Topical application of ex vivo expanded endothelial progenitor cells promotes vascularisation and wound healing in diabetic mice

Impaired wound healing leading to skin ulceration is a serious complication of diabetes and may be caused by defective angiogenesis. Endothelial progenitor cells (EPCs) can augment neovascularisation in the ischaemic tissue. Experiments were performed to test the hypothesis that locally administered EPCs can promote wound healing in diabetes. Full‐thickness skin wounds were created on the dorsum of diabetic mice. EPCs were obtained from bone marrow mononuclear cells (BMMNCs) and applied topically to the wound immediately after surgery. Vehicle and non‐selective BMMNCs were used as controls. Wound size was measured on days 5, 10 and 14 after treatment, followed by resection, histological analysis and quantification of vascularity. Topical application of EPCs significantly promoted wound healing, as assessed by closure rate and wound vascularity. Immunostaining revealed that transplanted EPCs induced increased expression of vascular endothelial growth factor and basic fibroblast growth factor. Few EPCs were observed in the neovasculature based on in vivo staining of the functional vasculature. Ex vivo expanded EPCs promote wound healing in diabetic mice via mechanisms involving increased local cytokine expression and enhanced neovascularisation of the wound. This strategy exploiting the therapeutic capacity of autologously derived EPCs may be a novel approach to skin repair in diabetes.     

Oxidant and antioxidant events during epidermal growth factor therapy to cutaneous wound healing in rats

Cutaneous wound healing is a highly complex process, which includes inflammation, cell proliferation, matrix deposition and remodelling phases. Various growth factors, like epidermal growth factor (EGF), play an important role during wound healing. However, little is known about relationship between EGF and oxidant-antioxidant events in cutaneous wound healing models. Thus we planned to evaluate the connection between EGF therapy and oxidative stress in dermal tissue followed by wounding. Fifty-four adult male Wistar-albino rats were randomly divided into three groups: control, untreated and topical EGF administrated group. A linear full-thickness excision of 40 mm in length on both sides of spinal cord was made on the back of each rat and sutured under anaesthesia and sterile conditions. Excision was closed with 4/0 atraumatic silk suture. EGF solution was freshly prepared at 10 ng/ml dose in thilotears gel under aseptic conditions. Following the surgery, 1 ml of EGF solution was administered to wound strips one time in everyday. The animals were euthanised and wound tissues were collected on days 1, 5, 7 and 14. Thiobarbituric acid reactive substans (TBARS), glutathione (GSH), reactive nitrogen oxide species (NOx), ascorbic acid levels and superoxide dismutase activity were measured spectrophotometrically. TBARS levels decreased and NOx levels increased on day 5 after operation, and GSH levels were increased on day 14 in EGF administered group compared with untreated group. Our data showed that EGF may act like an antioxidant by scavenging toxic oxidation products in wound tissue. In addition, it may contribute healing of the wound tissue in earlier stages and suggest a potential effective role for antioxidant therapies, especially until day 5.     

重组人表皮生长因子促进大鼠皮肤创面愈合的研究

目的观察重组人表皮生长因子(rhEGF)对皮肤创面愈合的作用。方法制作大鼠背部创伤模型,采用自身平行对照,将34只大鼠背部的68个创面分成rhEGF治疗组与盐水对照组,观察大体形态和组织学改变、创面愈合时间和愈合率,测定伤后不同时间创面羟脯氨酸(OHP)含量和Ⅰ型Ⅲ型胶原比例,进行细胞DNA周期分析。结果经rhEGF治疗的创面愈合速度较盐水对照明显加快,2组平均愈合时间为(17.2±1.3)d和(20.5±1.6)d(P<0.01);外用rhEGF使创面肉芽组织生成增多,再上皮化明显,显著增加创面中OHP含量,降低Ⅰ型Ⅲ型胶原比例,加速细胞DNA复制。结论外用rhEGF可缩短创面愈合时间,增加肉芽组织及OHP含量,降低Ⅰ型Ⅲ型胶原比例,加速细胞DNA复制,明显促进皮肤创面的修复。     

Effects of transcutaneous topical injection of oxygen on vascular endothelial growth factor gene into the healing ligament in rats.

