Effects of advanced platelet-rich fibrin on deep partial-thickness burn wounds in nude mice
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摘要:
目的 探讨改良型富血小板纤维蛋白（A-PRF）对裸鼠深Ⅱ度烧伤创面的作用及其机制。 方法 采用实验研究方法。招募苏北人民医院40名健康志愿者，包括32名女性、8名男性，年龄为60~72岁，抽取静脉血后制备富白细胞血小板纤维蛋白（L-PRF）和A-PRF膜片，采用场发射扫描电子显微镜观察2种富血小板纤维蛋白（PRF）膜片的微观结构。以下实验样本数均为3。将L-PRF、A-PRF膜片分别设为L-PRF组、A-PRF组并进行培养，采用酶联免疫吸附测定法检测培养1、3、7、14 d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。取小鼠L929成纤维细胞（Fb），分为L-PRF组、A-PRF组，并分别加入L-PRF或A-PRF条件培养基培养，于培养1、3、7 d采用噻唑蓝法检测细胞增殖活性，采用划痕试验检测并计算划痕后24 h细胞迁移率。取36只6~8周龄雄性BALB/c裸鼠，于一侧后腿皮肤制作1个深Ⅱ度烧伤创面后，按随机数字表法分为生理盐水组、L-PRF组、A-PRF组，每组12只。生理盐水组裸鼠创面仅行生理盐水冲洗，L-PRF组、A-PRF组裸鼠创面另覆盖相应PRF膜片，3组裸鼠创面均用敷料包扎固定。治疗4、7、14 d，观察创面愈合情况并计算创面愈合率，取创面组织行Masson染色观察新生胶原情况，行免疫组织化学染色检测创面CD31阳性细胞百分比。对数据行独立样本 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、LSD检验。 结果 L-PRF膜片由粗大的纤维蛋白束构成致密的网状结构，散在分布形态完整的白细胞及血小板。A-PRF膜片由细小的纤维蛋白束构成疏松的网状结构，散在分布少量变形的白细胞及血小板。培养1 d，A-PRF组PRF培养上清液中PDGF-AB释放浓度明显高于L-PRF组（ t=5.73， P<0.05），2组PRF培养上清液中VEGF释放浓度相近（ P>0.05）；培养3、7、14 d，A-PRF组PRF培养上清液中PDGF-AB、VEGF释放浓度均明显高于L-PRF组（ t值分别为6.93、7.45、5.49，6.97、8.97、13.64， P<0.05）。培养3、7、14 d，2组PRF培养上清液中PDGF-AB、VEGF释放浓度均明显高于组内前一时间点（ P<0.05）。培养1、3、7 d，A-PRF组小鼠Fb增殖活性分别为0.293±0.034、0.582±0.054、0.775±0.040，均明显强于L-PRF组的0.117±0.013、0.390±0.036、0.581±0.037（ t值分别为8.38、5.14、6.16， P<0.05）。划痕后24 h，A-PRF组小鼠Fb迁移率为（60.9±2.2）%，明显高于L-PRF组的（39.1±2.3）%（ t=11.74， P<0.05）。治疗4 d，L-PRF组和A-PRF组裸鼠创面渗液较少且未见明显感染迹象，生理盐水组裸鼠创面有较多渗液。治疗7 d，L-PRF组和A-PRF组裸鼠创面干燥结痂，生理盐水组裸鼠创面仍有少量渗液。治疗14 d，A-PRF组裸鼠创面趋于愈合，L-PRF组裸鼠仍残留小部分创面，生理盐水组裸鼠创面仍有未脱落焦痂。治疗4、7、14 d，L-PRF组裸鼠创面愈合率与CD31阳性细胞百分比均明显高于生理盐水组（ P<0.05）；与生理盐水组和L-PRF组相比，A-PRF组裸鼠创面愈合率均明显升高（ P<0.05），新生胶原排列有序、分布均匀且未见过度沉积，CD31阳性细胞百分比均明显升高（ P<0.05）。 结论 A-PRF稳定的纤维蛋白网状结构能维持生长因子持续释放、加快细胞增殖、促进细胞迁移，以缩短裸鼠深Ⅱ度烧伤创面愈合时间且改善愈合质量。 Abstract:Objective To explore the effects of advanced platelet-rich fibrin (A-PRF) on deep partial-thickness burn wounds in nude mice and its mechanism. Methods 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 t test, analysis of variance for repeated measurement, analysis of variance for factorial design, one-way analysis of variance, and least significant difference test. Results 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 ( t=5.73, P<0.05), while the release concentrations of VEGF in PRF culture supernatant in the two groups were similar ( P>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 t values of 6.93, 7.45, 5.49, 6.97, 8.97, and 13.64, respectively, P<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 ( P<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 t values of 8.38, 5.14, and 6.16, respectively, P<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 ( t=11.74, P<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 ( P<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 ( P<0.05), the newborn collagen was orderly and evenly distributed, with no excessive deposition, and the percentage of CD31 positive cells was significantly increased ( P<0.05). Conclusions 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. -
