Diabetic foot is a major end-stage complication of diabetes mellitus . It is estimated that 15% of all patients with diabetes develop lower extremity ulcers , and 15–20% of those patients require amputation . The prevalence of diabetic foot ulcers in the Arab world in general is higher, compared with Western countries .
Recently, few studies demonstrated efficacy and associated healing mechanisms (including angiogenesis) of local therapy of progenitor cells in a preclinical model of diabetic ischemic foot ulcer [5–8]. Previous studies have shown that CD34+ stem cells have the capacity to differentiate into progenitor cells that are further able to incorporate into newly forming blood vessels in pathological and nonpathological conditions [9–11]. Multipotent nonembryonic stem cells are available in large numbers in umbilical cord blood (UCB), and they are capable of giving rise to different types of cells that can be used in regenerative medicine applications [12,13].
As there are only few studies that used UCB CD34+ cells in healing of diabetic foot ulcers, this study was carried out with an objective to evaluate the efficacy of UCB-derived stem cells in the wound healing in diabetic animals. If such stem cells were proved to be effective in animals, then it should be studied in clinical use.
Materials and methods
The study was carried out at Histology, Surgery and Physiology Departments, Faculty of Medicine, Suez Canal University. Thirty apparently healthy male albino rats were used in this study. They were randomly divided into three groups, 10 rats each, which included control group, diabetic control (DC) group, and diabetic stem cell-treated (ST) group. Diabetes was induced in DC and ST groups by intraperitoneal injection of streptozotocin in 0.05 mol/l Na citrate, pH 4.5, at a dose of 50 mg/kg body weight . Streptozotocin was available in powder form; 1 gm produced by Sigma Company. In both the DC and ST groups, blood sugar was monitored daily using Glucometer by taking a drop of blood from the tail vein. After 1 week of having fasting blood sugar more than 250 mg/dl, a full thickness circular wound of approximately 10 mm in diameter was performed on the anterolateral side of right legs of rats of all groups using a sterile biopsy punch forceps. The wound size was chosen to be approximately 10 mm, as wound contraction decreases the size of the wound . The ST group was injected by UCB-derived CD34+ stem cells into the wound bed  (subcutaneously in the ulcer floor). No dressings were applied on the wounds throughout the duration of the experiment. All animals were provided with food and water, maintained at 25–28°C, and were housed in large cages with free mobility.
Preparation and transplantation of stem cells
Obtaining the umbilical cord blood
UCB (cells source) was collected from normal healthy volunteer donors undergoing full-term normal vaginal deliveries after obtaining informed consent. Women with a known history of hepatitis, infectious diseases, diabetes mellitus, severe hypertension, abortions, or with a bad obstetric history were excluded.
UCB was collected whereas the placenta was still in utero. Using strict aseptic techniques, 50 ml of UCB were withdrawn from the umbilical vein and collected in sterile tubes containing 5 ml of citrate phosphate dextrose adenine-l anticoagulant. Samples were collected separately, stored at 4°C, and processed within 24 h .
Separation and purification of CD34+ cells
Separation of UCB CD34+ stem cells was carried out according to the method described by the immunomagnetic separation technique . By mixing and incubation, CD34+ cells were bound to Dynabeads M-450 CD34. The formed rosettes were isolated from the suspension using a Dynal Magnetic Particle Concentrator. With subsequent incubation with DETACHaBEAD, CD34 gently detached isolated cells from the beads. A Dynal Magnetic Particle Concentrator was then used to separate the purified, positively selected CD34+ cells from the released Dynabeads M-450 CD34.
Assessment of quantity and quality of the separated cells
The quantity of the isolated CD34+ cells was assessed by putting the sample on an automated cell counter. The quality of the isolated CD34+ cells was determined by using trypan blue dye exclusion test in which the viable cells were not stained .
Transplantation of human umbilical cord blood stem cells
After preparation of UCB stem cells, a dose of 0.5×106 UCB stem cells dissolved in 1 ml isolation buffer/rat was injected locally in the wound bed.
Wound surface areas were measured immediately before killing the animals using a wound tracing method in which a pen was used to trace the outline of the wound directly onto a transparent film.
To detect early signs of healing, half of each group of rats was killed after 1 week (1W) and the rest after 2 weeks (2W):
- (1) One week subgroups: the control group (control 1W) and the diabetic groups (DC 1W group and ST 1W group).
- (2) Two week subgroups: control group (control 2W) and diabetic groups (DC 2W group and ST 2W group).
The wound areas were excised with a rim of 5 mm of normal surrounding skin and were used for histological and immunohistochemical studies. Skin specimens were fixed in 10% neutral-buffered formalin solution. They were then processed to prepare 5-μm thick paraffin sections for histological (hematoxylin and eosin and Masson's trichrome) and immunohistochemical stains.
