基于温敏性壳聚糖水凝胶的可注射性组织工程髓核的初步实验研究
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摘要
目的
验证自行制备的温敏性壳聚糖水凝胶作为髓核细胞支架材料构建组织工程髓核的可行性。
方法
1.温敏性壳聚糖水凝胶的制备及生物学评价
以壳聚糖、β-甘油磷酸钠水溶液和羟乙基纤维素溶液为原料制备壳聚糖水凝胶支架,观察其物理性状,并进行全身毒性、细胞毒性和组织相容性等生物学评价。
2.体外构建可注射性组织工程髓核及相关检测
将壳聚糖水凝胶支架与兔髓核细胞复合构建组织工程髓核。复合培养2 d,观察髓核细胞在支架中的存活情况;复合培养1周,扫描电镜观察髓核细胞在支架上的生长情况;复合培养3周后,行组织学、免疫组织化学检测,并用RT-PCR方法检测其分泌的聚集蛋白聚糖、Ⅱ型胶原mRNA表达。
3.可注射性组织工程髓核修复退变椎间盘的研究
18只新西兰大白兔手术建立腰椎间盘退行性变模型,随机分为3组:对照组、空白组和治疗组。在手术建立椎间盘退行性变模型结束时,在治疗组椎间隙,注射温敏性壳聚糖水凝胶与髓核细胞复合物,对照组注射壳聚糖水凝胶,空白组不予任何干预。观察椎间盘手术前后高度变化;术后12周取材大体观察椎间盘组织结构;免疫组织化学检测Ⅱ型胶原含量,分光光度法测量GAG含量,观察椎间盘退行性变修复效果。
结果
1.制备的温敏性壳聚糖水凝胶室温呈液态,37℃放置15min可发生交联反应成为固态凝胶。生物学评价证实温敏性壳聚糖水凝胶材料无全身毒性反应,具有良好的细胞相容性和组织相容性,在生物体内一定时间后可以降解。
2.吖啶橙/碘化丙啶(AO/PI)染色示壳聚糖水凝胶中大多数髓核细胞存活,少数死亡,细胞存活率> 90%;扫描电镜示髓核细胞分布于网状支架中,细胞表面有分泌的ECM;HE、番红O染色及免疫组织化学染色结果显示髓核细胞具有分泌ECM的功能;RT-PCR检测示髓核细胞在壳聚糖水凝胶支架中立体培养3周后Ⅱ型胶原、聚集蛋白聚糖基因表达水平较单纯培养高。
3.术后12周治疗组测量椎间隙高度与空白组和对照组比较差异有高度统计学意义
(P < 0.01)。取材大体观察,空白组椎间盘髓核与纤维环不清,中央区域的髓核为纤维组织所替代,对照组的椎间盘中度退行性变,组织结构尚可分清。治疗组的的椎间盘轻度退行性变,组织结构清晰,周围未发现炎症反应,壳聚糖材料完全降解。治疗组Ⅱ型胶原、GAG含量高于空白组和对照组(P < 0.05)。
结论
温敏性壳聚糖水凝胶材料具有良好生物相容性与安全性,髓核细胞与其复合培养后可保持正常形态及分泌功能,动物实验证实温敏性壳聚糖水凝胶复合髓核细胞具有修复退变椎间盘的能力,有望成为髓核细胞载体应用于组织工程髓核。
Objective: To investigate the feasibility of using thermo-responsive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP).
Methods: The temperature-responsive chitosan hydrogel scaffold was made of chitosan,disodiumβ-glycerophosphate and hydroxyethyl cellulose. Its physical properties and general condition were observed and its characters of the toxicity and histocompatibility were evaluated. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viabil ity of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by scanning electron microscopy 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of collagen II mRNA were analyzed by RT-PCR 21 days after compound culture. Models of degenerative intervertebral disc were established in eighteen New Zealand rabbits and the animals were randomly divided into three groups: in treatment group, The tissue engineered NP were transplanted into the degenerative disc; in control group, only chitosan hydrogel were transplanted; in blank group, nothing was transplanted. The changes in intervertebral disc height were detected. Gross histological observation was performed at 12 weeks postoperatively. TypeⅡcollagen content was detected by immunohistochemistry, and glycosaminoglycan was quantitated by spectrophotometer to observe the repair effect.
