Efficacy and long-term longitudinal follow-up of bone marrow mesenchymal cell transplantation therapy in a diabetic patient with recurrent lower limb bullosis diabeticorum
Chen et al. Stem Cell Research & Therapy
Efficacy and long-term longitudinal follow- up of bone marrow mesenchymal cell transplantation therapy in a diabetic patient with recurrent lower limb bullosis diabeticorum
Yan Chen 0
Yu Ma 0
Ning Li 0
Hongyan Wang 0
Bing Chen 2
Ziwen Liang 2
Rui Ren 2
Debin Lu 1
Johnson Boey 4
David G. Armstrong 3
Wuquan Deng 0
0 Department of Endocrinology and Nephrology, Diabetic Foot Center, Affliated Central Hospital of Chongqing University, Chongqing Emergency Medical Hospital , Chongqing , China
1 Department of Endocrinology, the 9th People's Hospital of Chongqing , Chongqing , China
2 Department of Endocrinology, Southwest Hospital, Army Medical University , Chongqing , China
3 Department of Surgery, Keck School of Medicine of the University of Southern California , Los Angeles, CA , USA
4 Department of Podiatry, Singapore General Hospital , Bukit Merah , Singapore
Bullosis diabeticorum is a rare presentation of cutaneous manifestation most commonly affecting the lower limbs in patients with diabetes. The appearance, often as insidious as its resolution, is characterized by tense blisters on the skin surfaces of the lower limbs and the feet. The cause still remains unclear, but it may relate to microangiopathy and neuropathy. In this report, we present a case of a 64-year-old male with multiple episodes of blistering in the left lateral lower limb after a traumatic fall who was subsequently diagnosed with type 2 diabetes mellitus. The patient had a history of poorly controlled blood glucose and subsequently developed vasculopathy and peripheral neuropathy. Despite appropriate glycemic control and antibiotics therapy, the patient developed recurrent bullosis diabeticorum on five separate occasions during a 2-year span from 2005 to 2007. Building on our success with ischemic diabetic foot, we used bone marrow mesenchymal stem cell (BMMSC) transplantation therapy for bullosis diabeticorum. After a 9-month treatment, this patient developed another episode of cellulitis in the same lower limb which was successfully treated with antibacterial therapy. It is interesting that the patient reported no recurrence in the next 10-year follow-up span. This study demonstrates that bullosis diabeticorum could appear even before the onset of diabetes, and vascular insufficiency predisposes to the occurrence of bullosis diabeticorum. Our findings suggest that autologous BMMSC transplantation therapy may be an effective measure for recurrent bullosis diabeticorum; however, this will require further investigation to be conclusive. Early identification of diabetes and its complications and appropriate treatment may improve clinical outcomes and prevent lower limb amputation. Trial registration: ClinicalTrials.gov Identifier: NCT00955669. Registered on August 10, 2009.
Diabetes mellitus; Bullosis diabeticorum; Diabetic peripheral arterial disease; Bone marrow mesenchymal stem cells
Dermatological diseases are relatively common in
diabetic patients, in which they manifest cutaneously as a
variety of conditions such as bacterial and fungal
infections, diabetic dermopathy, granuloma annulare, and
necrobiosis lipoidica diabeticorum [
diabeticorum is a rare cutaneous disease, with 100 cases or case
series reported in the literature [
], characterized by
spontaneous noninflammatory manifestations, painless
subcutaneous fluid-filled vesicles varying in size from a
few millimeters to a few centimeters. It is usually
distributed in the lower extremities, in which there is an
observed risk of developing secondary infection, including
diabetic skin ulcer (Fig. 1a), osteomyelitis (Fig. 1b), or
wet gangrene (Fig. 1c), even diabetic amputation.
Presently the exact etiology of bullosis diabticorum is not
well understood. Several studies revealed that its
occurrence is closely related to diabetic patients with
complications of microangiopathy [
], neuropathy, and poor
regulation of blood glucose [
]. Bullosis diabeticorum is
not uncommon in our clinical experience and
observation; an average of 250 people with diabetes per year
with foot problems (skin disease, ulcer, gangrene) were
treated in the past 10 years, including about 60 cases
with bullosis diabticorm. Thus, there is a bullosis
Fig. 1 a Bullosis diabeticorum with skin ulcer. b Bullosis diabticorum
with diabetic osteomyelitis. c Bullosis diabeticorum with wet gangrene
diabeticorum incidence rate of 2.4% per year in our clinic.
Bullosis diabticorum is prone to occur in patients with
local microcirculation dysfunction (Fig. 2a-d) and diabetic
neuropathy (Fig. 2e-h). Often, conservative treatment is
often considered, while aggressive surgical debridement
and subsequently skin grafting are indicated in more severe
]. Moreover, the elimination of causative factors is
imperative for prevention of its recurrence and
]. In our previous study, we successfully treated
diabetic critical limb ischemia with bone marrow
mesenchymal stem cells (BMMSCs) [
]. To further explore
their benefits, we have administered BMMSC
transplantation therapy to a patient with recurrent bullosis
diabeticorum in the left lower limb complicated by limb ischemia
and mild venous insufficiency.
