Fabrication and safety evaluation of collagen sponge

doi:10.12307/2022.271

Abstract

Abstract: BACKGROUND: Collagen could be extracted from the animal, and the collagen sponge could be fabricated by vacuum freeze-drying technology, which is used in clinical hemostasis. Besides, it would be degraded in vivo. However, there are some concerns about collagen biomaterials, owing to the potential risk of carrying viruses and immune rejection. The biological safety of collagen hemostatic sponge was achieved by removing the terminated peptide and inactivating the virus.
OBJECTIVE: To investigate the immunogenicity and the validity of virus inactivation of collagen hemostatic sponge in commercial process.
METHODS: The collagen was extracted from bovine tendons through acid-enzymatic hydrolysis. After the vacuum freeze-drying technology, the porous collagen hemostatic sponge was prepared. The immunogenicity was investigated by insert 1 or 4 pieces of collagen sponge in Wistar rats subcutaneously. The physical sign, blood routine examination, T lymphocyte proliferation, and immunoglobulin content were examined at 1, 4, 8 and 12 weeks. Pseudorabies virus, vesicular stomatitis virus, porcine parvovirus and porcine encephalomyocarditis virus were chose as indicator virus to investigate the validity of virus inactivation by three batches of crushed bovine tendons.
RESULTS AND CONCLUSION: (1) The collagen sponge was extracted and fabricated by freeze-drying technology in clean workshop in commercial process. The irradiation sterilization was conducted after the whole process of packaging of the collagen sponge. (2) After inserting 1 or 4 pieces of collagen sponge, the physiological signs, routine blood tests, NK cell killing activity, T lymphocyte proliferation, and immunoglobulin content of the rats were found to be normal. (3) There was no detectable virus, which could reduce the titer of these four kinds of indicator virus by more than four logs, after the acid-enzymatic process of collagen sponge. In addition, sensitive cells inoculation and blind cultivation for three generations were performed on the virus titer samples that could not be detected, and the results showed no cytopathic effect. (4) Immunogenicity and virus inactivation test results showed that the prepared collagen hemostatic sponge had good biological safety which could be used in clinical hemostasis.

Key words: collagen, sponge, hemostatic materials, terminated peptide, immunogenicity, freeze-drying, acid-enzymatic hydrolysis, virus inactivation

CLC Number:

Liu Chaoyuan, Gao Tianfang, Huang Wei. Fabrication and safety evaluation of collagen sponge[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(22): 3460-3466.

Figures/Tables 5

可以看出，各组大鼠体质量都随时间延长呈增加趋势，高剂量埋植组、低剂量埋植组、假手术对照组和空白对照组大鼠的体质量增长趋势一致，同时这4种处理类型的组间比较差异无显著性意义(P > 0.05)。结果表明医用胶原蛋白海绵的植入并未对大鼠的饮食、生长造成影响。 2.1.2 大鼠血常规分析借助血细胞计数仪对大鼠外周血进行了白细胞计数、淋巴细胞百分率、中性粒细胞百分率等进行了结果统计分析，其结果见图1。外周血白细胞计数：术后1，4，8，12周，医用胶原蛋白海绵高剂量埋植组、低剂量埋植组、假手术对照组和空白对照组的白细胞水平均在正常值范围[对应值为(2.9-20.9)×109 L-1]内，且4组间比较差异无显著性意义(P > 0.05)。此外，免疫抑制剂给药组大鼠白细胞数低于正常值范围，且与免疫刺激剂给药组的白细胞计数比较差异有显著性意义(P < 0.05)。淋巴细胞检测：术后1，4，8，12周，医用胶原海绵高剂量埋植组、低剂量埋植组、假手术对照组和空白对照组的淋巴细胞百分率均在正常值范围(56.7%-93.1%)，且组间比较差异无显著性意义(P > 0.05)。此外，在第8周时，免疫抑制剂给药组较其他各组低，并且低于正常值范围，与免疫刺激剂给药组比较差异有显著性意义(P < 0.05)。外周血的中性粒细胞百分率：术后1，4，8，12周，医用胶原蛋白海绵高剂量埋植组、低剂量埋植组、假手术对照组、空白对照组的中性粒细胞百分率相对稳定，基本都在正常值范围(5.3%-38.1%)内。此外，第8周时，免疫抑制剂给药组的淋巴细胞百分率低于其余各组，并且低于正常值范围，与免疫刺激剂给药组比较差异有显著性意义(P < 0.05)。以上血细胞检测结果表明，医用胶原蛋白海绵埋植大鼠体内后大鼠的外周血白细胞计数、淋巴细胞百分率、中性粒细胞百分率等数据结果同假手术对照组、空白对照组之间无明显差异，基本都维持在正常值范围内，表明了胶原蛋白海绵的植入未对血常规方面产生异常影响。 2.1.3 NK细胞杀伤活性测定自然杀伤细胞(NK细胞)杀伤活性结果，见图2A。可以看出，在术后1，4，8，12周，医用胶原海绵高剂量埋植组和低剂量埋植组的NK细胞杀伤活性同假手术对照组、空白对照组相近，组间比较差异无显著性意义(P > 0.05)。但另一方面从数据的均值上分析发现，第8-12周实验组的NK细胞杀伤活性有上升的趋势。研究表明，胶原蛋白是LAIR-1的高亲和性受体，而LAIR-1在机体中发挥着免疫抑制的作用[23]。将胶原蛋白与LAIR-1结合，对静止NK细胞及活化的NK细胞在行使对靶细胞杀伤时产生明显的抑制作用[24-26]。基于此，作者做出推断：胶原蛋白海绵植入前期对NK细胞活性产生抑制，因而表现为NK细胞杀伤活性较低；随着胶原蛋白在体内的降解(8周前)及胶原蛋白在植入位点的逐渐消失(8-12周)，在组织受损部位将重新表现出一定的杀伤活性；同时通过对比低剂量埋植和高剂量埋植组发现，高剂量埋植组在第8，12周的NK细胞杀伤活性较低剂量埋植组低，与其在体内降解仍有残留有关，也有效地支持此推论。结果表明，埋植医用胶原蛋白海绵的大鼠靶细胞表面自身MHC-I类分子与NK细胞表面抑制性受体之间的平衡仍能保持良好，故而胶原蛋白海绵的植入对大鼠NK细胞杀伤活性无明显影响。 2.1.4 T淋巴细胞增殖实验 T淋巴细胞增殖实验结果如图2B所示。可以看出，在术后第1，4，8，12周，医用胶原海绵高剂量埋植组、低剂量埋植组与假手术对照组、空白对照组的T细胞增殖刺激指数值接近(P > 0.05)；而前8周时，免疫抑制给药组的T淋巴细胞增殖刺激指数值同免疫刺激给药组比较差异有显著性意义(P < 0.05)。"

