聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架修复喉软骨缺损

doi:10.3969/j.issn.2095-4344.2015.52.003

摘要/Abstract

摘要：

背景：喉软骨缺损传统的修复方法受到供体来源、排斥反应等限制，因而难以推广。
目的：观察聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架修复喉软骨缺损的效果。
方法：将20只Wistar 大鼠随机分为聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架组和聚乳酸-乙醇酸共聚物支架组，建立喉软骨缺损模型后分别采用聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架和聚乳酸-乙醇酸共聚物支架修复。
结果与结论：聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架组大鼠造模后3，5，7 d时喉骨缺损直径显著小于聚乳酸-乙醇酸共聚物支架组；聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架组大鼠喉软骨缺损部位基本修复，表面平整，且与周围其他组织之间没有明显界限；而聚乳酸-乙醇酸共聚物支架组大鼠喉软骨缺损部位存在凹陷，表面粗糙，和周围组织存在明显界限。说明聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架能够促进喉软骨缺损部位修复，修复喉软骨缺损的效果更理想。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 软骨生物材料, 聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架, pickering乳液法, 修复, 聚乳酸-乙醇酸共聚物支架, 孔隙率

Abstract:

BACKGROUND: The traditional repair methods for laryngeal cartilage defect are restricted by donor source, rejection, which are difficult to be popularized.
OBJECTIVE: To investigate the effect of polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold in repair of laryngeal cartilage defect.
METHODS: A total of 20 Wistar rats were randomly divided into polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold and polylactic acid-glycolic acid copolymer scaffold groups. Polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold and polylactic acid-glycolic acid copolymer scaffold were respectively used for repair after the establishment of laryngeal cartilage defect models.
RESULTS AND CONCLUSION: The laryngeal cartilage defect diameter of rats at 3, 5 and 7 days after modeling in the polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold group was significantly smaller than that in the polylactic acid-glycolic acid copolymer scaffold group. The laryngeal cartilage defect of rats in the polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold group was basically repaired and presented with a smooth surface, and there were no clear boundaries with surrounding tissues; however, the laryngeal cartilage defect of rats in the polylactic acid-glycolic acid copolymer scaffold group had indentations with a rough surface, and there were obvious boundaries with surrounding tissues. These results demonstrate that polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold can promote the repair of laryngeal cartilage defect part, and its repair effect is more ideal.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

中图分类号:

R318

刘永刚，周香桃. 聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架修复喉软骨缺损[J]. 中国组织工程研究, 2015, 19(52): 8379-8383.

Liu Yong-gang, Zhou Xiang-tao. Polylactic acid-glycolic acid copolymer/hydroxyapatite composite scaffold repairs laryngeal cartilage defect[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(52): 8379-8383.

图/表（结果） 5

参考文献

[1] Gilpin DA, Weidenbecher MS, Dennis JE. Scaffold-free tissue-engineered cartilage implants for laryngotracheal reconstruction. Laryngoscope. 2010;120(3):612-617.
[2] 冷晔.聚羟基丁酸己酸酯及其在组织工程中的应用[J].生物医学工程学进展,2011,32(4):213-218.
[3] 孙安科,李万同,孟庆延,等.带蒂肌筋膜瓣充填与包裹构建喉支架形态组织工程软骨[J].中华耳鼻咽喉头颈外科杂志, 2011,46(12): 1019-1023.
[4] 刘清宇,王富友,杨柳.关节软骨组织工程支架的研究进展[J].中国修复重建外科杂志,2012,26(10):1247-1250.
[5] Delawi D, Kruyt MC, Huipin Y, et al. Comparing autograft, allograft, and tricalcium phosphate ceramic in a goat instrumented posterolateral fusion model. Tissue Eng Part C Methods. 2013;19(11):821-828.
[6] Dupont KM, Boerckel JD, Stevens HY, et al. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair. Cell Tissue Res. 2012;347(3):575-588.
[7] Park SH, Tofighi A, Wang X, et al. Calcium phosphate combination biomaterials as human mesenchymal stem cell delivery vehicles for bone repair. J Biomed Mater Res B Appl Biomater. 2011;97(2):235-244.
[8] Che JH, Zhang ZR, Li GZ, et al. Application of tissue-engineered cartilage with BMP-7 gene to repair knee joint cartilage injury in rabbits. Knee Surg Sports Traumatol Arthrosc. 2010;18(4):496-503.
[9] Wang H, Zhang LC, Shi T, et al. Properties evaluation of collagen-hydroxyapatite-chondroitin sulfate-bone morphogenetic protein bone substitute material. Beijing Da Xue Xue Bao. 20118;43(5):730-734.
[10] 江汕,刘勇,王秋实,等.组织工程骨神经化构建及其修复兔大段骨缺损的实验研究[J].中国修复重建外科杂志,2010,24(5):599-605.
[11] 李亮,张鑫鑫,姜晓锐,等.雪旺细胞促进组织工程骨修复大鼠股骨损的实验研究[J].中华创伤骨科杂志,2013,15(2):148-152.
[12] 邱耿韬.磷酸钙骨水泥在骨组织再生修复应用中的研究进展[J].中国矫形外科杂志,2013,21(14):1406-1408.
[13] 胡金龙.组织工程学技术治疗骨缺损的最新研究进展[J].中国矫形外科杂志,2013,21(2):150-152.
[14] 刘阳,曹延林,朱立新,等.新型可注射性nHA/CS骨修复材料修复兔股骨髁骨缺损的X线评估[J].中国矫形外科杂志, 2011,19(17): 1472-1475.
[15] 王岩松,刘丹平,张元和,等.纳米羟基磷灰石/聚酰胺 66 骨填充材料与自体骨修复良性骨肿瘤术后骨缺损的对比研究[J].军医进修学院学报,2011,32(4):373-374.
[16] 聂鹏飞,陈少文,应小洲,等.诱导膜技术治疗大段骨缺损的研究进展[J].中华创伤骨科杂志,2013,15(5):439-440.
[17] Villemagne T, Bonnard C, Accadbled F, et al. Intercalary segmental reconstruction of long bones after malignant bone tumor resection using primary methyl methacrylate cement spacer interposition and secondary bone grafting: the induced membrane technique. J Pediatr Orthop. 2011;31(5):570-576.

