Experimental study of tibial transverse bone transfer on wound healing of diabetic rabbit leg ulcer

Lin-hai CHEN,Zhi-wu CHEN,Zhuan YANG,Jun-shui ZHENG,Ming-jun GUO,Zhao-hui YE,Yang XIANG,Peng WEI

Department of Plastic and Reconstructive Surgery,Ningbo First Hospital,Ningbo City,Zhejiang Province 315000,China.

ABSTRACT Objective To investigate the efficacy and mechanism of tibial transverse bone transfer in the treatment of lower limb ulcer wounds in diabetic rabbits.Methods Animal models of diabetic foot lower extremity ischemia were made,and grouped control studies were designed to evaluate the time of wound healing and the expression level of vascular endothelial growth factor (VEGF),platelet derived growth factor (PDGF),and epidermal cell growth factor (EGF)in the wound during the same period of time.Results In animal experiments,transverse tibial bone transfer can shorten the healing time of diabetic ulcer and increase the expression of wound growth factor (VEGF,PDGF,EGF).Conclusion In animal experiments,diabetes can inhibit the expression of growth factors(VEGF,PDGF,EGF)in ulcer wounds,and tibial transverse bone transfer can promote wound healing.The mechanism may be related to the increased expression of VEGF,PDGF and EGF in the wound surface.

KEY WORDS Tibial transverse bone transfer;Animal experiment;Diabetic ulcer wound;Growth factor

INTRODUCTION

China is one of the countries with the highest incidence of diabetes in the world[1],the incidence of diabetes in adults is 10.9%,and the incidence of pre diabetes is 35.7%[2].The risk of foot ulcer in diabetic patients is up to 25%.14%-24% of foot ulcer patients need amputation,which has become the primary cause of amputation beyond trauma[3].At present,the treatment of diabetic foot includes anti infection,debridement,wound repair and amputation.According to the literature reports,the use of tibial transverse bone transfer technology can effectively reduce the amputation rate,improve the blood and nerve function of the lower extremity,and promote wound healing,but the mechanism of treatment has not been clear[4].The purpose of this study is to investigate the effectiveness and primary mechanism of tibial lateral bone transfer in animal experiments.

MATERIALS AND METHODS

32 healthy SD male New Zealand white rabbits,6 months old,with a body mass of about 2-2.5kg,were purchased from Zhejiang Academy of Medical Sciences,raised in a quiet and temperature controlled environment,free to eat and water.The rabbits were divided into four groups (n= 8):normal rabbit+sham operation group (a),normal rabbit+tibial transverse bone transfer group (b),diabetic rabbit+combined sham operation group (c),diabetic rabbit+tibial transverse bone transfer group (d)

The rabbits were fed adaptively for at least one week,and fasted for 12h before administration.Prepare 5%alloxan solution with sterile normal saline.The model was made by twice Administration[5].Generally,observe 72 hours after injection,and check the blood glucose every 8 hours,which can be managed by injecting glucose and insulin [6].One week after the model was established successfully,one side of the femoral artery was ligated to form ischemic ulcer of the lower extremity(Fig.1).All the experimental animals were designed a wound about 2cm×4cm in size on the dorsum of one foot.In the tibial transverse bone transfer group,the operation method was general anesthesia in rabbits.The inner side of the affected side of the lower limb was cut about 5cm,the subcutaneous tissue and muscle were peeled off carefully,and the inner side of the tibia was exposed to the lower part of the tibial plateau.Two bone removal windows were designed:the upper tibial was moved to the tibial shaft area,about 0.5cm×1cm;the medial side of the medial ridge in the middle tibia,about 0.5cm×0.5cm,and the distance between the two bone blocks was more than 2cm.The periosteum was peeled off with micro osteotomy,the lateral periosteum was carefully and completely pushed open,the medial periosteum was preserved,the medial cortex of tibia was exposed,the center of the bone mass was located with 0.8mm Kirschner wire,the bone mass was cut with micro osteotomy device at low speed to form an open and movable bone mass,then two screw needles with diameter of 2.0mm were respectively turned into the far and near ends of the bone window on the same axis,and finally each device was installed and adjusted to close Tighten the screws (Fig.2).To sew up the soft tissue layer by layer,bandage and complete the operation.After the operation,the patients began to move immediately.Three times a day,about 0.3mm/day in total.Six days after the operation,the patients moved outward.Six days later,the patients moved back to the bone block at the same speed.Every three days,the patients were photographed to observe the movement (Fig.3).Sham operation group:no bone removal operation was performed,but the same incision and tissue peeling were performed (Fig.4).

