手持静电纺丝可降解纳米纤维原位修复全层皮肤缺损
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摘要
背景:传统静电纺丝纳米纤维制造过程相对复杂,制造条件要求高,无法适应创伤、烧/烫伤等组织缺损的应急事件中快速组织修复的需求。目的:观察手持式静电纺丝聚乳酸/明胶可降解纳米纤维膜对小鼠皮肤缺损原位修复的效果。方法:(1)采用自制3D打印的手持式静电纺丝设备制备聚乳酸/明胶可降解纳米纤维膜,检测其接触角、水蒸气透过率;(2)以浓度100%,50%,20%的聚乳酸/明胶可降解纳米纤维膜浸提液培养胎鼠成纤维细胞,采用CCK-8细胞毒实验评估材料残留溶剂毒性;将胎鼠成纤维细胞与聚乳酸/明胶可降解纳米纤维膜共培养(实验组),设置单独细胞培养为对照,Alamar blue法检测细胞增殖,活/死染色观察细胞存活率,扫描电镜观察细胞形态;(3)在18只Balb/c小鼠背部制作直径2cm皮肤全层缺损,实验组进行手持静电纺丝原位聚乳酸/明胶可降解纳米纤维膜修复后纱布包扎,对照组进行纱布包扎,术后8周进行缺损部位苏木精-伊红和Masson染色,观察缺损皮肤修复效果。结果与结论:(1)聚乳酸/明胶可降解纳米纤维膜的接触角为(32.68±5.68)°,属亲水材料,适宜细胞黏附;24 h水蒸气透过率为(4.21±0.11)×10~3 g/m~2,可满足皮肤外敷料的要求;不同浓度的聚乳酸/明胶可降解纳米纤维膜浸提液无明显的细胞毒性;(2)实验组胎鼠成纤维细胞具有与对照组细胞等同的细胞活性,但具有更快的增殖速度与更长的增殖时间;(3)苏木精-伊红和Masson染色显示,实验组小鼠皮肤伤口全层愈合,材料降解完全,毛囊再生;对照组小鼠皮肤未全层愈合;(4)结果表明,手持式静电纺丝聚乳酸/明胶可降解纳米纤维实现小鼠皮肤全层缺损的原位修复。
BACKGROUND: The traditional electrospinning nanofiber manufacturing process is relatively complicated, which requires high manufacturing conditions and cannot meet the needs of rapid tissue repair in emergency events such as trauma and burn/scald. OBJECTIVE: To observe the effect of handheld electrospun polylactic acid/gelatin degradable nanofiber membrane on the in situ repair of mouse skin defects.METHODS: Handheld electrospun polylactic acid/gelatin degradable nanofiber membranes were prepared by self-made 3 D printing handheld electrospinning device, and the contact angle and water vapor transmission rate were then measured. Fetal rat fibroblasts were cultured with 100%, 50%, 20% polylactic acid/gelatin degradable nanofiber membrane extracts, and the residual solvent toxicity of the materials was evaluated by cell counting kit-8 cytotoxicity assay. Fetal rat fibroblasts were co-cultured with polylactic acid/gelatin degradable nanofiber membrane(experimental group), and cells cultured alone were set as control. Cell proliferation was detected by Alamar blue method, cell viability was observed by live/dead staining, and cell morphology was observed by scanning electron microscope. A full-thickness skin defect of 2 cm in diameter was made on the back of 18 Balb/c mice. The experimental group was inlaid with handheld electrospun polylactic acid/gelatin degradable nanofiber membrane for in situ repair followed by gauze dressing. The control group was treated with gauze dressing at the defect site. Eight weeks after operation, hematoxylin-eosin and Masson staining were used to observe the repair of skin defects. RESULTS AND CONCLUSION:(1) The contact angle of polylactic acid/gelatin degradable nanofiber membrane was(32.68±5.68)°, indicating a hydrophilic material suitable for cell adhesion. The 24-hour water vapor transmission rate was(4.21±0.11)×103 g/m2, which met the requirements of external skin dressing. Different concentrations of polylactic acid/gelatin degradable nanofiber membrane extract had no obvious cytotoxicity.(2) In the experimental group, fetal rat fibroblasts had the cell viability equivalent to control cells, but exhibited faster proliferation rate and longer proliferation time.(3) Results from the hematoxylin-eosin and Masson staining showed that the full-thickness skin defect healed in the experimental group, with the material being completely degraded and the hair follicles being regenerated. In the control group, the defect healed incompletely. To conclude, handheld electrospun polylactic acid/gelatin degradable nanofibers can implement the in situ repair of mouse full-thickness skin defects.
