The hemostasis effect and cell compatibility of a chitosan-calcium alginate-laponite nanosheet composite membrane: in vitro and in vivo evaluation

doi:10.19439/j.sjos.2022.06.002

Abstract

Abstract: PURPOSE: To explore the feasibility of a chitosan-calcium alginate-laponite nanosheet composite membrane being used as a new hemostatic membrane for wounds in oral cavity. METHODS: The composite membrane was prepared in a layered fashion: the lower layer of chitosan membrane was made through self-evaporation and the upper layer of calcium alginate-laponite nanosheet sponge was made via freeze-drying. The microstructure of the composite membrane was observed under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). X-ray diffraction was used as a means of identifying the compounds. Clotting time of composite membrane, medical gauze and Chitin dressing in vitro was measured by the plate method for blood coagulation. Cytotoxicity tests were quantified through co-culturing NIH/3T3 cells with chitosan-calcium alginate extract, composite hemostatic membrane extract and DMEM. Superficial buccal mucosal wound models and tooth extraction models were created on beagles dogs, the hemostatic effect and adhesion to oral mucosa were evaluated on these models. Statistical analysis was performed using SPSS 18.0 software package. RESULTS: The composite hemostatic membrane consisted of double-layer in microstructure, the upper layer was a foam layer consisting of calcium alginate and laponite nanosheet, the substratum was formed by uniform chitosan film. X-ray diffraction results showed that laponite nanosheet can be found in the composite membrane. Coagulation test showed that the composite hemostatic membrane group significantly shortened clotting time in vitro compared to pure calcium alginate group, commercial hemostatic membrane and blank control group(P<0.001). CCK-8 test of NIH/3T3 cell showed that there was no significant difference in absorbance between the experimental group, negative control group and blank control group (P>0.05). In addition, composite hemostatic membrane displayed a good hemostatic effect and strong adhesion to oral mucosa in animal models. CONCLUSIONS: The composite hemostatic membrane showed great hemostatic effects and had no significant cytotoxicity, which has the potential for clinical application as hemostatic membrane for wounds in oral cavity.

Key words: Chitosan, Calcium alginate, Laponite nanosheet, Hemostatic membrane for oral mucosal wounds, Coagulation

CLC Number:

R783.1

WU Jing, ZOU Duo-hong, ZHANG Zhi-yuan. The hemostasis effect and cell compatibility of a chitosan-calcium alginate-laponite nanosheet composite membrane: in vitro and in vivo evaluation[J]. Shanghai Journal of Stomatology, 2022, 31(6): 569-575.

