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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (3): 557-564. doi: 10.19723/j.issn.1671-167X.2022.03.024

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Effects of different crosslinking treatments on the properties of decellularized small intestinal submucosa porous scaffolds

Yi DENG1,Yi ZHANG2,Bo-wen LI1,Mei WANG1,Lin TANG1,Yu-hua LIU1,*()   

  1. 1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
    2. Department of General Dentistry Ⅱ, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
  • Received:2020-10-10 Online:2022-06-18 Published:2022-06-14
  • Contact: Yu-hua LIU E-mail:liuyuhua@bjmu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(81801027)

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Abstract:

Objective: To compare the effects of three different crosslinkers on the biocompatibility, physical and chemical properties of decellularized small intestinal submucosa (SIS) porous scaffolds. Methods: The SIS porous scaffolds were prepared by freeze-drying method and randomly divided into three groups, then crosslinked by glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide (EDC) and procyanidine (PA) respectively. To evaluate the physicochemical property of each sample in different groups, the following experiments were conducted. Macroscopic morphologies were observed and recorded. Microscopic morphologies of the scaffolds were observed using field emission scanning electron microscope (FESEM) and representative images were selected. Computer software (ImageJ) was used to calculate the pore size and porosity. The degree of crosslinking was determined by ninhydrin experiment. Collagenase degradation experiment was performed to assess the resistance of SIS scaffolds to enzyme degradation. To evaluate the mechanical properties, universal mechanical testing machine was used to determine the stress-strain curve and compression strength was calculated. Human bone marrow mesenchymal cells (hBMSCs) were cultured on the scaffolds after which cytotoxicity and cell proliferation were assessed. Results: All the scaffolds remained intact after different crosslinking treatments. The FESEM images showed uniformed interconnected micro structures of scaffolds in different groups. The pore size of EDC group[(161.90±13.44) μm] was significantly higher than GA group [(149.50±14.65) μm] and PA group[(140.10±12.06) μm] (P < 0.05). The porosity of PA group (79.62%±1.14%) was significantly lower than EDC group (85.11%±1.71%) and GA group (84.83%±1.89%) (P < 0.05). PA group showed the highest degree of crosslinking whereas the lowest swelling ratio. There was a significant difference in the swelling ratio of the three groups (P < 0.05). Regarding to the collagenase degradation experiment, the scaffolds in PA group showed a significantly lower weight loss rate than the other groups after 7 days degradation. The weight loss rates of GA group were significantly higher than those of the other groups on day 15, whereas the PA group had the lowest rate after 10 days and 15 days degradation. PA group showed better mechanical properties than the other two groups. More living cells could be seen in PA and EDC groups after live/dead cell staining. Additionally, the proliferation rate of hBMCSs was faster in PA and EDC groups than in GA group. Conclusion: The scaffolds gained satisfying degree of crosslinking after three different crosslinking treatments. The samples after PA and EDC treatment had better physicochemical properties and biocompatibility compared with GA treatment. Crosslinking can be used as a promising and applicable method in the modification of SIS scaffolds.

Key words: Decellularized small intestinal submucosa, Porous scaffolds, Glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, Procyanidine

CLC Number: 

  • R783.1

Figure 1

Macroscopic morphology of different crosslinked SIS porous scaffolds"

Figure 2

The surface and cross-section morphology of the crosslinked scaffolds by FESEM A, B and C, the top view of different SIS porous scaffolds crosslinked by GA, EDC and PA respectively; D, E and F, the side view of different SIS porous scaffolds crosslinked by GA, EDC and PA respectively.A to C, FESEM images of surface morphology of the scaffolds crosslinked by GA, EDC and PA respectively; D to F, FESEM images of cross-section morphology of the scaffolds crosslinked by GA, EDC and PA respectively (Magnification=500, scale bar=300 μm). GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide."

Figure 3

The pore size and porosity of crosslinked SIS porous scaffolds A, pore size; B, porosity; GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. * P<0.05."

Figure 4

The crosslinking degreeof SIS porous scaffolds GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. * P<0.05."

Figure 5

The swelling ratio of crosslinked SIS porous scaffolds GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. * P<0.05."

Figure 6

Weight losses rates of different crosslinked SIS porous scaffolds GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. * P<0.05."

Figure 7

Macroscopic morphology of different crosslinked SIS porous scaffolds in digestion solution on day 7, day 10 and day 15 A, B and C, the SIS scaffold of GA group degraded in digestion solution on day 7, day 10 and day 15 respectively; D, E and F, the SIS scaffold of EDC group degraded in digestion solution on day 7, day 10 and day 15 respectively; G, H and I, the SIS scaffold of PA group degraded in digestion solution on day 7, day 10 and day 15 respectively."

Figure 8

Compressive strengthand stress-strain curves of different crosslinked SIS porous scaffolds A, compressive strength; B, stress-strain curves. GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. * P<0.05."

Figure 9

Image of live/dead assay staining of hBMSCs indifferent crosslinked SIS scaffolds A-C, images of live/dead assay staining of hBMCSs in GA group after 24 h culture time (A, living cells, green; B, dead cells, red; C, merge of A and B); D-F, images of live/dead assay staining of hBMCSs in EDC group after 24 h culture time (D, living cells, green; E, dead cells, red; F, merge of D and E); G-I, images of live/dead assay staining of hBMCSs in PA group after 24 h culture time (G, living cells, green; H, dead cells, red; I, merge of G and H)."

Figure 10

The proliferation curves of hBMSCs cultured on different crosslinked SIS porous scaffolds GA, glutaraldehyde; PA, procyanidine; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide."

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