Dm Ulkus Jurnal

7/26/2019 Dm Ulkus Jurnal

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Open access publishingThe Journal of Diabetic Foot Complications

Role of combination cell therapy in non-healing diabetic ulcers in

patients with severe peripheral arterial disease a preliminary report

on ve casesAuthors:

The Journal of Diabetic Foot Complications, 2013; Volume 5, Issue 1, No. 1, Pages 1-14 All rights reserved.

Chandra Viswanathan1, Prathibha Shetty1, Shabari Sarang1, Khushnuma Cooper1, Deepa Ghosh1,

Arun Bal2

Abstract:

Key words: Mesenchymal stem cells, Haematopoietic stem cells, Combinational stem cell therapy,

Peripheral arterial disease

Corresponding author:

Dr. Chandra Viswanathan

Reliance Life Sciences Pvt Ltd,

Dhirubhai Ambani Life Sciences Centre,

R-282, TTC Area of MIDC,

Thane Belapur Road,

Rabale, Navi Mumbai- 400701.

Tel: +91-22-67678352; Fax: +91-22-67678099;

Email:[email protected]

Afliations:

1. Regenerative Medicine Group, Reliance life Sciences Pvt Ltd,

Dhirubhai Ambani Life Sciences Centre, R-282 TTC area of MIDC, Thane

Belapur Road, Rabale, Navi Mumbai-400701.

2. Dhanvantari Hospital and Research Center, D. L. Vaidya Road, Shivaji

Park, Dadar (West), Mumbai-400028.

1

Background:Peripheral arterial disease (PAD) is a growing medical problem and its management can be

a clinical challenge. Complications like non-healing ulcers are more challenging. Despite recent advances

in surgical and radiologic procedures, a large number of patients are not eligible for revascularization

procedures. In many such cases, amputation becomes inevitable. Recent evidences indicate that adult stem

cells are potentially new therapeutic targets. The present report is a summary of responses to combination

stem cell administration on 5 cases of PAD with non-healing ulcers. This is the rst report, depicting the

usefulness of different adult stem cell type combination, to address this unmet need.

Methods: Five patients with severe PAD and non-healing ulcers, in whom amputation was the only option,were considered for this short study. Here, we present short-term results of multiple intramuscular injections of

mesenchymal stem cells from umbilical cord matrix (UCMSC) and haematopoietic stem cells (HSCs, CD34+)

from the human umbilical cord blood, into the affected lower limbs, while allogeneic neonatal broblasts

were directly applied on the non-healing foot ulcers, Ankle brachial indices (ABI) were calculated before and

after the procedure, improvement in pain scores, wound closure, and prevention of limb loss were taken as

indicators of success.

Results:An average improvement of 0.2 to 0.45 in ABI measurements was noted, with no amputation of

the affected limb. Complete wound healing was achieved within three months in all patients. No recurrence

of ulcer was evident during this period. One patient voluntarily reported 14 months later, showing sustained

improvement. All patients demonstrated a decrease in the severity of symptoms as evidenced by alleviation

of rest pains and improvement by at least one level in Rutherford classication. There were also no adverse

events reported.

Conclusions: Thus, usefulness and effectiveness of this combination therapy to prevent limb loss in patients

with severe PAD, and non healing ulcer was demonstrated. A large multicenter study will be necessary to get

answers on the optimal dose, frequency and the ideal cell type to be used.


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Open access publishingThe Journal of Diabetic Foot Complications, 2013; Volume 5, Issue 1, No. 1, Pages 1-14

2

Peripheral arterial disease (PAD), arte-

riosclerosis obliterans and thromboangitis

commonly affect the arteries supplying blood

to the leg. It is one of the major manifestations

of systemic atherosclerosis affecting the lowerextremities and often culminating in critical limb

ischemia (CLI). Based on the severity of the

symptoms, usually two clinical presentations

are distinguished: intermittent claudicating (IC),

which is characterized by pain upon walking

while CLI is a more severe form in which pain

occurs at rest and is accompanied by necrosis

and ulceration1. CLI is characterized by a more

than 50% risk of major amputation within one

year without revascularization

2

and a poorprognosis with regard to survival. A substantial

number of patients with CLI remain unresponsive

to pharmacological therapies and are also un-

suitable candidates for endovascular or surgical

revascularization3, 4, 5.

