The Effect of MEBT on Epidermal Regenerative Stem Cells
Xu Ronxiang, Xu Zenglu
China National Science and Technology Center for Burns, Wounds and Ulcers, Beijing, 100053
[Abstract] Objective: To investigate the mechanism of the effect of MEBT on the spontaneous repair of fatty layer burn wound of superficial third degree burn. Method: Immunocytochemistry method---biotin-avidin DCS system, indirect immunofluo rescence technique (IFT) was applied. Mouse antihuman type 19-keratin monoclonal antibody (McAb) was used to detect epidermal regenerative stem cells. Results: Epidermal regenerative stem cells emerged in 24 hr post burn and the number of epidermal regenerative stem cell increased on day 4 post burn. On days 7 and 14 post burn, the number of epidermal stem cells increased to the peak. On days 21 and 28 post burn, the number of epidermal stem cells decreased. Conclusion: MEBT has the effect of promoting the activation and proliferation of regenerative epidermal stem cell in the residual surviving tissue of superficial third degree burn wounds.
[Key words] Burn; MEBT; Epidermal stem cell; Immunocytochemistry
As an innovative therapeutic system in burn therapy, BRT with MEBT/MEBO has enjoyed wide clinical acceptance as part of a protocol including the topical drug MEBO. This therapeutic system successfully solved four major clinical problems: pain, wound infection, progressive necrosis, and scarring of deep II-degree burn wounds. Recently, a new landmark innovation, the regeneration and replication of skin tissue in the subcutaneous fat tissue of full-thickness burn wound, has been accomplished by this innovative protocol. We know that no stem cells remain in the basal layer of epidermis of deep second-degree and superficial third-degree burn wounds. Therefore, we investigated the source of the regenerative epidermal stem cells which makes the fatty layer burn wound repair spontaneously. This study was designed to observe dynamic changes in the regenerative epidermal stem cells of deep burn wound tissues using the immunocytochemistry method.
MATERIALS AND METHODS
Tissue samples were taken from both normal skin and burn wounds of the following two burn patients who received BRT with MEBT/MEBO treatment as first aid immediately after the burn incident.
Case 1: A 6 year-old boy was scalded by hot water at his back and both lower limbs with area of 33 % TBSA, and depth of deep second-degree.
Case 2: A 24-year-old male sustained flame burn at his four extremities with area of 25 % TBSA, and depth of deep second-degree and superficial third-degree.
Tissue samples of the injured areas were taken from wounds of the 2 patients at 24h and on 4, 7, 14, 21 and 28 days postburn. The samples were placed in plastic tubes and frozen immediately in liquid nitrogen, then embedded in Tissue-Tek OCT Compound and frozen with liquid nitrogen. Sections of a 10 ?m thickness were made in a constant freezing microtome.
Indirect immunofluorescence staining with biotin-avidin DCS system was performed. The frozen section was incubated with 10% horse serum at 4oC for 20 min, then a diluted (1:20) solution of mouse anti-human keratin type 19 monoclonal antibody (the 1st antibody) was added and the mix was again incubated overnight at 4oC. Subsequent to washing with phosphate buffer solution, the section was added 7.5 ?g/ml of biotinized horse anti-mouse IgG antibody (the 2nd antibody, Vector Laboratories Burlingame, CA, USA) and incubated at 4oC for1h. After another washing, 10 ?g/ml of biotin-avidin DCS (Vector Laboratories Burlingame) was added and incubated at 4oC for 1h. The section was rinsed and then mounted with glycerin containing 10% PBS and 1% para-phenylenediamine. A control section of normal skin was stained in the same way, but without the adding of the 1st antibody. All specimens were observed under an Olympus reflecting fluorescence microscope (Japan) and photos were taken using ASA400 KODAK films.
RESULTS
Immunocytochemical examinations were made on normal skin and burn wound tissue sections treated with specific mouse anti-human keratin 19 monoclonal antibody. The results revealed that in normal controls of both cases, there were few positive number of epidermal stem cells with keratin type 19 (Figure 1). Wound tissue at 24h postburn showed moderate amount of positive epidermal regenerative stem cells (Figure 2), and on day 4 postburn, the number of positive epidermal stem cells around the sweat gland, capillaries and hair follicles increased (Figure 3). On days 7 (Figure 4) and 14 (Figure 5), epidermal stem cells contained human keratin type 19 continually increased and exceeded the level attained at day 4 postburn, before gradually reaching a peak level. Prior to days 21 (Figure 6) and 28 (Figure 7) postburn, the number of positive regenerative stem cells decreased to a certain level as burn wound progressed to healing. The observation showed that, after treated with BRT with MEBT/MEBO, the proliferation status of the potential regenerative stem cell of the burn patients changed at a regular rate. We propose that regenerative stem cells may be the source of epidermal regenerative stem cell. The glowing fluorescent cells found observed under the microscope represented the potential regenerative stem cells in the wound tissues of deep second and superficial third-degree burns. After treatment with MEBO, these stem cells may aid the deep partial thickness burn wounds to heal without scar formation and aid the superficial full-thickness burn wound to regenerate skin while healing spontaneously.
