Abstract
目的
探讨人脂肪来源蛋白复合物（ADPC）对人皮肤成纤维细胞（HSF）和人脐静脉内皮细胞（HUVEC）增殖和迁移能力的影响, 以及含ADPC的三维生物打印墨水（Bioink）在裸鼠全层皮肤缺损创面中的修复效应。
方法
采用实验研究方法。收集解放军总医院2020年10月—2021年3月收治的3例行腹部皮瓣转移修补术的女性慢性创面患者（年龄29~34岁）的废弃皮下脂肪组织和同期收治的3名行腹部抽脂术的健康女性（年龄24~36岁）的废弃抽脂术脂肪组织, 分别制备正常ADPC（nADPC）及抽脂术ADPC（lADPC）。采用二辛丁酸法测定2种ADPC的蛋白浓度, 计算2种ADPC的提取效率, 样本数均为3。取对数生长期HSF和HUVEC进行后续实验。取2种细胞均按随机数字表法（分组方法下同）分为磷酸盐缓冲液（PBS）对照组、4 μg/mL nADPC组、20 μg/mL nADPC组、100 μg/mL nADPC组和200 μg/mL nADPC组, 每组5孔。PBS对照组细胞采用PBS培养, 余4组分别采用含相应终质量浓度的nADPC培养。常规培养24 h, 采用细胞计数试剂盒8法检测细胞增殖活力。取HSF和HUVEC, 分为PBS对照组、单纯nADPC组、单纯lADPC组、单纯肿瘤坏死因子α（TNF-α）组、TNF-α+nADPC组、TNF-α+lADPC组。PBS对照组与单纯TNF-α组细胞分别加入PBS, 单纯nADPC组、单纯lADPC组、TNF-α+nADPC组及TNF-α+lADPC组中分别加入终质量浓度100 μg/mL的nADPC或lADPC, 单纯TNF-α组、TNF-α+nADPC组及TNF-α+lADPC组再加入终质量浓度20 ng/mL的TNF-α。进行细胞划痕试验后计算划痕后24 h细胞迁移率（样本数为3）, 同前检测培养24 h细胞增殖活力（样本数为5）。取明胶-海藻酸钠复合Bioink（Bioink AG）, 制备含100 μg/mL lADPC的Bioink AG（lADPC-Bioink AG）, 观察二者在室温下及冷凝后的形态和三维生物打印且交联后的形态, 采用流变仪检测流变性能时记录低温成胶时间（样本数为3）。取20只8~10周龄雌性BALB/c-NU裸鼠, 建立背部全层皮肤缺损创面模型后, 分为常规换药组、单纯lADPC组、单纯Bioink AG组和lADPC-Bioink AG组, 每组5只。常规换药组裸鼠创面仅覆盖水胶体敷料和常规换药, 其余3组裸鼠创面另予相应lADPC、Bioink AG、lADPC-Bioink AG处理。从治疗0 d起, 行大体观察, 计算治疗2、6、10 d的创面愈合率。治疗10 d, 采用苏木精-伊红染色行创面组织病理学观察。对数据行独立样本t检验、单因素方差分析、重复测量方差分析、SNK-q检验及LSD-t检验。
结果
lADPC的蛋白浓度、提取效率分别为（1.306±0.011）mg/mL、（11.1±1.5）%, 明显低于nADPC的（2.039±0.042）mg/mL、（22.2±2.0）%（t=23.83、6.38, P＜0.05或P＜0.01）。培养24 h, 与PBS对照组比较, 100 μg/mL nADPC组和200 μg/mL nADPC组HSF（q=6.943、6.375, P＜0.01）和HUVEC（q=6.301、6.496, P＜0.01）增殖活力均明显下降；与100 μg/mL nADPC组比较, 200 μg/mL nADPC组HSF和HUVEC增殖活力无明显变化（P＞0.05）。划痕后24 h, 与PBS对照组比较, 单纯nADPC组、单纯lADPC组、单纯TNF-α组HSF和HUVEC迁移率均明显降低（q=5.642、6.645、11.480, 4.772、6.298、10.420, P＜0.05或P＜0.01）；与单纯nADPC组比较, 单纯lADPC组HSF和HUVEC迁移率无明显变化（P＞0.05）；与单纯TNF-α组比较, TNF-α+nADPC组、TNF-α+lADPC组HSF迁移率无明显变化（P＞0.05）, HUVEC迁移率均明显升高（q=8.585、7.253, P＜0.01）；与TNF-α+nADPC组比较, TNF-α+lADPC组HSF和HUVEC迁移率无明显变化（P＞0.05）。培养24 h, 与PBS对照组比较, 单纯nADPC组、单纯lADPC组、单纯TNF-α组HSF和HUVEC增殖活力均明显降低（q=5.803、5.371、9.136, 11.580、9.493、13.510, P＜0.05或P＜0.01）；与单纯nADPC组比较, 单纯lADPC组HSF和HUVEC增殖活力均无明显变化（P＞0.05）；与单纯TNF-α组比较, TNF-α+nADPC组、TNF-α+lADPC组HSF（q=14.990、10.850, P＜0.01）和HUVEC（q=7.066、8.942, P＜0.01）增殖活力均明显升高；与TNF-α+nADPC组比较, TNF-α+lADPC组HSF和HUVEC增殖活力均无明显变化（P＞0.05）。室温及冷凝状态下, lADPC-Bioink AG比Bioink AG外观稍显浑浊；三维生物打印且交联后lADPC-Bioink AG与Bioink AG形态类似。在10 ℃时, lADPC-Bioink AG的凝固时间为（76.6±0.4）s, 明显慢于Bioink AG的（74.4±0.6）s（t=4.64, P＜0.01）。治疗2 d, 常规换药组裸鼠创面渗出较多, 其余3组无明显渗出；治疗8 d, lADPC-Bioink AG组裸鼠残余创面面积最小, 有明显上皮覆盖。治疗2 d, lADPC-Bioink AG组裸鼠创面愈合率明显高于单纯lADPC组（t=3.59, P＜0.05）, 与常规换药组、单纯Bioink AG组相近（P＞0.05）；治疗6 d, lADPC-Bioink AG组裸鼠创面愈合率明显高于常规换药组、单纯lADPC组、单纯Bioink AG组（t=18.70、15.70、3.15, P＜0.05或P＜0.01）；治疗10 d, lADPC-Bioink AG组裸鼠创面愈合率明显高于常规换药组、单纯lADPC组（t=12.51、4.84, P＜0.01）, 与单纯Bioink AG组相近（P＞0.05）。治疗10 d, lADPC-Bioink AG组裸鼠创面组织血管化程度适中, 上皮化充分, 愈合效果最好。
结论
抽脂术相关操作会降低ADPC蛋白浓度、提取效率等表征, 但lADPC与nADPC具有相同的生物学作用, 可在非炎症环境中抑制HSF与HUVEC的增殖与迁移能力, 在炎症环境中提高HSF与HUVEC的增殖活力, 同时提高HUVEC的迁移能力；将lADPC加入Bioink AG中后, 不会明显影响Bioink AG的物理性质及打印性能, 并可以提升裸鼠全层皮肤缺损创面的修复效果。
Keywords: 再生医学, 组织工程, 多蛋白复合物, 三维生物打印墨水, 创面修复
Abstract
Objective
To investigate the effects of human adipose-derived protein complex (ADPC) on the proliferation and migration ability of human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs), and the repairing effects of ADPC-containing three-dimensional (3D) bioprinting ink (Bioink) in full-thickness skin defect wounds of nude mice.