PAD is estimated to develop in 500-1000

individuals per million persons per year. Seventy

to eighty percent of affected individuals are

asymptomatic; only a minority ever requires

revascularization or amputation6. One in 3diabetics over the age of 50 are affected by PAD.

Diagnosis is critical, as people with PAD have

a four to ve times higher risk of heart attack or

stroke. Despite its prevalence and cardiovascu-

lar risk implications, only 25% of patients with

PAD actually seek treatment. The incidence of

symptomatic PAD increases with age and as-

sociated risk factors, from about 0.3% per year

for men aged 4055 years to about 1% per year

for men aged over 75 years. As PAD progresses,

leg pains at rest and/or ischemic ulceration

are hallmark presentations. Treatments vary

from simple conservative lifestyle alterations to

complex invasive endovascular or open surgical

interventions in severe cases. Experience shows

that the end stage PAD is difcult to treat by tra-

ditional methods, leaving major limb amputation

as the only treatment option7.

The development of novel therapies to

stimulate neovascularization, a strategy known

as therapeutic angiogenic factors or recently

the use of stem cells, may represent an option

to promote revascularization and/or remodel-ing of collaterals, with the aim of ameliorat-

ing symptoms, promoting the regeneration of

damaged tissues and preventing amputation8,9.

Mesenchymal stem cells (MSCs) have

dependable differentiating properties, and can

change the milieu that promotes and helps

regeneration10. The hematopoietic and mesen-

chymal cells secrete growth factors that promote

neoangiogenesis and endothelialization leadingto development of collateral vascular networks11,

12, 13.

Our previously published studies on animal

models with ischemic limb disease [ILD] have

demonstrated that implantation of MSCs

improved tissue perfusion. MSCs signicantly

enhanced perfusion of ischemic tissue and

collateral remodeling, reduced tissue damage

and improved limb function14. A large number

of experiments in mice and larger animals havedemonstrated the feasibility and efcacy of cells

both MSCs and HSCs in restoring blood ow to

the ischemic limb15, 16, 17 .

MSCs are also immunomodulatory in nature

and hence are very good candidates for re-

generative medicine applications even in the

allogeneic setting. Autologous stem cells have

several advantages; however limitations such

as bone marrow collection, patients age, stem

cell isolation, preparation, and the consequent

variations in quality and quantity cannot be

overlooked. To overcome these inconsistencies,

several groups, including ours have investigated

the use of an alternative source for deriving

MSCs that overcomes all the above shortcom-

ings and consider their usage in the allogeneic

mode. Mesenchymal stem cells derived from the

Introduction


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3

umbilical cord have distinct advantages be-

cause of their good growth kinetics, differen-

tiation potentials, banking ability, naivety and

immunomodulatory functions, as compared to

most other adult stem cell types18. Similarly,

CD34+ cells from ones own bone marrow

would have been desirable, but not feasible inall cases. This limitation could be due to rea-

sons such as advancing age, debility, anemia

and infection that decrease bone marrow aspira-

tion opportunities. Hence both CD34+ cells and

MSCs from allogeneic sources are more desir-

able.

Fibroblasts derived from an allogeneic

source like the neonatal foreskin have been

extensively used in the regeneration of wounds.

Dermal broblasts of allogeneic origin do not

evoke an immune response, making it possible

to use allogeneic dermal broblasts as skin

substitutes19, 20. The lack of immune response

to allogeneic dermal broblasts has been attrib-

uted primarily to the absence of expression of

human leukocyte antigen (HLA)DR by the cells

and co-stimulatory molecules such as CD40 and

CD8021.

We have also previously reported that

the neonatal broblasts did not stimulate T-cellproliferation even in the presence of Interferon

gamma (IFN-), thus indicating the safety of the

broblasts in wound treatment22.