[18] 李广恒,金丹,郑波,等.骨骼肌来源的细胞在骨和软骨组织修复与再生中的应用[J]中华创伤骨科杂志,2013,15(3):266-268.
[19] Blum AL, BongioVanni JC, Morgan SJ, et al. Complications associated with distraction osteogenesis for infected nonunion of the femoral shaft in the presence of a bone defect: a retrospective series. J Bone Joint Surg Br. 2010;92(4):565-570.
[20] 金绍林,佘迪,王辉,等.指端缺损的5种同指皮瓣修复方法[J].安徽医学,2010,31(10):1174 -1176.
[21] 陈伟盛.急诊应用 V-Y 皮瓣治疗软组织缺损病例分析[J].中国现代药物应用,2010,4(16):104 -105.
[22] 范大礼,钟小明.带血管神经蒂的食指近节背侧岛状皮瓣修复拇指皮肤缺损18例[J].重庆医学,2008,37(10):1044-1045.
[23] Tan HL, Tan DY, Zhao JK. Treatment of thumb soft-tissue defects using a bipedicle island flap of the index finger: anatomical basis and clinical application. Arch Orthop Trauma Surg. 2013;133(5):721-728.
[24] Greene EL, Houghton O, Collinsworth G, et al. 5-HT(2A) receptors stimulate mitogen-activated protein kinase via H(2)O(2) generation in rat renal mesangial cells. Am J Physiol Renal Physiol. 2000;278(4):F650-658.
[25] Grewal JS, Mukhin YV, Garnovskaya MN, et al. Serotonin 5-HT2A receptor induces TGF-beta1 expression in mesangial cells via ERK: proliferative and fibrotic signals. Am J Physiol. 1999;276(6 Pt 2):F922-930.
[26] Banes A, Florian JA, Watts SW. Mechanisms of 5-hydroxytryptamine(2A) receptor activation of the mitogen-activated protein kinase pathway in vascular smooth muscle. J Pharmacol Exp Ther. 1999;291(3):1179-1187.
[27] Shum JK, Melendez JA, Jeffrey JJ. Serotonin-induced MMP-13 production is mediated via phospholipase C, protein kinase C, and ERK1/2 in rat uterine smooth muscle cells. J Biol Chem. 2002;277(45):42830-42840.
[28] Florian JA, Watts SW. Integration of mitogen-activated protein kinase kinase activation in vascular 5-hydroxytryptamine2A receptor signal transduction. J Pharmacol Exp Ther. 1998; 284(1):346-355.
[29] Quinn JC, Johnson-Farley NN, Yoon J, et al. Activation of extracellular-regulated kinase by 5-hydroxytryptamine(2A) receptors in PC12 cells is protein kinase C-independent and requires calmodulin and tyrosine kinases. J Pharmacol Exp Ther. 2002;303(2):746-752.
[30] Gorin Y, Ricono JM, Wagner B, et al. Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells. Biochem J. 2004;381(Pt 1):231-239.
[31] Yang CM, Yo YL, Hsieh JT, et al. 5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells. Br J Pharmacol. 1994; 111(3):777-786.
