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[富血小板纤维蛋白进阶版对裸鼠深Ⅱ度烧伤创面的影响]

[Effects of advanced platelet-rich fibrin on deep partial-thickness burn wounds in nude mice].

作者信息

Tang L J, Li X M, Zhang X W, Luo Y, Xu G

机构信息

Department of Burn Rehabilitation, Shanghai Yangzhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), Tongji University, Shanghai 201613, China.

Department of Burns and Plastic Surgery of Subei People's Hospital, Clinical Medical College of Yangzhou University, Yangzhou 225001, China.

出版信息

Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2023 Aug 20;39(8):771-778. doi: 10.3760/cma.j.cn501225-20220804-00334.

Abstract

To explore the effects of advanced platelet-rich fibrin (A-PRF) on deep partial-thickness burn wounds in nude mice and its mechanism. The experimental study method was adopted. Forty healthy volunteers in Subei People's Hospital were recruited, including 32 females and 8 males, aged 60 to 72 years. Leukocyte platelet-rich fibrin (L-PRF) and A-PRF membranes were prepared after venous blood was extracted from them. The microstructure of two kinds of platelet-rich fibrin (PRF) membranes was observed by field emission scanning electron microscope. The number of samples was 3 in the following experiments. The L-PRF and A-PRF membranes were divided into L-PRF group and A-PRF group and cultured, and then the release concentrations of platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) in culture supernatant were determined by enzyme-linked immunosorbent assay on culture day 1, 3, 7, and 14. Mice L929 fibroblasts (Fbs) were divided into L-PRF group and A-PRF group, and cultured with L-PRF or A-PRF conditioned medium, respectively. On culture day 1, 3, and 7, the cell proliferation activity was detected by thiazole blue method. The cell migration rate was detected and calculated at 24 h after scratching by scratch test. Thirty-six male BALB/c nude mice aged 6-8 weeks were selected to make a deep partial-thickness burn wound on one hind leg, and then divided into normal saline group, L-PRF group, and A-PRF group, according to the random number table, with 12 mice in each group. The wounds of nude mice in normal saline group were only washed by normal saline, while the wounds of nude mice in L-PRF group and A-PRF group were covered with the corresponding membranes in addition. The wounds of nude mice in the 3 groups were all bandaged and fixed with dressings. On treatment day 4, 7, and 14, the wound healing was observed and the wound healing rate was calculated. Masson staining was used to observe the new collagen in wound tissue, and immunohistochemical staining was used to detect the percentage of CD31 positive cells in the wound. Data were statistically analyzed with independent sample test, analysis of variance for repeated measurement, analysis of variance for factorial design, one-way analysis of variance, and least significant difference test. L-PRF membrane's dense network structure was composed of coarse fibrin bundles, with scattered white blood cells and platelets with complete morphology. A-PRF membrane's loose network structure was composed of fine fibrin bundles, with scattered small amount of deformed white blood cells and platelets. On culture day 1, the release concentration of PDGF-AB in PRF culture supernatant in A-PRF group was significantly higher than that in L-PRF group (=5.73, <0.05), while the release concentrations of VEGF in PRF culture supernatant in the two groups were similar (>0.05). On culture day 3, 7, and 14, the release concentrations of PDGF-AB and VEGF in PRF culture supernatant in A-PRF group were significantly higher than those in L-PRF group (with values of 6.93, 7.45, 5.49, 6.97, 8.97, and 13.64, respectively, <0.05). On culture day 3, 7, and 14, the release concentrations of PDGF-AB and VEGF in PRF culture supernatant in the two groups were all significantly higher than those in the previous time points within the group (<0.05). On culture day 1, 3, and 7, the proliferation activity of mice Fbs in A-PRF group was 0.293±0.034, 0.582±0.054, and 0.775±0.040, respectively, which were significantly stronger than 0.117±0.013, 0.390±0.036, and 0.581±0.037 in L-PRF group (with values of 8.38, 5.14, and 6.16, respectively, <0.05). At 24 h after scratching, the migration rate of mice Fbs in A-PRF group was (60.9±2.2)%, which was significantly higher than (39.1±2.3)% in L-PRF group (=11.74, <0.05). On treatment day 4, the wound exudates of nude mice in L-PRF group and A-PRF group were less with no obvious signs of infection, while the wounds of nude mice in normal saline group showed more exudation. On treatment day 7, the wounds of nude mice in L-PRF group and A-PRF group were dry and crusted, while there was still a small amount of exudate in the wounds of nude mice in normal saline group. On treatment day 14, the wounds of nude mice in A-PRF group tended to heal; a small portion of wounds remained in nude mice in L-PRF group; the wound of nude mice was still covered with eschar in normal saline group. On treatment day 4, 7, and 14, the wound healing rate and percentage of CD31 positive cells of nude mice in L-PRF group were all significantly higher than those in normal saline group (<0.05); compared with those in normal saline group and L-PRF group, the wound healing rate of nude mice in A-PRF group was significantly increased (<0.05), the newborn collagen was orderly and evenly distributed, with no excessive deposition, and the percentage of CD31 positive cells was significantly increased (<0.05). The stable fibrin network structure of A-PRF can maintain the sustained release of growth factors, accelerate cell proliferation, and promote cell migration, so as to shorten the healing time and improve the healing quality of deep partial-thickness burn wounds in nude mice.

