含人脐血来源富血小板血浆的三维生物打印墨水在裸鼠全层皮肤缺损治疗中的应用

宋薇; 李曌; 朱世钧; 张超; 姚斌; 孔毅; 梁莉婷; 恩和吉日嘎拉; 付小兵; 黄沙

doi:10.3760/cma.j.cn501225-20220618-00243

含人脐血来源富血小板血浆的三维生物打印墨水在裸鼠全层皮肤缺损治疗中的应用

doi: 10.3760/cma.j.cn501225-20220618-00243

解放军总医院医学创新研究部创伤修复与组织再生研究中心，北京　　100048

基金项目:

国家重点研发计划 2017YFC1103303

国家自然科学基金青年科学基金项目 32000969

上海王正国创伤医学发展基金会生长因子复兴计划 SZYZ-TR-03

详细信息

通讯作者:
黄沙，Email：stellarahuang@sina.com

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出版历程
- 收稿日期: 2022-06-18

Application of three-dimensional bioprinting ink containing platelet-rich plasma derived from human umbilical cord blood in the treatment of full-thickness skin defects in nude mice

Research Center for Wound Repair and Tissue Regeneration, Medical Innovation Research Department, the PLA General Hospital, Beijing 100048, China

Funds:

National Key Research and Development Program of China 2017YFC1103303

Youth Science Foundation Project of National Natural Science Foundation of China 32000969

Shanghai Wang Zhengguo Foundation for Traumatic Medicine Growth Factor Rejuvenation Plan SZYZ-TR-03