[32] Sellers LA, Simon J, Lundahl TS, et al. Adenosine nucleotides acting at the human P2Y1 receptor stimulate mitogen-activated protein kinases and induce apoptosis. J Biol Chem. 2001;276(19):16379-16390.
[33] Labasque M, Meffre J, Carrat G, et al. Constitutive activity of serotonin 2C receptors at G protein-independent signaling: modulation by RNA editing and antidepressants. Mol Pharmacol. 2010;78(5):818-826.
[34] Song HJ, Lee TS, Jeong JH, et al. Hydrogen peroxide-induced extracellular signal-regulated kinase activation in cultured feline ileal smooth muscle cells. J Pharmacol Exp Ther. 2005;312(1):391-398.
[35] Goppelt-Struebe M, Stroebel M. Signaling pathways mediating induction of the early response genes prostaglandin G/H synthase-2 and egr-1 by serotonin via 5-HT2A receptors. J Cell Physiol. 1998;175(3):341-347.
[36] Goppelt-Struebe M, Fickel S, Reiser CO. The platelet-derived-growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells. Biochem J. 2000;345 Pt 2:217-224.
[37] Breshnahan BA, Kelefiotis D, Stratidakis I, et al. PGF2alpha- induced signaling events in glomerular mesangial cells. Proc Soc Exp Biol Med. 1996;212(2):165-173.
[38] Huwiler A, van Rossum G, Wartmann M, et al. Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells. Br J Pharmacol. 1997;120(5):807-812.
[39] Watts SW, Yang P, Banes AK, et al. Activation of Erk mitogen-activated protein kinase proteins by vascular serotonin receptors. J Cardiovasc Pharmacol. 2001;38(4): 539-551.
[40] Errico M, Crozier RA, Plummer MR, et al. 5-HT(7) receptors activate the mitogen activated protein kinase extracellular signal related kinase in cultured rat hippocampal neurons. Neuroscience. 2001;102(2):361-367.
[41] Mukhin YV, Garnovskaya MN, Collinsworth G, et al. 5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts. Biochem J. 2000;347 Pt 1:61-67.
[42] 陈建良,黄冬梅.充气式硅橡胶赝复体修复单侧上颌骨缺损的研究[J].口腔颌面修复学杂志,2009,10(3):157-161
[43] 杜兵,陈巨峰,符志峰.计算机辅助设计与制作在磁性固位分体式上颌骨赝复体修复中的应用[J].中华口腔医学研究杂志(电子版), 2011,5(2):178-183.
[44] 毛恩亮,戴红莲,雷军,等.热致相分离/粒子滤出法制备多孔支架的研究[J].武汉理工大学学报,2010,(12):15-18.
[45] 魏学磊,董福慧.计算机辅助成型技术制备骨组织工程支架的研究进展[J].中国修复重建外科杂志,2011,(12):1508-1512.
[46] 范伟,刘亮,孙海钰,等.以Ⅰ型胶原缓释重组人骨形态发生蛋白-2复合新型多孔酸钙支架修复兔大段桡骨缺损的实验研究[J].中国药物与临床,2008,8(8):627-629.
[47] 刘冰,陈鹏,臧晓霞,等.活性纳米羟基磷灰石复合胶原/聚乳酸材料对拔牙创早期愈合及牙槽嵴吸收影响的实验研究[J].口腔医学,2010,30(1):38-41.
[48] 袁道英,杨佑成,张彬,等.VEGF在脱细胞骨基质复合富血小板血浆修复颅骨缺损中的表达[J].中国口腔颌面外科杂志,2010,8(4): 347-351.