Immunostaining using CD31 antihuman (Ventana Medical Systems Inc., Strasburg, France) was carried out using the labeled streptavidin–biotin technique. CD31 antigen is a surface glycoprotein of 130 kDa with a broad cellular distribution. It gives brownish discoloration in the plasma membrane of capillary endothelial cells in addition to macrophages and fibroblasts .
Quantitative measurements were carried out using the image analyzer (Leica Imaging System) at Histology Department, Faculty of Medicine, Cairo University to measure: (i) epidermal thickness in hematoxylin and eosin-stained sections, (ii) color area percentage of collagen fibers, in the wound area, in Masson's trichrome-stained sections, (iii) vascular area density : mean percentage of vascular lumina per wound area, and (iv) vessel index  (vessels/mm2: number of blood vessels per mm2). Vascular area density and vessel index were measured in the dermal granulation tissue in CD31-immunostained sections. For morphometric analysis, five nonoverlapping fields from five different sections of five different rats were examined in each group at ×400.
Results were summarized using descriptive statistics. The quantitative measurements were presented as mean±standard deviation and compared using Student's t-test. Significance was set at P value of less than 0.05 for all comparisons. All statistical analyses were conducted with the aid of SPSS 15 software (SPSS Inc, Chicago, Illinois, USA).
Subgroup control 1W
Examination of sections stained with hematoxylin and eosin revealed that the skin of most of the animals showed healing of the wound with intact epidermis (although it was thinner than that of the control 2W group) and dermis. The epidermal cells were arranged in four ill-defined strata. The dermis showed both the papillary layer, in which there was inflammatory cell infiltration, and the reticular layer, which was formed of dense collagen fiber bundles (Fig. 1). The wound's mean surface area in this group was 4.63±3.34 mm2 and the mean epidermal thickness was 9.15±1.23 μm (Table 1).
Masson's trichrome-stained sections showed that the mean color area percentage of collagen in this group was 24.34±4.78. Immunostained sections using CD31 antihuman antibody showed that the granulation tissue of the wound had a vascular area density of 26.19±9.63 and a vessel index of 54.7±17.4 (Table 1).
Subgroup control 2W
Examination of hematoxylin and eosin-stained sections revealed that the skin of most of the animals showed intact epidermis and dermis, which were more or less normal in structure (Fig. 2). The epidermis (stratified squamous epithelium) was composed mainly of keratinocytes that seemed to be arranged into four layers; first, the basal layer (stratum basale; columnar cells that rested on basement membrane and had basal oval nuclei), second, the prickle layer (stratum spinosum; polygonal cells with central nuclei), third, the granular layer (stratum granulosum; spindle shaped cells with basophilic keratohyalin granules), and fourth and outermost layer, the horny layer (stratum corneum; many flattened noncellular acidophilic scales). The dermis was formed of an outer papillary layer and an inner reticular layer. The papillary layer seemed highly cellular in which there was mild inflammatory cellular infiltration. The wound mean surface area in this group was 1.4±0.89 mm2 and the mean epidermal thickness was apparently more than the control 1W group (11.79±2.51 μm; Table 1).
With Masson's trichrome stain, the skin sections showed fine collagen fiber bundles in the papillary layer and coarse bundles in the reticular layer and around hair follicles (Fig. 3). The mean color area percentage of collagen in this group was 25.45±4.52 (Table 1). Immunostained sections using CD31 antihuman antibody showed granulation tissue in the dermis of the wound area. The granulation tissue was formed of blood vessels, proliferated fibroblasts, and inflammatory cells, such as macrophages. Areas of edema were also seen in the granulation tissue. The antibody labeled the endothelial cells, some fibroblasts, and macrophages (Fig. 4). The vascular area density was 27.66±7.57 and the vessel index was 79.1±31.6 (Table 1).
Diabetic control group
Subgroup diabetic control group 1W
Examination of hematoxylin and eosin-stained sections revealed delayed healing of wounds in most of the animals (Fig. 5). The epidermis on the sides of the wound areas was very thin with incomplete differentiation where its cells appeared flat and migratory starting healing. Some migratory cells also appeared in hair follicles. The dermis showed less-dense collagen fibers, compared with the control 1W group. The wound's mean surface area was 12.86±9.58 mm2, which was significantly higher than the control 1W group. The mean epidermal thickness in this group was 1.12±0.1 μm, which was significantly decreased, compared with the control 1W group (Table 1).