A 64-year-old male presented to hospital in July 2004 for
the sudden occurrence of cutaneous blisters of varying
sizes with associated signs and symptoms of cellulitis in
the left lateral lower limb, including erythema, edema,
increased skin temperature, and tenderness. In May 2005,
the patient was re-admitted to the hospital with similar
signs and symptoms in the left lower limb when he was
diagnosed with diabetes mellitus based on fasting serum
glucose level and 2-h postprandial serum glucose (10.
06 mmol/L and 14.6 mmol/L, respectively), even though
he was not aware of having diabetes mellitus prior to this
admission. In addition, this patient had a medical history
of hypertension and was previously a smoker. In the
following 2 years, the patient had four other hospital
admissions due to recurring episodes of cutaneous blistering
at the same anatomical location. During the physical
examination, multiple clear fluid-filled vesicles of variable
dimensions were observed in the left lower limb,
surrounded by subcutaneous cellulitis (Fig. 3d). The fluid
within bullae was aspirated and cultured; it showed no
bacterial growth, consistent with the literature for the
diagnosis of bullosis diabeticorum [
Peripheral vascular assessment was performed to
establish the vascular status in the bilateral lower extremities.
Pedal pulses, both the posterior tibialis and the dorsalis
pedis artery, were found to be palpable and regular. The
left and right ankle brachial index (ABI) was 0.72 and 0.85,
respectively. Lower extremity ultrasound revealed
atheromatous plaque formation in the tunica intima of the left
femoral and popliteal artery, and atherosclerotic plaque
formation in the anterior wall of the left femoral artery.
Doppler ultrasound demonstrated no vascular
abnormalities in major vessels of the left lower limb. Evaluation of
the bilateral peripheral arterial system was also achieved
with magnetic resonance angiography (MRA). There were
no abnormalities in the bilateral internal iliac, external iliac,
and femoral artery, except for vessel wall thickening of the
left femoral and perforating arteries and enlargement of the
left posterior tibial artery. Venography of the left lower limb
showed delay in the venous return compared to the
contralateral limb. Plain X-ray showed no soft tissue swelling and
no bony cortical erosion or periosteal reaction and no
subcutaneous gas or osteomyelitis was detected. Combined
with the clinical assessments and literature review,
recurrent bullosis diabeticorum and cellulitis could be associated
with lower limb ischemia and venous stasis. Diabetic
neuropathy was excluded through nerve conduct velocity, a gold
standard for diagnosis of diabetic neuropathy.
Preparation of BMMSCs
With reference to our previous research technique
], 30 ml bone barrow was extracted from the
patient under aseptic and anesthetic conditions (Fig. 3a).
Mononuclear stem cells were isolated using density
gradient centrifugation (Fig. 3b). The harvested autologous
BMMSCs, with a total cell count of 7.8 × 107, were
cultured with alpha-modified minimum essential medium
(α-MEM; Invitrogen-Life Technologies Corp., USA)
supplemented with 10% autologous serum. The culture
medium was replaced every 3 days. Once approximately
70% confluence was achieved, the cultured stem cells were
resuspended in trypsin-EDTA. Cells with an approximately
adjusted cell density of 4000 cells per cm2 were transferred
once every 5 days. The above procedures were done in
accordance with the criteria for cell transplantation: (1)
negative results of the microbiological tests; (2) endotoxin
content of ≤ 5 EU/kg patient body weight; and (3) cell
viability of ≥ 95% [
Transplantation procedure and follow-up results
At the same time, the bullae were de-roofed to drain the
wound fluid and uncover the underlying wound bed.
After which, the wound was dressed with appropriate
wound dressing and the patient received combination
antibiotics therapy. As we described in a previous study [
the harvested autologous BMMSCs amount to 8.6 × 108
cells suspended in normal saline after 21 days of culture at
three passages (Fig. 3c). Subsequently, the prepared
BMMSCs, without cryopreservation, were injected into the
left calf muscle with 1 cm intervals after the local infection
had subsided (Fig. 3e). Nine months after treatment, the
patient developed another episode of bullosis diabeticorum
with accompanying cellulitis in the same lower limb and
they were successfully treated similarly tot he previous
measures. At a 10-year follow-up period, the patient
reported no recurrence of bullosis diabeticorum in the left
lower limb (Fig. 3f). Peripheral vascular assessment was
performed again in October 2017 and the left and right
ABI was 1.03 and 0.89, respectively.
Without history of trauma, skin manifestations have been
suggested to be probable cutaneous markers for early
detection of overt diabetes or prediabetes [
]. As has been
reported, bullosis diabecticorum could precede the
diagnosis of diabetes mellitus , consistent with our reported
case. Bullosis diabecticorum is also commonly known as
diabetic bullous disease, which is a rare, spontaneous, and
non-inflammatory diabetic complication with an
incidence rate of 0.16% [
]. The pathogenesis is largely
ambiguous, with some researchers believing that a long
duration of microvascular disease results in skin dystrophy
with capillary basement membrane thickening, which
eventually leads to blister formation with tissue hypoxia
and microcirculation ischemia [
]. The predisposition to
develop bullosis diabeticorum, as we learnt from this case,
seems to be associated with varying degrees of limb
ischemia. Notably, both Doppler ultrasonography and magnetic
resonance angiography showed that this patient had
suffered multiple vascular stenoses in the lower extremity.