References

[1] 邵小钧, 庞恋苏, 袁仕廷, 等. 异种脱细胞真皮基质在口腔颌面部创面修复中的应用[J]. 解放军医学院学报, 2015, 36(9): 900-903, 907.
[2] 李晓彬, 刘禄斌, 刘丹彦. 口腔颌面部创伤的急救和麻醉处理[J]. 创伤外科杂志, 2003, 5(2): 89-90.
[3] 张智杰, 王瑜, 陈晓凤, 等. 口腔可溶性羟乙基止血纱布的止血性能及生物相容性研究[J]. 口腔医学, 2014, 34(1): 31-34.
[4] Nooh N, Graves DT.Healing is delayed in oral compared to dermal excisional wounds[J]. J Periodontol, 2003, 74(2): 242-246.
[5] Dhivya S, Padma VV, Santhini E.Wound dressings - a review[J]. Biomedicine (Taipel), 2015, 5(4): 22-22.
[6] Becher TB, Braga CB, Bertuzzi DL, et al.The structure-property relationship in LAPONITE^® materials: from Wigner glasses to strong self-healing hydrogels formed by non-covalent interactions[J]. Soft Matter, 2019, 15(6): 1278-1289.
[7] Gaharwar AK, Cross LM, Peak CW, et al.2D nanoclay for biomedical applications: regenerative medicine, therapeutic delivery, and additive manufacturing[J]. Adv Mater, 2019, 31(23): e1900332.
[8] Dawson JI, Kanczler JM, Yang XB, et al.Clay gels for the delivery of regenerative microenvironments[J]. Adv Mater, 2011, 23(29): 3304-3308.
[9] Velu R, Calais T, Jayakumar A, et al.A comprehensive review on bio-nanomaterials for medical implants and feasibility studies on fabrication of such implants by additive manufacturing technique[J]. Materials (Basel), 2019, 13(1): 92-92.
[10] Taketa T, Beppu M.Layer-by-layer thin films of alginate/chitosan and hyaluronic acid/chitosan with tunable thickness and surface roughness[J]. Mater Sci Forum, 2014, 783: 1226-1231.
[11] Cunha VRR, Lima FCDA, Sakai VY, et al.LAPONITE^®-pilocarpine hybrid material: experimental and theoretical evaluation of pilocarpine conformation[J]. RSC Advances, 2017, 7(44): 27290-27298.
[12] Freedman M, Stassen LF.Commonly used topical oral wound dressing materials in dental and surgical practice-a literature review[J]. J Ir Dent Assoc, 2013, 59(4): 190-195.
[13] 王静娟, 胡开进, 刘平, 等. 拔牙窝止血类覆盖及充填材料的选择及应用[J]. 中国实用口腔科杂志, 2017, 10(10): 586-589.
[14] Emami F, Vatanara A, Park EJ, et al.Drying technologies for the stability and bioavailability of biopharmaceuticals[J]. Pharmaceutics, 2018, 10(3): 131-131.
[15] He Q, Ao Q, Gong Y, et al.Preparation of chitosan films using different neutralizing solutions to improve endothelial cell compatibility[J]. J Mater Sci Mater Med, 2011, 22(12): 2791-2802.
[16] Khutoryanskiy VV.Advances in mucoadhesion and mucoadhesive polymers[J]. Macromol Biosci, 2011, 11(6): 748-764.
[17] Sogias IA, Williams AC, Khutoryanskiy VV.Why is chitosan mucoadhesive?[J]. Biomacromolecules, 2008, 9(7): 1837-1842.
[18] Agarwal A, Mcanulty JF, Schurr MJ, et al.Advanced wound repair therapies [M]. Sawston: Woodhead Publishing Limited, 2011: 186-208.
[19] Gaharwar AK, Avery RK, Assmann A, et al.Shear-thinning nanocomposite hydrogels for the treatment of hemorrhage[J]. ACS Nano, 2014, 8(10): 9833-9842.
[20] Baker SE, Sawvel AM, Zheng N, et al.Controlling bioprocesses with inorganic surfaces: layered clay hemostatic agents[J]. Chem Mater, 2007, 19(18): 4390-4392.
[21] Lokhande G, Carrow JK, Thakur T, et al.Nanoengineered injectable hydrogels for wound healing application[J]. Acta Biomater, 2018, 70: 35-47.
[22] 高广宇, 陈美玲, 李明媛, 等. 纳米技术转化医学发展现状及前景展望[J]. 药学学报, 2015, 50(8): 919-924.
[23] Gaharwar AK, Mihaila SM, Swami A, et al.Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells[J]. Adv Mater, 2013, 25(24): 3329-3336.
[24] Liu B, Li J, Lei X, Miao S, et al.Cell-loaded injectable gelatin/alginate/LAPONITE^® nanocomposite hydrogel promotes bone healing in a critical-size rat calvarial defect model[J]. RSC Adv, 2020, 10(43): 25652-25661.
[25] Tomás H, Alves CS, Rodrigues J.Laponite^®: a key nanoplatform for biomedical applications?[J]. Nanomedicine, 2018, 14(7): 2407-2420.
[26] Cho YD, Kim KH, Lee YM, et al.Periodontal wound healing and tissue regeneration: a narrative review[J]. Pharmaceuticals(Basel), 2021, 14(5): 456-456.