可注射羟乙基壳聚糖基水凝胶理化性能及其对人牙髓细胞增殖和成牙本质向分化的作用

doi:10.19723/j.issn.1671-167X.2020.01.002

北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (1): 10-17. doi: 10.19723/j.issn.1671-167X.2020.01.002

可注射羟乙基壳聚糖基水凝胶理化性能及其对人牙髓细胞增殖和成牙本质向分化的作用

曹春玲¹,杨聪翀¹,屈小中²,韩冰^1,^△(),王晓燕^1,^△()

1. 北京大学口腔医学院·口腔医院,牙体牙髓科　国家口腔疾病临床医学研究中心　口腔数字化医疗技术和材料国家工程实验室　口腔数字医学北京市重点实验室,北京　100081
2. 中国科学院大学材料科学与光电技术学院,北京　100049

收稿日期:2019-10-10 出版日期:2020-02-18 发布日期:2020-02-20
通讯作者: 韩冰,王晓燕 E-mail:hanbing822@126.com;wangxiaoyan@pkuss.bjmu.edu.cn
基金资助:
国家自然科学基金(81771061);国家自然科学基金(81400562)

Effects of the injectable glycol-chitosan based hydrogel on the proliferation and differentiation of human dental pulp cells

Chun-ling CAO¹,Cong-chong YANG¹,Xiao-zhong QU²,Bing HAN^1,^△(),Xiao-yan WANG^1,^△()

1. Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
2. College of Materials Science and Opto-electronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China

Received:2019-10-10 Online:2020-02-18 Published:2020-02-20
Contact: Bing HAN,Xiao-yan WANG E-mail:hanbing822@126.com;wangxiaoyan@pkuss.bjmu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(81771061);Supported by the National Natural Science Foundation of China(81400562)

摘要/Abstract

摘要：

目的:制备羟乙基壳聚糖(glycol-chitosan, GC)基单/双网络水凝胶,比较人牙髓细胞(human dental pulp cells,hDPCs)在水凝胶内三维培养增殖和分化情况,探究GC基单/双网络水凝胶及其理化性能对hDPCs生物学行为的影响。方法:制备不同组成配比的GC基单网络水凝胶(GC31)和GC基双网络水凝胶(DN3131、DN6262)。用双联注射器在恒力下推注GC基单/双网络水凝胶,测定其可注射性能。用失重法测定水凝胶的体外耐降解性能,并用万能力学试验机测定材料断裂应力。在水凝胶内包封hDPCs进行三维培养,用CCK-8法和Calcein-AM/PI活死细胞染色法测定hDPCs的增殖情况。成牙本质向诱导培养14 d后,用实时荧光定量PCR(real-time quantitative reverse transcription PCR, RT-PCR)测定各组成牙本质向分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein, DSPP)、牙本质基质蛋白(dentin matrix protein-1, DMP-1)和矿化相关基因碱性磷酸酶(alkaline phosphatase, ALP)的表达变化,用Von Kossa染色法观察矿化结节的形成。结果:3组水凝胶均具有良好的可注射性能,GC31推注时间最短,DN6262推注时间较DN3131长(P<0.05)。GC31水凝胶在体外降解速率高于双网络水凝胶组(P<0.05), DN3131和DN6262的降解速率差异无统计学意义(P>0.05)。GC31断裂应力为1.10 kPa,DN3131和DN6262断裂应力分别为7.33 kPa和43.30 kPa,双网络水凝胶的抗压缩性能较单网络水凝胶有明显增强。hDPCs在3种水凝胶内均处于良好的增殖状态,GC31组的增殖能力高于DN3131和DN6262组(P<0.05),DN3131组的增殖能力高于DN6262组(P<0.05)。在成牙本质向诱导培养14 d后,DN3131和DN6262组的DSPP、DMP-1、ALP表达水平较GC31组升高(P<0.05), DN3131和DN6262组的DMP-1和ALP表达水平差异无统计学意义(P>0.05)。DN3131和DN6262组体外培养2周后均可见明显生成棕黑色的团块状矿化结节,而GC31组仅观察到浅棕色钙沉积染色,为零星颗粒状散在分布。结论:不同组成配比的GC基单/双网络水凝胶均满足可注射需求,GC单网络水凝胶力学性能较低,hDPCs在其中表现出更好的增殖能力;而双网络水凝胶具备更好的耐降解性能和更高的力学性能,hDPCs在其中表现出更好的成牙本质向分化潜力和矿化潜力。

