| Background: Diabetic foot(DF)is based on lower extremity skin lesions caused by nerve and capillary damage in patients with diabetes mellitus(DM),which can be induced by fractures.The incidence of DF is high.At least about a quarter of all DM patients in their lives are due to skin microcirculation disorders and hyperkeratosis in diabetic patients,which often leads to chronic prolongation of the wound and a refractory ulcer.Not only does the long-term treatment cost become too high,the prognosis of conventional surgical and internal medical treatments such as common debridement and vacuum suction is not good,especially for patients with severe diabetic foot above Wagner III,the amputation rate is between 20% and 40%.At present,it is still the main composition of hospital amputation patients.The tibial transverse transport(TTT)technique of our research group is a clinical treatment technique based on the Ilizarov tension stress law.It is found that bone distraction has a promoting effect on bone tissue and surrounding biological tissues such as nerves and muscle tissues,and significantly improves the microcirculation and microcirculation of the treatment situation.In clinical studies,blood supply found that the wound healing rate was high,the skin structure was intact,and the basal cells proliferated,suggesting regenerative healing.Therefore,on the basis of this study,the main concern TTT skin stem cell activation and distribution,in order to explore the skin stem cell activation is involved in the promotion of TTT mechanism DF severe wound healing regeneration.Objective: On the basis of observed TTT promote DF wound healing on focusing expression and distribution of activated wounds of skin cells and the Wnt signaling pathway associated protein before and after surgery,in order to investigate the skin stem cells activation is involved TTT promote severe DF wound regeneration healing mechanism.Method: A total of 126 patients with severe diabetic foot who underwent tibial lateral bone transfer from June 2014 to January 2018 were selected.The patients were admitted to the Department of Orthopedics and Joint Surgery of the First Affiliated Hospital of Guangxi Medical University.Firstly,the basic information of the patient is counted,including gender,Wagner classification,Texas classification,age,disease course and so on.Follow-up observation and record the patient’s wound macroscopic view and wound healing,and the healing rate and amputation rate were counted.Randomly select patients for preoperative and postoperative clinical index evaluation and statistical results.Evaluation indexes include TCSS score,dorsal artery pulsation detection,skin temperature,10 g nylon wire test,VAS score,and the patient’s lower limb nerve and blood flow recovery judge.Secondly,take the patient’s wound edge tissue before treatment and 1 month after treatment,fix it into slices,and HE staining was performed to observe the histological changes of the wound,and apply immunohistochemical staining technology to analyze the CK19,CK15,LGR5 and so on in the patient’s wound edge tissue to carry out location detection to mark skin stem cells to detect the degree of activation of skin stem cells.Results:(1)Statistics of basic clinical conditions: A total of 126 patients were included in the study,aged 61.66±9.16 years old,94 males and 32 females,of which 67 were in Wagner grade III,57 were in grade IV,and 2 were in grade V,duration of diabetes 8.39 ± 6.67 years,and the course of diabetic foot was107.63±282.63 days.(2)Clinical curative effect: 1 case of Wagner grade 5 patient was amputation,within 2-9 months after TTT,except for amputation patients,all other patients had wounds with ulcers,and the limb salvage rate was 99%.The skin completely covered the wound,leaving only linear scar;the pain of the patient’s limb has been basically relieved or even disappeared,and the numbness symptoms have gradually improved,and there is a feeling of relief.(3)Clinical indicators: The overall TCSS score after treatment was lower than that before treatment(P<0.05,n=15);after treatment,the pulsation of the dorsal artery of the feet of the patient increased,and the skin temperature of the patient’s foot increased after the treatment(P<0.05,n=15);the measured value of10 g nylon filament after surgery is greater than that before surgery(P<0.05,n=15);the VAS score after surgery is less than that before surgery(P<0.05,n=15).The results show that TTT can not only heal the patient’s wound,but also improve the blood flow and neuropathy of the patient’s lower limbs.(4)HE staining observation of skin morphology structure before and after TTT: the epidermal structure is disordered,the epidermis becomes thinner,part of the epidermis disappears,the number of spinous cells is significantly reduced and severe vacuolation is seen,and the dermal papilla in the dermal papilla is atrophy,dermis and subcutaneous blood vessels,blockage,decrease,thickening of the vessel wall,infiltration of scores of inflammatory cells,clusters into clusters;postoperative staining results in the naked eye can be seen under the optical microscope complete skin structure,skin full-thickness inflammatory cell infiltration phase compared with the preoperative reduction,the structure of the epidermis is intact,scores of new blood vessels and collagen fibers can be observed in the dermis,scores of glands can be observed under the skin,and hair follicle tissue is occasionally seen.(5)Immunohistochemical staining of skin stem cells before and after TTT:Before the operation,CK19 showed low positive rate in the epidermis,dermis and subcutaneous tissues.After the operation,CK19 was distributed in the upper basal layer of the epidermis,showing a cluster of expression in the glands.The postoperative CK19 positive staining area ratio was(9.349±2.715)%,and the preoperative CK19 positive staining area ratio was(1.658±0.765)%.The comparison of the two by t test was t=10.559,P<0.05,n=15.Before surgery,LGR5 was occasionally positively expressed in several places in the epidermis,dermis and subcutaneous tissues.After the operation,LGR5 was positive in the glandular structure in the dermis and in the hair follicles,and expressed in clusters,but no positive expression was found in the epidermis.The ratio of positive staining area of LGR5 after operation was(6.710±2.057)%,and the ratio of positive staining area of LGR5 before operation was(1.318±0.594)%.The comparison of the two by t test was t=9.754,P<0.05,n=15.There was no positive staining of CK15 before operation.After treatment,CK15 is localized in the cytoplasm,mainly expressed in the basal cells of the epidermis,and the hair follicles in the dermis can be expressed in clusters with deep staining.There was no significant difference in the expression levels of integrin β and integrin α6 in tissues before and after surgery.CD34 positive staining was occasionally seen in the dermis before the operation,and the color was light and discontinuous.Postoperative CD34 positive staining was localized in vascular endothelial cells,mainly expressed in small arteries and venules in the dermis.There was no positive staining of ki67 before operation.After the operation,the positive staining of ki67 was located in the nucleus,mainly expressed in the basal layer of the epidermis,and distributed substantially continuously along the basal layer.Before the operation,F4/80 was expressed in the epidermis and dermis,and it was distributed in patches.After the operation,F4/80 positive staining was localized in the cytoplasm,and weak positive manifestations were seen in the epidermis.No Wnt7 a expression was seen before surgery.After operation,Wnt7 a was localized in the nucleus,mainly expressed in the epidermal layer,with a scattered distribution,and positive expression in the blood vessels of the dermis.No expression of Wnt10 a was seen before surgery.After treatment,Wnt10 a positive staining was localized in the cytoplasm,and expressed in the epidermis and dermis.Before the operation,the expression of β-catenin was weakly positive,scattered in the dermal blood vessels.After the operation,β-catenin was localized in the cell membrane and expressed in a large amount in the epidermal layer,in a circular distribution,and slightly expressed in the blood vessels and glands of the dermis,and the color was dark.In preoperative tissues,BMP2 was localized in the nucleus and scattered in large numbers.There was no expression of BMP2 in postoperative tissues.Conclusion: 1.TTT can significantly promote DF ulcer wound healing.2.TTT may activate tissue skin stem cells through Wnt and other signal pathways to promote wound regeneration and healing. |
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