BACKGROUND: The traditional electrospinning nanofiber manufacturing process is relatively complicated, which requires high manufacturing conditions and cannot meet the needs of rapid tissue repair in emergency events such as trauma and burn/scald. OBJECTIVE: To observe the effect of handheld electrospun polylactic acid/gelatin degradable nanofiber membrane on the in situ repair of mouse skin defects.METHODS: Handheld electrospun polylactic acid/gelatin degradable nanofiber membranes were prepared by self-made 3 D printing handheld electrospinning device, and the contact angle and water vapor transmission rate were then measured. Fetal rat fibroblasts were cultured with 100%, 50%, 20% polylactic acid/gelatin degradable nanofiber membrane extracts, and the residual solvent toxicity of the materials was evaluated by cell counting kit-8 cytotoxicity assay. Fetal rat fibroblasts were co-cultured with polylactic acid/gelatin degradable nanofiber membrane(experimental group), and cells cultured alone were set as control. Cell proliferation was detected by Alamar blue method, cell viability was observed by live/dead staining, and cell morphology was observed by scanning electron microscope. A full-thickness skin defect of 2 cm in diameter was made on the back of 18 Balb/c mice. The experimental group was inlaid with handheld electrospun polylactic acid/gelatin degradable nanofiber membrane for in situ repair followed by gauze dressing. The control group was treated with gauze dressing at the defect site. Eight weeks after operation, hematoxylin-eosin and Masson staining were used to observe the repair of skin defects. RESULTS AND CONCLUSION:(1) The contact angle of polylactic acid/gelatin degradable nanofiber membrane was(32.68±5.68)°, indicating a hydrophilic material suitable for cell adhesion. The 24-hour water vapor transmission rate was(4.21±0.11)×103 g/m2, which met the requirements of external skin dressing. Different concentrations of polylactic acid/gelatin degradable nanofiber membrane extract had no obvious cytotoxicity.(2) In the experimental group, fetal rat fibroblasts had the cell viability equivalent to control cells, but exhibited faster proliferation rate and longer proliferation time.(3) Results from the hematoxylin-eosin and Masson staining showed that the full-thickness skin defect healed in the experimental group, with the material being completely degraded and the hair follicles being regenerated. In the control group, the defect healed incompletely. To conclude, handheld electrospun polylactic acid/gelatin degradable nanofibers can implement the in situ repair of mouse full-thickness skin defects.
引文
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[2]Sanford JA,Gallo RL.Functions of the skin microbiota in health and disease.Semin Immunol.2013;25(5):370-377.
[3]Swann G.The skin is the body's largest organ.J Vis Commun Med.2010;33(4):148-149.
[4]Na M,Mohammad M,Fei Y,et al.Lack of Receptor for Advanced Glycation End products leads to less severe staphylococcal skin infection but more skin abscesses and prolonged wound healing.J Infect Dis.2018.doi:10.1093/infdis/jiy007.[Epub ahead of print]
[5]Albright V,Xu M,Palanisamy A,et al.Micelle-Coated,Hierarchically Structured Nanofibers with Dual-Release Capability for Accelerated Wound Healing and Infection Control.Adv Healthc Mater.2018:e1800132.