Results: The chitosan hydrogel was temperature-responsive : it was liquid when be put on room temperature , and became solid hydrogel within 10 - 15 minutes when be put on 37℃. The toxic and histocompatibility test suggested that this material has good biocompatibility. Acridine orange-propidiumiodide staining showed that the viability rate of NP cells was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of type II collagen and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold was 0.631±0.064and 0.832±0.052, respectively, showing more strengths of producing matrix than that of monolayer culture (0.528±0.039,0.773±0.046) with a significant difference (P < 0.05). the changes in disc height in postoperative 12 weeks were significantly different compared with the blank group and control group (P < 0.01). In gross observation, the nucleus pulposus and annulus fibrosus of intervertebral disc in blank and control groups were not clear, and the nucleus pulposus in central region were replaced by fibrous tissues. While in treatment group, the degeneration was relieved, and the tissue structure was still clear. By spectrophotometer and immunohistochemistry, the content of glycosaminoglycan and typeⅡcollagen in treatment was significantly higher (P < 0.05) than that in blank and control group in the twelfth postoperative week.
Conclusion: With good biocompatibility, the temperature-responsive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion in the compound culture. The tissue engineered NP can repair the degenerative disc in animal study. the temperature-responsive chitosan hydrogel can be used as scaffold in the tissue engineered study on the repair of degenerative disc.
验证自行制备的温敏性壳聚糖水凝胶作为髓核细胞支架材料构建组织工程髓核的可行性。
方法
1.温敏性壳聚糖水凝胶的制备及生物学评价
以壳聚糖、β-甘油磷酸钠水溶液和羟乙基纤维素溶液为原料制备壳聚糖水凝胶支架,观察其物理性状,并进行全身毒性、细胞毒性和组织相容性等生物学评价。
2.体外构建可注射性组织工程髓核及相关检测
将壳聚糖水凝胶支架与兔髓核细胞复合构建组织工程髓核。复合培养2 d,观察髓核细胞在支架中的存活情况;复合培养1周,扫描电镜观察髓核细胞在支架上的生长情况;复合培养3周后,行组织学、免疫组织化学检测,并用RT-PCR方法检测其分泌的聚集蛋白聚糖、Ⅱ型胶原mRNA表达。
3.可注射性组织工程髓核修复退变椎间盘的研究
18只新西兰大白兔手术建立腰椎间盘退行性变模型,随机分为3组:对照组、空白组和治疗组。在手术建立椎间盘退行性变模型结束时,在治疗组椎间隙,注射温敏性壳聚糖水凝胶与髓核细胞复合物,对照组注射壳聚糖水凝胶,空白组不予任何干预。观察椎间盘手术前后高度变化;术后12周取材大体观察椎间盘组织结构;免疫组织化学检测Ⅱ型胶原含量,分光光度法测量GAG含量,观察椎间盘退行性变修复效果。
结果
1.制备的温敏性壳聚糖水凝胶室温呈液态,37℃放置15min可发生交联反应成为固态凝胶。生物学评价证实温敏性壳聚糖水凝胶材料无全身毒性反应,具有良好的细胞相容性和组织相容性,在生物体内一定时间后可以降解。
2.吖啶橙/碘化丙啶(AO/PI)染色示壳聚糖水凝胶中大多数髓核细胞存活,少数死亡,细胞存活率> 90%;扫描电镜示髓核细胞分布于网状支架中,细胞表面有分泌的ECM;HE、番红O染色及免疫组织化学染色结果显示髓核细胞具有分泌ECM的功能;RT-PCR检测示髓核细胞在壳聚糖水凝胶支架中立体培养3周后Ⅱ型胶原、聚集蛋白聚糖基因表达水平较单纯培养高。
3.术后12周治疗组测量椎间隙高度与空白组和对照组比较差异有高度统计学意义
(P < 0.01)。取材大体观察,空白组椎间盘髓核与纤维环不清,中央区域的髓核为纤维组织所替代,对照组的椎间盘中度退行性变,组织结构尚可分清。治疗组的的椎间盘轻度退行性变,组织结构清晰,周围未发现炎症反应,壳聚糖材料完全降解。治疗组Ⅱ型胶原、GAG含量高于空白组和对照组(P < 0.05)。
结论
温敏性壳聚糖水凝胶材料具有良好生物相容性与安全性,髓核细胞与其复合培养后可保持正常形态及分泌功能,动物实验证实温敏性壳聚糖水凝胶复合髓核细胞具有修复退变椎间盘的能力,有望成为髓核细胞载体应用于组织工程髓核。
Objective: To investigate the feasibility of using thermo-responsive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus (NP).