The effects of intermittent exposure to oxygen injection on an experimentally induced ligament tear were studied in the right hind limb of 17 male Sprague-Dawley rats. Two rats were used for monitoring the partial oxygen pressure (pO(2)) of subcutaneous tissue and 15 rats were divided into the following three groups of 5 after an experimentally induced ligament tear: Group A, control group; Group B, injection of 0.5 ml hyaluronan to the wound transcutaneously; Group C, injection of 0.5 ml hyaluronan mixed with haemoglobin and oxygen (n=5). At 7 days post-ligament injury, we compared the ligaments of the three treatment groups for gross appearance, histology and expression of vascular endothelial growth factor (VEGF) mRNA by RT-PCR. Our results indicate that the pO(2) was immediately elevated to 334.6 mmHg by topical oxygen injection and this method was effective in promoting vessel formation in comparison to the control group (p<0.01). However, the expression of VEGF mRNA in the topical oxygen injection group (Group C) was lower than that in control group (p<0.05). Our results suggest that oxygen is able to accelerate vessel formation in spite of its effect of decreasing VEGF mRNA. Our method of using topical injection proved to be useful in healing the ligament and the wound.     

Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats

Background

Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types.

Methods

To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry.

Results

There was a significant decrease in wound area in diabetic rats treated with 125 and 60 μg/wound pcDNA3-FLAG-SPK1 (P < 0.001–0.01). The mean sizes of the wounds were 0.67 ± 0.15 cm², 0.83 ± 0.18 cm², and 1.09 ± 0.23 cm² in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2 ± 5.7% and 66 ± 7.3% in test group of 125 and 60 μg/wound respectively, and 55.4 ± 9.9% in diabetic control group (P < 0.001–0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats.

Conclusions

SPK1 gene therapy may represent a novel approach to cutaneous wound healing.     

创面愈合过程中内源性EGF变化的研究

为探讨创面愈合的机理，本实验采用免疫组织化学方法，对大鼠中厚皮片供皮区创面愈合过程中第４、８、１２和１６天创面内源性表皮生长因子（Ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）的变化进行了研究。结果表明，创面愈合过程中内源性ＥＧＦ含量表现有规律性变化，以伤后第８天含量最多，伤后早期和晚期次之，创面愈合后其含量进一步减少。结论：创面愈合过程中，内源性ＥＧＦ的变化促进了创面愈合，是创面愈合的机理之一，结合内源性ＥＧＦ变化，合理外用ＥＧＦ对创面愈合可能会取得促进的效果。     

An investigation on burn wound healing in rats with chitosan gel formulation containing epidermal growth factor

Various studies have shown that chitosan is effective in promoting wound healing. In this study, we aimed to develop an effective chitosan gel formulation containing epidermal growth factor (EGF), and to determine the effect on healing of second-degree burn wounds in rats. Ten micrograms per millilitre EGF in 2% chitosan gel was prepared. In an in vitro study to investigate release of EGF from the formulations, the release rate was 97.3% after 24 h. In in vivo studies, animals were divided into six groups as follows: silver sulfadiazine [Silverdin cream (SIL)], chitosan gel with and without EGF (EJ, J), EGF solution (ES) and untreated control groups [unburned (S) and untreated (Y) rats] applied groups, respectively. A uniform deep second-degree burn of the backskin was performed with water heated to 94+/-1 degrees C during a 15-s exposure. The EGF formulations were repeatedly applied on the burned areas with a dose of 0.160 microg/cm2 for 14 days (one application per day). Healing of the wounds was evaluated immunohistochemically, histochemically and histologically on the tissue samples. When the results were evaluated immunohistochemically, there were significant increases in cell proliferation observed in the EGF containing gel applied group (p<0.001). The histochemical results showed that the epithelization rate in the EJ group was the highest compared to the ES group results (p<0.001). The histological results indicated and supported these findings. It can be concluded that a better and faster epithelization was observed in the EJ group compared to the other groups.     