[6]Sadiq A,Shah A,Jeschke MG,et al.The Role of Serotonin during Skin Healing in Post-Thermal Injury.Int J Mol Sci.2018;19(4).pii:E1034.
[7]Shi L,Zhao Y,Xie Q,et al.Moldable Hyaluronan Hydrogel Enabled by Dynamic Metal-Bisphosphonate Coordination Chemistry for Wound Healing.Adv Healthc Mater.2018;7(5).doi:10.1002/adhm.201700973.
[8]Shin YC,Shin DM,Lee EJ,et al.Hyaluronic Acid/PLGACore/Shell Fiber Matrices Loaded with EGCG Beneficial to Diabetic Wound Healing.Adv Healthc Mater.2016;5(23):3035-3045.
[9]Wang S,Yang H,Tang Z,et al.Wound Dressing Model of Human Umbilical Cord Mesenchymal Stem Cells-Alginates Complex Promotes Skin Wound Healing by Paracrine Signaling.Stem Cells Int.2016;2016:3269267.
[10]Hsu LC,Peng BY,Chen MS,et al.The potential of the stem cells composite hydrogel wound dressings for promoting wound healing and skin regeneration:In vitro and in vivo evaluation.J Biomed Mater Res B Appl Biomater.2018.doi:10.1002/jbm.b.34118.[Epub ahead of print]
[11]Zarei F,Soleimaninejad M.Role of growth factors and biomaterials in wound healing.Artif Cells Nanomed Biotechnol.2018;15:1-6.
[12]Sheikholeslam M,Wright MEE,Jeschke MG,et al.Biomaterials for Skin Substitutes.Adv Healthc Mater.2018;7(5).doi:10.1002/adhm.201700897.
[13]Zhang W,Chen L,Chen J,et al.Silk Fibroin Biomaterial Shows Safe and Effective Wound Healing in Animal Models and a Randomized Controlled Clinical Trial.Adv Healthc Mater.2017;6(10).doi:10.1002/adhm.201700121.
[14]Paschoalin RT,Traldi B,Aydin G,et al.Solution blow spinning fibres:New immunologically inert substrates for the analysis of cell adhesion and motility.Acta Biomater.2017;51:161-174.
[15]Su K,Wang C.Recent advances in the use of gelatin in biomedical research.Biotechnol Lett.2015;37(11):2139-2145.
[16]Deng K,Yang Y,Ke Y,et al.A novel biomimetic composite substitute of PLLA/gelatin nanofiber membrane for dura repairing.Neurol Res.2017;39(9):819-829.
[17]Liu Y,Cui H,Zhuang X,et al.Electrospinning of aniline pentamer-graft-gelatin/PLLA nanofibers for bone tissue engineering.Acta Biomater.2014;10(12):5074-5080.
[18]陈亮,许运.静电纺丝微米/纳米纤维材料在硬膜组织损伤修复中的应用[J].中华实验外科杂志,2016,33(7):1880-1883.
[19]Xu SC,Qin CC,Yu M,et al.A battery-operated portable handheld electrospinning apparatus.Nanoscale.2015;7(29):12351-12355.
[20]Dong RH,Jia YX,Qin CC,et al.In situ deposition of a personalized nanofibrous dressing via a handy electrospinning device for skin wound care.Nanoscale.2016;8(6):3482-3488.
[21]Dong RH,Qin CC,Qiu X,et al.In situ precision electrospinning as an effective delivery technique for cyanoacrylate medical glue with high efficiency and low toxicity.Nanoscale.2015;7(46):19468-19475.
[22]Lv FY,Dong RH,Li ZJ,et al.In situ precise electrospinning of medical glue fibers as nonsuture dural repair with high sealing capability and flexibility.Int J Nanomedicine.2016;11:4213-4220.
[23]Deng K,Ye X,Yang Y,et al.Evaluation of efficacy and biocompatibility of a new absorbable synthetic substitute as a dural onlay graft in a large animal model.Neurol Res.2016;38(9):799-808.