Methods
The experimental research method was used. Discarded subcutaneous adipose tissue from 3 female patients with chronic wounds (aged 29-34 years) admitted to PLA General Hospital for abdominal flap transfer from October 2020 to March 2021 and discarded liposuction adipose tissue from 3 healthy female (aged 24-36 years) for abdominal liposuction during the same period were collected to prepare normal ADPC (nADPC) and liposuction-derived ADPC (lADPC), respectively. The protein concentration of the two kinds of ADPC was measured by bicinchoninic acid method, and the extraction efficiency of them was calculated. The sample numbers were 3. HSFs and HUVECs in logarithmic growth phase were taken for the subsequent experiments. HSFs and HUVECs were divided into phosphate buffered saline (PBS) control group, 4 μg/mL nADPC group, 20 μg/mL nADPC group, 100 μg/mL nADPC group, and 200 μg/mL nADPC group according to the random number table (the same grouping method below), with 5 wells in each group. Cells in PBS control group were cultured with PBS, and the cells in the 4 remaining groups were cultured with the corresponding final mass concentration of nADPC. After 24 h of conventional culture, the cell proliferation viability was detected by cell counting kit 8 method. HSFs and HUVECs were taken and divided into PBS control group, nADPC alone group, lADPC alone group, tumor necrosis factor-α (TNF-α) alone group, TNF-α+nADPC group, and TNF-α+lADPC group. Cells in PBS control group and TNF-α alone group were added with PBS. nADPC or lADPC was added to the cells in nADPC alone group, lADPC alone group, TNF-α+nADPC group, and TNF-α+lADPC group with a final mass concentration of 100 μg/mL, respectively. TNF-α with a final mass concentration of 20 ng/mL was added to the cells in TNF-α alone group, TNF-α+nADPC group, and TNF-α+lADPC group. The cell migration rate was calculated after the scratch test at 24 h after scratching (n=3), and the cell proliferation viability was detected after 24 h of culture as above (n=5). Gelatin-alginate composite Bioink (Bioink AG) was taken. Bioink AG containing 100 μg/mL lADPC (lADPC-Bioink AG) was prepared. The morphology of the two at room temperature and after condensation was observed. The morphology after 3D bioprinting and cross-linking was observed. The low-temperature gel formation time was recorded when detecting rheological properties using rheometer (n=3). Twenty BALB/c-NU female nude mice of 8-10 weeks old were taken to establish the full-thickness skin defect wounds on the back, and then they were divided into routine dressing change group, lADPC alone group, Bioink AG alone group, and lADPC-Bioink AG group, with 5 nude mice in each group. The wounds of nude mice in routine dressing change group were covered with hydrocolloid dressings and performed with routine dressing changes only, while the wounds of nude mice in the remaining 3 groups were treated with lADPC, Bioink AG, and lADPC-Bioink AG accordingly in addition. General observation was performed from treatment day (TD) 0, and the wound healing rate was calculated on TD 2, 6, and 10. On TD 10, histopathological observation of wounds was performed with hematoxylin eosin staining. Data were statistically analyzed with independent samples t test, one-way analysis of variance, analysis of variance for repeated measurement, Student-Newman-Keuls q test, and least significant difference t test.
Results
The protein concentration and extraction efficiency of lADPC were respectively (1.306±0.011) mg/mL and (11.1±1.5)%, which were significantly lower than (2.039±0.042) mg/mL and (22.2±2.0)% of nADPC (t=23.83, 6.38, P < 0.05 or P < 0.01). After 24 h of culture, compared with those in PBS control group, the proliferation viabilities of HSFs (q=6.943, 6.375, P < 0.01) and HUVECs (q=6.301, 6.496, P < 0.01) were significantly decreased in 100 μg/mL nADPC group and 200 μg/mL nADPC group; compared with those in 100 μg/mL nADPC group, the proliferation viabilities of HSFs and HUVECs in 200 μg/mL nADPC group did not change significantly (P > 0.05). At 24 h after scratching, compared with those in PBS control group, the HSF and HUVEC migration rates were significantly lower in nADPC alone group, lADPC alone group, and TNF-α alone group (q=5.642, 6.645, 11.480, 4.772, 6.298, 10.420, P < 0.05 or P < 0.01); compared with those in nADPC alone group, there were no significant changes in the HSF and HUVEC migration rates in lADPC alone group (P > 0.05); compared with those in TNF-α alone group, there were no significant changes in the HSF migration rates in TNF-α+nADPC group or TNF-α+lADPC group (P > 0.05), the HUVEC migration rates were significantly higher in TNF-α+nADPC group and TNF-α+lADPC group (q=8.585, 7.253, P < 0.01); compared with those in TNF-α+nADPC group, there were no significant changes in the HSF and HUVEC migration rates in TNF-α+lADPC group (P > 0.05). After 24 h of culture, compared with those in PBS control group, the HSF and HUVEC proliferation viabilities were significantly lower in nADPC alone group, lADPC alone group, and TNF-α alone group (q=5.803, 5.371, 9.136, 11.580, 9.493, 13.510, P < 0.05 or P < 0.01); compared with those in nADPC alone group, the HSF and HUVEC proliferation viabilities in lADPC alone group did not change significantly (P > 0.05); compared with those in TNF-α alone group, the HSF (q=14.990, 10.850, P < 0.01) and HUVEC (q=7.066, 8.942, P < 0.01) proliferation viabilities were significantly higher in TNF-α+nADPC group and TNF-α+lADPC group; compared with those in TNF-α+nADPC group, the HSF and HUVEC proliferation viabilities in TNF-α+lADPC group did not change significantly (P > 0.05). At room temperature and in the condensed state, lADPC-Bioink AG had a more slightly turbid appearance than Bioink AG. lADPC-Bioink AG had a similar morphology to Bioink AG after 3D bioprinting and cross-linking. At 10 ℃, the coagulation time of lADPC-Bioink AG was (76.6±0.4) s, which was significantly slower than (74.4±0.6) s of Bioink AG (t=4.64, P < 0.01). On TD 2, the nude mice in routine dressing change group had more wound exudation, while the nude mice in the remaining 3 groups had no significant exudation. On TD 8, the nude mice in lADPC-Bioink AG group had the smallest residual wound area and obvious epithelial coverage. On TD 2, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than that in lADPC alone group (t=3.59, P < 0.05) and similar to the rates in routine dressing change group and Bioink AG alone group (P > 0.05). On TD 6, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than the rates in routine dressing change group, lADPC alone group, and Bioink AG alone group (t=18.70, 15.70, 3.15, P < 0.05 or P < 0.01). On TD 10, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than the rates in routine dressing change group and lADPC alone group (t=12.51, 4.84, P < 0.01) but similar to that in Bioink AG alone group (P > 0.05). On TD 10, the wounds of nude mice in lADPC-Bioink AG group had moderate vascularization of the traumatic tissue, adequate epithelialization, and the best healing effect.
