改性化学交联脱细胞真皮基质材料的生物相容性
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摘要
背景:脱细胞真皮基质存在降解速率较快且不可调控、力学性能不佳等天然材料固有的缺点,对其进行戊二醛交联改性是常用到的改善措施,然而戊二醛本身具有较大的细胞毒性,会影响脱细胞真皮基质的生物相容性。目的:利用甘氨酸中和封闭戊二醛分子中未参与反应的醛基,改善脱细胞真皮基质材料的生物相容性。方法:对脱细胞猪真皮基质依次进行戊二醛改性、甘氨酸中和处理,作为实验组,以单纯戊二醛改性的脱细胞真皮基质为对照,利用DNA试剂盒检测实验组样品的DNA残留量。将未交联脱细胞猪真皮基质与实验组、对照组样品浸泡于胶原酶溶液中,观察材料降解情况。分别以细胞培养基、高密度聚乙烯材料浸提液、实验组样品浸提液与对照组样品浸提液培养小鼠成纤维细胞,培养24h后,采用MTT法检测细胞增殖率。将小鼠成骨细胞分别接种到实验组与对照组样品膜表面,培养7d后,共聚焦显微镜下观察细胞状态。将实验组与对照组样品膜片分别植入新西兰兔(深圳市医疗器械检测中心提供)皮下,2周后取试样及其周围组织,进行苏木精-伊红染色观察。动物实验经深圳市医疗器械检测中心伦理委员会审批。结果与结论:(1)实验组样品DNA残留量为(3.12±0.7)μg/g;(2)酶解8 h,实验组与对照组样品的失重率无显著差异,为18%-21%,未进行戊二醛交联脱细胞猪真皮基质的失重率为100%;(3)实验组与对照组样品浸提液中小鼠成纤维细胞的增殖率分别为98.1%,90.3%,细胞毒性均为1级;(4)实验组膜片表面的小鼠成骨细胞繁殖旺盛,分布较为均匀,细胞骨架铺展较为充分;对照组膜片表面的成骨细胞数量较少且细胞团聚在一起,细胞骨架未充分铺展开;(5)植入皮下2周后,两组膜片的胶原纤维结构均基本完整且清晰可见,实验组植入部位周围组织炎症反应轻微,对照组炎症反应较为严重;(6)结果表明,甘氨酸封端可在保证降解性能的前提下,提高戊二醛交联改性脱细胞真皮基质材料的生物相容性。
BACKGROUND: Acellular dermal matrix has the disadvantages of natural materials such as rapid and unregulated degradation and poor mechanical properties. It is a commonly used improvement measure for glutaraldehyde cross-linking modification. However, glutaraldehyde itself has a high cytotoxicity, which affects the biocompatibility of acellular dermal matrix.OBJECTIVE: To improve the biocompatibility of the glutaraldehyde cross-linked acellular dermal matrix by using glycine to neutralize remaining aldehyde groups.METHODS: As an experimental group, the acellular porcine dermal matrix was cross-linked by glutaraldehyde and then neutralized by glycine.The control group was that acellular dermal matrix which was cross-linked by glutaraldehyde but not neutralized by glycine. The residual exogenous DNA of materials of the experimental group was detected by DNA kit. The uncrosslinked acellular porcine dermal matrix and the samples of the experimental group and control group were immersed in collagenase solution to observe degradation performance. Mouse fibroblasts were cultured in culture medium, high-density polyethylene extracts, sample extracts of the experimental group and control group,respectively. After 24 hours of culture, cell proliferation rate was determined by MTT assay. Mouse osteoblasts were seeded onto the membrane surface of the experimental group and the control group and cultured for 7 days. The cell status was observed under confocal microscope. The membrane materials of the experimental group and the control group were respectively implanted beneath the skin of New Zealand rabbits, and the samples with surrounding tissues were removed at 2 weeks later for hematoxylin-eosin staining observation.RESULTS AND CONCLUSION:(1) The amount of DNA residues in samples of the experimental group was(3.12±0.7) μg/g.(2) After enzymolysis for 8 hours, there was no significant difference in the weight loss rate of samples between the experimental group and the control group(18%-21%), while the weight loss rate of samples without glutaraldehyde cross-linking was 100%.(3) After culture for 24 hours, the proliferation rates of fibroblasts cultured in the leaching liquor of samples in the experimental and the control groups were 98.1% and 91.3%,respectively. The results showed the level of cytotoxicity was both grade 1.(4) Osteoblasts spread evenly and multiplied vigorously on the membrane surface in the experimental group, while curled up and were few in number in the control group.(5) After membranes implanted beneath the skin of the rabbit for 2 weeks, tissues around the implant site of the experimental group showed a slight inflammatory response, while those in the control group showed severe inflammatory response.(6) These results suggest that glycine neutralizing remaining aldehyde groups could improve the biocompatibility of glutaraldehyde cross-linked acellular dermal matrix material on the premise of guaranteeing the degradation performance.