Previous studies have revealed that peripheral arterial
disease (PAD) of the lower limb was one of significant and
independent risk factors for diabetic foot infection
]. Therefore, lower extremity cellulitis followed
by bullosis diabecticorum was considered as a
complication of diabetic PAD in this case.
So far as we know, there is no clinical practice guidance
for the appropriate treatment of bullosis diabeticorum.
Based on the case findings and literature reports,
appropriate management of lower limb ischemia is fundamental
for preventing its recurrence. Conventionally, the
treatment of severe ischemic lower extremity vascular disease
mainly involves both medical and/or early
revascularization intervention, either endovascular or open surgery. In
some cases, pharmacotherapy alone could not fully
address the lower limb ischemia while open surgery or
endovascular treatment is contradicted in others due to
pre-existing cardiovascular and cerebrovascular diseases.
In this case, the patient declined any revascularization
treatment but was willing to try conservative
treatment. As such, the novel cell therapy was
assigned to this patient to eradicate its potential
recurrence. Since 2005, our team has been dedicated to
the therapeutic use of different stem cells for diabetic
wound repair and limb salvage [
6, 7, 12–14
BMMSCs and bone marrow-derived mononuclear cells
have been used for diabetics with critical limb ischemia
and foot ulcer, the former producing better clinical
]. In a basic study, we explored the regulatory
mechanism of peroxisome proliferator-activated
receptorγ coactivator-1α (PGC-1α) and found that it could
enhance engraftment and angiogenesis of mesenchymal
stem cells [
]. In another study by Wang et al., the authors
observed that PGC-1α also regulates the expression of
Bcell lymphoma/leukemia-2 (Bcl-2) and stimulates the
survival of mesenchymal stem cells via PGC-1α–ERRα
interaction without the influence of serum, hypoxia, and high
glucose conditions [
]. In addition, adipose-derived stem
cells were also employed for the regulation of diabetic
In view of the above theoretical and practical
experience, autologous BMMSCs were isolated, cultured, and
transplanted into the patient’s left ischemic lower
extremities. Other than one reported recurrence at 9 months,
bullosis diabeticorum and the associated cellulitis went
into remission after transplantation therapy in the
following 10 years. In a meta-analysis study by Rigato et al., they
showed that cell therapy reduced the risk of amputation
by 37% and improved limb salvage by 18% and wound
healing by 59%. Cell therapy also significantly increased
ABI and transcutaneous oxygen pressure and reduced rest
]. We speculated that the effects of transplanted
stem cells might be gradual, starting from cellular
differentiation, proliferation, and formation of new capillaries,
and eventually improve the lower limb blood flow. At the
start of the therapy period, formation of capillary networks
and establishment of collateral circulation would not be
adequate in ischemic tissue, which may induce another
recurrence of bullosis diabecticorum. As blood flow and
lower limb ischemia improve, the occurrence slowly tapers
off. Thus, stem cell therapy provides an additional, viable
option for patients who are ineligible for revascularization
procedures. In another meta-analysis study by Fadini et al.
, the authors reported that traditional revascularization is
not suitable for more than one-third of patients and these
may benefit from stem cell therapies [
This case describes a patient with a repeated blister disease
and cellulitis that were resistant to various treatment
modalities but eventually improved with the application of
autologous BMMSCs. Autologous bone marrow cell
therapy is a feasible, safe, and potentially effective therapeutic
strategy for PAD patients who are considered unsuitable
candidates for traditional revascularization. This case
highlights the potential effect of autologous BMMSCs to relieve
repeated cellulitis and blister disease. Early identification of
diabetes and its complications and appropriate treatment
may improve clinical outcomes and prevent lower limb
ABI: ankle brachial index; Bcl-2: B-cell lymphoma/leukemia-2; BMMSCs: bone
marrow mesenchymal stem cells; MRA: magnetic resonance angiography;
PAD: peripheral arterial disease; PGC-1α: peroxisome proliferator-activated
receptor-γ coactivator-1α; α-MEM: alpha-modified minimum essential
This work was supported by the National Natural Science Foundation of
China (number 81500596) and Cultivation Project of Chongqing Youth
Medical High-end Reserve Talents (number 2017HBRC015) awarded to Dr.
Availability of data and materials
All data generated and/or analyzed during this study are included in the
YM, NL, HW, RR, ZL, and DL contributed to data collection; YC and WD were
major contributors to data collection and the writing of the manuscript; JB,
CB, and DGA participated in data interpretation and contributed to the
writing of the manuscript. All authors have read and approved the final
Ethics approval and consent to participate
This clinical study was approved by the ethical committee board of
Southwest Hospital. The patient provided written informed consent prior to
participation in this study.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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