Conclusions
Liposuction-related operations have little effect on the characterization of ADPC protein concentration and extraction efficiency. LADPC and nADPC have the same biological effects, which can inhibit the proliferation and migration ability of HSFs and HUVECs in non-inflammatory environment and improve the proliferation viabilities of HSFs and HUVECs in inflammatory environment, while improving the migration ability of HUVECs. Adding lADPC to Bioink AG does not significantly affect the physical properties or printing performance of Bioink AG, but can enhance the wound repair effect of full-thickness skin defect wounds in nude mice.
Keywords: Regenerative medicine, Tissue engineering, Multiprotein complexes, Three-dimensional bioprinting ink, Wound repair
创面的瘢痕愈合或难愈合是严重的医学问题, 不仅会导致局部组织生长受限及功能丧失, 还会造成机体活动障碍和身心疾病, 给患者及社会带来沉重的经济负担[1-2]。再生医学和组织工程技术在创面治疗领域已取得良好成果[3-4]。其中, 三维生物打印技术可以构建功能化的活体组织, 因而被广泛应用于开发创面敷料及皮肤替代物[5-6]。该技术所使用的三维生物打印墨水（Bioink）可在体外为细胞构建仿生三维微环境, 精准调控多种类型细胞的生物学功能, 在三维生物打印技术的应用中起着决定性的作用[7-8]。Bioink中的成分和因子会显著改变皮肤创面的细胞行为[9-10], 但目前多数Bioink中的活性成分与正常环境比较仍过于简单, 无法有效调控创面微环境以加快愈合、减少瘢痕[11]。
近年来, 脂肪组织的非代谢功能得到了越来越多的研究与重视[12-13]。作为丰富的细胞及生物活性物质储存库, 皮下的脂肪组织有利于维持正常的皮肤生理状态, 也可以促进皮肤的创面愈合[14-15]。但外科抽脂手术抽取的脂肪组织多被当作医疗废物丢弃, 大量生物活性物质难以得到有效利用。目前, 已有学者将外科手术产生的脂肪组织回收利用, 制备出的脂肪提取液显示出良好的促进血管和脂肪组织生成效应, 一定程度上促进了创面的愈合[16-17-18], 但尚未见将脂肪组织来源的蛋白复合物应用于Bioink制备的报道。
本课题组在前期研究中已验证了明胶-海藻酸钠复合Bioink（Bioink AG）的优良物理性能[19-21], 但其中所含的生物化学信号（如细胞因子及生长因子）还十分有限, 因此本研究希望通过提取正常脂肪来源蛋白复合物（nADPC）及抽脂术脂肪来源蛋白复合物（lADPC）, 体外验证两者生物学功能后, 将lADPC加入Bioink AG, 用以提升Bioink AG的生物学性能。本研究同时拟明确ADPC对创面愈合的影响及相关机制, 为Bioink的研发及其临床转化应用提供新思路和新途径。
1. 材料与方法
本实验研究经解放军总医院伦理委员会审批通过, 批号：伦审第S2020-120-01号。本研究符合《赫尔辛基宣言》的基本原则, 患者均签署知情同意书, 遵循解放军总医院和国家有关实验动物管理和使用的相关规定。
1.1. 主要材料来源
人体正常脂肪组织来源于解放军总医院2020年10月—2021年3月收治的3例行腹部皮瓣转移修补术的女性慢性创面患者（年龄29~34岁）的废弃皮下脂肪组织, 人体抽脂术脂肪组织来源于解放军总医院同期收治的3名行腹部抽脂术的健康女性（年龄24~36岁）的废弃抽脂术脂肪组织。20只健康无特殊病原体级8~10周龄雌性BALB/c-NU裸鼠, 体重15~25 g, 由斯贝福（北京）生物技术有限公司提供, 许可证号：SCXK（京）2019-0010。人皮肤Fb（HSF, 永生化）及其专用培养基、人脐静脉内皮细胞（HUVEC）及其专用培养基均购自赛百慷（上海）生物技术股份有限公司。Bioink AG由国科瑞诺（中山）生物科技有限公司提供。氯化钙购于天津市福晨化学试剂厂, 重组人TNF-α购于美国PeproTech公司, 红细胞裂解液、二辛丁酸蛋白定量测定试剂盒购于北京索莱宝科技有限公司, 细胞计数试剂盒8（CCK-8）和HE染色试剂盒购于上海碧云天生物技术有限公司。BBD 6220型二氧化碳培养箱购于美国Thermo Fisher Scientific公司, Spark 10M型多功能酶标仪购于瑞士TECAN公司, Regenovo S/R型三维生物打印机购于杭州捷诺飞生物科技股份有限公司, BMI4000型倒置荧光显微镜购于德国莱卡公司, ARES-G2型旋转流变仪购于美国TA Instruments公司, S60型数字全景扫描仪购于日本滨松光子学株式会社。
1.2. nADPC和lADPC的制备及性质观测
1.2.1. nADPC和lADPC的制备
取人体正常脂肪组织及人体抽脂术脂肪组织, 用PBS多次（≥3次）清洗, 至洗涤液呈透明无色, 使用眼科剪去除筋膜组织, 并将脂肪组织剪成约0.1 cm×0.1 cm×0.1 cm小块。各取10 mL上述处理后2种脂肪组织, 分别加入双倍体积红细胞裂解液, 于冰上裂解红细胞15 min, 于离心半径8.0 cm、2 000 r/min、4 ℃（离心温度下同）离心5 min后, 取上层脂肪组织, 置于容积20 mL研磨器中研磨至脂肪全部变为淡黄色乳糜状液体。将研磨液于离心半径8.0 cm、4 500 r/min离心10 min, 可见研磨液分层, 取最下层液体部分, 于离心半径5.5 cm、15 000 r/min离心30 min, 取上清液, 经滤孔直径0.22 μm过滤器过滤, 制得nADPC和lADPC。