引言

材料方法

1.1 设计随机对照动物实验。

1.2 时间及地点实验于2015年1至8月河南大学医学院完成。

1.3 材料

实验动物：Wistar大鼠20只，雌雄不限，鼠龄4周，体质量0.13-0.35 kg，平均(0.26±0.01) kg，由杭州电子科技大学动物实验中心提供。对大鼠进行等条件喂养，对大鼠的处理符合《关于善待实验动物的指导下意见》中相关规则。实验均经过相关动物伦理学委员会批准同意。

1.4 方法

1.4.1 支架的制备

聚乳酸-乙醇酸共聚物支架：将纯石蜡微球模板与 0.12 g/mL聚乳酸-乙醇酸共聚物的二氧六环溶液进行混合，-20 ℃冷冻干燥6 h，获得石蜡微球与聚乳酸-乙醇酸共聚物混合物，正乙烷洗，去除石蜡后，获得聚乳酸-乙醇酸共聚物支架^[8-9]。

聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架：将羟基磷灰石覆盖石蜡微球模具中，45 ℃烘干30 min，冷却后取出，加入含0.12 g/mL聚乳酸-乙醇酸共聚物的二氧六环溶液，-20 ℃冷冻干燥6 h后，正乙烷洗，去除石蜡后干燥，获得聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架^[10-11]。

1.4.2 支架形态观察两组支架治疗3 d及制备10 d后在扫描电镜下观察2种支架的形态，分析不同材料支架的生物力学性能，采用排乙醇体积方法测定2种支架的孔隙率。

1.4.3 动物分组将20只Wistar大鼠随机分为聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架组和聚乳酸-乙醇酸共聚物支架组，各10只。

1.4.4 喉软骨缺损模型的建立将入选大鼠均采用浓度为3.6%水合氯醛进行全身麻醉，待麻醉生效后进行消毒、铺巾。将2组所需要的支架等进行辐射消毒，麻醉成功后在显微外科技术操作下，造成大鼠喉软骨大小为8 mm的全层缺损，建立喉软骨缺损模型^[12-13]。

1.4.5 喉软骨修复聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架组采用聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架修复，聚乳酸-乙醇酸共聚物支架组采用聚乳酸-乙醇酸共聚物支架修复，大鼠手术完毕后采用小针缝线进行固定，并将所有动物逐层缝合颈前组织和皮肤，并经过耳缘静脉注射160万单位青霉素^[14-15]。

1.4.6 喉骨缺损直径的测量造模后1，3，5，7 d，利用游标卡尺测量2组大鼠喉骨缺损直径^[16-17]，记录喉骨愈合情况。

1.4.7 喉骨缺损部位的大体形态造模后8周，大鼠麻醉后处死，分离出缺损喉骨部位，观察喉骨修复情况。

1.5 主要观察指标聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架的形态。大鼠喉骨缺损直径。喉骨缺损部位的大体形态。

1.6 统计学分析采用SPSS 18.0软件对采集到的数据进行分析，其中符合正态分布的数据进行单因素方差分析，进一步两两比较采用LSD法。P < 0.05为差异有显著性意义。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

讨论

软骨缺损是耳鼻喉科中最为常见的疾病之一，也是医学上难以攻破的难题。众所周知：软骨细胞由于再生能力相对较差，当软骨组织出现缺损或创伤后修复相对比较困难。作者通过利用聚乳酸-乙醇酸共聚物的二氧六环溶液制备出羟基磷灰石纳米粒子涂敷的石蜡微球，并将其作为孔剂，制备出高孔隙率、生物性能较好的聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架^[18-19]。本实验制成的聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架大孔孔径为450-600 μm，大孔内壁上存在一层致密的纳米羟基磷灰石粒子，孔隙率为(95.1±4.9)%；聚乳酸-乙醇酸共聚物支架大孔内径比较光滑，孔隙率为(90.3±4.6)%。与单纯的聚乳酸-乙醇酸共聚物支架相比，聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架具有更强的诱导磷灰石沉积能力，随着其沉积的不断增多，能够提高支架的生物力学性能^[20-21]。同时，聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架还能够有效的促进细胞的体外成骨分化，能够更好的促进骨髓间充质干细胞在机体内成骨^[22-23]。2种支架制成10 d后，大孔结构均保持比较良好，聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架形成了致密的类骨磷灰石层，并且表面形成磷灰石粒子，具有纳米结构^[24-41]；而聚乳酸-乙醇酸共聚物支架表面带有的羟基和羧基较少，无法为骨磷灰颗粒生长提供位点^[42-43]。
将聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架运用于大鼠喉软骨缺损修复中效果理想，该支架在缺损部位与正常的软骨间相互融合，支架对机体也不会产生明显的伤害，可以在一定时间内分解，并参与机体内代谢而排出^[44-45]。还有人在裸鼠体内采用聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架成功制备出组织工程化软骨^[46]；刘冰等^[47]则采用材料符合软骨细胞对兔的膝关节软骨进行修复，并取得成功。聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架的运用在细胞接种后分布状况较好，能够均匀附着，且分泌的基质比较旺盛，更加容易塑性，且这种复合支架更加容易获得，是目前软组织工程中比较合适的支架材料之一^[48]。本实验结果显示，聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架组修复后喉软骨缺损部位基本修复，表面平整，且与周围其他组织之间没有明显界限；而聚乳酸-乙醇酸共聚物支架组修复存在凹陷，表面粗糙，和周围组织存在明显界限。
综上所述，聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架制备方法相对比较简单，材料容易获得，将聚乳酸-乙醇酸共聚物/羟基磷灰石复合支架运用于喉软骨缺损修复中效果理想，能够促进喉软骨缺损部位修复，具有重要的研究意义和价值。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程