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Figure 1. Normal skin. No keratin type 19 positive cell.
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Figure 2. At 24 hours postburn. After treated with burn regenerative therapy, moderate amount of keratin type 19 positive cells.
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Figure 3. On day 4 postburn after treated with MEBO, keratin type 19 positive cells increased.
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Figure 4. After treated with BRT with MEBT/MEBO, on day 7 postburn, the number of keratin type 19 positive cells reached the peak.
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Figure 5. On day 14 postburn after treated, the number of keratin type 19 positive cells remained the peak level as in day 7.
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Figure 6. After treated with BRT with MEBT/MEBO, on day 21 postburn, the number of keratin type 19 positive cells decreased. |
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Figure 7. After treated with BRT with MEBT/MEBO, on day 28 postburn, the number of keratin type 19 positive cells decreased significantly.
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[Figure 1 to 7 are photos of stained frozen sections using mouse anti-human keratin type 19 monoclonal antibody (Biotin-avidin DCS system, indirect immunofluorescence technique). ? 200].
DISCUSSION
Research on the cell cycle has revealed that cell division is closely related to physiological regeneration and wound repair. Some cells stay at phase Go or G1 for long time and will not proliferate unless the condition becomes favorable. But some cells can undergo continuous division yielding daughters destined to differentiate to mature cells, while others retain their ability to continuously proliferate. These are termed ¡°stem cells¡±. Stem cells in the basal layer of epidermis are capable of proliferating continuously. Newly proliferated cells move upwards until reaching the deep area of the stratum spinosum layer, where they replicate two or three times before losing their proliferative ability.
In deep second- and third-degree burn wounds, the whole epidermis and deep part of dermis are damaged so all resident stem cells in the basal layer of epidermis are destroyed. The residual viable mesenchymal cells around the hair follicles, sweat glands and capillaries in subcutaneous tissue may provide a source of available regenerative epidermal stem cells. These stem cells can synthesize specific cellular keratin type 19, therefore they can be identified by immunocytochemical method. In this study, anti-human keratin type 19 monoclonal antibody was used. With biotin-avidin DCS system and indirect immunofluorescence technique, specific and exact detection of residual epidermal regenerative stem cells in the subcutaneous tissue of deep second and superficial third-degree burn wounds is accomplished. We observed the number of potential regenerative stem cells with the positive label of human keratin type 19 reached the peak level on 7-14 days postinjury in burn wounds treated with BRT with MEBT/MEBO. The immunofluorescent cells were epidermal stem cells, which induced the fatty layer burn wound to repair spontaneously. Therefore we conclude that BRT with MEBT/MEBO treatment activates the dormant potential epidermal regenerative stem cell to proliferate thereby ensuring the spontaneous repair and healing of deep burn wound without scar formation. BRT with MEBT/MEBO treatment eliminates the lifetime of pain caused by the hyperplastic scar sof patients with deep burns.
After transformation and differentiation, the epidermal regenerative stem cells can yield cells capable of synthesizing other types of keratin, i.e. keratin types 9 and 16. These cells still have the ability of transformation and can transform into the cells capable of synthesizing harder keratin ---- type 1 and 10. These are the typical types of keratin contained in mature epidermal cells. This finding proved that MEBO has the effect of promoting the activation, proliferation and transformation of epidermal stem cells.
It has been reported that when the cells are awoken from their dormant phase, the first activated protein is cyclin D. We know that cyclin D can only expressed after being stimulated by growth factors. For eukaryotes, cells at phase G1 can either enter into the proliferation state or withdraw from the cell cycle. The main regulator of phase G1 limiting point is the complex of cyclin D1/CDK4. In order to further investigate the mechanisms of the effect of BRT with MEBT/MEBO, we will continue our research on the gene regulation for the proliferation cycle of epidermal regenerative stem cells.
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