More Information

Corresponding author: Huang Sha, Email: stellarahuang@sina.com

摘要

摘要: 目的探讨含人脐血来源富血小板血浆（HUCB-PRP）的海藻酸钠-明胶（AG）生物墨水（称为HUCB-PRP-AG生物墨水）的可打印性能和细胞相容性，及该生物墨水的三维打印组织治疗裸鼠全层皮肤缺损创面的效果。方法采用实验研究方法。制备含体积分数2.5%、5.0%、10.0%HUCB-PRP的HUCB-PRP-AG生物墨水，分别命名为1P-AG、2P-AG、4P-AG。取AG、1P-AG、2P-AG、4P-AG，观察室温下外观，采用旋转流变仪检测黏度、储能模量和损耗模量。利用以上4种生物墨水分别进行三维生物打印并观察打印组织外观（后续使用交联后打印组织），将4种打印组织分别与人脐静脉内皮细胞（HUVEC）在Transwell小室内用HUVEC专用培养基共培养24 h，采用细胞计数试剂盒8检测细胞增殖水平（样本数为3）；将4种打印组织分别用DMEM培养基培养12、24、48 h，进行干燥称重并计算降解率（样本数为3）；将1P-AG、2P-AG、4P-AG打印组织分别用磷酸盐缓冲液培养0.5、24.0、48.0 h，采用酶联免疫吸附测定法检测培养上清液中血管内皮生长因子（VEGF）的表达（样本数为5）。取16只6~8周龄雌性BALB/c-NU裸鼠建立背部全层皮肤缺损创面模型，分为创面仅覆盖医用水胶体敷料的常规对照组和另予HUCB-PRP滴加的HUCB-PRP组、AG打印组织覆盖的AG组、4P-AG打印组织覆盖的4P-AG组（每组4只），观察每组3只裸鼠伤后4、8、14 d创面愈合情况并计算创面愈合率；取每组剩余裸鼠伤后8 d创面组织，行苏木精-伊红染色后观察组织病理学改变，行免疫组织化学染色后观察CD31阳性新生血管情况。对数据行重复测量方差分析、LSD检验及Bonferroni校正。结果在室温下，AG、1P-AG、2P-AG、4P-AG均呈半透明液态；AG为淡黄色，1P-AG、2P-AG、4P-AG均为淡红色但颜色依次逐渐加深。在温度10 ℃和剪切率0.1~10.0 s^-1范围内，AG、1P-AG、2P-AG、4P-AG的黏度均随着剪切率的增加而减小；在温度10 ℃和角频率1~100 rad/s范围内，4种生物墨水的储能模量均大于损耗模量。4种生物墨水打印组织的分辨率与形态均相近。与1P-AG、2P-AG、4P-AG打印组织共培养24 h的HUVEC增殖水平分别为0.885±0.030、1.126±0.032、1.156±0.045，均明显高于与AG打印组织共培养的HUVEC的0.712±0.019（P<0.01）；与2P-AG、4P-AG打印组织共培养24 h的HUVEC增殖水平均明显高于与1P-AG打印组织共培养的HUVEC（P<0.01）。1P-AG、2P-AG、4P-AG打印组织培养12、24、48 h的降解率均明显高于AG打印组织（P<0.01）；2P-AG、4P-AG打印组织培养24、48 h的降解率均明显高于1P-AG打印组织（P<0.01）；4P-AG打印组织培养12 h的降解率明显高于1P-AG打印组织（P<0.01），培养24、48 h的降解率均明显高于2P-AG打印组织（P<0.01）。培养0.5、24.0、48.0 h，2P-AG打印组织培养上清液中VEGF表达量均明显高于1P-AG打印组织（P<0.01），1P-AG和2P-AG打印组织培养上清液中VEGF表达量均明显低于4P-AG打印组织（P<0.01）。常规对照组与HUCB-PRP组裸鼠伤后8 d创面较伤后4 d干燥且缩小，HUCB-PRP组裸鼠伤后14 d创面较常规对照组缩小且无结痂；AG组、4P-AG组裸鼠伤后4 d创面上打印组织明显降解且创面无明显渗出，伤后8 d创面明显上皮化且缩小，伤后14 d创面无结痂；4P-AG组裸鼠伤后14 d创面完全上皮化。与常规对照组比较，AG组裸鼠伤后4 d创面愈合率明显降低（P<0.05），HUCB-PRP组、4P-AG组裸鼠伤后各时间点及AG组裸鼠伤后8、14 d创面愈合率均明显升高（P<0.01）；与HUCB-PRP组比较，AG组裸鼠伤后4、8 d及4P-AG组裸鼠伤后4 d创面愈合率均明显降低（P<0.01），AG组裸鼠伤后14 d及4P-AG组裸鼠伤后8、14 d创面愈合率均明显升高（P<0.01）；4P-AG组裸鼠伤后各时间点创面愈合率均明显高于AG组（P<0.01）。伤后8 d，常规对照组裸鼠创面组织可见大量炎症细胞浸润、少量新生微血管以及少量CD31阳性新生血管，HUCB-PRP组裸鼠创面组织可见大量炎症细胞浸润、丰富新生微血管以及较多CD31阳性新生血管，AG组裸鼠创面组织可见轻度炎症浸润、少量新生微血管以及少量CD31阳性新生血管，4P-AG组裸鼠创面组织可见轻度炎症浸润、大量新生微血管以及大量CD31阳性新生血管。结论 HUCB-PRP-AG生物墨水具备良好的可打印性能和细胞相容性，其三维打印组织可促进裸鼠全层皮肤缺损创面血管化，加速创面愈合。
- 生物相容性材料 /
- 打印，三维 /
- 新生血管化，生理性 /
- 生物墨水 /
- 人脐血来源富血小板血浆 /
- 创面修复
Abstract: Objective To investigate the printability and cytocompatibility of sodium alginate-gelatin (AG) bioink containing platelet-rich plasma derived from human umbilical cord blood (HUCB-PRP), named HUCB-PRP-AG bioink, and the effect of the three-dimensionally printed tissue with the bioink on full-thickness skin defect wounds in nude mice. Methods The method of experimental research was used. HUCB-PRP-AG bioinks with 2.5%, 5.0%, and 10.0% of HUCB-PRP by volume were prepared and named 1P-AG, 2P-AG, and 4P-AG, respectively. The appearances of AG, 1P-AG, 2P-AG, and 4P-AG at room temperature were observed, and their viscosity and storage/loss modulus were measured by a rotational rheometer. The above four bioinks were used for three-dimensional bioprinting respectively, and the appearances of the printed tissue were observed (the printed tissue was subsequently cross-linked and used). The