烧伤创面与糖尿病溃疡创面的比较

目的比较烧伤创面与糖尿病溃疡创面的差异，初步分析糖尿病患者溃疡创面难愈的机制。方法分别切取非糖尿病烧伤患者的足部创面（对照组）和糖尿病患者的足部溃疡创面（试验组）组织，行组织块培养。用酶联免疫吸附测定（ELISA）法、反转录-PCR法分别检测创面组织释放的成纤维细胞生长因子2（FGF2）、血管内皮生长因子（VEGF）蛋白质及其mRNA水平；免疫组织化学法检测创面微血管密度（MVD）的变化。人脐静脉内皮细胞分别在含5mmol／L葡萄糖的培养液（正常培养液组）、含30mmol／L葡萄糖的培养液（高糖组）、含30mmol／L甘露醇的培养液（甘露醇组）中培养7d，以ELISA法测定VEGF蛋白质水平。结果对照组患者FGF2、VEGF蛋白质水平分别为（59±3）ng／ml、（56±7）pg／ml，试验组2种蛋白质水平分别为（89±6）ng／ml、（108±5）pg／ml，组间比较差异均有统计学意义（P〈0．05或P〈0．01），mRNA比较结果与蛋白质相似；2组的MVD水平，差异亦有统计学意义（P〈0．05）。体外细胞培养时当培养液含FGF2，高糖组与正常培养液组的VEGF蛋白质水平相近（P〉0．05）；移去FGF2后2、5d，正常培养液组该指标明显高于高糖组（P〈0．05或P〈0．01）。结论糖尿病患者溃疡创面难愈与血管化受到抑制以及调控血管生长的因子低表达密切相关。     

创面愈合过程中内源性FGF含量变化的研究

为探讨创面愈合的机制，本实验通过免疫组织化学方法，对大鼠断层供皮区创面愈合过程中伤后４天，８天，１２天和１６天创面内源性成纤维细胞生长因子（ＦｉｂｒｏｂｌａｓｔＧｒｏｗｔｈＦａｃｔｏｒ．ＦＧＦ）变化进行了研究。结果表明：创面愈合过程中内源性ＦＧＦ有规律性变化，以伤后８天时相对含量最多，伤后早期和伤后晚期次之，创面愈合后其内源性含量进一步减少。结论：创面愈合过程中，内源性ＦＧＦ的变化促进了创面愈合，是创面愈合的机制之一，结合内源性ＦＧＦ变化，合理外用ＦＧＦ对促进创面愈合可能会取得更好的效果。     

负压封闭引流促进糖尿病足溃疡的愈合

目的分析负压封闭引流(vacuum sealing drainage,VSD)能否促进糖尿病足溃疡的愈合。方法回顾分析自2015年1月至2019年12月,北部战区总医院烧伤整形科收治的糖尿病足溃疡患者60例,并根据患者的治疗方式分为常规治疗组(30例)和VSD治疗组(30例)。统计对比分析两组患者的平均换药次数、平均愈合时间、疼痛程度及满意度。搜集治疗前和治疗14 d后的肉芽组织进行HE染色和VEGF免疫组化染色,分析创面愈合情况以及VEGF的表达情况。结果VSD治疗组换药次数[(5.40±0.28)次]显著少于常规治疗组[(31.41±1.11)次],组间比较P<0.05;VSD治疗组平均愈合时间(29.38±0.63)d显著短于常规治疗组(50.81±2.15)d,组间比较P<0.05;VSD治疗组患者的痛苦程度明显轻于常规治疗组,满意度显著优于常规治疗组,组间比较P<0.05。结论VSD治疗能够促进创面成纤维细胞的增殖、减少炎性细胞的浸润、促进VEGF的表达及创面的愈合。     

