Effects Of Bone Marrow Derived Cells Mobilization By G-CSF During The Wound Healing Process

Massive skin defects caused by severe burn, trauma and chronic ulcer are very common in clinic work. It still remains the most important problem in wound healing. To handle this problem, the traditional approach is autologous skin graft, which often results in heavy pigmentation and scarring in both donor sites and recipient sites, furthermore donor sites are limited in patients with large area skin injuries. With the development of tissue engineering, the application of stem cell is expected to provide a new methods of wound repair. Due to ethic and technical reasons, embryonic stem cells still has a very long way to clinic use. Therefore, recent research focuses on adult stem cells. Studies have shown that epidermal stem cells under specific conditions can be renewable and differentiate to keratinocytes. However,in normal skin epidermal stem cells is limited in quantity and hardly to separate, purificate and culture. Recent evidence indicates that bone marrow derived cells with the potential to differentiation into a variety tissues, including bone, cartilage, fat, tendon, muscle,etc, can be an important complement progenitor cells of skin. Some experiments have shown that, in the absence of injury, BMDCs engraft at low levels as keratinocytes in the skin at low levels. Some results suggest that BMDCs do not significantly contribute to steady-state epidermal homeostasis and are not required or responsible for providing keratinocyte stem cells and keratinocyte repopulation following skin injury. These data indicate that BMDCs contribute to wound repair after skin injury or in the absence of injury at a very low level. Recent studies have suggested that bone marrow mobilization may be effective in mobilizing cells that contribute to liver repair after damage. We hypothesis that bone marrow mobilization could accelerate wound healing after skin injury. Granulocyte - colony stimulating factor (G-CSF) is among the most commonly used BMDC mobilizing agents because of its potency and lack of toxicity. The potential trophic effects of G-CSF on bone marrow stem cells to accelerate wound healing could have a dramatic clinical impact because G-CSF is one of the few growth factors approved for use in patients.To allow for tracking of the BMDC, we used a chimeric mouse model in which green fluorescent protein (GFP) expressing bone marrow cells were isolated from C57BL/6-Tg(ACTB-GFP) 1Osb/J donor mice and transplanted into lethally irradiated wild-type C57BL/6 mice to investigate the role of BMDCs in a full-thickness skin incisional wound. Using this mouse model, we investigated the total contribution of the bone marrow-derived cells after mobilization to cutaneous wound healing.PARTⅠEstablishment of syngeneicbone marrow transplantation miceObjective: To investigate the optimal irradiation dose for myeloablation in C57BL/6J mice and to detect the long-term effects.Methods: C57BL/6J mice were irradiated with 7 Gy, 8 Gy, 9 Gy and 10 Gy from 60Co respectively, then transplanted or not transplanted with C57BL/6J marrow. The survival rates were obtained. Peripheral leukocytes were dynamically examined in transplanted mouse undergoing 8.0 Gy irradiation. Recipient C57BL/6J mice with 8.0 Gy irradiation were transplanted with marrow from C57BL/6-Tg(ACTB-GFP) 1Osb/J donor mice. The overall level of engraftment was assessed by analysis of FACS in peripheral leukocytes. The change of hair appearance and expression of TRP-2 were observed up to 8 months in irradiated mice.Results: The non-transplanted mice with 8 Gy,9 Gy,10 Gy dosage irradiation were all death and mortality was 40% with 7 Gy irradiation. But in transplanted mice, the mortality were 0%, 0 %, 20% ,80% with 7 Gy, 8 Gy, 9 Gy,10 Gy respectively.The difference of the mortality was significant between 9 Gy and 10 Gy (p< 0.05). The body weight of mice after irradiation decreased early and begin to increase from the 9th day in BMT group while in control group the body weight kept decreasing to death. The mean positive rate of GFP cells was 94.1±2.8% in peripheral circulation. 