four kinds of bioprinted tissue were respectively co-cultured with human umbilical vein endothelial cells (HUVECs) in Transwell chambers with HUVEC special medium for 24 h, and the cell proliferation level was detected by cell counting kit 8 (n=3). The four kinds of bioprinted tissue were respectively cultured in Dulbecco's modified eagle medium for 12, 24, and 48 h, which were dried and weighed, and the degradation rate was calculated (n=3). The expression of vascular endothelial growth factor (VEGF) in the culture supernatant of 1P-AG, 2P-AG, or 4P-AG cultured in phosphate buffer solution at 0.5, 24.0, and 48.0 h was detected by enzyme-linked immunosorbent assay (n=5). Sixteen female BALB/c-NU nude mice aged 6-8 weeks were selected to establish a full-thickness skin defect wound model on the back and were divided into conventional control group with wounds being covered with medical hydrocolloid dressing alone, HUCB-PRP group with additional HUCB-PRP dripping to the wounds, AG group additionally covered with AG printed tissue, and 4P-AG group additionally covered with 4P-AG printed tissue, respectively (with 4 nude mice in each group). The wound healing of 3 nude mice in each group was observed on post injury day (PID) 4, 8, and 14, and the wound healing rate was calculated. The wound tissue of the remaining nude mouse in each group was collected on PID 8, the histopathological changes were observed after hematoxylin and eosin staining, and the CD31-positive new blood vessels were observed after immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, least significant difference test, and Bonferroni correction. Results At room temperature, AG, 1P-AG, 2P-AG, and 4P-AG were semi-transparent liquid, and AG was light yellow, while 1P-AG, 2P-AG, and 4P-AG were light red but the color successively deepened. The viscosity of AG, 1P-AG, 2P-AG, and 4P-AG decreased with the increase of shear rate at the temperature of 10 ℃ and shear rate of 0.1-10.0 s^-1; the storage moduli of the four bioinks were greater than the loss moduli at the temperature of 10 ℃ and angular frequency range of 1-100 rad/s. Both the resolution and morphology of the printed tissue of four bioinks were similar. The proliferation levels of HUVECs co-cultured with 1P-AG, 2P-AG, and 4P-AG printed tissue for 24 h were 0.885±0.030, 1.126±0.032, and 1.156±0.045, respectively, which were significantly higher than 0.712±0.019 of HUVECs co-cultured with AG printed tissue (P<0.01). The proliferation levels of HUVECs co-cultured with 2P-AG and 4P-AG printed tissue for 24 h were significantly higher than the level of HUVECs co-cultured with 1P-AG printed tissue (P<0.01). The degradation rates of 1P-AG, 2P-AG, and 4P-AG printed tissue were significantly higher than those of AG printed tissue at 12, 24, and 48 h of culture (P<0.01). The degradation rates of 2P-AG and 4P-AG printed tissue at 24 and 48 h of culture were significantly higher than those of 1P-AG printed tissue (P<0.01). The degradation rate of 4P-AG printed tissue at 12 h of culture was significantly higher than that of 1P-AG printed tissue (P<0.01), and the degradation