Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Simple, Non-invasive and Effective New generation Vascular And Tissue Regenerative Endothelial Progenitor Cell Therapy For Non-healing Diabetic Wounds
Rica Tanaka, MD, PhD1, Kayo Arita, PhD1, Satoshi Fujimura, PhD1, Kayoko Okada, PhD1, Makiko Kado, MD1, Takayuki Asahara, MD, PhD2, Hiroshi Mizuno, MD, PhD1.
1Juntendo University School of Medicine, Tokyo, Japan, 2Tokai University School of Medicine, Isehara, Japan.


PURPOSE:
The quality and quantity of endothelial progenitor cells (EPC), i.e.,
CD34+ cells is known to be impaired in diabetic patients, thereby raising
declined tissue repair in autologous EPC therapy. We have recently disclosed the newly developed a serum free ex vivo
expansion system called Hybrid Quantity and Quality Control Culture System
(HyQQc) using peripheral blood mononuclear cells (PbMNC) to potentiate the
vasculogenic property of diabetic EPCs for enhanced vasculogenesis and wound
healing from small amount of peripheral blood. The purpose of this study is to
evaluate the efficacy of HyQQc using diabetic PbMNCs for wound healing.
METHODS:
Mononuclear
cells (MNC) were isolated from 50 ml of peripheral blood in diabetic patients
(n=40) and healthy volunteers (n=40). Then underwent HyQQc for 7 days using
STEMLINE serum free medium with VEGF, Flt-3 ligand, TPO, IL-6 and SCF. The
vascular regeneration capability of HyQQ cells pre- or post QQc was evaluated
with EPC colony forming assay (EPC-CFA), FACS, EPC culture assay, and qRT-PCR.
For in vivo study, 1x104cells of pre and
post HyQQc cells were injected in a murine wound healing model. Morphometric analysis of % wound closure and angio-vasculogenesis by
anti CD31 staining and/or human specific mitochondria antigen (HMA), was
assessed for wound healing.
RESULTS:
Diabetic patients demonstrated significantly lower number of total
colonies pre HyQQc compared to healthy volunteers (totalEPC-CFU No./1000 cells:
1100.76±479.55 vs
151.92±317.92 p<0.001).
Post HyQQc, EPC-CFA disclosed predominant generation of functional total
EPC-CFU compared to pre HyQQc(151.92±317.92 vs 729.04±1241.78,
p<0.01)with higher RNA transcripts of VEGF,
Ang1, Ang2, MMP9. FACS analysis demonstrated significantly higher percentage of
CD34+ cells and CD206 cells post HyQQc in diabetic patients. Transplantation of post diabetic HyQQc cells consecutively unveiled
the greater closure compared to pre HyQQc diabetic cell Tx (% wound closure
post- vs pre- QQc Tx at day 14: 84.2±3.2vs 66.9±2.5, p<0.05, n=10) with
higher wound vascularity compared at day 14 (post- vs pre- QQc Tx for CD31+
cells/ hpf: 321.8±162.9 vs. 145.7±69.9; p < 0.05, n=10). Co-staining for HMA
and CD31 revealed vasculogenesis of Tx cells
Discussion and Conclusions:
In conclusion, HyQQc system of autologous
peripheral blood MNC provides the methodological clue to overcome the
insufficient efficacy of naïve mononuclear cell therapy for wound healing in
diabetic patients. From our data, we assume that 150cc of peripheral blood will
be necessary to replace the existing EPC therapy for diabetic patients for wound
healing. With this new technology, we will be able to establish outpatient
based simple, safe and effective vascular and regenerative therapy for diabetic
patients.


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