This brief report elucidates the role of

allogeneic stem cell combination in improving

the ischemic condition in PAD patients, who

otherwise had no treatment option but amputa-

tion. Autologous cell based studies have shown

favorable results, most of them being MSCs from

the bone marrow. This report is based on using

the combination cell type consisting of MSCs and

CD34+ positive cells derived from nave source,

which have proven immunomodulatory behavior.

This study was aimed at evaluating

the usefulness of a combination of allogeneic

stem cells (UCMSC + CD34+) implantation in5 consecutive patients with severe PAD with

an impending limb or a toe amputation; and to

evaluate the role of local application of neonatal

broblasts in the treatment of nonhealing ulcers.

Patient details:

At the time of commencement of this study,

all ve patients had received signicant medical

therapy for PAD, and were under the discretion

of the managing vascular surgeon. They wereincluded in the study if they had severe limb-

threatening PAD, dened as ABI less than 0.5

with presence of non-healing ischemic ulcers.

They had documented stenosis or occlusion of

any two of the following lower extremity arteries:

anterior tibial, posterior tibial, and/or peroneal.

Additional stenosis or occlusion may be present

proximal to these vessels. They may or may not

have undergone peripheral vascular angioplas-

ties or bypasses as a part of therapy.

All the biological samples used for the study

were obtained with approval from the Institution-

al Ethics Committee and informed consent from

donors.

A) Umbilical cord mesenchymal stem cell

preparation

Mesenchymal stem cells were prepared

from the human umbilical cord matrix .The

cord matrix was serially cut in a cross sectionalmanner and 4 to 5 explants of the matrix

ranging from 1-2 cm in size were placed in 100

mm tissue culture dishes with 2-3 ml of culture

medium. Adherent MSCs were harvested and

characterized by ow cytometry as per the inter-

national standards and cryopreserved in liquid

nitrogen until further requirement23.

METHODS


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B) CD34+ cell preparation

The CD34+ cells were sorted from Mono-

nuclear cells (MNCs) using sterile magnetic

beads (Miltenyi Biotech, Germany) following

the manufacturers protocol. For every 100x106

cells, 100l of FcR blocking reagent was added

to the MNC pellet and resuspended in 300l of

MACS Buffer (1xPBS with 4% serum) and mixed

well. One-hundred l of CD34+ microbeads

were also added to the cell suspension and

was incubated at 40C for 30-40 minutes. The

cells were then resuspended in MACS buffer

and passed through the magnetic column. The

positive fraction of CD34+ cells was collected

by depletion strategy in a fresh sterile centrifuge

tube. The cells thus collected, were counted and

characterized before expansion to conrm theirpurity. The pure population of CD34+ cells were

co-cultured with human mesenchymal stem cells

obtained from the human umbilical cord tissue

(UCMSCs) for a period of two weeks in Iscoves

Modied Dulbeccos Medium (IMDM) (Invitro-

gen, Singapore) with 10% Fetal Bovine Serum

(FBS) (Hyclone, USA), 10ng/ml SCF, 10ng/ml

Flt3, 10ng/ml TPO (Peprotech, USA) and 1ng/

ml bFGF (R&D systems, USA) with 1mM L-glu-

tamine (Invitrogen, Singapore)24. The expanded

CD34+ cells were then characterized by immu-nophenotyping for purity and viability.

C) Neonatal Fibroblast preparation

Human dermal broblasts were isolated

from neonatal foreskin biopsies. The dermis

was separated from the epidermis by treatment

with Dispase (Sigma-Aldrich, St. Louis, MO).

The dermis was digested with 0.017% collage-

nase (Gibco, Grand Island, NY) in DulbeccosModied Eagles Medium (DMEM) (SAFC Bio-

sciences, Lenexa, KS) with 10% Fetal FBS;

(Hyclone Labs, South Logan, UT) overnight. The

digested material was pelleted at 1000 rpm for

10 min, the pellet was resuspended in DMEM

10% FBS and then cells were allowed to attach

in a culture ask (Nunc, Roskilde, Denmark).

