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Volume 37 Issue 11
Nov.  2021
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Article Navigation >  CHINESE JOURNAL OF BURNS AND WOUNDS  > 2021  >  37(11): 1011-1023
Zhang C,Li Z,Song W,et al.Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes[J].Chin J Burns,2021,37(11):1011-1023.DOI: 10.3760/cma.j.cn501120-20210813-00282.
Citation: Zhang C,Li Z,Song W,et al.Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes[J].Chin J Burns,2021,37(11):1011-1023.DOI: 10.3760/cma.j.cn501120-20210813-00282.
shu

Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes

doi: 10.3760/cma.j.cn501120-20210813-00282
  • 1.

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

  • 2.

    Department for Wound Repair and Plastic Surgery, PLA Strategic Support Force Characteristic Medical Center, Beijing 100005, China

Funds:

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

More Information
  • Corresponding author: Huang Sha, Email: stellarahuang@sina.com
  • Received Date: 2021-08-13
    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.

     

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