery DOI 10.1002/micr
Time of sensory nerve function recovery (4–6 weeks)
and significant progress observed with time (up to 26
weeks) in vehicle and MSC treated groups are in agree-
ment with the previous reports on limb transplanta-
tion.43,46 The poor foot prints obtained in walking track
analysis in the present study was mainly due to progres-
sive foot flexion–contractures. This feature has been
well-documented.48,54 Several procedures to remedy the
development of foot flexion–contractures have been
attempted in limb transplantation and crushed/transected
sciatic nerve injury models with modest success. Stras-
berg and co-workers55 used wire mesh floor to house the
rats in addition to manual physiotherapy following sciatic
nerve repair that reduced foot flexion–contractures and
improved recovery. Endo and co-workers54 used tread-
mill training protocol for hind limb transplanted rats that
improved plantar surface contact and foot prints. How-
ever, in the present study, none of the above methods
were combined with MSC therapy. Nonetheless, in the
present study animals were given 5 min manual physio-
therapy 1–2 times per week.
Yeh and co-workers43 used distal nerve anastomosis
techniques (tibial, peroneal, sural) which enhanced motor
function and prevented flexion–contractures. It has been
well documented since the early development of periph-
eral nerve repair techniques that outcomes are directly
related to the level of the nerve injury; distal injuries
have superior functional recovery to proximal injuries.
The triple nerve repair technique, though it requires more
time and is technically more challenging, may provide
superior outcomes in this model.56–58
Because of poor foot prints we were unable to calcu-
late Sciatic Function Index (SFI), a conventional measure
to assess limb motor function.49 According to our novel
grading system (grades 0–4) there was a significant
reduction in print scores with time in both vehicle and
MSC treated groups, opposite to what we anticipated.
Low print scores due to foot flexion–contractures is an
inherent limitation of walking track analysis, particularly
in the setting of limb transplant. While SFI has been reli-
ably used in models of sciatic nerve injury, more sophis-
ticated gait analysis techniques, such as the CatWalkTM
XT system (Noldus, Wageningen, The Netherlands), may
be better suited for studies in limb transplantation.
The surgical procedures in orthotopic-hindlimb trans-
plantation are extensive and time-consuming. Strong
bone fixation is vital for the success of limb transplanta-
tion. Osteosynthesis performed provided stable fixation
and prevented torsion as confirmed by our X-ray imaging
is in agreement with previous report.43,46 The cuff tech-
nique44 used to anastomose femoral vessels was less time
consuming (compared to conventional suture technique),
effective, and had no vascularization problems. Short
vascular anastomosis time (<20 min) reduced warm

ischemia time to a minimum which is in agreement with
the previous report.44 The donor and recipient sciatic
nerve repair in the present study grossly looked well
aligned, yet nerve function recovery was suboptimal; this
was possibly due to improper axon growth, improper
apposition/alignment of the nerve fascicles14,15,17,18,21,52,59
or other unknown factors.
In the present study, tissue quality was maintained by
limiting exposure to desiccation and minimizing the total
ischemic period (cold and warm) of donor limb. Cell atro-
phy, necrosis and fibrosis are proportionate to ischemic
times. It’s known that longer ischemic times result in more
severe morphological changes to the muscle. Tsuji and co-
workers60 observed that cold ischemic period of <8 h and
warm ischemic period of <4 h would restore good function
of major muscular extremities. The short ischemic time
(<3 h) in the present study prevented any major ischemic
damage to limb muscles.
The significant reduction in transplant gastrocnemius
muscle weight (muscular atrophy) was possibly due to poor
peripheral nerve regeneration and reinnervation of the target
muscles, compounded by prolonged disuse of the limb,
which is in agreement with previous reports.12,13 Self-
mutilation of the transplanted limb occurs in certain strains
of rats more commonly than in others.61 In the present study,
one animal showed some symptoms of self-mutilation of the
transplanted limb and was euthanized humanely.
Advanced immunosuppressive drugs and novel surgical
procedures have enabled successful VCAs. Nonetheless,
there are many challenges to counter prior to its broad
scale applications in humans: long-term complications
such as chronic rejection, graft-versus-host disease, lack of
optimal functional recovery, and poor understanding of
tolerance induction. Research to identify novel therapeutic
modalities (stem cells and small molecules) is expected to
make a significant improvement in the field of VCA.
Following are the specific measures and recommenda-
tions to overcome the limitations of the present study in the
future. (1) Due to equipment limitations, we provided pas-
sive, but not active, physiotherapy. Including wire-mesh
therapy or tread mill exercises may reduce contractures and
enhance functional recovery. (2) Motor function data was
limited in part by the use of the walking track analysis and
SFI as our endpoint. Alternative gait analysis techniques
such as CATwalk XT system (Noldus Information Tech-
nology, Leesburg, VA), which automatically classifies and
analyzes footprints, will likely generate more reliable data.
(3) Nerve transection and repair at the level of the sciatic
nerve might have resulted in poor target nerve innervations
and poor motor function. Performing the nerve repair closer
to the target of innervations at the level of sciatic nerve
branches (tibial, sural, and peroneal nerves) might enhance
muscle reinnervation, reduce contractures, and improve
functional recovery.
Stem Cells and Limb Transplantation 11
In conclusion, the strategy used to improve limb
transplant function recovery utilizing bone marrow
derived MSC administration is attractive, feasible and
deserve further investigation. Physiotherapy seems to be
vital for optimal limb transplant functional recovery.
When clear foot prints are not available to calculate sci-
atic function index (SFI), alternate methodology and
grading system developed in this study could be used to
measure motor function recovery.
ACKNOWLEDGMENTS
The authors acknowledge Mr. Juan Tercero, Ms. Joanna
Dandeneau, and Mr. John Schaphorst, Department of
Clinical Investigation, for their assistance with animal
care, anesthesia, and behavioral testing.
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