Cells were cultured in DMEM 10 % FBS at 37oC

in 5% CO2 and subcultured using 0.25% Tryp-

sin-EDTA solution (SAFC Biosciences).

The cell derivation and nal characteristics

in all the above cell types are compliant with the

international recommendations.

D) Stem cell dose and administration of stem

cell combination:

The total cell dose of UCMSCs and CD34+

cells was calculated at 2 million per kg body

weight, in the ratio of 80:20 respectively, to be

given intramuscular, along the travel path of the

vascular track approximately at 2-inch distances

as shown in Figure 1.

MSCs and CD34+ cells are transported in

dry ice at -80o C. These cells were mixed after

rapid thawing at 37oC and diluted in 24 ml of

saline. The surgeon was handed over loaded

syringes of stem cells for transplantation. Pre-

caution was taken to waste no time between

thawing, loading the syringe, and the injection

process.

Administration of stem cells:

Local skin preparation was done as per

routine procedure. The injection sites were

selected according to the angiographic ndings

to localize and understand the level of the

occlusion; Intramuscular injections were given

above and below this site. Thus approximately

Figure 1- The points of injection marked above and

below the block along the vascular track

4



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1 to 1.5 ml of cell volume was injected per site,

and about 10 sites at a distance of 2-3 inches

from each other. Marking was done ahead of

time to help expedite the procedure. The intra-

muscular (IM) injection thus was made into the

calf muscles (soleus and gastrocnemius) and the

popliteal fossa along the path of the blood owas shown in Figure 1. The cells were delivered

intramuscularly into the ischemic limb using 25G

needle in all cases, except in one case where it

was administered using a blunt lumbar puncture

needle to help with better cell dispersion.

Light pressure was applied after every IM

injection to ensure that the cells were dispersed

well into the tissues. All procedures were

performed by the treating senior surgeon.

For broblast implantation, the surface of the

wound bed was debrided. The broblasts were

suspended in saline adequate to inject 0.1 ml at

multiple sites along the ulcer bed and the walls.

The wound was then covered with ReliHeal-G,

a hydrogel dressing (Reliance Life Sciences,

India).

The gel was secured using Tegaderm (3M Health

Sciences, USA). The dressing was changed at

regular intervals of 1, 4, 8 and 12 weeks until

complete wound closure.

Safety and efcacy parameters post

transplantation:

Success following cell implantation was

dened as fulllment of at least three of the

following four criteria:

1. Improvement of ABI measurements

2. Healing and decrease in the ulcer size (clas-

sied as per the Wagner classication, (Table 1)

and appearance of fresh granulation tissue

3. Minimum of a one grade improvement in Ruth-

erford classication (Table 2)

4. Prevention of major limb amputations at the

three month follow up

This short study had three female and

two male patients. The mean age of this group

was 66.5 years. None of them were smokers.

Four of them were diabetic for at least 15 years

(Patient1, 2, 4 & 5) and one female patient

(Patient 3) was not diabetic (Figure 2). Three

of them were on dialysis for at least 2 years

and two of them were not. Four had rest pain

and varying sizes of non-healing ulcers prior to

being included into our study, and one female

patient had an impending ulcer on the foot, with

gangrene setting in on the great toe (Patient 4).

(Table 3 and Table 4)

5

Table 1: WAGNER CLASSIFICATION OF DIABETIC

FOOT ULCERS

Table 2: The Rutherford classication for ischemic

limb

Grade Conditions

0 No ulcer in a high risk foot

I Supercial ulcer involving the full skin thickness

but not underlying tissues

II Deep ulcer, penetrating down to ligaments and

muscle, but no bone involvement or abscess formation

III Deep ulcer with cellulitis or abscess formation, often

with osteomyelitis

IV Localized gangrene

V Extensive gangrene involving the whole foot

Stages Conditions

0 Asymptomatic

1 Mild claudication

2 Moderate claudication The distance that delineates

mild, moderate and severe claudication is not specied

in the Rutherford classication, but is mentioned in the

Fontaine classication as 200 meters.