1.2.2. nADPC和lADPC蛋白浓度检测
采用二辛丁酸法检测。将二辛丁酸溶液与铜离子溶液以50∶1的体积比配制成二辛丁酸工作液, 充分混匀后备用。取10 μL牛血清白蛋白标准品用PBS稀释至100 μL, 使终质量浓度为0.5 mg/mL。将标准品按0、2、4、6、8、12、16、20 μL加到96孔板的蛋白标准品孔中, 不足20 μL的加PBS补足, 每个浓度梯度设置3个复孔。再将样品按0.5、1.0、2.0、4.0、20.0 μL加到96孔板的样品孔中, 同前补足至20 μL, 每个浓度设3个复孔, 再在各孔加入200 μL二辛丁酸工作液, 于37 ℃下放置30 min。采用多功能酶标仪测定562 nm波长下的吸光度值, 绘制标准曲线, 根据标准曲线计算出nADPC和lADPC的蛋白浓度。本实验重复3次, 结果选最具代表性的1次数据, 下同。
1.2.3. nADPC和lADPC提取效率计算
将1.2.1中每位研究对象经PBS清洗、修剪后的2种脂肪组织体积记为V0, 对应所获得的ADPC体积记为V, 分别计算2种ADPC的提取效率, ADPC提取效率=V÷V0×100%, 每种ADPC样本数为3。
1.2.4. nADPC最佳作用浓度筛选
将HSF和HUVEC加入各自专用培养基, 置于37 ℃含体积分数5%二氧化碳培养箱中恒温培养（常规培养条件下同）, 每2天换液1次, 细胞生长达80%融合时进行传代。取对数生长期（下同）的HSF和HUVEC, 分别用相应专用培养基调整细胞浓度为3×104个/mL, 接种于96孔板中, 每孔100 μL。细胞贴壁后弃去原培养基, PBS冲洗3次。按照随机数字表法（分组方法下同）分别将2种细胞分为PBS对照组、4 μg/mL nADPC组、20 μg/mL nADPC组、100 μg/mL nADPC组和200 μg/mL nADPC组, 每组5孔。PBS对照组细胞采用含PBS的专用培养基培养, 其余4组分别采用含相应终质量浓度的nADPC的专用培养基培养。各组细胞常规培养24 h后, 每孔加入CCK-8溶液10 μL, 常规培养2 h。采用多功能酶标仪测量波长450 nm处的吸光度值, 表示细胞增殖活力。本实验重复3次。
1.3. 非炎症及炎症环境中不同来源ADPC对HSF和HUVEC迁移及增殖的影响
1.3.1. 细胞迁移能力
取HSF和HUVEC, 分别用相应专用培养基调整细胞浓度为2×105个/mL, 接种于6孔板中, 每孔2 mL, 分为PBS对照组、单纯nADPC组、单纯lADPC组、单纯TNF-α组、TNF-α+nADPC组及TNF-α+lADPC组, 每组3孔。细胞贴壁后弃去原培养基, 使用规格为200 μL移液器枪头在培养孔正中垂直于培养孔划一竖痕, PBS洗涤3次以清除细胞碎片及杂质, 保证划痕细胞完全脱离。进行划痕试验后, 各组每孔先加入含终体积分数1%胎牛血清的HSF或HUVEC专用培养基, PBS对照组与单纯TNF-α组细胞中分别另加入PBS, 单纯nADPC组、单纯lADPC组、TNF-α+nADPC组及TNF-α+lADPC组细胞中分别另加入终质量浓度为100 μg/mL的nADPC或lADPC, 单纯TNF-α组、TNF-α+nADPC组及TNF-α+lADPC组细胞额外再加入终质量浓度为20 ng/mL的TNF-α。分别于划痕后0（即刻）、24 h, 于50倍倒置荧光显微镜下, 每组选取3个视野采集图片。采用Image J 1.8.0图像分析软件（美国国立卫生研究院）计算其相对面积, 将划痕后0 h面积记为S0, 划痕后24 h面积记为S, 计算细胞迁移率, 细胞迁移率=（S0-S）÷S0×100%。本实验重复3次。
1.3.2. 细胞增殖活力
取HSF和HUVEC, 分别用相应专用培养基调整细胞浓度为5×104个/mL, 接种于96孔板中, 每孔100 μL。同1.3.1分组及培养, 每组5孔, 培养24 h后同1.2.4检测细胞增殖活力。本实验重复3次。
1.4. 含lADPC Bioink的制备、性能检测及对裸鼠全层皮肤缺损创面的作用
1.4.1. 含lADPC Bioink的制备
在室温条件下, 取无菌Bioink AG, 37 ℃预热后, 无菌条件下与lADPC混匀, 制成含100 μg/mL lADPC的Bioink, 命名为lADPC-Bioink AG, 用于后续实验。
1.4.2. 室温、冷凝下及三维生物打印且交联后形态观察
取Bioink AG和制备后即刻lADPC-Bioink AG各5 mL, 分别装入15 mL离心管后, 观察二者室温下形态。将离心管放入4 ℃冰箱冷凝（冷凝条件下同）30 min后, 肉眼观察其形态。另取以上2种Bioink各5 mL, 分别装入无菌打印筒中, 冷凝后安装在温度为4 ℃的三维生物打印机打印臂上, 将打印平台温度调至4 ℃、喷头温度调至0 ℃, 打印喷嘴直径为420 μm, 在直径60 mm培养皿中行三维生物打印, 打印组织层高为0.6 mm、层数为4, 采用线性填充方法, 填充间距为2 mm, 挤出压强为0.08 MPa、挤出速度为10 mm/s。打印完成后, 用25 g/L氯化钙溶液（约6 mL）交联10 min, 之后肉眼观察打印组织形态。
1.4.3. 流变性能检测
取Bioink AG和制备后即刻lADPC-Bioink AG各5 mL, 分别在配备40 mm平行板（间隙宽度为1 mm）的旋转流变仪上进行流变性能检测, 记录2种Bioink由液态转变为固态时所对应的时间。测试过程中通过板式传感器系统将平行板温度保持在10 ℃, 旋转流变仪的动态频率扫描（频率范围在0.1~10.0 Hz）以5%的应变进行。每种Bioink检测3次。
1.4.4. 裸鼠背部全层皮肤缺损创面模型的建立与分组处理