摘要

探讨高级富血小板纤维蛋白(A-PRF)对裸鼠深Ⅱ度烧伤创面的影响及其机制。采用实验研究方法。选取苏北人民医院40例健康志愿者,其中女性32例,男性8例,年龄60~72岁。采集其静脉血后制备白细胞富血小板纤维蛋白(L-PRF)和A-PRF膜。通过场发射扫描电子显微镜观察两种富血小板纤维蛋白(PRF)膜的微观结构。以下实验样本数为3。将L-PRF和A-PRF膜分为L-PRF组和A-PRF组进行培养,然后在培养第1、3、7和14天通过酶联免疫吸附测定法测定培养上清液中血小板衍生生长因子-AB(PDGF-AB)和血管内皮生长因子(VEGF)的释放浓度。将小鼠L929成纤维细胞(Fbs)分为L-PRF组和A-PRF组,分别用L-PRF或A-PRF条件培养基培养。在培养第1、3和7天,采用噻唑蓝法检测细胞增殖活性。划痕试验于划痕后24 h检测并计算细胞迁移率。选取36只6~8周龄雄性BALB/c裸鼠,在其一侧后肢制作深Ⅱ度烧伤创面,然后按随机数字表法分为生理盐水组、L-PRF组和A-PRF组,每组12只。生理盐水组裸鼠创面仅用生理盐水冲洗,而L-PRF组和A-PRF组裸鼠创面除用生理盐水冲洗外还覆盖相应的膜。3组裸鼠创面均用敷料包扎固定。在治疗第4、7和14天,观察创面愈合情况并计算创面愈合率。采用Masson染色观察创面组织中的新生胶原,采用免疫组织化学染色检测创面中CD31阳性细胞百分比。数据采用独立样本t检验、重复测量方差分析、析因设计方差分析、单因素方差分析和最小显著差法进行统计学分析。L-PRF膜致密的网络结构由粗大的纤维蛋白束组成,有形态完整的散在白细胞和血小板。A-PRF膜疏松的网络结构由细小的纤维蛋白束组成,有少量变形的散在白细胞和血小板。在培养第1天,A-PRF组PRF培养上清液中PDGF-AB的释放浓度显著高于L-PRF组(t=5.73,P<0.05),而两组PRF培养上清液中VEGF的释放浓度相似(P>0.05)。在培养第3、7和14天,A-PRF组PRF培养上清液中PDGF-AB和VEGF的释放浓度显著高于L-PRF组(t值分别为6.93、7.45、5.49、6.97、8.97和13.64,P<0.05)。在培养第3、7和14天,两组PRF培养上清液中PDGF-AB和VEGF的释放浓度均显著高于组内前一时间点(P<0.05)。在培养第1、3和7天,A-PRF组小鼠Fbs的增殖活性分别为0.293±0.034、0.582±0.054和0.775±0.040,显著强于L-PRF组的0.117±0.013、0.390±0.036和0.581±0.037(t值分别为8.38、5.14和6.16,P<0.05)。划痕后24 h,A-PRF组小鼠Fbs的迁移率为(60.9±2.2)%,显著高于L-PRF组的(39.1±2.3)%(t=11.74,P<0.05)。在治疗第4天,L-PRF组和A-PRF组裸鼠创面渗出物较少,无明显感染迹象,而生理盐水组裸鼠创面渗出较多。在治疗第7天,L-PRF组和A-PRF组裸鼠创面干燥结痂,而生理盐水组裸鼠创面仍有少量渗出物。在治疗第14天,A-PRF组裸鼠创面趋于愈合;L-PRF组裸鼠创面有一小部分未愈合;生理盐水组裸鼠创面仍覆盖有焦痂。在治疗第4、7和14天,L-PRF组裸鼠的创面愈合率和CD31阳性细胞百分比均显著高于生理盐水组(P<

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