rates of 4P-AG printed tissue at 24 and 48 h of culture were significantly higher than those of 2P-AG printed tissue (P<0.01). At 0.5, 24.0, and 48.0 h of culture, the expressions of VEGF in the culture supernatant of 2P-AG printed tissue were significantly higher than those of 1P-AG printed tissue (P<0.01), and the expressions of VEGF in the culture supernatant of 1P-AG and 2P-AG printed tissue were significantly lower than those of 4P-AG printed tissue (P<0.01). The wounds of nude mice in conventional control group and HUCB-PRP group were dry and smaller on PID 8 compared with those on PID 4, and the wounds of nude mice in HUCB-PRP group were smaller with no scabs on PID 14 compared with those in conventional control group. The printed tissue on the wound of nude mice in AG and 4P-AG groups was significantly degraded with no obvious exudation being observed on the wounds on PID 4, the wounds were significantly epithelialized and smaller on PID 8, and there was no scab on the wound on PID 14. The wounds of nude mice in 4P-AG group were completely epithelialized on PID 14. Compared with those in conventional control group, the wound healing rate of nude mice in AG group was significantly decreased on PID 4 (P<0.05), and the wound healing rates of nude mice in HUCB-PRP group and 4P-AG group at all time points after injury and in AG group on PID 8 and 14 were significantly increased (P<0.01). Compared with those in HUCB-PRP group, the wound healing rates of nude mice were significantly decreased on PID 4 and 8 in AG group and on PID 4 in 4P-AG group (P<0.01), while the wound healing rates of nude mice were significantly increased on PID 14 in AG group and on PID 8 and 14 in 4P-AG group (P<0.01). The wound healing rate of nude mice in 4P-AG group was significantly higher than that in AG group at all time points after injury (P<0.01). On PID 8, a large number of inflammatory cells infiltration, a small amount of new microvessels, and a small amount of CD31-positive new blood vessels were observed in the wounds of nude mice in conventional control group; a large number of inflammatory cells infiltration, abundant new microvessels, and quite a lot CD31-positive new blood vessels were observed in the wounds of nude mice in HUCB-PRP group; light inflammatory inflammation, a small amount of new microvessels, and a small amount of CD31-positive new blood vessels were observed in the wounds of nude mice in AG group; light inflammatory inflammation, a large number of new microvessels, and a large number of CD31-positive new blood vessels were observed in the wounds of nude mice in 4P-AG group. Conclusions HUCB-PRP-AG bioink has good printability and cytocompatibility, and its three-dimensionally printed tissue can promote vascularization of full-thickness skin defect wounds in nude mice and accelerate wound healing.
- Biocompatible materials /
- Printing, three-dimensional /
- Neovascularization, physiologic /
- Bioink /
- Human umbilical cord blood-derived platelet-rich plasma /
- Wound repair
宋薇、李曌：实验设计、实验操作以及论文撰写；朱世钧：实验操作、数据采集；张超：数据整理与统计分析；姚斌、孔毅、梁莉婷、恩和吉日嘎拉：技术和材料支持；付小兵、黄沙：获取研究经费、对文章的知识性内容作批评性审阅