恒磁场对SD大鼠深创面愈合的影响

目的：探讨恒磁场对SD大鼠深创面愈合的影响,恒磁场对创面愈合过程中VEGF表达的影响,以及不同强度恒磁场治疗SD大鼠深创面的区别。方法：将48只清洁级SD大鼠随机分成0．16T、0．32T磁疗组和对照组。分别在术后第3、6、9、12天每组处死4只大鼠,测定创面愈合指数,免疫组化检测肉芽组织VEGF表达。比较各组的创面愈合指数及VEGF表达情况。结果：④愈合指数。0．16T磁疗组在第6、9天时的愈合指数高于对照组,差异有统计学意义;0．32T磁疗组在第3、6、9、12天时的愈合指数高于对照组,差异有统计学意义。0.32T磁疗组与0．16T磁疗组相比差异无统计学意义。②创面修复不同时期VEGF表达观察结果。在术后第3、6天磁疗组VEGF表达强于对照组,差异有统计学意义;第9、12天磁疗组与对照组相比差异无统计学意义（P〉0．05）。0．32T磁疗组与0．16T磁疗组相比差异无统计学意义。两个磁疗组在第6天时VEGF阳性率达到整个愈合过程中的峰值,而对照组在第9天时才达到高峰,且VEGF表达强度低于两个磁疗组。结论：0．16T及0．32T恒磁片均能促进SD大鼠深创面的愈合,恒磁场促进深创面愈合的机制可能和在创面愈合早、中期增强VEGF的表达有关。     

成骨细胞移植促进老龄鼠骨质疏松性骨折愈合过程中VEGF的动态变化

目的：动态观察在幼龄成骨细胞移植促进骨质疏松性骨折愈合过程中，VEGF在不同时相的表达及其生物学意义。方法：通过建立老龄骨质疏松SD大鼠骨折的动物模型，并将体外培养的SD雄性乳鼠成骨细胞移动到SD雌性鼠老年骨质疏松性骨缺损部位，利用免疫组化及原位杂交检测骨折愈合过程中不同时间相的移植标本VEGF、VEGFmRNA的表达，并作图像分析、绘出其动态变化图。结果：实验组VEGF、VEGFmRNA均在7d左右可见有阳性表达的细胞，14d有分泌,高峰其中以软骨细胞中阳性最强，21 d分泌量开始下降，56d后基本消失。而对照且未见明显分泌高峰。结论；成骨细胞细胞促进老龄鼠骨质疏松性骨折愈合，其机制可能是通过促进VEGF的转录和表达，从而促进骨折部位建立良好的血液循环，加速骨形成。     

Epidermal growth factor‐containing wound closure enhances wound healing in non‐diabetic and diabetic rats

Background: This study was designed to elucidate the in vivo efficacy of epidermal growth factor (EGF) on wound healing in non diabetic and diabetic rats. Methods: Ninety‐six male Wistar‐Albino rats were randomly divided into six groups. Saline‐moistened gauze, pure gelatin or EGF in gelatin‐microsphere dressings were used in a dermal excision model in both normal and streptomycin‐induced diabetic rats. Wound healing was evaluated on day 7 and 14. Reduction in wound area, hydroxypyroline content and tensile strength of the wound were evaluated in each rat. Tissue samples taken from the wounds were examined histopathologically for reepithelialisation, cellular infiltration, number of fibroblasts, granulation and neovascularisation. Results: On day 7, the use of EGF‐containing dressing was observed to reduce the wound area better when compared with the other dressings tested. This effect was significant in normal rats rather than diabetic rats. The difference in reduction of wound area did not persist on day 14. No significant effect on hydroxyproline content of the wound was found with EGF‐containing dressing in either normal or diabetic rats. There was a statistically significant increase in tensile strength values of EGF‐applied non diabetic rats over the 14 day period. An increase in tensile strength was prominent in also EGF‐applied diabetic rats on day 14. Histological examination revealed higher histopathologic scores in EGF‐applied diabetic and non diabetic rats. Conclusion: These findings implicate that use of EGF in gelatin‐microsphere dressings improves wound healing both in normal and diabetic rats.     