3 Severe claudication

4 Rest pain

5 Ischemic ulceration not exceeding ulcer of

the digits of the foot

6 Severe ischemic ulcers or frank gangrene

RESULTS



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Patient

NumberAge and Gender (years) Grade of ulcer Conditions

1 70/F I 5

2 58/F II 4

3 75/M III 4

4 75/M III 5

5 65/M III 5

Patient

Number

Cerebrovascular

eventsCAD, RAD

Other risk

factorsHTN

Hypercholesterolae-

mia, hyperlipidaemia

Prior vascular

procedures,

Major procedures

Open endovascular

1 Old history of Transient

ischemic attack (TIA)

CAD, RAD Non Smoker,

Diabetic

Y Present Yes No

2 Absent On dialysis non Diabetic,

Non Smoker

Y Present Yes No

3 Old Cerebrovascular

accident (CVA), H/O

transient ischemia

CABG

(1 year) Non Smoker,

Diabetic

Y Present Yes, plasty

on the cur-

rent leg

By pass

done, on the

other leg

4 Ejection fraction-19%,

lacunar infarcts

Absent Diabetic ,non

smoker

Y Present Plasty ad-

vised,

None

5 No history On dialysis,

hyperbaric O2

for non healing

ulcers

Non Smoker,

Diabetic

Y Present Present Vascular

bypass

done, plasty

performed

on one leg

Table 3: Clinical details about

the patients pretreatment

Table 4: Co-morbidities and prior vascular procedures in the patients

CAD (Coronary artery disease), RAD (Renal artery disease), HTN (hypertension)

A:Great toe showing marked discoloration, swelling, edema.B: Same toe after three months of stem cell

injection showing no further deterioration and there is a marked decrease in swelling and discoloration.

CDE: Left sole of the heel showing an impending ulcer; The heel area also shows varying grades of

discoloration. F: Three months post stem cell transplant, heel showing near normal foot with no swelling, or

ulcer formation.

Figure 2: Patient 1

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Ulcers were present in three of them and

one female patient had an impending gangrene

of the great toe with a large impending ulcer on

the other foot (Figure 2). Co-morbidities and

prior vascular procedures are listed on a per-

patient basis in Table 4. Both the male patients

had previous endovascular procedures. Allpatients had hypertension, and diabetes was

present in four of the 5 for over 15 years. All

patients with diabetes were insulin dependent.

One female patient who was non-diabetic

showed a complete improvement of the wound

within three months post stem cell treatment.

The improvement involved good wound closure

on the plantar side accompanied by granulation

(Figure 3). The oldest patient had undergone

coronary bypass surgery, had old cerebrovascu-

lar episode, and was on dialysis for over a year.

Two of the patients had undergone peripheral

angioplasty on the same extremity under discus-sion, and angioplasty was being contemplated

for the contra lateral extremity at the time of this

study. Impending gangrene, inevitable ampu-

tation decision and non-healing large wounds

prompted their enrollment into the study.

In all ve subjects, no immediate or delayed

local or systemic complications were reported,

after stem cell injections. The ulcers weremanaged and monitored more closely and

frequently, whereas the other post-procedure

objective evaluations were done at week 4, 8

and 12. In all cases, Doppler-guided arterial

segmental pressure of the dorsalis pedis artery

and posterior tibial artery were measured prior

to the procedure. Ankle brachial indices were

calculated separately for each of the lower

extremity arteries by dividing the ankle systolic

pressure of the individual artery by the brachial

artery systolic pressure. The differences werecompared with the pre-implantation measure-

ments. Although nominal improvement in ABI

measurements was observed at three months

after stem cell implantation, none reached

normal values. A clear improvement, albeit non

uniform rise in the ankle pressure was certainly

a positive development. There was no rest pain

in all ve patients at 3 months follow up

A:Wound with gangrene reaching the centre of the foot, three toes seen, two toes were amputated,B: Wound cleaned debridement done

prior to stem cell treatment. C: Stem cell application, IM injection and topical instillation, Cell Injection, Cell Instillation. D: Wound 7 days post

stem cell injection, clean wound granulation tissue, E: Wound one month post transplant showing good healing. F: ( >) Wound almost closed

from the plantar side, ( >) very good granulation tissue, ( >) hard bone, No slough or foul smell.