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1 4种生物墨水的表征和流变性能与储能模量/损耗模量比较。1A.大体外观图，室温下均为半透明液态；1B.10 ℃下黏度-剪切率曲线；1C.10 ℃下储能模量/损耗模量-角频率曲线

注：图1A中1、2、3、4与图1B、1C中红色、紫色、蓝色、绿色线均分别指示海藻酸钠-明胶（AG）和含体积分数2.5%、5.0%、10.0%人脐血来源富血小板血浆的AG生物墨水1P-AG、2P-AG、4P-AG；图1C中上方4条虚线为储能模量，下方4条虚线为损耗模量；图1C为经过lg 处理的数据形成的描记图，坐标轴数据为未经lg处理的原始数据

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2 4种生物墨水的三维生物打印组织外观相近。2A、2B、2C、2D.分别为AG、1P-AG、2P-AG、4P-AG打印组织

注：AG为海藻酸钠-明胶，1P-AG、2P-AG、4P-AG分别为含体积分数2.5%、5.0%、10.0%人脐血来源富血小板血浆的AG生物墨水

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3 4组裸鼠全层皮肤缺损创面伤后各时间点愈合情况。3A、3B、3C与3D、3E、3F.分别为常规对照组与HUCB-PRP组伤后4、8、14 d创面，图3B、3E创面分别较图3A、3D创面干燥且缩小，图3F创面明显小于图3C且无结痂；3G、3H、3I与3J、3K、3L.分别为AG组与4P-AG组伤后4、8、14 d创面，图3G与3J创面上打印组织明显降解且创面无明显渗出，图3H与3K创面上皮化明显（乳白色区域）且图3K创面较图3H与3J明显缩小，图3I创面无结痂且基底鲜红，图3L创面上皮化完全且无结痂