[24]van Wachem PB,Hogt AH,Beugeling T,et al.Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge.Biomaterials.1987;8(5):323-328.
[25]Kailani MH,Jafar H,Awidi A.Synthetic Biomaterials for Skin Tissue Engineering[M]//Skin Tissue Engineering and Regenerative Medicine,2016:163-183.
[26]郑崇亮,陆树良.创伤后表皮更新和修复的研究进展[J].中华创伤杂志,2017,33(2):185-188.
[27]杨道朋,夏旭.3D打印生物材料研究及其临床应用优势[J].中国组织工程研究,2017,22(18):2927-2933.
[28]Dash BC,Xu Z,Lin L,et al.Stem Cells and Engineered Scaffolds for Regenerative Wound Healing.Bioengineering(Basel).2018;5(1).pii:E23.doi:10.3390/bioengineering5010023.Review.
[29]Sun L,Gao W,Fu X,et al.Enhanced wound healing in diabetic rats by nanofibrous scaffolds mimicking the basketweave pattern of collagen fibrils in native skin.Biomater Sci.2018;6(2):340-349.
[30]Lien SM,Ko LY,Huang TJ.Effect of pore size on ECMsecretion and cell growth in gelatin scaffold for articular cartilage tissue engineering.Acta Biomater.2009;5(2):670-679.
[31]Hoveizi E,Ebrahimi-Barough S,Tavakol S,et al.In Vitro Differentiation of Human iPS Cells into Neural like Cells on a Biomimetic Polyurea.Mol Neurobiol.2017;54(1):601-607.
[32]Kim HW,Yu HS,Lee HH.Nanofibrous matrices of poly(lactic acid)and gelatin polymeric blends for the improvement of cellular responses.J Biomed Mater Res A.2008;87(1):25-32.
[33]Wang S,Zhang Y,Wang H,et al.Fabrication and properties of the electrospun polylactide/silk fibroin-gelatin composite tubular scaffold.Biomacromolecules.2009;10(8):2240-2244.
[34]Chiou BS,Jafri H,Avena-Bustillos R,et al.Properties of electrospun pollock gelatin/poly(vinyl alcohol)and pollock gelatin/poly(lactic acid)fibers.Int J Biol Macromol.2013;55:214-220.
[35]Shi Z,Xu T,Yuan Y,et al.A New Absorbable Synthetic Substitute With Biomimetic Design for Dural Tissue Repair.Artif Organs.2016;40(4):403-413.
[36]Haik J,Kornhaber R,Blal B,et al.The Feasibility of a Handheld Electrospinning Device for the Application of Nanofibrous Wound Dressings.Adv Wound Care(New Rochelle).2017;6(5):166-174.
[37]Sofokleous P,Stride E,Bonfield W,et al.Design,construction and performance of a portable handheld electrohydrodynamic multi-needle spray gun for biomedical applications.Mater Sci Eng C Mater Biol Appl.2013;33(1):213-223.
[38]林青,郝宏,王坤,等.逆向设计在制造工程中的应用[J].新技术新工艺,2017,37(12):65-67.
[39]Wong JY,Pfahnl AC.3D Printed Surgical Instruments Evaluated by a Simulated Crew of a Mars Mission.Aerosp Med Hum Perform.2016;87(9):806-810.
[40]Azimifar F,Hassani K,Saveh AH,et al.A medium invasiveness multi-level patient's specific template for pedicle screw placement in the scoliosis surgery.Biomed Eng Online.2017;16(1):130.
[2]Sanford JA,Gallo RL.Functions of the skin microbiota in health and disease.Semin Immunol.2013;25(5):370-377.
[3]Swann G.The skin is the body's largest organ.J Vis Commun Med.2010;33(4):148-149.