I-型胰岛素样生长因子基因转移对烫伤大鼠创面愈合作用的实验研究

目的探讨I-型胰岛素样生长因子(insulin-likegrowthfactorI,IGF-I)基因转移对烫伤大鼠创面愈合的作用效果。方法利用我们成功构建的重组真核表达质粒pcDNA3.1/IGF-I,通过脂质体介导转染烫伤大鼠皮肤组织,用免疫组化方法检测IGF-I基因在皮肤及肝脏组织的表达情况,观察烫伤大鼠体重、创面愈合情况等指标。结果免疫组化结果显示,脂质体介导的IGF-I基因转移可在创面注射点周围皮肤成纤维细胞中出现阳性表达,而肝脏组织中未出现阳性表达,实验组大鼠创面注射IGF-I重组质粒后,创面愈合与对照组比较差异有统计学意义(P<0.05),实验组大鼠体重与对照组大鼠比较未出现减轻现象(P<0.05)。结论脂质体介导IGF-I基因转移,可促进创面愈合,并可避免IGF-I全身应用所带来的副作用。     

Transplantation of BMSCs expressing hVEGF165/hBD3 promotes wound healing in rats with combined radiation‐wound injury

The combined radiation‐wound injury is a refractory wound with decreased number or dysfunction of repairing cells and growth factors. This remains a challenge in clinical practice. The object of this study is to evaluate the therapeutic efficacy of a combination of human vascular endothelial growth factor 165 (hVEGF₁₆₅) and human beta‐defensin 3 (hBD3) in the treatment of such wounds. A plasmid‐carrying hVEGF₁₆₅ gene and hBD3 gene was used to transfect rat bone‐marrow‐derived mesenchymal stem cells (BMSCs). The supernatant from the modified BMSCs significantly promoted the proliferation and cell migration of human endothelial cells and it also inhibited the growth of bacteria and fungus, demonstrating the successful expression of the transfected genes. The hVEGF₁₆₅/hBD3‐modified BMSCs were then injected into the sites of combined radiation‐wound injury on rats. It demonstrated that wound‐healing time was shortened significantly in the treated rats. The granulation tissue formation/maturation, skin appendage regeneration and collagen deposition were also improved significantly. Strong expression of hVEGF₁₆₅ and hBD3 was detected in the wound surface at early stage of the healing. The results indicate that topical transplantation of hVEGF₁₆₅/hBD3‐modified BMSCs promoted wound healing, and this gene therapy strategy presents a promising approach in the treatment of refractory wounds such as the combined radiation‐wound injury.     

精氨酸促进糖尿病伤口愈合的机制

目的 观察精氨酸促进糖尿病伤口愈合的机制。方法 成年雄性Lewis大鼠，以链脲佐菌素复制糖尿病模型，7d后在大鼠背部制作伤口，置入PVA海绵，每天腹腔注射L-精氨酸1g／kg体重，10d后动物安乐死取样，观察伤口液精氨酸、鸟氨酸、瓜氨酸、NOx、尿素氮及蛋白含量的变化。结果与对照组大鼠比较，糖尿病大鼠伤口液总蛋白、NOx水平显著升高，精氨酸、鸟瓜氨酸、瓜氨酸及尿素氮水平无明显变化。结论 精氨酸促进糖尿病伤口愈合的机制可能与iNOS途径有关，与精氨酸酶途径无关。     