BACKGROUND: Acellular dermal matrix has the disadvantages of natural materials such as rapid and unregulated degradation and poor mechanical properties. It is a commonly used improvement measure for glutaraldehyde cross-linking modification. However, glutaraldehyde itself has a high cytotoxicity, which affects the biocompatibility of acellular dermal matrix.OBJECTIVE: To improve the biocompatibility of the glutaraldehyde cross-linked acellular dermal matrix by using glycine to neutralize remaining aldehyde groups.METHODS: As an experimental group, the acellular porcine dermal matrix was cross-linked by glutaraldehyde and then neutralized by glycine.The control group was that acellular dermal matrix which was cross-linked by glutaraldehyde but not neutralized by glycine. The residual exogenous DNA of materials of the experimental group was detected by DNA kit. The uncrosslinked acellular porcine dermal matrix and the samples of the experimental group and control group were immersed in collagenase solution to observe degradation performance. Mouse fibroblasts were cultured in culture medium, high-density polyethylene extracts, sample extracts of the experimental group and control group,respectively. After 24 hours of culture, cell proliferation rate was determined by MTT assay. Mouse osteoblasts were seeded onto the membrane surface of the experimental group and the control group and cultured for 7 days. The cell status was observed under confocal microscope. The membrane materials of the experimental group and the control group were respectively implanted beneath the skin of New Zealand rabbits, and the samples with surrounding tissues were removed at 2 weeks later for hematoxylin-eosin staining observation.RESULTS AND CONCLUSION:(1) The amount of DNA residues in samples of the experimental group was(3.12±0.7) μg/g.(2) After enzymolysis for 8 hours, there was no significant difference in the weight loss rate of samples between the experimental group and the control group(18%-21%), while the weight loss rate of samples without glutaraldehyde cross-linking was 100%.(3) After culture for 24 hours, the proliferation rates of fibroblasts cultured in the leaching liquor of samples in the experimental and the control groups were 98.1% and 91.3%,respectively. The results showed the level of cytotoxicity was both grade 1.(4) Osteoblasts spread evenly and multiplied vigorously on the membrane surface in the experimental group, while curled up and were few in number in the control group.(5) After membranes implanted beneath the skin of the rabbit for 2 weeks, tissues around the implant site of the experimental group showed a slight inflammatory response, while those in the control group showed severe inflammatory response.(6) These results suggest that glycine neutralizing remaining aldehyde groups could improve the biocompatibility of glutaraldehyde cross-linked acellular dermal matrix material on the premise of guaranteeing the degradation performance.
引文
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[14]Sheikh Z,Qureshi J,Alshahrani AM,et al.Collagen based barrier membranes for periodontal guided bone regeneration applications.Odontology.2017;105(1):1-12.
[15]达静姝,陈武.异种脱细胞真皮基质对新生血管作用的研究[J].口腔生物医学,2016,7(4):187-190.
[16]张丽娜.不同口腔修复膜材料在牙种植中引导骨再生的应用效果对比[J].系统医学,2017,2(23):117-119.
[17]Lutz R,Neukam FW,Simion M,et al.Long-term outcomes of bone augmentation on soft and hard-tissue stability:a systematic review.Clin Oral Implants Res.2015;26(11):103-122.
[18]Parma-Benfenati S,Roncati M,Galletti P,et al.Resorbable dome device and guided bone regeneration:an alternative bony defect treatment around implants.A case series.Int J Periodontics Restorative Dent.2014;34(6):749-755.
[19]Porcaro G,Busa A,Bianco E,et al.Use of a Partial-thickness Flap for Guided Bone Regeneration in the Upper Jaw.The journal of contemporary dental practice.2017;18(12):1117-1121.