Masson's trichrome-stained sections revealed that the mean color area percentage of collagen was 19.03±2.83, which was significantly decreased, compared with the control 1W group. Immunostained sections using the CD31 antihuman antibody showed that the vascular area density was 10.85±2.88 and the vessel index was 33±13. Both were significantly decreased, compared with the control 1W group (Table 1).
Subgroup diabetic control group 2W
Examination of hematoxylin and eosin-stained sections revealed healing of the wounds in some animals; however, the epidermis was thin, in which most of its cells were flat, less-differentiated, and were laid on an area of the dermis deficient in collagen fibers (Fig. 6). The wound's mean surface area was 3.8±2.07 mm2, which was significantly higher than the control 2W group. The mean epidermal thickness in this group was 7.15±1.45 μm, which was significantly decreased, compared with the control 2W group (Table 1).
Masson's trichrome-stained sections revealed decrease in collagen fibers in the papillary dermis of the wound area (Fig. 7). The mean color area percentage of collagen, in this group, was 20.89±3.81, which was significantly decreased, compared with the control 2W group. Immunostained sections using the CD31 antihuman antibody showed decrease in the number of blood vessels in the dermal granulation tissue. The endothelial cells of capillaries showed positive reaction. Fibroblast proliferation was also noticed (Fig. 8). The vascular area density was 13.49±6.92 and the vessel index was 36±11.6; both were significantly decreased, compared with the control 2W group (Table 1).
Stem cell-treated group
Subgroup stem cell-treated group 1W
Hematoxylin and eosin-stained sections revealed that the skin of most of the animals showed healing of the wound with intact epidermis and dermis (Fig. 9). The epidermis was still thinner than that of the control groups (1W and 2W); however, all layers were present. The wound's mean surface area was 5.88±5.19 mm2, which was significantly lower compared with the DC 1W group. The mean epidermal thickness in this group was 8.55±1.78 μm, which was significantly increased, compared with the DC 1W group (Table 1).
Masson's trichrome-stained sections revealed that the mean color area percentage of collagen in this group was 22.12±5.99, which was significantly increased, compared with the DC 1W group. Immunostained sections using the CD31 antihuman antibody showed improvement of the vascular area density (29.29±11.71) and the vessel index (51.6±21); both were significantly increased compared with the DC 1W group (Table 1).
Subgroup stem cell-treated group 2W
Hematoxylin and eosin-stained sections revealed that the skin of most of the animals in this group showed intact epidermis and dermis, which were more or less normal in appearance (Fig. 10). The epidermis seemed to be of normal thickness showing all the strata with more differentiation of its cells. The wound's mean surface area was 1±1.4 mm2, which was significantly lower, compared with the DC 2W group. The mean epidermal thickness was 10.73±2.47 μm, which was significantly increased, compared with the DC 2W group (Table 1).
Masson's trichrome-stained sections showed fine collagen fibers in the papillary dermis and coarse fibers in the reticular dermis and around hair follicles (Fig. 11). The mean color area percentage of collagen in this group was 24.13±6.46, which was significantly increased compared with the DC 2W group. Immunostained sections using the CD 31 antihuman antibody revealed an increased number of blood vessels in the dermal granulation tissue. Most of the vessels were functioning and contained red blood cells. There was a strong positive reaction in the endothelial cells lining these vessels, in many of the proliferated fibroblast, and in some macrophages. Areas of edema are also apparent in the granulation tissue (Fig. 12). The vascular area density was 32.13±5.03 and the vessel index was 73.7±23.1; both were significantly increased compared with the DC 2W group (Table 1).
Skin ulcers are a severe and frequent complication of diabetes and constitute a significant burden to health-care systems all over the world . Numerous studies demonstrated that a subset of hematopoietic cells can function as adult stem cells, and it seems that leukocytes expressing the cell surface antigen CD34+ are enriched for these stem cells . Blood-derived stem cells are capable of differentiating into a variety of cell types, including endothelial cells [21,22]. Impaired wound healing in diabetic patients results from multifactorial deficits, including inefficient reparative angiogenesis and aberrant control of cell survival. Thus, it may be clinically relevant that transplantation of stem cells restored reparative angiogenesis in diabetic ulcers through stimulation of endothelial cells survival, proliferation, and migration [23–26].
Wound healing pass through three different phases; the inflammatory phase which was evident in this study as inflammatory cell infiltration seen in the control 1W group and decreased to mild infiltration in the 2W group and finally subsides within a month, the proliferative phase in which fibroblasts proliferate and migrate to the site of injury to deposit collagen in addition to proliferation and migration of keratinocytes as well as activation of endothelial cells of preexisting vessels to send out capillary sprouts to produce new vessels, and then the remodeling phase in which balance between collagen synthesis and degradation takes place .
