The present study aimed to observe the role of mesenchymal stem cells (MSCs) in the repair of acute skin damage caused by radiation. Rat bone marrow MSCs (BMSCs) were isolated and cultured
Nuclear technology has been widely used in a number of fields, including industry, agriculture, military and medicine. Since the Chernobyl nuclear accident (
Bone mesenchymal stem cells (BMSCs) are a type of somatic stem cell with self-renewal and multi-directional differentiation potential. Owing to the progress in cell engineering and tissue engineering, the application of BMSCs has gained increasing attention (
Transforming growth factor (TGF)-β1, stromal cell-derived factor (SDF)-1 and prostaglandin E2 (PGE2) are important factors involved in inflammatory and anti-inflammatory responses (
Healthy male Sprague-Dawley rats (n=43; weight, 60–80 g) from Shanghai Laboratory Animal Center (Shanghai, China), were sacrificed by neck dislocation. Under aseptic conditions, bone marrow from the femur and tibia was collected by flushing the marrow cavity with physiological saline and was isolated using Ficoll separation solution (Hyclone, Logan, UT, USA). Bone marrow mononuclear cells prepared were then grown in complete Dulbecco's modified Eagle's medium (DMEM; HyClone Laboratories, Inc., Logan, UT, USA) with low glucose supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml penicillin (Sigma-Aldrich) and 100 U/ml streptomycin (Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China) at 37°C in an atmosphere of 5% CO2/95% air/100% humidity. The medium was changed every three days. As the cells grew to 80–90% confluence, they were subcultured at a ratio of 1:3. This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Fuzhou General Hospital (Fuzhou, China).
Third passage BMSCs were seeded in 6-well plates at a density of 3×105 cells/well and were grown in osteoinduction medium (DMEM with high glucose supplemented with 10% FBS, 10−7 M dexamethasone, 10 mM β-sodium glycerophosphate and 50 mg/l vitamin C) or adipoinduction medium (DMEM with high glucose supplemented with 10% FBS, 10−6 M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10
Third passage BMSCs were harvested and adjusted to a concentration of 1×106/ml. The cell suspension (100
Sprague-Dawley rats (n=40) were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Then their right hip skin was irradiated using a linear accelerator (ZL00C-SN428, Varian Medical Systems, Inc., Palo Alto, CA, USA) with a total dose of 45 Gy (999 cGy/min). The irradiation area was 2×2 cm. After irradiation, 40 rats were randomly divided into two groups: BMSC group and control group. Each rat in the BMSC group received tail vein injection of 2×106 BMSCs immediately after irradiation and local multipoint injection of 2×106 BMSCs around the damaged skin 2 weeks after irradiation. Rats in the control group received a tail vein injection of 1 ml physiological saline.
After irradiation, the skin wounds of each rat were observed every day for a total of 8 weeks. The damaged skin was scored according to the acute radioactive skin reaction criteria of the International Union Against Cancer (
At 2, 4, 6 and 8 weeks after irradiation, the wounded skin was sampled from 3 rats of each group after the rats were anesthetized with 1% pentobarbital sodium by intraperitoneal injection. Then the skin tissues were fixed in 4% paraformaldehyde solution and embedded into paraffin. Thereafter, the tissues were cut into slices, mounted on slides and stained with hematoxylin and eosin or Masson's trichrome stain.
At two, four, six and eight weeks after irradiation, the wounded skin tissue samples were obtained from three rats from each group under anesthesia by intraperitoneal injection of 0.4 ml/100 g 1% pentobarbital sodium. The tissues were sliced and mounted on 3-aminopropyltriethoxysilane-coated slides (Fuzhou Maixin Biotechnology Development Co., Ltd.). Following normal deparaffinization, enzyme closure with 3% hydrogen peroxide was performed, followed by antigen retrieval with citrate buffer (Sigma-Aldrich). The immunohistochemistry for PGE2, TGF-β1 and SDF-1 was performed according to the manufacturer's instructions. The primary antibodies, including mouse anti-PGE2 monoclonal antibody (cat. no. mx18829; dilution, 1:100), mouse anti-TGF-β1 monoclonal antibody (cat. no. mx36721; dilution, 1:200) and mouse anti-SDF-1 monoclonal antibody (cat. no. mx22617; dilution, 1:200) were provided by Fuzhou Maixin Biotechnology Development Co., Ltd. The secondary antibody goat anti-mouse immunoglobulin G-horseradish peroxidase (cat. no. sc-2031; dilution 1:500) was provided by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
All statistical processes were performed using SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Comparisons between two groups were conducted using a t-test. P<0.05 was considered to indicate a statistically significant difference.