[4]Na M,Mohammad M,Fei Y,et al.Lack of Receptor for Advanced Glycation End products leads to less severe staphylococcal skin infection but more skin abscesses and prolonged wound healing.J Infect Dis.2018.doi:10.1093/infdis/jiy007.[Epub ahead of print]
[5]Albright V,Xu M,Palanisamy A,et al.Micelle-Coated,Hierarchically Structured Nanofibers with Dual-Release Capability for Accelerated Wound Healing and Infection Control.Adv Healthc Mater.2018:e1800132.
[6]Sadiq A,Shah A,Jeschke MG,et al.The Role of Serotonin during Skin Healing in Post-Thermal Injury.Int J Mol Sci.2018;19(4).pii:E1034.
[7]Shi L,Zhao Y,Xie Q,et al.Moldable Hyaluronan Hydrogel Enabled by Dynamic Metal-Bisphosphonate Coordination Chemistry for Wound Healing.Adv Healthc Mater.2018;7(5).doi:10.1002/adhm.201700973.
[8]Shin YC,Shin DM,Lee EJ,et al.Hyaluronic Acid/PLGACore/Shell Fiber Matrices Loaded with EGCG Beneficial to Diabetic Wound Healing.Adv Healthc Mater.2016;5(23):3035-3045.
[9]Wang S,Yang H,Tang Z,et al.Wound Dressing Model of Human Umbilical Cord Mesenchymal Stem Cells-Alginates Complex Promotes Skin Wound Healing by Paracrine Signaling.Stem Cells Int.2016;2016:3269267.
[10]Hsu LC,Peng BY,Chen MS,et al.The potential of the stem cells composite hydrogel wound dressings for promoting wound healing and skin regeneration:In vitro and in vivo evaluation.J Biomed Mater Res B Appl Biomater.2018.doi:10.1002/jbm.b.34118.[Epub ahead of print]
[11]Zarei F,Soleimaninejad M.Role of growth factors and biomaterials in wound healing.Artif Cells Nanomed Biotechnol.2018;15:1-6.
[12]Sheikholeslam M,Wright MEE,Jeschke MG,et al.Biomaterials for Skin Substitutes.Adv Healthc Mater.2018;7(5).doi:10.1002/adhm.201700897.
[13]Zhang W,Chen L,Chen J,et al.Silk Fibroin Biomaterial Shows Safe and Effective Wound Healing in Animal Models and a Randomized Controlled Clinical Trial.Adv Healthc Mater.2017;6(10).doi:10.1002/adhm.201700121.
[14]Paschoalin RT,Traldi B,Aydin G,et al.Solution blow spinning fibres:New immunologically inert substrates for the analysis of cell adhesion and motility.Acta Biomater.2017;51:161-174.
[15]Su K,Wang C.Recent advances in the use of gelatin in biomedical research.Biotechnol Lett.2015;37(11):2139-2145.
[16]Deng K,Yang Y,Ke Y,et al.A novel biomimetic composite substitute of PLLA/gelatin nanofiber membrane for dura repairing.Neurol Res.2017;39(9):819-829.
[17]Liu Y,Cui H,Zhuang X,et al.Electrospinning of aniline pentamer-graft-gelatin/PLLA nanofibers for bone tissue engineering.Acta Biomater.2014;10(12):5074-5080.
[18]陈亮,许运.静电纺丝微米/纳米纤维材料在硬膜组织损伤修复中的应用[J].中华实验外科杂志,2016,33(7):1880-1883.
[19]Xu SC,Qin CC,Yu M,et al.A battery-operated portable handheld electrospinning apparatus.Nanoscale.2015;7(29):12351-12355.
[20]Dong RH,Jia YX,Qin CC,et al.In situ deposition of a personalized nanofibrous dressing via a handy electrospinning device for skin wound care.Nanoscale.2016;8(6):3482-3488.
[21]Dong RH,Qin CC,Qiu X,et al.In situ precision electrospinning as an effective delivery technique for cyanoacrylate medical glue with high efficiency and low toxicity.Nanoscale.2015;7(46):19468-19475.