Diabetes affects the three phases of wound healing; the inflammatory phase through compromising the immune system, the proliferative phase through suppression of collagen deposition and formation of new vessels (angiogenesis) , and the remodeling phase in which reorganization of collagen occurs to restore the tissue structural integrity [27–29]. In this study, diabetes (in groups; DC 1W and DC 2W) caused delayed wound healing in most of the animals. There was also a significant increase in wound's mean surface area and significant decrease in each of the epidermal thickness, color area percentage of collagen, vascular area density, and vessel index indicating impaired angiogenesis, compared with the control groups (1W and 2W). These data were in agreement with other studies . Previous studies have reported diminished proliferative capacity and abnormal morphology of fibroblasts derived from diabetic ulcers . Other researchers  reported impaired keratinocytes and fibroblast migration to wound in diabetic patients. All these factors interfere with healing of diabetic wounds through impairment of both reepithelization and collagen deposition. In addition, impaired angiogenesis, which was also evident in this study, is a clinically significant problem in diabetic patients , and clinical trials indicate that therapies designed to improve vascularization can improve outcomes in patients with severe skin wounds and diabetic ulcers .
In this study, the skin epidermis of diabetic wounds was formed of flat, less-differentiated cells that appeared migratory in an attempt of healing. Migratory cells also appeared in the dermal hair follicles. It was reported that hair follicles are the primary source of regenerating epithelium and during establishment of epithelial continuity, keratinocytes detach from neighboring cells and basement membrane to migrate along the matrix in which proteolytic cleavage of stromal collagen at focal adhesion contacts enables them to migrate and cover the wound .
In this study, the use of CD34+ cells (groups; stem cells 1W and 2W) revealed marked improvement and healing of wounds in most of the animals. There was a significant decrease in wound's mean surface area in addition to the significant increase in all other parameters (epidermal thickness, color area percentage of collagen, vascular area density, and vessel index) compared with the diabetic control groups (DC 1W and DC 2W). During the first week of this study, the epidermal cells increased in size and number and the thickness was significantly higher compared with the DC 1W group. During the second week, the increase in epidermal thickness in the stem cell group was minimal. Nevertheless, it was maintained significantly higher compared with the DC 2W group and the epidermal cells showed more differentiation. The same finding was shown in other studies .
The immunostained sections in this study revealed that the dermal granulation tissue under the wound was formed of a large number of blood vessels, which were mostly functioning and contained red blood cells. There was a strong positive reaction in the endothelial cells lining the vessels, in many of the proliferated fibroblasts, and in some macrophages. Areas of edema were also apparent in the granulation tissue, because the newly formed vessels were leaky . These findings supported the efficacy of CD34+ stem cells in stimulation of granulation tissue formation as a part of the healing process.
Previous studies demonstrated that injection of CD34+ enriched peripheral blood mononuclear cells into the ischemic limbs of diabetic mice could rapidly and significantly improve blood flow to the limbs . In this study, we tested the potential of these same cells to improve vascularization of skin wounds in diabetic rats. Our data indicated that, compared with the diabetic controls, treatment with UCB enriched for CD34+ cells dramatically enhanced revascularization of the wound by 7 days after wounding and injection of stem cells. In the initial 7-day wound healing period, the vessel index was significantly higher in the ST group compared with the DC group. This was different from another study, which showed that the index was similar in DC and ST wounds . Nevertheless, there were dramatic differences in vascular area density in both studies between DC and ST groups. At 2 weeks, both studies showed that vascular area density and vessel index were significantly higher in the ST group compared with the DC group.
Different mechanisms were suggested for the action by which CD34+ stem cells transplantation leads to tissue repair. One of these mechanisms is differentiation into mature endothelial cells leading to angiogenesis, or expression of angiogenic growth factors in a paracrine way to stimulate neovascularization at the site of the cell graft . Immunohistochemical staining with CD31 antihuman antibody in this study would agree with this mechanism. In addition to the increase in vasculature, the immunostained sections from the ST 2W group showed a strong positive reaction for the CD31 antihuman antibody in the endothelial cells of the newly formed vessels.
Another mechanism suggested by other researchers  was that CD34+ stem cells have transdifferentiation potential and can contribute to the formation of the newly formed keratinocytes and dermal fibroblasts, which deposit collagen and share in the healing process. Our study revealed that stem cells improved keratinocyte formation and differentiation, and also collagen deposition, and hence promoted diabetic wound healing.
In conclusion, CD34+ stem cells derived from the UCB are a simple, safe, and effective therapy for diabetic wounds. They stimulate blood vessel proliferation and collagen deposition. Further studies are recommended to evaluate the efficacy of this therapy in clinical trials.
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