注：4组均用医用水胶体敷料覆盖创面，人脐血来源富血小板血浆（HUCB-PRP）组、海藻酸钠-明胶（AG）组、4P-AG组另分别行HUCB-PRP滴加、AG打印组织覆盖、4P-AG打印组织覆盖，4P-AG为含体积分数10.0% HUCB-PRP的AG生物墨水

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4 4组裸鼠全层皮肤缺损创面伤后8 d组织病理学变化与CD31阳性新生血管（棕色）情况。4A、4B、4C、4D.分别为常规对照组、HUCB-PRP组、AG组、4P-AG组创面组织，图4A创面基底有大量炎症细胞浸润和少量新生微血管，图4B创面基底可见大量炎症细胞浸润和丰富新生微血管，图4C创面基底可见轻度炎症浸润和少量新生微血管，图4D创面基底可见轻度炎症浸润和大量新生微血管苏木精-伊红（HE）×50；4E、4F、4G、4H.分别为图4A、4B、4C、4D中方框处放大图，可明显观察到新生微血管情况 HE×400；4I、4J、4K、4L.分别为常规对照组、HUCB-PRP组、AG组、4P-AG组创面组织，图4I和4K均有少量CD31阳性新生血管，图4J有较多CD31阳性新生血管，图4L有大量CD31阳性新生血管 CD31-二氨基联苯胺×400

注：4组均用医用水胶体敷料覆盖创面，人脐血来源富血小板血浆（HUCB-PRP）组、海藻酸钠-明胶（AG）组、4P-AG组另分别行HUCB-PRP滴加、AG打印组织覆盖、4P-AG打印组织覆盖，4P-AG为含体积分数10.0% HUCB-PRP的AG生物墨水；图4A~4D中D为创缘真皮组织，E为创缘表皮组织，N/E为新生表皮组织，N/D为新生真皮组织，黑色虚线为N、E、N/E与N/D的分界线

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表1 4种生物墨水三维打印组织培养各时间点降解率比较（%，x¯±s）

材料名称	样本数	12 h	24 h	48 h
AG打印组织	3	1.23±0.07	3.57±0.12	7.47±0.06
1P-AG打印组织	3	1.53±0.06^a	4.07±0.06^a	7.77±0.06^a
2P-AG打印组织	3	1.63±0.06^a	4.27±0.06^ab	8.07±0.06^ab
4P-AG打印组织	3	1.77±0.06^ab	4.53±0.06^abc	8.90±0.10^abc
注：AG为海藻酸钠-明胶，1P-AG、2P-AG、4P-AG分别为含体积分数2.5%、5.0%、10.0%人脐血来源富血小板血浆的AG生物墨水；处理因素主效应，F=41 941.00，P<0.001；时间因素主效应，F=316.70，P<0.001；两者交互作用，F=63.69，P<0.001；与AG打印组织比较，^aP<0.01；与1P-AG打印组织比较，^bP<0.01；与2P-AG打印组织比较，^cP<0.01

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表2 3种生物墨水三维打印组织培养各时间点上清液中VEGF的表达比较（pg/mL，x¯±s）

材料名称	样本数	0.5 h	24.0 h	48.0 h
1P-AG打印组织	5	29.6±0.6	185.4±1.8	199.9±1.0
2P-AG打印组织	5	117.3±1.0^a	387.4±3.2^a	408.0±1.6^a
4P-AG打印组织	5	314.2±2.6^ab	547.2±3.3^ab	569.4±1.5^ab
注：AG为海藻酸钠-明胶，1P-AG、2P-AG、4P-AG分别为含体积分数2.5%、5.0%、10.0%人脐血来源富血小板血浆的AG生物墨水；处理因素主效应，F=14 613.00，P<0.001；时间因素主效应，F=66 236.00，P<0.001；两者交互作用，F=1 275.00，P<0.001；与1P-AG打印组织比较，^aP<0.01；与2P-AG打印组织比较，^bP<0.01

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表3 4组裸鼠全层皮肤缺损创面伤后各时间点愈合率比较（%，x¯±s）