Figure 3: Patient 2

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Patient Number ABI index

CAD, RAD

Other risk factors

HTN

Rutherford

Category

Pre Post Size Pre Post @3 months Amputation Pre Post

1 0.36 0.9 Impending ulcer, on the heel;

4x5cm;blackened great toe,

amputation of the left small toe

in the past.

At 3 months, great toe

shows reduced blacken-

ing and heel looking

normal

None 5 4

2 0.50 0.8 10x12x2cm

Heel area, severe pain in the leg,

ulcer area etc.

100% wound totally

healed

None 4 1

3 0.3 0.7 15x10x5cm,

Calcaneum exposed, deep sub-

cutaneous and muscular tissue

exposed, infected, foul smelling.

Amputation of small toe done 1year ago

100% wound closure, skin

grafting done. Patient well

and ambulatory

At 14 months

visit no new

ulcers seen.

No amputa-

tion

4 2

4 0.36 0.8 Heel area- 6x6cm No

amputation

Good granulation tissue,

size reduced to 2cmx2cm

None 5 3

5 0.45 0.7 80% wound healing , 2

mm area remaining at 6

months

At 6 months

no new

lesions, no

amputation

5 1

evaluation. All subjects exhibited an improve-

ment by at least one grade increase from the

pre-procedure evaluation as per the Rutherford

classication. In addition, improvement in wound

healing in all the patients ranged between 80-

90% within three months. The granulation tissue

was healthy and was seen along the sides andat the base of the ulcer. There was no evidence

of infection, abscess or accumulation of pus in

any of these cases at this time point. No local

itching, pain or swelling was reported. One of

the 5 patients was reported 14 months later to

show sustained improvement. (Figure 4). He is

ambulatory and well. At six months follow up, be-

yond the planned time point, the second patient

had presented with a completely healed wound.

No new amputation decision or new ulcer was

reported in the follow up period. The other 3 pa-tients are also doing well, and are under obser-

vation of the senior surgeon even after the stated

time point (Table 5).

Table 5: ABI measurements, rest pain, and ischemic ulceration status before and after stem cell implantation and current amputation

status listed patient wise

CAD (Coronary artery disease), RAD (Renal artery disease), HTN (hypertension)

A: Ulcer, bone deep, exposing tissues underneath, calcaneus exposed prior to stem cell treatment

B: Same ulcer showing less inammation, healthy granulated tissue, but calcaneus still exposed.

C: Three months post calcaneal shaving, an almost closed wound, 5% remaining area showing healthy granulation tissue

D: Completely healed wound showing a signicantly changes in contour 8


Figure 4: Patient 3


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9


Chronic non-healing wounds are a cause

of signicant morbidity and mortality and pose a

large nancial burden on the healthcare system.

Such patients with underlying severe PAD in

whom conservative management and surgicalinterventions such as endovascular or open

procedures have failed, are advised to undergo

limb amputation as a nal option. Proper

cutaneous wound repair requires a well coordi-

nated response of inammation, neovasculariza-

tion, extracellular matrix formation, and epithe-

lialization. Failure of any of this process due to

ischemia, reperfusion injury, bacterial infection,

or aging can result in chronic inammation and

a non-healing wound25. Despite the most recent

advances in wound management, up to 50% of

chronic wounds still fail to heal26. One hypothesis

for this problem is that resident cells in non-heal-

ing wounds are intrinsically impaired and dem-

onstrate increased senescence and decreased

response to growth factors27.

Beginning in 2000, several animal model

studies of limb ischemia reported successful

outcomes using stem cell therapy to improve

peripheral blood circulation. These studies sub-sequently spurred early clinical trials in Asia and

Europe involving injection of mononuclear bone

marrow stem cells to treat severe PAD patients.