去神经支配对大鼠创面愈合的影响

目的通过制备去神经支配大鼠创面模型,观察去神经大鼠创面愈合率、创面血管和皮肤增殖改变情况,为去神经创面Ⅹ合机制提供理论依据。方法分离并切断SD大鼠胸9～腰1右侧脊神经,造模后在背部对称制作两个直径为1.0cm的圆形创面,分别观察两侧创面愈合时间、创面大体和组织形态学、创面细胞增殖活性、创面修复过程中血管再生情况,并对数据进行统计学分析。结果实验组愈合时间在4周,对照组为3周;实验组创面上皮化速度慢于对照组;实验组愈合表皮层较薄,上皮细胞大小不一;愈合时真皮和表皮层细胞增殖活跃,2～4周时实验组增殖活性低于对照组(P0.05);创面微血管主要分布于真皮层,密度随Ⅹ合时间增加,但2～4周实验组表现较对照组弱(P0.05)。结论去神经后皮肤创面细胞增殖低下,愈合时间延长,血管再生现象受到抑制。     

封闭负压引流技术对失感觉神经支配创伤愈合中Bcl-2与NGF/NGF mRNA表达的影响

目的　研究封闭负压引流技术 (vacuum assistedclosure ,VAC)对失感觉神经支配的创面愈合过程中Bcl 2与NGF表达的影响。方法　将 80只SD大鼠随机分成四组 (每组 2 0只 ) ,即实验组(T组 ) :施加VAC的失去神经支配创面 ;对照组 1(C1) :未施加VAC的失去神经支配的创面 ;对照组2 (C2 ) :施加VAC的正常神经支配的创面 ;对照组 3(C3) :未施加VAC的正常神经支配的创面。T、C2组施加间断性VAC每日 3次 ,负压 80mmHg。伤后 1、3、6、9、12d ,四组同时取材 ,采用免疫组化和原位杂交检测各组各时间点Bcl 2与NGF/NGFmRNA表达。结果　在伤后C2组、C3组Bcl 2与NGF/NGFmRNA表达明显 ,水平逐渐升高 ,至第 9天达最高 ,然后降低 ,C2组较C3组Bcl 2和NGF/NGFmRNA维持相对高的状态 (P <0 .0 5 )。T、C1组创缘和肉芽组织中Bcl 2与NGF/NGFmRNA表达相对C2、C3组处于较低水平 (P <0 .0 5 )。结论　VAC通过增强创面组织抑制凋亡相关基因蛋白的表达 ,影响内源性NGF表达 ,具有明显的促进创面愈合的作用。     

Sustained local application of low-dose epidermal growth factor on steroid-inhibited colonic wound healing

Background/purpose

The effects of locally administered low-dose epidermal growth factor in a steroid-inhibited wound healing were investigated in a rat model.

Methods

Long-acting release of epidermal growth factor was enabled using microspheres embedded in gelatin sponge. Study groups consisted of 60 rats with 10 in each: colonic anastomosis only (C), plus pure gelatin sponge (CG), plus epidermal growth factor loaded sponge (CE), colonic anastomosis and steroid (S), plus gelatine sponge (SG), and plus epidermal growth factor-loaded gelatine sponge (SE) groups. Bursting pressure and wound hydroxy-proline content were measured. Bursting sites were recorded. Collagen deposits, inflammation, and foreign body reactions were evaluated.

Results

Bursting pressure and hydroxy-proline contents were found lowest in the S and highest in the CE groups (P < .01). There was almost no difference between C and SE groups. Bursts were encountered in peri-anastomotic normal colon sites in the nonsteroid-treated C, CG, and CE groups. They were noted overwhelmingly at the anastomosis in steroid-inhibited S, SG, and SE groups. Histopathology results showed a standstill at the inflammatory phase of healing in S and SG groups. The best healing was observed in the CE group. Degree of collagen accumulation was well correlated with bursting pressure and hydroxy-proline content data with a negligible foreign body reaction to gelatine sponge.

Conclusions

Continuous local epidermal growth factor administration by microspheres in gelatin increases wound collagen and further enhances healing in colonic anastomoses even with steroid inhibition.