Before and 3,6,9 and 12 days after the operation,about 1g of new tissue around the wound was cut with surgical blade.If the wound healed 12 days after the operation,cut the wound tissue.Wash the cut tissue with clean pure water and dry it with absorbent paper,put it into the test tube immediately and store it in the refrigerator at-80℃.The obtained tissue was homogenized with lysate,and the supernatant was centrifuged.The expression levels of VEGF,PDGF and EGF were detected by enzyme-linked immunosorbent assay.VEGF,PDGF and EGF ELISA kits(Hangzhou Lianke Biotechnology Co.,Ltd.)were used strictly in accordance with the instructions of the kits.

Fig.1 diabetes mellitus with lower limb ischemic ulcer model

Fig.2 transverse tibial bone removal

Fig.3 observation of bone moving in X-ray

Fig.4 sham operation group

Graphpad prism 7 was used for statistical analysis.The normal distribution measurement data were expressed in X±S.single way ANOVA or two way ANOVA was used for comparison between groups and within groups.By calculating Spearman’s correlation coefficient,the difference of growth factor level between the conventional treatment group and the tibial lateral bone transfer treatment group in the same time period was compared.P< 0.05,the difference was statistically significant.

RESULTS

All the operations were performed by the same senior doctor.There were no complications such as infection,ulcer and necrosis.After the operation,the operation of the moving group was smooth,and the healing days of the four groups were observed visually (Fig.5),(Table 1)(P < 0.05).The results showed that the tibial transverse bone transfer was effective in promoting the healing of ulcer wounds.

Compared with group C,VEGF,PDGF,EGF and EGF were all P < 0.0001 (Table 2,Figure 6),P < 0.05 (Table 3,Figure 7)and P < 0.05 (Table 4,figure 8).Group B and group D control,VEGF in each time period,except the 12th day P > 0.05,others all P < 0.05 (Table 2,Figure 6);PDGF in each time period,all P < 0.01 (Table 3,Figure 7); EGF in each time period,all P < 0.05 (table 4,figure 8).The above results indicate that the expression level of tissue factors in diabetic ulcer wound is lower than that in normal wound.

Compared with group B,the expression level of tissue factors in group A and group B was different (P >0.05)(table 2-4,figure 6-8),VEGF in each period after operation,except for the 12th day (P > 0.05),others (P <0.0001)(Table 2,Figure 6); PDGF in each period after operation,except for the 12th day (P > 0.05),others (P< 0.0001)(Table 3,Figure 7); EGF in each period after operation,except for the 3rd day (P < 0.01),others (P >0.05)( Table 4,figure 8).Compared with group C and group D,the difference of expression level of various tissue factors in preoperative stage (P > 0.05)(table 2-4,figure 6-8),VEGF in postoperative period (P < 0.001)(Table 2,Figure 6); PDGF in postoperative period (P <0.001)(Table 3,figure 7); EGF in postoperative period(excluding the 12th day P > 0.05),other p < 0.01 (Table 4,figure 8).These results suggest that the tibial transverse bone transfer can promote the expression of tissue factors.

DISCUSSION

Diabetic ulcer is the most common clinical manifestation of diabetic foot.There are 4 million new diabetic ulcers in the world every year,with a mortality rate of 11% and a amputation rate of 22%.It costs about 40% of medical resources,which is the main cause of death and disability of diabetic patients,and also brings social medical burden and other problems[7].

Fig.5 observation of wound healing

Table 1 average healing time of wound in four groups of rabbits(n=8,)

Table 1 average healing time of wound in four groups of rabbits(n=8,)

Note:normal rabbit+sham operation group (A),normal rabbit+tibial transverse bone transfer group (B),diabetic rabbit+combined sham operation group (C),diabetic rabbit+tibial transverse bone transfer group (D)compared with group A,a P < 0.01; compared with group C,b P < 0.01;compared with group B,c P < 0.01; compared with group C,d P < 0.05

groups average healing time (day)A 13.86±1.55d 10.25±1.04a C 16.38±2.20 D 13.25±1.83bc B F 17.37 P＜0.05

Table 2 Comparison of VEGF expression levels of four groups of rabbits in different time periods(n=8,)

Table 2 Comparison of VEGF expression levels of four groups of rabbits in different time periods(n=8,)

Note:normal rabbit+sham operation group (A),normal rabbit+tibial transverse bone transfer group (B),diabetic rabbit+combined sham operation group(C),diabetic rabbit+tibial transverse bone transfer group (D); compared with group A,a P > 0.05,b P < 0.0001; compared with group C,c P > 0.05,d P <0.001,e P < 0.0001; compared with group B,f P < 0.0001,g P < 0.05,h P > 0.05; compared with group C,i P < 0.0001