Microscopically, the primary rat BMSCs were round and of slightly different sizes. They were suspended in the medium with strong refractivity (
As the rat BMSCs grew in the osteoinduction medium, they gradually fused. With the extension of induction time, cells overlapped and the matrix gradually accumulated. In addition, mineral salt was deposited to form multiple nodules, which gradually merged. After 3 weeks of cultivation, the small-flake calcium nodules formed, which were stained black using Von Kossa stain (
Third passage BMSCs showed positive expression of CD29 and CD90, but almost no expression of CD34 and CD45, as determined by flow cytometry. CD29-positive cells accounted for 99.25% and CD90-positive cells accounted for 98.37%, while CD34-positive and CD45-positive cells only accounted for 1.12 and 1.03%, respectively (
There were two fatalities in the control group following irradiation. The other rats presented with depilation, red swelling, mild erosion and slight seepage on the skin at 2 weeks after irradiation. At 4 weeks after irradiation, the wounded skin was erosive and formed into ulcers. After 6 weeks, the wounded skin began to dry and heal gradually, but the healing was slow. At 8 weeks after irradiation, new epithelium could be observed around the ulcer, but the wound healing was still incomplete (
No rats in the BMSC group died following irradiation. At 4 weeks after irradiation, rats presented with exudation and erosion of the skin, but the depth and area of ulcers was significantly milder than that in the control group. At 6 weeks after irradiation, new epithelial growth could be observed at the injured site. After 8 weeks, the wound was almost healed and sparse hairs grew on the new skin (
The wounded skin in the control group revealed epidermal shedding and ulcer formation and a large number of inflammatory cells infiltrated 2 weeks after irradiation. The surface layer was made up of necrotic tissue, in which the hair follicle, sebaceous glands and other adnexa disappeared (
Prostaglandin E2 (PGE2) is important in inflammation. As detected by immunohistochemistry (IHC), PGE2 was predominantly expressed in fibroblasts and inflammatory cells (
TGF-β1 is involved in the inflammatory response at the early stages of damage and the tissue fibrosis process. The immunohistochemical staining of TGF-β1 revealed that TGF-β1 was predominantly distributed in the epithelium, hair follicle cells, basal vascular endothelial cells and fibroblasts around the ulcer (
SDF-1 is one of the predominant chemotactic factors
MSCs are characterized by their potential for self-renewal and multi-directional differentiation, migrating towards damaged tissues to exert a reparative role and secrete a variety of growth factors. A large body of studies has shown that MSCs are involved in tissue repair in intestinal injury caused by radiation (
It is reported that the reason that radioactive skin damage is difficult to heal is that the local vascular injury leads to micro-circulation disturbance and tissue collagen fiber damage (
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TGF-β1 is involved in the inflammatory response at the early stages of injury (
In conclusion, BMSCs effectively promote the repair of radiation-induced acute skin injury by secreting a variety of cytokines to communicate with the microenvironment of the wounded skin and lead to inhibition of the inflammatory response, increased chemotaxis, proliferation and prevent fibrosis. With the development of tissue engineering and cell engineering, BMSCs may become a novel method for the treatment of wounds caused by radioactivity. However, the appropriate BMSC treatment timing, treatment pathway and administration quantity requires further investigation in future studies.
The present study was supported by grants from the key project of the Army 'Twelfth Five-year' Science and Technology Plan (grant no. BWS11J004), the key project of the Science and Technology Plan of Nanjing Military Command (grant no. 10z031) and the Science and Technology Innovation Platform Construction Project of Fujian province, China.
Morphology and differentiation of cultured rat BMSCs (magnification, ×200). (A) Primary rat BMSCs suspended in medium, with strong refractivity. (B) Third passage rat BMSCs, had a uniform spindle shape, with abundant cytoplasm and clear nucleoli. (C) Osteo-induced BMSCs had calcium nodules demonstrated by Von Kossa staining. (D) Adipo-induced BMSCs presented with lipocytes as observed by Oil Red O staining. BMSCs, bone marrow mesenchymal stem cells.
Surface markers of third passage rat BMSCs detected by flow cytometry. (A) CD29-positive and (B) CD90-positive cells accounted for 99.25 and 98.37% of the total cells, respectively. However, (C) CD34-positive and (D) CD45-positive cells only accounted for 1.12 and 1.03%, respectively. BMSCs, bone marrow mesenchymal stem cells.
Wounded skin in rats after radiation exposure. In the BMSC group, the radioactive wound healed with time at a faster rate than the wounds in the rats form the control group.
Histopathological examination of wound skin tissues in the BMSC group (H&E staining and Masson's trichrome staining). Wounded skin 2 weeks after irradiation stained with H&E (A, ×100) and Masson's trichrome staining (B, ×100). H&E staining showed a large number of infiltrated inflammatory cells and the disappearance of hair follicles and sebaceous glands; collagen fibers were stained blue by Masson's trichrome staining, which demonstrated collagen fiber degeneration, decomposition and fracture. Wounded skin at 6 weeks after irradiation was stained with H&E (C, ×100) and Masson's trichrome staining (D, ×100). H&E staining showed epidermis proliferation and migration towards the ulcerated area; subcutaneous fibroblasts migrated and aggregated and new granulation tissue could be seen. Masson's trichrome staining showed hyperplasia of collagen fibers and disordered arrangement of collagen. Wounded skin at 8 weeks after irradiation stained with H&E (E, ×100) and Masson's trichrome staining (F, ×100). The wound had been completely covered with epithelium. Hair follicles and sebaceous glands were observed and collagen fibers arranged neatly. H&E, hematoxylin and eosin.
Expression of cytokines in wounded skin in the BMSC group (immunohistochemical staining, A, C and E, ×100; B, D and F, ×200). (A and B) PGE2 mainly expressed in fibroblasts and inflammatory cells; (C and D) TGF-β1 mainly expressed in the hair follicle cells, vascular endothelial cells, fibroblasts and inflammatory cells; (E and F), SDF-1 was mainly distributed in new skin cells, fibroblasts and capillary endothelial cells. PGE2, prostaglandin E2; TGF-β1, transforming growth factor-β1; SDF-1, stromal cell-derived factor-1.
Wound scores of radioactively damaged skin.
Group | n | Time after radiation injury (weeks)
| |||
---|---|---|---|---|---|
2 | 4 | 6 | 8 | ||
Control | 20 | 2.6±0.5 | 3.0±0.0 | 2.3±0.5 | 1.2±0.4 |
MSCs | 20 | 2.1±1.0 | 2.4±0.7 |
1.5±0.5 |
0.3±0.5 |
Data are presented as the mean ± standard deviation.
P<0.05 vs. the control.