取20只裸鼠, 称重后按50 mg/kg腹腔注射20 g/L戊巴比妥钠麻醉, 待完全麻醉后, 于每只裸鼠背部正中制备直径1 cm的圆形全层皮肤缺损创面。将裸鼠分为常规换药组、单纯lADPC组、单纯Bioink AG组和lADPC-Bioink AG组, 每组5只。常规换药组除水胶体敷料覆盖和常规换药以外不予其他治疗措施；单纯lADPC组裸鼠另于伤后即刻滴加100 μg/mL的lADPC 200 μL；单纯Bioink AG组和lADPC-Bioink AG组裸鼠另于伤后即刻分别予Bioink AG和lADPC-Bioink AG打印组织覆盖创面, 并使其与创面完全贴合, 外用敷料覆盖并包扎固定。各组均持续治疗14 d。
1.4.5. 创面大体观察和愈合率
从治疗0 d起, 创面每2天换药1次, 换药时肉眼观察创面颜色、渗出及愈合情况并拍照。采用Image J图像分析软件测量创面面积, 并计算治疗2、6、10 d的创面愈合率, 创面愈合率=（治疗0 d创面面积-治疗各时间点创面面积）÷治疗0 d创面面积×100%。
1.4.6. 组织病理学观察
治疗10 d, 每组取2只裸鼠, 换药前处死并收集背部创面及距创缘0.5 cm内的组织, 用40 g/L多聚甲醛固定。常规石蜡包埋、切片（厚度为5 μm）, HE染色后用数字全景扫描仪进行扫片, 并在NDP图像分析软件（日本滨松光子学株式会社）上进行观察、测量和截图等处理。
1.5. 统计学处理
采用SPSS 24.0统计软件进行分析。所有计量资料数据均符合正态分布, 以x±s表示。2组数据的组间比较行独立样本t检验；对单一时间点单一指标组间总体比较行单因素方差分析、多个时间点组间总体比较行重复测量方差分析, 组间两两比较采用SNK-q检验或LSD-t检验。P＜0.05为差异有统计学意义。
2. 结果
2.1. nADPC和lADPC蛋白浓度、提取效率及nADPC最佳作用浓度
lADPC蛋白浓度为（1.306±0.011）mg/mL, 明显低于nADPC的（2.039±0.042）mg/mL（t=23.83, P＜0.01）。lADPC的提取效率为（11.1±1.5）%, 明显低于nADPC的（22.2±2.0）%（t=6.38, P＜0.05）。
培养24 h, 与PBS对照组比较, 4 μg/mL nADPC组和20 μg/mL nADPC组HSF和HUVEC增殖活力均无明显变化（P＞0.05）, 而100 μg/mL nADPC组和200 μg/mL nADPC组HSF和HUVEC增殖活力均明显下降（P＜0.01）；与100 μg/mL nADPC组比较, 200 μg/mL nADPC组HSF和HUVEC增殖活力无明显变化（P＞0.05）。见表 1。故后续实验中选择用终质量浓度为100 μg/mL的nADPC。
表 1.
5组HSF和HUVEC常规培养24 h细胞增殖活力比较（x±s）
| 组别 | 样本数 | HSF | HUVEC |
| 注：HSF为人皮肤成纤维细胞, HUVEC为人脐静脉内皮细胞, PBS为磷酸盐缓冲液, nADPC为正常脂肪来源蛋白复合物；F值、P值为2种细胞5组间总体比较所得；q1值、P1值, q2值、P2值, q3值、P3值, q4值、P4值分别为PBS对照组与4 μg/mL nADPC组、20 μg/mL nADPC组、100 μg/mL nADPC组、200 μg/mL nADPC组比较所得；q5值、P5值为100 μg/mL nADPC组与200 μg/mL nADPC组比较所得 | |||
| PBS对照组 | 5 | 1.013±0.034 | 0.821±0.027 |
| 4 μg/mL nADPC组 | 5 | 1.109±0.065 | 0.822±0.083 |
| 20 μg/mL nADPC组 | 5 | 0.886±0.019 | 0.648±0.101 |
| 100 μg/mL nADPC组 | 5 | 0.792±0.064 | 0.516±0.049 |
| 200 μg/mL nADPC组 | 5 | 0.810±0.017 | 0.506±0.063 |
| F值 | 18.26 | 10.32 | |
| P值 | ＜0.01 | ＜0.01 | |
| q1值 | 3.015 | 0.030 | |
| P1值 | ＞0.05 | ＞0.05 | |
| q2值 | 4.007 | 3.560 | |
| P2值 | ＞0.05 | ＞0.05 | |
| q3值 | 6.943 | 6.301 | |
| P3值 | ＜0.01 | ＜0.01 | |
| q4值 | 6.375 | 6.496 | |
| P4值 | ＜0.01 | ＜0.01 | |
| q5值 | 0.568 | 0.195 | |
| P5值 | ＞0.05 | ＞0.05 | |
2.2. 非炎症及炎症环境中不同来源ADPC对HSF和HUVEC迁移及增殖的影响
2.2.1. 细胞迁移能力
划痕后24 h, PBS对照组HSF剩余划痕面积最小, 单纯nADPC组、单纯lADPC组HSF剩余划痕面积相近, 单纯TNF-α组、TNF-α+nADPC组、TNF-α+lADPC组HSF剩余划痕面积相近且均大于单纯nADPC组及单纯lADPC组。见图 1。划痕后24 h, PBS对照组HUVEC剩余划痕面积最小, 单纯nADPC组、单纯lADPC组HUVEC剩余划痕面积相近, 单纯TNF-α组HUVEC剩余划痕面积最大, TNF-α+nADPC组、TNF-α+lADPC组HUVEC剩余划痕面积相近。见图 2。
图 1.
划痕试验观察6组人皮肤成纤维细胞划痕后各时间点迁移情况 倒置荧光显微镜×50, 图中标尺为500 μm。1A、1B、1C、1D、1E、1F.分别为磷酸盐缓冲液（PBS）对照组、单纯正常脂肪来源蛋白复合物（nADPC）组、单纯抽脂术脂肪来源蛋白复合物（lADPC）组、单纯肿瘤坏死因子α（TNF-α）组、TNF-α+nADPC组、TNF-α+lADPC组划痕后即刻；1G、1H、1I、1J、1K、1L.分别为PBS对照组、单纯nADPC组、单纯lADPC组、单纯TNF-α组、TNF-α+nADPC组、TNF-α+lADPC组划痕后24 h, 图1G剩余划痕面积最小, 图1H、1I剩余划痕面积相近, 图1J、1K、1L剩余划痕面积相近且均大于图1H、1I
图 2.
