Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes
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摘要:
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。 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. -
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
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
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
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
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
表1 5组HSF和HUVEC常规培养24 h细胞增殖活力比较（
组别 样本数 HSF HUVEC 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 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 
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表2 6组HSF和HUVEC划痕后24 h细胞迁移率比较（%，
组别 样本数 HSF HUVEC 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 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
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表3 6组HSF和HUVEC常规培养24 h细胞增殖活力比较（
组别 样本数 HSF HUVEC 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 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
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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（%，
组别 鼠数（只） 2 d 6 d 10 d 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 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
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