Pre-op Post-op 3days Post-op 6days Post-op 9days Post-op 12days A 7404.13±801.67i 8176.13±357.79i 8449.63±663.60j 8044.38±515.92i 7618.13±907.37i B 7814.13±566.06a 11767.13±1672.78b 20579.75±2139.10b 32714.50±3576.20b 8667.00±496.01a C 2204.17±218.50 4219.97±348.50 5783.43±708.19 4463.93±831.73 3942.58±513.01 D 3202.90±674.17cf 6945.25±1205.08df 23460.45±2588.94eg 28088.82±2901.52ef 12043.15±1074.26eh 132.20 136.20 178.40 25.59 19.68 P＜0.0001 ＜0.001 ＜0.05 ＜0.0001 ＜0.001 F

Fig.6 Comparison of VEGF expression in animal experiments.

Table 3 Comparison of PDGF expression levels of four groups of rabbits in different time periods(n=8,)

Table 3 Comparison of PDGF expression levels of four groups of rabbits in different time periods(n=8,)

Note:normal rabbit+sham operation group (A),normal rabbit+tibial transverse bone transfer group (B),diabetic rabbit+combined sham operation group (C),diabetic rabbit+tibial transverse bone transfer group (D); compared with group C,c P > 0.05,d P < 0.001,e P < 0.0001;compared with group B,f P < 0.0001,g P < 0.01,h P < 0.001; compared with group C,i P < 0.0001; j P <0.05

Pre-op Post-op 3days Post-op 6days Post-op 9days Post-op 12days A 2024.00±86.84i 1962.63±47.02i 2003.50±94.69j 1962.88±42.52i 1937.00±75.33i B 1996.50±99.26a 2423.88±162.74b 2858.63±111.84b 2539.13±218.17b 1842.38±74.85a C 1440.95±102.66 1517.08±76.99 1302.49±68.08 1390.14±473.34 1242.32±379.26 D 1353.21±52.48cf 1939.87±130.24df 3156.13±317.19eg 1910.92±50.72ef 1813.75±81.71eh F 179.40 69.75 200.60 288.80 143.20 P＜0.0001 ＜0.001 ＜0.05 ＜0.0001 ＜0.0001

Fig.7 Comparison of PDGF expression in animal experiments.

Table 4 Comparison of EGF expression levels of four groups of rabbits in different time periods(n=8,)

Table 4 Comparison of EGF expression levels of four groups of rabbits in different time periods(n=8,)

Note:normal rabbit+sham operation group (A),normal rabbit+tibial transverse bone transfer group (B),diabetic rabbit+combined sham operation group (C),diabetic rabbit+tibial transverse bone transfer group (D); Compared with group A,a P >0.05,bP <0.0001; compared with group C,c P > 0.05,d P < 0.0001,e P < 0.01; compared with group B,f P < 0.0001,g P < 0.05,h P < 0.01; compared with group C,i P < 0.0001; j P <0.05

Pre-op Post-op 3days Post-op 6days Post-op 9days Post-op 12days A 6928.38±448.19i 7756.63±410.86i 8833.75±450.64i 7662.50±289.51i 6213.50±406.37j 7125.50±353.95a 10565.13±1231.25b 9106.38±205.55a 7338.00±328.82a 5851.88±501.02a C 5090.31±747.56 6042.87±807.28 5454.86±390.34 4911.38±441.80 5213.64±726.19 D 5035.13±790.10cf 9484.70±515.85dg 7269.84±961.18df 6083.63±719.97eh 4817.09±183.15cg B F 27.42 48.42 79.52 55.96 12.87 P＜0.0001 ＜0.05 ＜0.0001 ＜0.01 ＜0.05

Fig.7 Comparison of EGF expression in animal experiments.

Diabetic patients often form ulcer wounds due to lower extremity skin ischemia or lack of nerve nutrition.Comprehensive evaluation and multidisciplinary comprehensive treatment should be carried out in treatment,including comprehensive treatment:blood glucose management,anti-infection,improvement of peripheral nerve and microcirculation,etc.At the same time,we should pay attention to the treatment of wound:debridement,skin grafting,skin flap,etc.[4],but these treatments can not completely solve the pathological basis of diabetic ulcer wound.Literature reports:tibial transverse bone transfer technology can stimulate the regeneration of peripheral capillaries by moving the bone block with blood supply,improve blood supply and promote wound healing.Animal experiments show that appropriate mechanical stress can stimulate the regeneration of capillaries and surrounding soft tissues[8].But in the wound,the mechanism of its action is not clear.In this experiment,rabbits were selected as experimental animals.By comparing the different operation methods of different groups,and detecting the expression level of wound growth factor (VEGF,PDGF,EGF)in the same time period,the mechanism of tibial transverse bone transfer technology in diabetic foot ulcer wound was preliminarily discussed.