[20]陈一宁,但卫华,但年华.脱细胞真皮基质的改性及应用概述[J].材料导报,2018,32(13):2311-2319.
[21]Gielkens PF,Schortinghuis J,de Jong JR,et al.Vivosorb,Bio-Gide,and Gore-Tex as barrier membranes in rat mandibular defects:an evaluation by microradiography and micro-CT.Clin Oral Implants Res.2008;19(5):516-521.
[22]Ferreira AM,Gentile P,Chiono V,et al.Collagen for bone tissue regeneration.ActaBiomaterialia.2012;8(9):3191-3200.
[23]Calciolari E,Ravanetti F,Strange A,et al.Degradation pattern of a porcine collagen membrane in an in vivo model of guided bone regeneration.J Periodontal Res.2018;53(3):430-439.
[24]Lu HK,Lee SY,Lin FP.Elastic modulus,permeation time and swelling ratio of a new porcine dermal collagen membrane.J Periodontal Res.1998;33(5):243-248.
[25]Rothamel D,Schwarz F,Sager M,et al.Biodegradation of differently cross-linked collagen membranes:an experimental study in the rat.Clin Oral Implants Res.2005;16(3):369-378.
[26]Speer DP,Chvapil M,Eskelson CD,et al.Biological effects of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials.JBiomed Mater Res.1980;14:753-764.
[27]Gilbert TW,Freund JM,Badylak SF.Quantification of DNA in biologic scaffold materials.J Surg Res.2009;152(1):135-139.
[28]Crapo PM,Gilbert TW,Badylak SF.An overview of tissue and whole organ decellularization processes.Biomaterials.2011;32(12):3233-3243.
[29]Harriger MD,Supp AP,Warden GD,et al.Glutaraldehyde crosslinking of collagen substrates inhibits degradation in skin substitutes grafted to athymic mice.J Biomed Mater Res.1997;35(2):137-145.
[30]Rothamel D,Schwarz F,Sculean A,et al.Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblast-like cells.Clin Oral Implants Res.2004;15(4):443-449.
[31]Cheung HY,Brown MR.Evaluation of glycine as an inactivator of glutaraldehyde.J Pharm Pharmacol.1982;34(4):211-214.
[32]李盼盼.活性羰基化合物诱导的氧化应激致细胞能量代谢障碍和毒性的机制[D].咸阳:西北农林科技大学,2015.
[2]Dimitriou R,Mataliotakis GI,Calori GM,et al.The role of barrier membranes for guided bone regeneration and restoration of large bone defects:current experimental and clinical evidence.BMCMed.2012;10(1):81-104.
[3]Crump TB,Rivera-Hidalgo F,Harrison JW,et al.Influence of three membrane type on healing of bone defects.Oral Surg Oral Med Oral Histol.1996;82(4):365-374.
[4]Bottino MC,Thomas V,Schmidt G,et al.Recent advances in the development of GTR/GBR membranes for periodontal regeneration-A materials perspective.Dent Mater.2012;28:703-721.
[5]Cucchi A,Vignudelli E,Napolitano A,et al.Evaluation of complication rates and vertical bone gain after guided bone regeneration with non-resorbable membranes versus titanium meshes and resorbable membranes.A randomized clinical trial.Clin Implant Dent Relat Res.2017;19(5):821-832.
[6]Miron RJ,Fujioka-Kobayashi M,Buser D,et al.Combination of Collagen Barrier Membrane with Enamel Matrix Derivative-Liquid Improves Osteoblast Adhesion and Differentiation.Int J Oral Maxillofac Implants.2017;32(1):196-203.
[7]Kaushal S,Kumar A,Khan MA,et al.Comparative study of nonabsorbable and absorbable barrier membranes in periodontal osseous defects by guided tissue regeneration.J Oral Biol Craniofac Res.2016;6(2):111-117.
[8]Jung RE,Fenner N,H?mmerle CH,et al.Long-term outcome of implants placed with guided bone regeneration(GBR)using resorbable and non-resorbable membranes after 12-14 years.Clin Oral Implants Res.2013;24(10):1065-1073.
[9]Caballé-Serrano J,Munar-Frau A,Ortiz-Puigpelat O,et al.On the search of the ideal barrier membrane for guided bone regeneration.J Clin Exp Dent.2018;10(5):e477-e483.