关键词: 可注射水凝胶, 牙髓再生, 支架, 双网络水凝胶

Abstract:

Objective: To prepare glycol-chitosan (GC)-based single /dual-network hydrogels with different composition ratios (GC31, DN3131 and DN6262) and to investigate the effects of hydrogel scaffolds on biological behavior of human dental pulp cell (hDPC) encapsulated. Methods: GC-based single-network hydrogels (GC31) and GC-based dual-network hydrogels (DN3131, DN6262) with different composition ratios were prepared. The injectability was defined as the average time needed to expel a certain volume of hydrogel under a constant force. The degradation of the hydrogel was determined by the weight loss with time. The fracture stress was measured using a universal testing machine. The proliferation of hDPCs in hydrogels was detected using the cell counting kit-8 (CCK-8) method and Calcein-AM/PI Live/Dead assay. After 14 days of odontoblastic induction, the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) was detected by real-time quantitative reverse transcription PCR (real-time RT-PCR) and the mineralized nodules was observed by Von Kossa staining. Results: The injectability of all three groups of hydrogels was acceptable. The time of injection of GC31 was the shortest, and that of DN6262 was longer than DN3131 (P<0.05). The degradation rate of GC31 hydrogel in vitro was significantly faster than that of the dual-network hydrogel groups (P<0.05). There was no significant difference between DN3131 and DN6262 (P>0.05). The compressive resistance failure point of GC31 group was 1.10 kPa, while it was 7.33 kPa and 43.30 kPa for DN3131 and DN6262. The compressive strength of dual-network hydrogel was significantly enhanced compared with single-network hydrogel. hDPCs were in continuous proliferation in all the three groups,and the GC31 group showed a higher proliferation rate (P<0.05). The expression levels of DSPP, DMP-1 and ALP in the dual-network hydrogel groups (DN3131, DN6262) were significantly higher than that of GC31 after culturing for 14 days (P<0.05), there was no difference in the expression levels of DMP-1 and ALP between DN3131 and DN6262 (P>0.05); Von Kossa staining showed that more mineralization deposition and mass-shaped mineralized nodules formed in DN3131 and DN6262, while only light brown calcium deposition staining was observed in GC31 group, which was scattered in granular forms. Conclusion: GC-based single/dual network hydrogels with different composition ratios met the injectable requirements. GC31 group had a lower mechanical properties, in which hDPCs exhibited a higher proliferation rate. Dual-network hydrogels had slower degradation rate and higher mechanical properties, in which hDPCs exhibited better odontoblastic differentiation potential and mineralization potential.

Key words: Hydrogel, Pulp regeneration, Scaffold, Dual-network hydrogel

中图分类号:

R783.1

曹春玲,杨聪翀,屈小中,韩冰,王晓燕. 可注射羟乙基壳聚糖基水凝胶理化性能及其对人牙髓细胞增殖和成牙本质向分化的作用[J]. 北京大学学报（医学版）, 2020, 52(1): 10-17.

Chun-ling CAO,Cong-chong YANG,Xiao-zhong QU,Bing HAN,Xiao-yan WANG. Effects of the injectable glycol-chitosan based hydrogel on the proliferation and differentiation of human dental pulp cells[J]. Journal of Peking University(Health Sciences), 2020, 52(1): 10-17.

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Group abbreviation	GC	Component concentration/%
Group abbreviation	GC	OHC-PEO-CHO	Alg	CaCl₂
GC31	3	1	-	-
DN3131	3	1	3	1
DN6262	6	2	6	2

Gene	Gene sequence (5' to 3')
DSPP	Forward: ATATTGAGGGCTGGAATGGGGA Reverse: TTTGTGGCTCCAGCATTGTCA
DMP-1	Forward: AGGAAGTCTCGCATCTCAGAG Reverse: TGGAGTTGCTGTTTTCTGTAGAG
β-actin	Forward: CATGTACGTTGCTATCCAGGC Reverse: CCATCCAATCGGTAGTAGCG
ALP	Forward: AGCACTCCCACTTCATCTGGAA Reverse: GAGACCCAATAGGTAGTCCACATTG

可注射羟乙基壳聚糖基水凝胶理化性能及其对人牙髓细胞增殖和成牙本质向分化的作用

Effects of the injectable glycol-chitosan based hydrogel on the proliferation and differentiation of human dental pulp cells

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