There are three stages of normal wound healing:inflammatory stage,proliferative stage and remodeling stage.Many growth factors are involved and play an important role,especially VEGF,PDGF and EGF.In the inflammatory phase,PDGF chemotaxis a large number of neutrophils and macrophages infiltrate the wound,phagocytosis of self necrotic tissues and pathogens,and promote the formation of positive feedback regulation of secretion of other tissue factors,including PDGF,EGF,VEGF,etc.[9].VEGF can release the adhesion between endothelial cells and surrounding tissues,protect endothelial cells from apoptosis,and enhance the permeability of blood vessels,which is conducive to blood supply and oxygen supply[10].In the proliferative stage,it is mainly to reconstruct extracellular matrix,construct vascular network and epithelialization[11].VEGF can induce collagenase and no,clear and inhibit angiogenesis matrix,relax vascular smooth muscle cells,promote the migration and germination of endothelial cells,construct microvascular network and promote the recovery of blood flow[12].PDGF chemotaxis and stimulates the proliferation and division of fibroblasts,migrates smooth muscle cells and fibroblasts to the injured site,regulates the deposition of extracellular matrix,and promotes the formation of new granulation tissue[13].In the aspect of epithelialization,EGF is the main one.EGF can stimulate the replication and transcription of DNA and RNA in keratinocytes,further complete the construction of collagen tissue,inhibit inflammatory cells,improve wound microenvironment,and accelerate the formation of wound granulation and epithelial tissue[14].In the remodeling stage,extracellular matrix remodeling and scar formation are the main factors.PDGF can up regulate the level of metalloproteinase to degrade extracellular matrix,and EGF can recombine collagen tissue of scar,thus completing the reconstruction of wound[15].

Diabetic ulcer wound is based on pathological metabolic disorder.Peripheral vascular disease causes high glucose in local wound to form advanced glycosylation end products and accumulate under hypoxia conditions,which induces changes in protein properties of active tissue factors,leading to the lack of active growth factors[16].Studies have shown that diabetic ulcer wound may be related to the decreased secretion of growth factors such as VEGF,PDGF and EGF,which may lead to the insufficient ability of cell proliferation and migration and colonization[17].At the same time,in vitro experiments,accumulated glycosylation end products can inhibit the proliferation and adhesion of keratinocytes,endothelial cells and fibroblasts[18].Further study found that the peripheral vascular disease caused the wound to be in a state of continuous hypoxia and decreased responsiveness to hypoxia,which is an important mechanism of diabetic wound non healing[19]

The data of this experiment showed that the expression level of growth factors in diabetic ulcer wound was lower than that in normal wound in animal experiment,and the transverse bone transfer of tibia could promote the expression of growth factors,so as to promote the healing of ulcer wound.However,there are some non statistically significant data,especially on the 12th day after operation,which may be related to the basic healing of the wound,and the rest of the data differences may be related to sample contamination and measurement errors.In view of the fact that experimental rabbits are used as animal models to simulate the clinical symptoms of diabetes mellitus,and the formation of spontaneous ulcer is induced by ligation of blood vessels,which has the advantages of controllable ulcer area,high modeling rate,and convenient for quantitative analysis,but it is not completely consistent with the pathological state of chronic diabetic ulcer in clinical[20],and further research is needed in combination with clinical data.

CONCLUSION

The mechanism may be to promote the regeneration of peripheral blood vessels,increase the vascular network density of the wound,change the hypoxia state of the wound,accelerate the metabolism of high sugar in the wound,reduce the accumulation of advanced glycosylation end products,and increase the expression of growth factors to promote the wound healing.

ACKNOWLEDGEMENTS

This study is currently receiving the grant(Ningbo Natural Science Project No.2017A610183 )and the grant(Ningbo Natural Science Project No.2019C50046)and the grant(Ningbo Science and technology benefiting people project No.2017C50046)from Ningbo Science and Technology Bureau.

Disclosure of Conflicts of Interest:The authors have no financial interest to declare in relation to the content of this article

Ethical Approval:This study was conducted in accordance with the Helsinki Declaration