划痕试验观察6组人脐静脉内皮细胞划痕后各时间点迁移情况 倒置荧光显微镜×50, 图中标尺为500 μm。2A、2B、2C、2D、2E、2F.分别为磷酸盐缓冲液（PBS）对照组、单纯正常脂肪来源蛋白复合物（nADPC）组、单纯抽脂术脂肪来源蛋白复合物（lADPC）组、单纯肿瘤坏死因子α（TNF-α）组、TNF-α+nADPC组、TNF-α+lADPC组划痕后即刻；2G、2 H、2I、2J、2K、2L.分别为PBS对照组、单纯nADPC组、单纯lADPC组、单纯TNF-α组、TNF-α+nADPC组、TNF-α+lADPC组划痕后24 h, 图2G剩余划痕面积最小, 图2H、2I剩余划痕面积相近, 图2J剩余划痕面积最大, 图2K、2L细胞剩余划痕面积相近
划痕后24 h, 与PBS对照组比较, 单纯nADPC组、单纯lADPC组、单纯TNF-α组HSF和HUVEC迁移率均明显降低（P＜0.05或P＜0.01）；与单纯nADPC组比较, 单纯lADPC组HSF和HUVEC迁移率无明显变化（P＞0.05）；与单纯TNF-α组比较, TNF-α+nADPC组、TNF-α+lADPC组HSF迁移率无明显变化（P＞0.05）, HUVEC迁移率均明显升高（P＜0.01）；与TNF-α+nADPC组比较, TNF-α+lADPC组HSF和HUVEC迁移率无明显变化（P＞0.05）。见表 2。
表 2.
6组HSF和HUVEC划痕后24 h细胞迁移率比较(%, x±s)
| 组别 | 样本数 | HSF | HUVEC |
| 注：HSF为人皮肤成纤维细胞, HUVEC为人脐静脉内皮细胞, PBS为磷酸盐缓冲液, nADPC为正常脂肪来源蛋白复合物, lADPC为抽脂术脂肪来源蛋白复合物, TNF-α为肿瘤坏死因子α；F值、P值为2种细胞6组间总体比较所得；q1值、P1值, q2值、P2值, q3值、P3值分别为PBS对照组与单纯nADPC组、单纯lADPC组、单纯TNF-α组比较所得；q4值、P4值为单纯nADPC组与单纯lADPC组比较所得；q5值、P5值, q6值、P6值分别为单纯TNF-α组与TNF-α+nADPC组、TNF-α+lADPC组比较所得；q7值、P7值为TNF-α+nADPC组与TNF-α+lADPC组比较所得 | |||
| PBS对照组 | 3 | 68.1±2.7 | 56.8±3.3 |
| 单纯nADPC组 | 3 | 55.3±2.4 | 44.3±4.6 |
| 单纯lADPC组 | 3 | 53.0±5.5 | 40.3±2.8 |
| 单纯TNF-α组 | 3 | 42.0±2.6 | 29.6±5.1 |
| TNF-α+nADPC组 | 3 | 46.0±2.7 | 52.0±2.9 |
| TNF-α+lADPC组 | 3 | 47.6±2.2 | 48.5±2.5 |
| F值 | 16.48 | 13.48 | |
| P值 | ＜0.01 | ＜0.01 | |
| q1值 | 5.642 | 4.772 | |
| P1值 | ＜0.05 | ＜0.05 | |
| q2值 | 6.645 | 6.298 | |
| P2值 | ＜0.01 | ＜0.01 | |
| q3值 | 11.480 | 10.420 | |
| P3值 | ＜0.01 | ＜0.01 | |
| q4值 | 1.003 | 1.526 | |
| P4值 | ＞0.05 | ＞0.05 | |
| q5值 | 1.760 | 8.585 | |
| P5值 | ＞0.05 | ＜0.01 | |
| q6值 | 2.486 | 7.253 | |
| P6值 | ＞0.05 | ＜0.01 | |
| q7值 | 0.726 | 1.331 | |
| P7值 | ＞0.05 | ＞0.05 | |
2.2.2. 细胞增殖活力
培养24 h, 与PBS对照组比较, 单纯nADPC组、单纯lADPC组、单纯TNF-α组HSF和HUVEC增殖活力均明显降低（P＜0.05或P＜0.01）；与单纯nADPC组比较, 单纯lADPC组HSF和HUVEC增殖活力均无明显变化（P＞0.05）；与单纯TNF-α组比较, TNF-α+nADPC组、TNF-α+lADPC组HSF和HUVEC增殖活力均明显升高（P＜0.01）；与TNF-α+nADPC组比较, TNF-α+lADPC组HSF和HUVEC的增殖活力均没有明显变化（P＞0.05）。见表 3。
表 3.
6组HSF和HUVEC常规培养24 h细胞增殖活力比较(x±s)
| 组别 | 样本数 | HSF | HUVEC |
| 注：HSF为人皮肤成纤维细胞, HUVEC为人脐静脉内皮细胞, PBS为磷酸盐缓冲液, nADPC为正常脂肪来源蛋白复合物, lADPC为抽脂术脂肪来源蛋白复合物, TNF-α为肿瘤坏死因子α；F值、P值为2种细胞6组间总体比较所得；q1值、P1值, q2值、P2值, q3值、P3值分别为PBS对照组与单纯nADPC组、单纯lADPC组、单纯TNF-α组比较所得；q4值、P4值为单纯nADPC组与单纯lADPC组比较所得；q5值、P5值, q6值、P6值分别为单纯TNF-α组与TNF-α+nADPC组、TNF-α+lADPC组比较所得；q7值、P7值为TNF-α+nADPC组与TNF-α+lADPC组比较所得 | |||
| PBS对照组 | 5 | 1.01±0.03 | 0.821±0.027 |
| 单纯nADPC组 | 5 | 0.79±0.06 | 0.516±0.049 |
| 单纯lADPC组 | 5 | 0.81±0.04 | 0.571±0.028 |
| 单纯TNF-α组 | 5 | 0.66±0.06 | 0.465±0.046 |
| TNF-α+nADPC组 | 5 | 1.24±0.05 | 0.651±0.031 |
| TNF-α+lADPC组 | 5 | 1.08±0.08 | 0.700±0.037 |
| F值 | 31.24 | 24.53 | |
| P值 | ＜0.01 | ＜0.01 | |
| q1值 | 5.803 | 11.580 | |
| P1值 | ＜0.05 | ＜0.01 | |
| q2值 | 5.371 | 9.493 | |
| P2值 | ＜0.05 | ＜0.01 | |
| q3值 | 9.136 | 13.510 | |
| P3值 | ＜0.01 | ＜0.01 | |
| q4值 | 0.432 | 2.088 | |
| P4值 | ＞0.05 | ＞0.05 | |
| q5值 | 14.990 | 7.066 | |
| P5值 | ＜0.01 | ＜0.01 | |
| q6值 | 10.850 | 8.942 | |
| P6值 | ＜0.01 | ＜0.01 | |
| q7值 | 4.138 | 1.876 | |
| P7值 | ＞0.05 | ＞0.05 | |
以上结果说明nADPC与lADPC作用相近, 后续实验选用终质量浓度为100 μg/mL的lADPC。
2.3. 含lADPC Bioink的制备、性能检测及对裸鼠全层皮肤缺损创面的作用
2.3.1. 含lADPC Bioink的性能检测
在室温及冷凝状态下, lADPC-Bioink AG的外观比Bioink AG稍显浑浊；三维生物打印且交联后lADPC-Bioink AG与Bioink AG的形态类似, 见图 3。在10 ℃时, lADPC-Bioink AG和Bioink AG均在80 s内转变为固态, 其中lADPC-Bioink AG的凝固时间为（76.6±0.4）s, 明显慢于Bioink AG的（74.4±0.6）s（t=4.64, P＜0.01）。
图 3.