References

[1] BEHRENS AM, SIKORSKI MJ, KOFINAS P. Hemostatic strategies for traumatic and surgical bleeding. Journal of Biomed Mater Res Part A. 2014;102(11):4182-4194.
[2] SHEN W, ZHENG Z, RAN J, et al. Long-term effects of knitted silk-collagen sponge scaffold on anterior cruciate ligament reconstruction and osteoarthritis prevention. Biomaterials. 2014;35(28):8154-8163.
[3] BARNARD J, MILLNER R. A review of topical hemostatic agents for use in cardiac surgery. Ann Surg. 2009;88(4):1377-1383.
[4] TOMPECK A, GAJDHAR AUR, DOWLING M, et al. A comprehensive review of topical hemostatic agents: the good, the bad, and the novel. J Trauma Acute Care. 2019;88(1):e1-e21.
[5] 孙国飞,刘建恒,张里程,等.院前创伤急救止血敷料:优势与问题及应用前景[J].中国组织工程研究,2018,22(22):3575-3582.
[6] SLEZAK P, MONFORTE X, FERGUSON J, et al. Properties of collagen-based hemostatic patch compared to oxidized cellulose-based patch. J Mater Sci Mater M. 2018;29(6):71.
[7] JIN J, JI Z, XU M, et al. Microspheres of carboxymethyl chitosan, sodium alginate, and collagen as a hemostatic agent in vivo. ACS Biomater Sci Eng. 2018;4(7):2541-2551.
[8] CHENG X, SHAO Z, LI C, et al. Isolation, Characterization and evaluation of collagen from jellyfish rhopilema esculentum kishinouye for use in hemostatic applications. Plos One. 2017;12(1):e0169731.
[9] 景沛.胶原蛋白质(Ⅰ-XⅫ型)[J].生命的化学,1995,15(6):30-31.
[10] FRIESS W. Collagen–biomaterial for drug delivery. Eur J Pharm Biopharm. 1998;45:113-136.
[11] ACHNECK HE, SILESHI B, JAMIOLKOWSKI RM, et al. A comprehensive review of topical hemostatic agents: efficacy and recommendations for use. Ann Surg. 2010;251(2):217-228.
[12] PALLASKE F, PALLASKE A, HERKLOTZ K, et al. The significance of collagen dressings in wound management: a review. J Wound Care. 2018;27(10):692-702.
[13] SILESHI B, ACHNECK HE, LAWSON JH. Management of surgical hemostasis: topical agents. Vascular. 2008;16 Suppl 1:S22-S28.
[14] MANON-JENSEN T, KJELD NG, KARSDAL MA. Collagen-mediated hemostasis. J Thromb Haemost. 2016;14:438-448.
[15] FLECK CA, SIMMAN R. Modern collagen wound dressings: function and purpose. J Am Coll Cert Wound Spec. 2010;2(3):50-54.
[16] AN B, LIN Y, BRODSKY B. Collagen interactions: drug design and delivery. Adv Drug Deliver Rev. 2016;97:69-84.
[17] SHEEHY EJ, CUNNIFFE GM, O’BRIEN FJ. 5-Collagen-based biomaterials for tissue regeneration and repair. Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair. 2018:27-150.
[18] GELSE K, PÖSCHL E, AIGNER T. Collagens--structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55(12):1531-1546.
[19] 陆美娇.动物源性修复材料的病毒灭活与免疫原的观察[D].烟台:烟台大学,2015.
[20] No. 97D-0411, Guidance for Industry: The sourcing and processing of gelatin to reduce the potential risk by bovine spongiform encephalopathy (BSE) in FDA-regulated products for human.1997.
[21] 史新立,谭芳奕,王召旭,等疯牛病病原体研究及动物源性医疗器械产品安全性思考[J].中国修复重建外科杂志,2006,20(20):1138-1144.
[22] 国家食品药品监督管理局.血液制品去除/灭活病毒技术方法及验证指导原则[S].2002.
[23] LEBBINK RJ. Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1. J Exp Med. 2006;203(6):1419-1425.