[10]李栎,张恒,唐历波.膜生物材料及在组织工程中的应用:“引导膜再生理论”的展望[J].中国组织工程研究,2018,22(22):3595-3601.
[11]Gentile P,Chiono V,Tonda-Turo C,et al.Polymeric membranes for guided bone regeneration.Biotechnol J.2011;6(10):1187-1197.
[12]Wang J,Wang L,Zhou Z,et al.Biodegradable polymer membranes applied in guided bone/tissue regeneration:a review.Polymers.2016;8(4):115.
[13]Behring J,Junker R,Walboomers XF,et al.Toward guided tissue and bone regeneration:morphology,attachment,proliferation,and migration of cells cultured on collagen barrier membranes.Asystematic review.Odontology.2008;96(1):1-11.
[14]Sheikh Z,Qureshi J,Alshahrani AM,et al.Collagen based barrier membranes for periodontal guided bone regeneration applications.Odontology.2017;105(1):1-12.
[15]达静姝,陈武.异种脱细胞真皮基质对新生血管作用的研究[J].口腔生物医学,2016,7(4):187-190.
[16]张丽娜.不同口腔修复膜材料在牙种植中引导骨再生的应用效果对比[J].系统医学,2017,2(23):117-119.
[17]Lutz R,Neukam FW,Simion M,et al.Long-term outcomes of bone augmentation on soft and hard-tissue stability:a systematic review.Clin Oral Implants Res.2015;26(11):103-122.
[18]Parma-Benfenati S,Roncati M,Galletti P,et al.Resorbable dome device and guided bone regeneration:an alternative bony defect treatment around implants.A case series.Int J Periodontics Restorative Dent.2014;34(6):749-755.
[19]Porcaro G,Busa A,Bianco E,et al.Use of a Partial-thickness Flap for Guided Bone Regeneration in the Upper Jaw.The journal of contemporary dental practice.2017;18(12):1117-1121.
[20]陈一宁,但卫华,但年华.脱细胞真皮基质的改性及应用概述[J].材料导报,2018,32(13):2311-2319.
[21]Gielkens PF,Schortinghuis J,de Jong JR,et al.Vivosorb,Bio-Gide,and Gore-Tex as barrier membranes in rat mandibular defects:an evaluation by microradiography and micro-CT.Clin Oral Implants Res.2008;19(5):516-521.
[22]Ferreira AM,Gentile P,Chiono V,et al.Collagen for bone tissue regeneration.ActaBiomaterialia.2012;8(9):3191-3200.
[23]Calciolari E,Ravanetti F,Strange A,et al.Degradation pattern of a porcine collagen membrane in an in vivo model of guided bone regeneration.J Periodontal Res.2018;53(3):430-439.
[24]Lu HK,Lee SY,Lin FP.Elastic modulus,permeation time and swelling ratio of a new porcine dermal collagen membrane.J Periodontal Res.1998;33(5):243-248.
[25]Rothamel D,Schwarz F,Sager M,et al.Biodegradation of differently cross-linked collagen membranes:an experimental study in the rat.Clin Oral Implants Res.2005;16(3):369-378.
[26]Speer DP,Chvapil M,Eskelson CD,et al.Biological effects of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials.JBiomed Mater Res.1980;14:753-764.
[27]Gilbert TW,Freund JM,Badylak SF.Quantification of DNA in biologic scaffold materials.J Surg Res.2009;152(1):135-139.
[28]Crapo PM,Gilbert TW,Badylak SF.An overview of tissue and whole organ decellularization processes.Biomaterials.2011;32(12):3233-3243.
[29]Harriger MD,Supp AP,Warden GD,et al.Glutaraldehyde crosslinking of collagen substrates inhibits degradation in skin substitutes grafted to athymic mice.J Biomed Mater Res.1997;35(2):137-145.
[30]Rothamel D,Schwarz F,Sculean A,et al.Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblast-like cells.Clin Oral Implants Res.2004;15(4):443-449.
[31]Cheung HY,Brown MR.Evaluation of glycine as an inactivator of glutaraldehyde.J Pharm Pharmacol.1982;34(4):211-214.
[32]李盼盼.活性羰基化合物诱导的氧化应激致细胞能量代谢障碍和毒性的机制[D].咸阳:西北农林科技大学,2015.