Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats

Abstract

Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.

Figures

Figure 1

Treatment of diabetic wounds with BMSCs increases wound‐breaking strength (WBS). A 5‐cm long midline abdominal fascial incision was made with a scalpel in normal and diabetic rats. One day after wounding, diabetic rats were treated with syngeneic bone marrow‐derived mesenchymal stromal cells (BMSCs) systemically or locally. For systemic administration, 1·5 × 106 BMSCs cells in 1 ml of phosphate‐buffered saline (PBS) were injected once daily over a 2‐minute period for four consecutive days into each animal via the tail vein (n = 7). For local treatment with BMSCs at the wound site, 6 × 106 viable cells were suspended in 0·5 ml PBS and 50 μl cell suspension was injected at 10 different sites 1 mm lateral to the wound edge along the entire length of the incision immediately after closing (n = 6). Normal control (n = 8) and diabetic control (n = 8) animals were treated with PBS without BMSCs. Wounds were harvested on day 7 and WBS measured using a tensiometer as described in Materials and Methods. (A) Data shown are means ± SD. *P < 0·05, **P < 0·01. (B) Photomicrographs of histological sections of untreated normal control (a), untreated diabetic control (b) and diabetic wounds treated with BMSCs intravenously (c). Wounds were harvested on day 7 and processed for histological examination of haematoxylin and eosin‐stained tissue sections. Original magnification ×40 (a,b,c).

Figure 2

Treatment with bone marrow‐derived mesenchymal stromal cells (BMSCs) increases collagen content of diabetic wounds. Diabetic animals were wounded and treated with BMSCs intravenously as described in Figure 1. Wound tissue was harvested on days 7, homogenised in lysis buffer and clarified by centrifugation. Total soluble collagen was measured colorimetrically at 540 nm using the Sircol Collagen Assay kit. Bar graphs represent mean collagen concentration (μg/g) ± SD.

Figure 3

Effect of bone marrow‐derived mesenchymal stromal cells (BMSC) treatment on growth factor expression in diabetic wounds. (A) Reverse transcriptase polymerase chain reaction (RT–PCR) analysis of growth factor expression in wound tissue of normal and diabetic rats. Total cellular RNA was isolated and reverse transcribed to generate cDNAs from wound tissue harvested 7 days after wounding as described in Materials and Methods. cDNA (0·5–1·0 μg) was amplified using growth factor‐specific upper and lower primers and amplified products were separated on 1·5% DNA agarose gel. Gels were stained with ethidium bromide and amplified DNA fragments were identified by base pair (bp) sizes. Top panel shows pattern of growth factor expression in wounds of normal and diabetic rats. Bar graph compares band densities of various growth factors in normal and diabetic wounds (n = 5). (B) RT–PCR analysis of growth factor expression in diabetic wounds treated with BMSCs. Untreated diabetic wounds and diabetic wounds treated with BMSCs intravenously (i.v.) or locally as described in Figure 1 were analysed for growth factor expression by RT–PCR as described above. Data presented compare pattern (top panel) and relative band densities (bar graph) of growth factors in treated and untreated diabetic rats (n = 3). Similar results were obtained in two independent experiments. (C) Measurement of growth factor secretion by enzyme‐linked immunosorbent assays (ELISA). Levels of TGF‐β and EGF in tissue homogenates prepared from normal control, diabetic control and diabetic wounds treated with BMSCs systemically or locally were measured by ELISA following the instructions provided in the kit. Data shown are mean ± SD of three replicate measurements with three rats in each group. EGF, epidermal growth factor; GADPH, glyceraldehyde‐3‐phosphate dehydrogenase; KGF, keratinocyte growth factor; PDGF‐BB, platelet‐derived growth factor; TGF‐β, transforming growth factor; VEGF, vascular endothelial growth factor.