Key words:
- Burns /
- Wound healing /
- Cell proliferation /
- Platelet-rich fibrin /
- Growth factor
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参考文献
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1 2种富血小板纤维蛋白膜片微观结构 场发射扫描电子显微镜×5 000。1A.富白细胞血小板纤维蛋白膜片可见致密的纤维蛋白网状结构；1B.改良型富血小板纤维蛋白膜片可见疏松的纤维蛋白网状结构
2 2组小鼠L929成纤维细胞划痕后各时间点划痕情况 光学显微镜×200。2A、2B.分别为富白细胞血小板纤维蛋白（L-PRF）组和改良型富血小板纤维蛋白（A-PRF）组划痕后0 h（即刻），划痕面积相近；2C、2D.分别为L-PRF组和A-PRF组划痕后24 h，图2C剩余划痕面积大于图2D
3 3组裸鼠深Ⅱ度烧伤创面治疗各时间点愈合情况。3A、3B、3C.分别为生理盐水组治疗4、7、14 d创面情况，图3A创面湿润、焦痂下较多渗液、创缘红肿，图3B创面仍有少量渗液，图3C创面仍有少量焦痂覆盖、未见脱落迹象；3D、3E、3F.分别为富白细胞血小板纤维蛋白组治疗4、7、14 d创面情况，图3D相较图3A创面相对干燥，图3E创面被焦痂覆盖，图3F创面表面痂皮大部分脱落；3G、3H、3I.分别为改良型富血小板纤维蛋白组治疗4、7、14 d创面情况，图3G相较图3D创面渗液少，图3H创面干燥，图3I创面趋于愈合
4 3组裸鼠深Ⅱ度烧伤创面治疗各时间点组织病理学观察 Masson×200。4A、4B、4C.分别为生理盐水组治疗4、7、14 d创面胶原沉积情况，图4A、4B可见少量胶原新生，图4C中胶原排列疏松、分布不均；4D、4E、4F.分别为富白细胞血小板纤维蛋白组治疗4、7、14 d创面胶原沉积情况，图4E胶原新生较图4D增多，图4F胶原排列整齐、分布均匀；4G、4H、4I.分别为改良型富血小板纤维蛋白组治疗4、7、14 d创面胶原沉积情况，图4G、4H中胶原分别较图4D、4E致密，图4I相比图4C、4F新生胶原数量更多，排列有序、分布均匀致密且完整度高，未见胶原过度沉积
5 3组裸鼠深Ⅱ度烧伤创面治疗各时间点CD31阳性细胞（棕色）情况 二氨基联苯胺×200。5A、5B、5C.分别为生理盐水组治疗4、7、14 d创面CD31阳性细胞情况，图5C中CD31阳性细胞数明显多于图5A、5B；5D、5E、5F.分别为富白细胞血小板纤维蛋白组治疗4、7、14 d创面CD31阳性细胞情况，数量分别较图5A、5B、5C多；5G、5H、5I.分别为改良型富血小板纤维蛋白组治疗4、7、14 d创面CD31阳性细胞情况，图5I中CD31阳性细胞数明显多于图5C、5F
表1 2组富血小板纤维蛋白培养各时间点上清液中生长因子释放浓度比较（pg/mL，
组别 样本数 PDGF-AB VEGF 1 d 3 d 7 d 14 d 1 d 3 d 7 d 14 d L-PRF组 3 2 594±133 3 859±269 a 4 841±188 a 5 363±165 a 124±12 153±10 a 172±9 a 185±8 a A-PRF组 3 3 310±171 5 123±167 a 6 194±252 a 6 951±473 a 161±22 199±6 a 235±8 a 265±6 a t值 5.73 6.93 7.45 5.49 2.57 6.97 8.97 13.64 P值 0.005 0.002 0.002 0.005 0.062 0.002 0.001 <0.001 注：L-PRF为富白细胞血小板纤维蛋白，A-PRF为改良型富血小板纤维蛋白，PDGF-AB为血小板衍生生长因子AB，VEGF为血管内皮生长因子；PDGF-AB、VEGF处理因素主效应， F值分别为109.04、313.26， P值均<0.001；时间因素主效应， F值分别为213.26、52.41， P值均<0.001；两者交互作用， F值分别为3.71、3.58， P值分别为0.043、0.047；与组内前一时间点比较， a P<0.05 
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表2 3组裸鼠深Ⅱ度烧伤创面治疗各时间点愈合率比较（%，
组别 样本数 4 d 7 d 14 d 生理盐水组 3 12.9±2.4 26.0±2.0 39.4±3.2 L-PRF组 3 19.8±2.6 32.4±3.3 57.5±4.5 A-PRF组 3 32.9±4.8 54.7±4.5 81.0±6.2 F值 26.03 58.36 56.33 P值 0.001 <0.001 <0.001 P 1值 0.026 0.047 0.005 P 2值 0.003 0.001 0.001 P 3值 0.014 0.002 0.006 注：L-PRF为富白细胞血小板纤维蛋白，A-PRF为改良型富血小板纤维蛋白；时间因素主效应， F=290.35， P<0.001；处理因素主效应， F=98.71， P<0.001；两者交互作用， F=13.37， P<0.001； P 1值、 P 2值分别为生理盐水组与L-PRF组、A-PRF组各时间点比较所得； P 3值为L-PRF组与A-PRF组各时间点比较所得
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表3 3组裸鼠深Ⅱ度烧伤创面治疗各时间点CD31阳性细胞百分比比较（%，
组别 样本数 4 d 7 d 14 d 生理盐水组 3 12.1±1.2 25.2±3.1 45.8±3.8 L-PRF组 3 20.3±3.3 37.5±2.3 63.1±1.3 A-PRF组 3 32.8±4.6 53.6±5.4 80.3±2.3 F值 29.84 41.44 124.02 P值 0.001 <0.001 <0.001 P 1值 0.015 0.005 0.002 P 2值 0.002 0.001 <0.001 P 3值 0.018 0.009 <0.001 注：L-PRF为富白细胞血小板纤维蛋白，A-PRF为改良型富血小板纤维蛋白；时间因素主效应， F=355.10， P<0.001;处理因素主效应， F=160.29， P<0.001;两者交互作用， F=3.43， P=0.030； P 1值、 P 2值分别为生理盐水组与L-PRF组、A-PRF组各时间点比较所得； P 3值为L-PRF组与A-PRF组各时间点比较所得
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