These preliminary trials were very promising,

which then led investigators to believe that stem

cells have a role in PAD.

Promising results have been shown in

patients with PAD using autologous BM-MNC

(bone marrow mononuclear cells), BM-MSC

(bone marrow mesenchymal cells) and G-CSF(granulocyte colony-stimulating factor) mobilized

PB-MNC (peripheral blood mononuclear cells), a

new treatment option for these patients. Several

clinical trials are consistent in their ndings on

clinical benets including improvements of ABI,

TcPO2, reduction of pain and reduced need for

amputation26. While the advantage of using au-

tologous MNCs and MSCs cannot be disputed,

there are several challenges in getting consistent

bone marrow and cell yields from the type of

patients who present with PAD. Hence the need

to look for easier and dependable sources for

these cells.

Wound healing studies have also looked at

MSCs as contributing to cutaneous regeneration

Studies in both mice and humans have consis-

tently demonstrated enhanced wound repair

following treatment with bone marrow derived

MSCs. This is yet more evidence supporting

MSC-based therapies for cutaneous wound

healing and future directions to bring their poten-

tials to the clinical setting.

The application of MSCs for the tissue repair

had ranged from intravenous infusion to reduce

the size of brain infarcts in a rat stroke model28 to

implantation of cells in the myocardium to reduce

left ventricular dysfunction in a swine model of

myocardial ischemia29. Our own experience in

preclinical models of PAD, Diabetes and Par-

kinsons diseases in small animals has showed

substantial improvement when mesenchymal

stem cells alone or in combination with CD34+

cells were used14

.

A recent report hypothesized that implanted

cells stimulate muscle cells to produce angio-

genic factors, thereby promoting neovascular-

ization30. Marrow and peripheral blood CD34+

haematopoietic stem cells express (Vascular

Endothelial Growth Factor Receptors) VEGFR

and Tie 2 (tyrosine kinase-2), when cultured ex

vivo, these cells differentiate into endothelial

cells expressing the Von Willebrand factor31.

Vascular development is regulated by growthfactors such as VEGF, angiopoietin-1 that bind a

tyrosine kinase receptor Tie-2 involved in com-

pleting the vascular architecture32. Asahara et al

demonstrated that co-culture of CD34+ cells with

the CD34- cells in an in-vitro 3-D matrix model

using microvascular endothelial cells signicantly

enhanced neovascularization11.

DISCUSSION


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Fibroblasts are increasingly used in various

cosmetic applications for their cytokine prole.

Autologous broblasts are difcult to harvest, but

not impossible. Allogeneic broblasts give equal

efcacy and their immunomodulatory nature

makes it much easier for ready use22. These

patients with non-healing foot ulcers were admin-istered 2 million per cm2neonatal broblasts by

inltration and local application.

Thus, mesenchymal stem cells alone or in

combination with the haematopoietic stem cells

represent yet another promising modality sup-

porting new concepts in cellular therapy. This

report on ve patients with non-healing foot

ulcers was to assess the efcacy and feasibil-

ity of an allogeneic stem cell combination com-

prising the MSCs and CD34+ cells with useof broblasts cells for local application. These

ve patients showed decrease in severity of

symptoms three months after the procedure,

as evidenced by alleviation of rest pain and

improvements in Rutherford scores. Two of the

patients had a three level improvement, while

two others had 2 level improvements and the

remaining one patient showed a one level im-

provement in the Rutherford category.

Prior to the therapy, there was a distinct

demarcation of the cold and the warm area of

the leg, which improved after stem cell injection.

We saw good granulation along the borders

and the oor of the ulcer, and overall, the tissue

had a healthy look. No infection was observed

and borders of the ulcers were all normal and

patients were advised to wear special shoes

before weight bearing. The patient with the

longest follow up period of about 14 months

is ambulatory, and goes for frequent dialysis;cannot walk much due to the low cardiac ejection

fraction. The second patient at 6 months follow

up is walking within the house and is very

energetic and feeling well. The third patient

at 3-months follow up is better and has been

advised to walk but is still wheelchair bound due

to fear. The ulcer has completed healed but a toe

still has some residual blackening. The rest of

the patients at 3 months are feeling very comfort-

able, with reduced pain and no new ulcers. No

special post stem cell treatment Doppler exami-

nations were performed.