明胶-海藻酸钠复合三维生物打印墨水（Bioink AG）与含抽脂术脂肪来源蛋白复合物的Bioink AG（lADPC-Bioink AG）室温、冷凝下、三维生物打印且交联后形态。3A、3B.分别为Bioink AG与lADPC-Bioink AG室温下形态, 图3B较图3A浑浊；3C、3D.分别为Bioink AG与lADPC-Bioink AG冷凝下形态, 图3D较图3C浑浊；3E、3F.分别为Bioink AG与lADPC-Bioink AG三维生物打印且交联后形态, 二者外观无明显区别
2.3.2. 裸鼠创面大体情况
治疗2 d, 常规换药组裸鼠可见大量淡黄色渗出物覆盖创面；单纯lADPC组裸鼠创面湿润, 未见明显渗出；单纯Bioink AG组和lADPC-Bioink AG组裸鼠创面无明显渗出, 打印组织有降解。治疗4、6 d, 常规换药组与单纯lADPC组裸鼠创面逐渐干燥、缩小, 单纯Bioink AG组与lADPC-Bioink AG组裸鼠创面打印组织进一步降解, 创面缩小。治疗8 d, 常规换药组裸鼠创面干燥结痂, 基底呈淡红色, 无明显上皮覆盖；单纯lADPC组裸鼠创面基底呈鲜红色, 无明显上皮覆盖；单纯Bioink AG组裸鼠创面基底呈淡粉色, 有明显上皮覆盖；lADPC-Bioink AG组裸鼠创面面积最小, 基底呈淡红色, 有明显上皮覆盖。治疗10 d, lADPC-Bioink AG组裸鼠创面基本愈合。治疗12 d, 单纯Bioink AG组裸鼠创面也基本愈合。治疗14 d, 仅常规换药组裸鼠创面仍未愈。见图 4。
图 4.
4组全层皮肤缺损裸鼠治疗各时间点创面大体情况。4A.常规换药组治疗2 d可见大量淡黄色渗出物覆盖创面；4B.单纯抽脂术脂肪来源蛋白复合物（lADPC）组治疗2 d创面湿润, 无明显渗出；4C.单纯明胶-海藻酸钠复合三维生物打印墨水（Bioink AG）组治疗2 d创面打印组织有降解, 无明显渗出；4D.lADPC-Bioink AG组治疗2 d打印组织覆盖创面, 有降解, 创面无明显渗出；4E.常规换药组治疗8 d创面干燥结痂, 面积较图4A缩小, 创面基底呈淡红色, 无明显上皮覆盖；4F.单纯lADPC组治疗8 d创面面积较图4E缩小, 创面基底呈鲜红色, 无明显上皮覆盖；4G.单纯Bioink AG组治疗8 d创面面积较图4F缩小, 创面基底呈淡粉色, 有明显上皮覆盖；4H.lADPC-Bioink AG组治疗8 d创面面积最小, 创面基底呈淡红色, 有明显上皮覆盖
2.3.3. 裸鼠创面愈合率
治疗2 d, lADPC-Bioink AG组裸鼠创面愈合率明显高于单纯lADPC组（P＜0.05）, 而与常规换药组、单纯Bioink AG组相近（P＞0.05）；治疗6 d, lADPC-Bioink AG组裸鼠创面愈合率明显高于其余3组（P＜0.05或P＜0.01）；治疗10 d, lADPC-Bioink AG组裸鼠创面愈合率明显高于常规换药组、单纯lADPC组（P＜0.01）, 与单纯Bioink AG组相近（P＞0.05）。见表 4。
表 4.
4组全层皮肤缺损裸鼠治疗各时间点创面愈合率比较(%, x±s)
| 组别 | 鼠数（只） | 2 d | 6 d | 10 d |
| 注：lADPC为抽脂术脂肪来源蛋白复合物, Bioink AG为明胶-海藻酸钠复合三维生物打印墨水；处理因素主效应, F=109.30, P＜0.01；时间因素主效应, F=7 273.00, P＜0.01；两者交互作用, F=97.48, P＜0.01；F值、P值为各时间点组间总体比较所得；t1值、P1值, t2值、P2值, t3值、P3值分别为常规换药组、单纯lADPC组、单纯Bioink AG组与1ADPC-Bioink AG组各时间点比较所得 | ||||
| 常规换药组 | 3 | 11.4±2.0 | 45.2±2.2 | 77.3±1.9 |
| 单纯lADPC组 | 3 | 4.2±1.6 | 51.0±1.9 | 90.5±1.6 |
| 单纯Bioink AG组 | 3 | 7.4±0.7 | 75.5±2.1 | 98.1±2.4 |
| lADPC-Bioink AG组 | 3 | 9.0±0.7 | 81.6±1.4 | 99.4±0.9 |
| F值 | 10.01 | 169.10 | 65.83 | |
| P值 | ＜0.01 | ＜0.01 | ＜0.01 | |
| t1值 | 1.74 | 18.70 | 12.51 | |
| P1值 | ＞0.05 | ＜0.01 | ＜0.01 | |
| t2值 | 3.59 | 15.70 | 4.84 | |
| P2值 | ＜0.05 | ＜0.01 | ＜0.01 | |
| t3值 | 1.19 | 3.15 | 0.72 | |
| P3值 | ＞0.05 | ＜0.05 | ＞0.05 | |
2.3.4. 裸鼠创面组织病理学观察
治疗10 d, 常规换药组裸鼠创面上皮化不完全, 表面有痂, 痂下有一定程度血管化和炎症细胞浸润；单纯lADPC组裸鼠创面上皮化不完全, 创面基底可见较多新生血管和炎症细胞浸润；单纯Bioink AG组裸鼠创面上皮化完全, 但创面基底血管化不足；lADPC-Bioink AG组裸鼠创面上皮化完全, 创面基底血管化程度适中。见图 5。
图 5.