[22]Lv FY,Dong RH,Li ZJ,et al.In situ precise electrospinning of medical glue fibers as nonsuture dural repair with high sealing capability and flexibility.Int J Nanomedicine.2016;11:4213-4220.
[23]Deng K,Ye X,Yang Y,et al.Evaluation of efficacy and biocompatibility of a new absorbable synthetic substitute as a dural onlay graft in a large animal model.Neurol Res.2016;38(9):799-808.
[24]van Wachem PB,Hogt AH,Beugeling T,et al.Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge.Biomaterials.1987;8(5):323-328.
[25]Kailani MH,Jafar H,Awidi A.Synthetic Biomaterials for Skin Tissue Engineering[M]//Skin Tissue Engineering and Regenerative Medicine,2016:163-183.
[26]郑崇亮,陆树良.创伤后表皮更新和修复的研究进展[J].中华创伤杂志,2017,33(2):185-188.
[27]杨道朋,夏旭.3D打印生物材料研究及其临床应用优势[J].中国组织工程研究,2017,22(18):2927-2933.
[28]Dash BC,Xu Z,Lin L,et al.Stem Cells and Engineered Scaffolds for Regenerative Wound Healing.Bioengineering(Basel).2018;5(1).pii:E23.doi:10.3390/bioengineering5010023.Review.
[29]Sun L,Gao W,Fu X,et al.Enhanced wound healing in diabetic rats by nanofibrous scaffolds mimicking the basketweave pattern of collagen fibrils in native skin.Biomater Sci.2018;6(2):340-349.
[30]Lien SM,Ko LY,Huang TJ.Effect of pore size on ECMsecretion and cell growth in gelatin scaffold for articular cartilage tissue engineering.Acta Biomater.2009;5(2):670-679.
[31]Hoveizi E,Ebrahimi-Barough S,Tavakol S,et al.In Vitro Differentiation of Human iPS Cells into Neural like Cells on a Biomimetic Polyurea.Mol Neurobiol.2017;54(1):601-607.
[32]Kim HW,Yu HS,Lee HH.Nanofibrous matrices of poly(lactic acid)and gelatin polymeric blends for the improvement of cellular responses.J Biomed Mater Res A.2008;87(1):25-32.
[33]Wang S,Zhang Y,Wang H,et al.Fabrication and properties of the electrospun polylactide/silk fibroin-gelatin composite tubular scaffold.Biomacromolecules.2009;10(8):2240-2244.
[34]Chiou BS,Jafri H,Avena-Bustillos R,et al.Properties of electrospun pollock gelatin/poly(vinyl alcohol)and pollock gelatin/poly(lactic acid)fibers.Int J Biol Macromol.2013;55:214-220.
[35]Shi Z,Xu T,Yuan Y,et al.A New Absorbable Synthetic Substitute With Biomimetic Design for Dural Tissue Repair.Artif Organs.2016;40(4):403-413.
[36]Haik J,Kornhaber R,Blal B,et al.The Feasibility of a Handheld Electrospinning Device for the Application of Nanofibrous Wound Dressings.Adv Wound Care(New Rochelle).2017;6(5):166-174.
[37]Sofokleous P,Stride E,Bonfield W,et al.Design,construction and performance of a portable handheld electrohydrodynamic multi-needle spray gun for biomedical applications.Mater Sci Eng C Mater Biol Appl.2013;33(1):213-223.
[38]林青,郝宏,王坤,等.逆向设计在制造工程中的应用[J].新技术新工艺,2017,37(12):65-67.
[39]Wong JY,Pfahnl AC.3D Printed Surgical Instruments Evaluated by a Simulated Crew of a Mars Mission.Aerosp Med Hum Perform.2016;87(9):806-810.
[40]Azimifar F,Hassani K,Saveh AH,et al.A medium invasiveness multi-level patient's specific template for pedicle screw placement in the scoliosis surgery.Biomed Eng Online.2017;16(1):130.