No procedure-related complications, local or

systemic were reported during the procedure or

at the follow-up evaluations. Mean improvements

of dorsalis pedis artery (DPA) and posteriortibial artery (PTA) 0.13 and 0.09 in ABI were

observed after the cell implantation at three

months. Although only nominal improvement was

observed in the ABI measurements before and

after one dose of the stem cell product, there

was denite progress. All important biochemical

parameters remained within normal range during

follow up, and there was no signicant differ-

ence from the pretreatment values, indicating no

adverse effect of MSC implantation on plasma

glucose, liver, or renal functions.

To our knowledge, this is the rst report of

an early clinical experience of this type of stem

cell combination therapy for PAD with ulcers.

While there are several reports of autologous

bone marrow derived mononuclear cells into

various low-oxygenated ischemic sites of the

lower extremity, a combination cell type using

allogeneic cells has greater signicance. Readily

available and immunomodulatory properties

make UCMSCs very attractive for clinical ap-

plications. CD34+ cells are normally used for

hematopoietic reconstitution after an HLA match.

But local application of CD34+ cells in such smal

quantities does not require HLA match33. The

goal of this type of combination stem cell therapy

thus probably helped promote neoangiogen-

esis, endothelial formation, thereby increasing

the collaterals to the affected organs, reducing

symptoms, and facilitating wound healing in

patients with PAD.

Our study population is more heterogeneous

than those in the other studies, and therefore,

may better represent the general PAD patient

population as a whole. A signicant improve-

ment in ABI and TcPO2 along with the pain free

walking time was reported in trials in which au-

tologous bone marrow mononuclear cells were

transplanted to bring out the therapeutic10



11/14

In conclusion, all patients showed at

least one level of clinical improvement after stem

cell implantation, suggesting the therapeutic

goal of improving limb perfusion is truly possible.

Complete wound healing was achieved within

three months in several patients. These short-

term results indicate the immense potential of

MSC+HSC combination, with broblasts for

local repair. In the light of these encouraging

observations, and based on results in the litera-

ture, the authors feel the need to initiate stem

cell procedure earlier on in the treatment plan

in patients with severe ischemia or non-healing

ulcers to achieve best results.

The authors acknowledge Reliance

Life Sciences Pvt. Ltd (www.rellife.com), for

providing the infrastructure and nancial support

to work on this project. The authors would also

like to thank Dr. S. K Rane and Dr. D. Hattanga-

di for their support during the conduct of this

study.

11


CONCLUSION

ACKNOWLEDGEMENTS

angiogenesis in patients with critical limb isch-

emia.

All these studies reported proved that

the autologous implantation of bone marrow-

mononuclear cells could be safe and effective

for achievement of therapeutic angiogenesis,because of the natural ability of marrow cells to

supply endothelial progenitor cells and to secrete

various angiogenic factors or cytokines34-38.

A clinical trial using autologous mesenchy-

mal stem cells alone for limb ischemia has been

reported in the clinical trial.govsite. Similarly,

another trial using CD34+ cell alone for critical

limb ischemia has been reported in the clinical

trial website. Both the studies have been

completed but results of both these trials have

not yet been published39-40.

The present study demonstrated that alloge-

neic implantation which includes the MSCs and

the CD34+ cells combination is a simple, safe,

and effective tool to treat chronic non healingulcers in cases of PAD. Pain relief was satisfac-

tory and there was a signicant decrease in ulcer

size and increase in pain-free walking distance in

the stem cell treated group as compared to the

untreated group. The role played by broblasts

needs to be noted. Feasibility of MSC and hae-

matopoietic stem cells (HSC) application, their

availability, safety, and absence of any adverse

effects is encouraging.

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