4组全层皮肤缺损裸鼠治疗10 d组织形态 苏木精-伊红, 图中标尺为1 mm。5A.常规换药组创面表面有痂覆盖, 痂下见较多新生血管和炎症细胞浸润；5B.单纯抽脂术脂肪来源蛋白复合物（lADPC）组创面可见少量上皮细胞增殖, 创面基底中有较多新生血管和炎症细胞浸润；5C.单纯明胶-海藻酸钠复合三维生物打印墨水（Bioink AG）组创面可见明显上皮细胞增殖, 但创面基底中的新生血管较少；5D.lADPC-Bioink AG组创面可见明显上皮细胞增殖, 创面基底新生血管较图5C多, 较图5A、5B少
3. 讨论
发现具有促进再生特性的生物活性物质的合理来源, 以刺激多种类型细胞的生长活动是组织工程的一项巨大挑战[22-23]。理想情况下, 这些生物活性物质还应该具有低廉的成本, 易于制备, 并具有良好的安全性[17]。在本研究中, lADPC的来源是临床废弃脂肪组织, 并通过快捷高效的物理手段获取, 其蛋白浓度可以满足应用需求, 无化学及生物污染, 大大提高了安全性, 降低了经济负担。理论上lADPC既可以作为基于天然组织来源的药物, 也可以作为生物添加剂应用于三维生物打印等新兴技术, 应用前景广泛。
创建适宜微环境调控愈合过程中功能性细胞的行为活动是解决创面异常愈合的有效途径之一[22, 24]。正常的创面愈合过程包括止血、炎症、增殖和伤口重塑4个阶段, 其中炎症和增殖阶段是愈合的关键, Fb与血管内皮细胞在这2个阶段中发挥着重要作用[25-26]。因此, 本研究以HSF与HUVEC为研究对象, 通过在细胞培养环境中添加TNF-α以模拟创面愈合早期的炎症微环境, 不添加TNF-α模拟增殖阶段的微环境, 研究ADPC的生物学效应。最终观察到, 炎症环境中HSF与HUVEC的增殖与迁移率较非炎症环境降低, 这可能是由于高浓度的TNF-α促进了这2种细胞的凋亡[27-29]。加入ADPC后, 2种细胞的增殖活力较单纯TNF-α处理有所提升, 血管内皮细胞的迁移功能得到改善。这提示ADPC可以通过促进血管新生和胶原沉积达到转变创面微环境为非炎症状态, 对抗感染并推动愈合进入增殖阶段。而在非炎症环境中添加ADPC后, HSF与HUVEC的增殖与迁移受到了抑制。既往研究表明, 过度愈合（增生性瘢痕及瘢痕疙瘩）的特点是大量ECM的沉积以及过度局部血管化[30], 因此ADPC将功能细胞的增殖与迁移控制在适宜范围有利于避免过度愈合的发生。脂肪组织可以促进创面愈合、抑制瘢痕组织形成的结果在其他研究中也被观察到, 但其具体生物学机制有待进一步阐明[31-33], 本研究提示这可能是由于脂肪组织中的生物活性成分时序性调控了皮肤功能细胞生物学行为, 具体机制仍有待进一步研究。
三维生物打印技术通过构建类似天然组织的三维环境同样为调控创面微环境提供了途径, 充当ECM功能的Bioink决定了微环境的理化性质[34]。研究表明, 无论是Bioink的硬度、黏性、孔隙率等物理性质, 还是其所包含的各类生物活性因子, 都影响着Bioink的使用效果[35-37]。为了提高Bioink AG的生物活性, 并将其运用于创面治疗的相关研究, 本研究团队将lADPC与Bioink AG相结合, 通过对Bioink室温及冷凝状态下外观、凝固时间及可打印性的检测, 证实lADPC除了稍延长Bioink AG的凝固时间外, 对其打印相关性能无明显影响。
最后, 本研究团队运用裸鼠创面模型探究lADPC-Bioink AG的创面治疗作用。结果显示, lADPC-Bioink AG治疗的裸鼠创面愈合速度最快, 病理切片HE染色结果提示愈合过程中组织炎症细胞浸润相对较少, 创面血管化程度适中, 愈合效果最好。创面修复是多种细胞相互协调与配合的结果, 它们的功能发挥既需要合适的三维空间以供黏附和增殖, 也需要合适的生化信息调控其不同时期的生物学行为[25], 三维生物打印技术很好地解决了以上2点需求, 目前多种Bioink取得了良好的促进创面愈合效果[38-40]。本研究同样验证了Bioink AG具有良好的促进创面愈合效果；而lADPC通过调控Fb及血管内皮细胞的功能, 促进了真皮组织的修复, 进一步提升了Bioink AG的功能。
综上所述, 在这项研究中, 本研究团队以临床废弃抽脂术后脂肪组织为主要研究对象, 首先创新性提出了一种ADPC的提取方法, 重点是通过物理手段最大限度保留脂肪组织中的蛋白活性成分, 并证明抽脂术相关操作对ADPC的蛋白浓度、提取效率影响不大；其次, 通过体外实验探究了nADPC与lADPC对创面愈合相关细胞的生物学效应, 验证两者作用无明显差异后, 将lADPC加入Bioink AG中, 在不影响Bioink AG打印性能的前提下, 在体内实验中证明了lADPC可以提高Bioink AG促进创面愈合的效果。本研究的创新点在于将临床废弃脂肪组织提取的蛋白复合物用于提升Bioink AG的生物学性能。当然, 创面愈合本身是一个高度动态化、影响因素众多的过程, ADPC的作用机制仍需要深入研究探索, 确定ADPC中的精确活性成分及其含量是必要的；此外, 需要建立更标准化的提取方法与步骤, 以保证提取出的ADPC的稳定性。不可否认的是, ADPC为今后Bioink的研发与临床转化提供了新的思路, 为解决创面瘢痕愈合和难愈合提供了新的解决途径。
Funding Statement
国家自然科学基金创新研究群体科学基金项目（81721092）；国家自然科学基金重点项目（81830064）；国家自然科学基金青年科学基金项目（32000969、82002056）；中国医学科学院医学与健康科技创新工程项目（2019-I2M-5-059）；解放军总医院军事医学创新研究项目（CX19026）；王正国创伤医学发展基金会生长因子复兴计划（SZYZ-TR-03）
Science Fund for Creative Research Groups of National Natural Science Foundation of China (81721092); Key Program of National Natural Science Foundation of China (81830064); Youth Science Foundation Project of National Natural Science Foundation of China (32000969, 82002056); Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-059); Military Medical Innovation Research Project of PLA General Hospital (CX19026); Wang Zhengguo Foundation for Traumatic Medicine Growth Factor Rejuvenation Plan (SZYZ-TR-03)
Footnotes
利益冲突 所有作者均声明不存在利益冲突
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