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Journal of Peking University(Health Sciences) ›› 2020, Vol. 52 ›› Issue (1): 10-17. doi: 10.19723/j.issn.1671-167X.2020.01.002

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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 HAN1,△(),Xiao-yan WANG1,△()   

  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)

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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

CLC Number: 

  • R783.1

Table 1

Component concentration of each group"

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

Figure 1

Preparation and utilization of three GC-based hydrogels as three-dimensional (3D) scaffolds for culture of hDPCs"

Table 2

The chain of reaction primers"

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

Figure 2

The hydrogel formed a cylinder shape using 48-wells plate as a mold"

Figure 3

Demonstration of the injection of hydrogel into root canal mold through a connected mixing syringe of 21G needle"

Figure 4

Injectability of GC-based hydrogels *P<0.05."

Figure 5

Compressive stress-strain curve of hydrogels A, GC31; B, DN3131; C, DN6262."

Figure 6

Degradation observation of hydrogels A, residual weight of GC31, DN3131, DN6262 hydrogels within 3 weeks; B, remaining size of hydrogels within 3 weeks in vitro. * P<0.05, compared with GC31."

Figure 7

The proliferation of hDPCs cultured in GC-based hydrogels A, CCK-8 assay in hydrogels; B, images of live/dead assay staining of hDPCs in 3D hydrogel scaffolds for different culture time (living cells: green, dead cells: red). * P<0.05, compared with DN6262, # P<0.05, compared with DN3131. Images were taken at ×100 magnification."

Figure 8

Quantitative determination of mRNA expression of ondontoblastic differentiation marker genes (DMP-1, DSPP, ALP) by real-time RT-PCR * P<0.05."

Figure 9

Von Kossa staining of histologic sections for hDPCs cultured in GC-based hydrogels in vitro"

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