组别	样本数	4 d	8 d	14 d
常规对照组	3	10.2±0.8	52.3±1.2	71.3±0.8
HUCB-PRP组	3	35.4±1.7^a	72.3±0.3^a	85.8±0.5^a
AG组	3	6.7±1.0^bc	60.4±1.1^ac	89.6±0.6^ac
4P-AG组	3	24.2±2.9^acd	78.3±0.8^acd	98.6±0.8^acd
注：4组均用医用水胶体敷料覆盖创面，人脐血来源富血小板血浆（HUCB-PRP）组、海藻酸钠-明胶（AG）组、4P-AG组另分别行HUCB-PRP滴加、AG打印组织覆盖、4P-AG打印组织覆盖，4P-AG为含体积分数10.0% HUCB-PRP的AG生物墨水；处理因素主效应，F=9 258.00，P<0.001；时间因素主效应，F=650.70，P<0.001；两者交互作用，F=105.10，P<0.001；与常规对照组比较，^aP<0.01，^bP<0.05；与HUCB-PRP组比较，^cP<0.01；与AG组，^dP<0.01

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参考文献(29)

[1]	ChenS,ShiY,LuoY,et al.Layer-by-layer coated porous 3D printed hydroxyapatite composite scaffolds for controlled drug delivery[J].Colloids Surf B Biointerfaces,2019,179:121-127.DOI: 10.1016/j.colsurfb.2019.03.063.
[2]	da SilvaLP,ReisRL,CorreloVM,et al.Hydrogel-based strategies to advance therapies for chronic skin wounds[J].Annu Rev Biomed Eng,2019,21:145-169.DOI: 10.1146/annurev-bioeng-060418-052422.
[3]	HuH,XuFJ.Rational design and latest advances of polysaccharide-based hydrogels for wound healing[J].Biomater Sci,2020,8(8):2084-2101.DOI: 10.1039/d0bm00055h.
[4]	XiaS,WengT,JinR,et al.Curcumin-incorporated 3D bioprinting gelatin methacryloyl hydrogel reduces reactive oxygen species-induced adipose-derived stem cell apoptosis and improves implanting survival in diabetic wounds[J/OL].Burns Trauma,2022,10:tkac001[2022-06-18].https://pubmed.ncbi.nlm.nih.gov/35291229/.DOI: 10.1093/burnst/tkac001.
[5]	YaoB,WangR,WangY,et al.Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration[J].Sci Adv,2020,6(10):eaaz1094.DOI: 10.1126/sciadv.aaz1094.
[6]	张超,李曌,宋薇,等.含人脂肪来源蛋白复合物的三维生物打印墨水的创面修复效应初探[J].中华烧伤杂志,2021,37(11):1011-1023.DOI: 10.3760/cma.j.cn501120-20210813-00282.
[7]	SongW,YaoB,ZhuD,et al.3D-bioprinted microenvironments for sweat gland regeneration[J/OL].Burns Trauma,2022,10:tkab044[2022-06-18].https://pubmed.ncbi.nlm.nih.gov/35071651/.DOI: 10.1093/burnst/tkab044.
[8]	LopesSV,CollinsMN,ReisRL,et al.Vascularization approaches in tissue engineering: recent developments on evaluation tests and modulation[J].ACS Appl Bio Mater,2021,4(4):2941-2956.DOI: 10.1021/acsabm.1c00051.
[9]	BelderbosME,LevyO,MeyaardL,et al.Plasma-mediated immune suppression: a neonatal perspective[J].Pediatr Allergy Immunol,2013,24(2):102-113.DOI: 10.1111/pai.12023.
[10]	CoxST,DanbyR,HernandezD,et al.Functional characterisation and analysis of the soluble NKG2D ligand repertoire detected in umbilical cord blood plasma[J].Front Immunol,2018,9:1282.DOI: 10.3389/fimmu.2018.01282.
[11]	CoxST,MadrigalJA,SaudemontA.Three novel allelic variants of the RAET1E/ULBP4 gene in humans[J].Tissue Antigens,2012,80(4):390-392.DOI: 10.1111/j.1399-0039.2012.01933.x.
[12]	SambergM,StoneR2nd,NatesanS,et al.Platelet rich plasma hydrogels promote in vitro and in vivo angiogenic potential of adipose-derived stem cells[J].Acta Biomater,2019,87:76-87.DOI: 10.1016/j.actbio.2019.01.039.
[13]	SteinleA,LiP,MorrisDL,et al.Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family[J].Immunogenetics,2001,53(4):279-287.DOI: 10.1007/s002510100325.
[14]	VersuraP,ProfazioV,BuzziM,et al.Efficacy of standardized and quality-controlled cord blood serum eye drop therapy in the healing of severe corneal epithelial damage in dry eye[J].Cornea,2013,32(4):412-418.DOI: 10.1097/ICO.0b013e3182580762.
[15]	EvertsP,OnishiK,JayaramP,et al.Platelet-rich plasma: new performance understandings and therapeutic considerations in 2020[J].Int J Mol Sci,2020,21(20):7794.DOI: 10.3390/ijms21207794.
[16]	LjubimovAV,SaghizadehM.Progress in corneal wound healing[J].Prog Retin Eye Res,2015,49:17-45.DOI: 10.1016/j.preteyeres.2015.07.002.
[17]	NagarajaS,ChenL,DiPietroLA,et al.Computational analysis identifies putative prognostic biomarkers of pathological scarring in skin wounds[J].J Transl Med,2018,16(1):32.DOI: 10.1186/s12967-018-1406-x.
[18]	FréchetteJP,MartineauI,GagnonG.Platelet-rich plasmas: growth factor content and roles in wound healing[J].J Dent Res,2005,84(5):434-439.DOI: 10.1177/154405910508400507.
[19]	SirchiaG,RebullaP,MozziF,et al.A quality system for placental blood banking[J].Bone Marrow Transplant,1998,21 Suppl 3:S43-47.
[20]	BuzziM,VersuraP,GrigoloB,et al.Comparison of growth factor and interleukin content of adult peripheral blood and cord blood serum eye drops for cornea and ocular surface diseases[J].Transfus Apher Sci,2018,57(4):549-555.DOI: 10.1016/j.transci.2018.06.001.
[21]	RebullaP,PupellaS,SantodiroccoM,et al.Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood[J].Blood Transfus,2016,14(1):73-79.DOI: 10.2450/2015.0122-15.
[22]	ReddyLVK,MuruganD,MullickM,et al.Recent approaches for angiogenesis in search of successful tissue engineering and regeneration[J].Curr Stem Cell Res Ther,2020,15(2):111-134.DOI: 10.2174/1574888X14666191104151928.
[23]	KoleskyDB,TrubyRL,GladmanAS,et al.3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs[J].Adv Mater,2014,26(19):3124-3130.DOI: 10.1002/adma.201305506.
[24]	KobayashiY,SaitaY,TakakuT,et al.Platelet-rich plasma (PRP) accelerates murine patellar tendon healing through enhancement of angiogenesis and collagen synthesis[J].J Exp Orthop,2020,7(1):49.DOI: 10.1186/s40634-020-00267-1.
[25]	BerndtS,CarpentierG,TurziA,et al.Angiogenesis is differentially modulated by platelet-derived products[J].Biomedicines,2021,9(3):251.DOI: 10.3390/biomedicines9030251.
[26]	中国老年医学学会烧创伤分会.浓缩血小板制品在创面修复中应用的全国专家共识(2020版)[J].中华烧伤杂志,2020,36(11):993-1002.DOI: 10.3760/cma.j.cn501120-20200507-00256.
[27]	LiY,MouS,XiaoP,et al.Delayed two steps PRP injection strategy for the improvement of fat graft survival with superior angiogenesis[J].Sci Rep,2020,10(1):5231.DOI: 10.1038/s41598-020-61891-6.
[28]	MartinoMM,BriquezPS,RangaA,et al.Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix[J].Proc Natl Acad Sci U S A,2013,110(12):4563-4568.DOI: 10.1073/pnas.1221602110.
[29]	ZhangX,YaoD,ZhaoW,et al.Engineering platelet-rich plasma based dual-network hydrogel as a bioactive wound dressing with potential clinical translational value[J]. Adv Funct Mater,2021,31(8):2009258.DOI: 10.1002/adfm.202009258.