Methods: The temperature-responsive chitosan hydrogel scaffold was made of chitosan,disodiumβ-glycerophosphate and hydroxyethyl cellulose. Its physical properties and general condition were observed and its characters of the toxicity and histocompatibility were evaluated. The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viabil ity of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by scanning electron microscopy 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of collagen II mRNA were analyzed by RT-PCR 21 days after compound culture. Models of degenerative intervertebral disc were established in eighteen New Zealand rabbits and the animals were randomly divided into three groups: in treatment group, The tissue engineered NP were transplanted into the degenerative disc; in control group, only chitosan hydrogel were transplanted; in blank group, nothing was transplanted. The changes in intervertebral disc height were detected. Gross histological observation was performed at 12 weeks postoperatively. TypeⅡcollagen content was detected by immunohistochemistry, and glycosaminoglycan was quantitated by spectrophotometer to observe the repair effect.
Results: The chitosan hydrogel was temperature-responsive : it was liquid when be put on room temperature , and became solid hydrogel within 10 - 15 minutes when be put on 37℃. The toxic and histocompatibility test suggested that this material has good biocompatibility. Acridine orange-propidiumiodide staining showed that the viability rate of NP cells was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of type II collagen and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold was 0.631±0.064and 0.832±0.052, respectively, showing more strengths of producing matrix than that of monolayer culture (0.528±0.039,0.773±0.046) with a significant difference (P < 0.05). the changes in disc height in postoperative 12 weeks were significantly different compared with the blank group and control group (P < 0.01). In gross observation, the nucleus pulposus and annulus fibrosus of intervertebral disc in blank and control groups were not clear, and the nucleus pulposus in central region were replaced by fibrous tissues. While in treatment group, the degeneration was relieved, and the tissue structure was still clear. By spectrophotometer and immunohistochemistry, the content of glycosaminoglycan and typeⅡcollagen in treatment was significantly higher (P < 0.05) than that in blank and control group in the twelfth postoperative week.
Conclusion: With good biocompatibility, the temperature-responsive chitosan hydrogel makes it possible for NP cells to maintain their normal morphology and secretion in the compound culture. The tissue engineered NP can repair the degenerative disc in animal study. the temperature-responsive chitosan hydrogel can be used as scaffold in the tissue engineered study on the repair of degenerative disc.
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63. Nomura T, Mochida J, Okuma M, Nishimura K, Sakabe K: Nucleus pulposus allograft retards intervertebral disc degeneration. Clin Orthop Relat Res 2001(389):94-101.
64. Nishida K, Kang JD, Suh JK, Robbins PD, Evans CH, Gilbertson LG: Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration. Spine 1998, 23(22):2437-2442; discussion 2443.
65. Sato M, Asazuma T, Ishihara M, Ishihara M, Kikuchi T, Kikuchi M, Fujikawa K: An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method. Spine 2003, 28(6):548-553.
66. Sakai D, Mochida J, Yamamoto Y, Nomura T, Okuma M, Nishimura K, Nakai T, Ando K, Hotta T: Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials 2003, 24(20):3531-3541.
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7. Hutton WC, Murakami H, Li J, Elmer WA, Yoon ST, Minamide A, Akamaru T, Tomita K: The effect of blocking a nutritional pathway to the intervertebral disc in the dog model. J Spinal Disord Tech 2004, 17(1):53-63.
8. Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG: Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 2002, 27(23):2631-2644.
9. Yamazaki S, Weinhold PS, Graff RD, Tsuzaki M, Kawakami M, Minchew JT, Banes AJ: Annulus cells release ATP in response to vibratory loading in vitro. J Cell Biochem 2003, 90(4):812-818.
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62. Gruber HE, Johnson TL, Leslie K, Ingram JA, Martin D, Hoelscher G, Banks D, Phieffer L, Coldham G, Hanley EN, Jr.: Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat. Spine 2002, 27(15):1626-1633.
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66. Sakai D, Mochida J, Yamamoto Y, Nomura T, Okuma M, Nishimura K, Nakai T, Ando K, Hotta T: Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials 2003, 24(20):3531-3541.
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2. Buckwalter JA, Pedrini-Mille A, Pedrini V, Tudisco C: Proteoglycans of human infant intervertebral disc. Electron microscopic and biochemical studies. J Bone Joint Surg Am 1985, 67(2):284-294.
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9. Yamazaki S, Weinhold PS, Graff RD, Tsuzaki M, Kawakami M, Minchew JT, Banes AJ: Annulus cells release ATP in response to vibratory loading in vitro. J Cell Biochem 2003, 90(4):812-818.
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