[24] MEYAARD L, ADEMA G, CHANG C, et al. LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes. Immunity. 1997;7:283-290.
[25] POGGI A, TOMASELLO E, REVELLO V, et al. p40 molecule regulates NK cell activation mediated by NK receptors for HLA class I antigens and TCR-mediated triggering of T lymphocytes.Int Immunol. 1997;9:1271-1279.
[26] 付强.LAIRs和胶原分子调节人早孕蜕膜NK细胞功能的分子机制[D].上海:复旦大学,2014.
[27] 国家药品监督管理局.动物源性医疗器械注册技术审查指导原则(2017年版)[S]. 2017.
[28] NAGHSHINEH N, TAHVILDARI K, NOZARI M. Preparation of chitosan, sodium alginate, gelatin and collagen biodegradable sponge composites and their application in wound healing and curcumin delivery. J Polym Environ. 2019;27:2819-2830.
[29] FUKUSHIMA SI,YONETSU M, YASUI T. Polarization-resolved second-harmonic-generation imaging of dermal collagen fiber in prewrinkled and wrinkled skins of ultraviolet-B-exposed mouse. J Biomed Opt. 2018;24(3):1-8.
[30] ARAGONA F. Is bovine collagen safe? J Duro. 1991;97(6):279-281.
[31] LYNN AK, YANNAS IV, BONFIELD W. Antigenicity and immunogenicity of collagen. J Biomed Mater Res Part B. 2010;71B(2):343-354.
[32] BONDIOLI E, FINI M, VERONESI F, et al. Development and evaluation of a decellularized membrane from human dermis. J Tissue Eng Regen Med. 2014;8(4):325-336.
[33] INABA S, NAGAHARA S, MAKITA N, et al. Atelocollagen-mediated systemic delivery prevents immunostimulatory adverse effects of siRNA in mammals. Mol Ther. 2012;20(2):356-366.
[34] ZHAO W, CHI C, ZHAO Y, et al. Preparation, physicochemical and antioxidant properties of acid- and pepsin-soluble collagens from the swim bladders of miiuycroaker (miichthysmiiuy). Mar Drugs. 2018; 16(5):161.
[35] BAE JE, KIM CK, KIM S, et al. Virus inactivation during the manufacture of a collagen type I from bovine hides. Korean J Microbio. 2012;48(4): 314-318.
[36] LI L, ZHAO Y,HE Y, et al. Physicochemical and antioxidant properties of acid- and pepsin-soluble collagens from the scales of miiuycroaker (miichthysmiiuy). Mar Drugs. 2018;16(10): 394.
[37] BLANCO M, SOTELO CG, RICARDO I, et al. New strategy to cope with common fishery policy landing obligation: collagen extraction from skins and bones of undersized hake (merlucciusmerluccius). Polymers. 2019;11(9):1485.
[38] MONIRUZZAMAN SM, TAKAHASHI K, NESA NU, et al. Characterization of acid- and pepsin-soluble collagens extracted from scales of carp and lizardfish caught in Japan, Bangladesh and Vietnam with a focus on thermostability. Food Sci and Tech Res. 2019;25(2):331-340.
[39] CALIGIURI MA. Human natural killer cells. Blood. 2008;112(3):461-469.
[40] MELISSA, A, GELLER, JEFFREY J, et al. Use of allogeneic NK cells for cancer immunotherapy. Immunotherapy. 2011;3(12):1445-1459.
[41] SNYDER MR, WEYAND CM, GORONZY JJ. The double life of NK receptors: stimulation or co-stimulation? Trends Immunol. 2004; 25(1):25-32.
[42] ALI RI, KUMAR S, ALI RA, et al. B and T cell epitope mapping and study the humoral and cell mediated immune response to B-T constructs of YscF antigen of Yersinia pestis. Comp Immunol Microbiol Infect Dis. 2013;36(4):365-378.