1 month after irradiation the hair changed to grayish-white color and up to eight months later, the most of hair became white with a little richomadesis. Quantitative histomorphometry of TRP2 positive cells revealed significantly decreased expression level of TRP2 at different time points of one month, two month and eight month after irradiation.Conclusion: 8.0 Gy is the optimal irradiation dose for myeloablation in C57BL/6J mice. Depigmentation, which might be associated with the decreased expression of TRP2, is the long term effect of irradiation.PARTⅡResearch of effect on wound healing of G-CSF mobilization in vivoObjective: To investigate the total contribution of the bone marrow-derived cells after G-CSF mobilization to cutaneous wound healing.Methods: Chimeric mice were splenectomized and 2 weeks later were injected s.c. with recombinant human G-CSF, 50ug/kg/day, once a day for 5 days. Then made a wound injury (1.3×1.3 cm2). G-CSF were given for 3 more days. Others of splenectomized wounded mice injected with saline as vehicle-only control. Peripheral leukocytes were dynamically examined. Shrinkage and wound healing time were studied. On days 0, 3, 6, 9,12,15, 18 and 21, fluorescence imaging was conducted using a whole-animal imaging system. On days 0, 2, 4, 6, 8, 10, 12, 16, 18 and 20 (6 rats/time point), the wounds were collected for histological analysis.Results: Results: Peripheral leukocytes kept increasing, and the top was on the sixth day after mobilization, with the peak value 27.87±2.34/L. Then peripheral leukocytes decreased to normal value within 15d. Remarkably, epidermal healing, measured by wound closure, proceeded significantly faster in mobilization group than control mice. The mean time of wound healing in the G-CSF–treated mice was 12.5±0.9 d while in the control mice was 18.3±0.8 d. Control mice show lower GFP signal intensity compared with mobilized group during the whole wound healing process. The difference was statistically significant. Morphological examination showed that after bone marrow mobilization a large number of fluorescent cells could been observed on the wounds tissue (on the 5th day and one week after healing).Some fluorescent cells were spindle-shape fibroblast-like withα-SMA expression; Some fluorescent cells in the dermis and subcutaneous were around the blood vessels, enveloping the blood vessels or in vascular endothelial with F-Ⅷexpression; Some fluorescent cells in the dermis and subcutaneous were around the newborn hair follicles , located in outer root sheath with CK10 expression. In the process of hair follicle regeneration, GFP+ cells located in outer root sheath cells of the hair follicle could different to epidermal cell around hair follicle.Conclusion: This study indicated: 1, Bone marrow mobilization by G-CSF could accelerate wound healing after skin injury and bone marrow may be a rich source of cells that re-establish the healed skin. 2, BMDCs participate in wound healing not only during inflammatory response,but also contribute to wound repair and remodeling; 3, BMDCs could differentiation to the hair follicle cells, epidermal cells, vascular endothelial cells and myofibroblasts,etc in vivo after injury; 4, BMDC can localize to a known stem cell niche: outer root sheath of mouse hair follicles, as a "transit niche of stem cell ."PARTⅢResearch on the effect of mobilization by G-CSF on growth factor expression during wound healing Objective: To explore the possible mechanism of accelerated wound healing after mobilization by G-CSF through the expression of growth factor.Methods: Experimental animals were divided and managed as described before. Tissue of wounds were excised termly and changes of VEGF, bFGFand EGF were detected by real time quantitive RT-PCR. The correlations between GFP fluorescence signal intensity and growth factor expression level were analysized by Spearman rank correlation analysis.Results: In control group, expression of EGF/bFGF increased , reached the peak on the 3th day, then dropped to normal range.In G-CSF mobilization group, EGF/bFGF expression increased on the 3th,6th days, then dropped to normal range. There were no statistically difference of the peak values between two groups. In control group, expression of VEGF increased , reached the peak on the 6th day, then dropped to normal range. On 0,3,6,9 day after G-CSF mobilization the expression of VEGF were significantly higher than that of control group. Spearman rank correlation analysis showed that the fluorescence intensity of the wound site have a correlation between the growth factor expression level.Conclusion: The results showed: 1, After G-CSF mobilization the expression level of EGF, bFGF and VEGF increased, which have a correlation with fluorescence intensity of the wound site. 2, Accelerated wound repair by G-CSF mobilization might be associated with increased growth factor production.