SUPERCRITICAL FLUID TREATMENT OF THREE-DIMENSIONAL HYDROGEL MATRICES, COMPOSED OF CHITOSAN DERIVATIVES

Aim. Controlled treatment of the physico-chemical and mechanical properties of a three-dimensional crosslinked matrix based on reactive chitosan. Materials and methods. The three-dimensional matrices were obtained using photosensitive composition based on allyl chitosan (5 wt%), poly(ethylene glycol) diacrylate (8 wt%) and the photoinitiator Irgacure 2959 (1 wt%) by laser stereolithography setting. The kinetic swelling curves were constructed for structures in the base and salt forms of chitosan using gravimetric method and the contact angles were measured using droplet spreading. The supercritical fl uid setting (40 °C, 12 MPa) was used to process matrices during 1.5 hours. Using nanohardness Piuma Nanoindenter we calculated values of Young’s modulus. The study of cytotoxicity was performed by direct contact with the culture of the NIH 3T3 mouse fi broblast cell line. Results. Architectonics of matrices fully repeats the program model. Matrices are uniform throughout and retain their shape after being transferred to the base form. Matrices compressed by 5% after treatment in supercritical carbon dioxide (scCO2 ). The elastic modulus of matrices after scCO2 treatment is 4 times higher than the original matrix. The kinetic swelling curves have similar form. In this case the maximum degree of swelling for matrices in base form is 2–2.5 times greater than that of matrices in salt form. There was a surface hydrophobization after the material was transferred to the base form: the contact angle is 94°, and for the salt form it is 66°. The basic form absorbs liquid approximately 1.6 times faster. The fi lm thickness was increased in the area of contact with the liquid droplets after absorption by 133 and 87% for the base and the salt forms, respectively. Treatment of samples in scCO2 reduces their cytotoxicity from 2 degree of reaction (initial samples) down to 1 degree of reaction. Conclusion. The use of supercritical carbon dioxide for scaffolds allows improving biocompatibility of the applied material for 1 degree and increasing the elastic modulus of the material more than 3 times. Allyl chitosan forms stable three-dimensional networks during laser photopolymerization. This enables desorbing toxic low molecular weight component without destruction of the matrix structure. 

1. Биосовместимые материалы (учебное пособие). Под ред. В.И. Севастьянова и М.П. Кирпичникова. М.: МИА, 2011: 544 с. Biosovmestimye materialy (uchebnoe posobie). Pod red. V.I. Sevast’yanova i M.P. Kirpichnikova. M.: MIA, 2011: 544 s.

2. Kim IY, Seo SJ, Moon HS, Yoo MK, Park IY, Kim BC et al. Chitosan and its derivatives for tissue engineering applications. Biotechnology advances. 2008; 26 (1): 1–21. doi: 10.1016/j.biotechadv.2007.07.009.

3. Stella JA, D’Amore A, Wagner WR, Sacks MS. On the biomechanical function of scaffolds for engineering load-bearing soft tissues. Acta biomaterialia. 2010; 6 (7): 2365–2381. doi: 10.1016/j.actbio.2010.01.001.

4. Chen G, Ushida T, Tateishi T. Scaffold design for tissue engineering. Macromolecular Bioscience. 2002; 2 (2): 67– 77. doi: 10.1002/1616-5195(20020201)2:23.0.CO;2-F.

5. Волошин АИ, Шехтер АБ, Попов ВК. Тканевая реакция на акриловые пластмассы, модифицированные сверхкритической экстракцией двуокисью углерода. Стоматология. 1998; 4: 4–9. Voloshin AI, Shehter AB, Popov VK. Tkanevaja reakcija na akrilovye plastmassy, modifi cirovannye sverhkriticheskoj jekstrakciej dvuokis’ju ugleroda. Stomatologija. 1998; 4: 4–9.

6. Huang GP, Shanmugasundaram S, Masih P, Pandya D, Amara S, Collins G et al. An investigation of common crosslinking agents on the stability of electrospun collagen scaffolds. Journal of Biomedical Materials Research Part A. 2015; 103 (2): 762–771. doi: 10.1002/ jbm.a.35222.

7. Martínez A, Blanco MD, Davidenko N, Cameron RE et al. Tailoring chitosan/collagen scaffolds for tissue engineering: Effect of composition and different crosslinking agents on scaffold properties. Carbohydrate polymers. 2015; 132: 606–619. doi: 10.1016/j.carbpol.2015.06.084.

8. Глыбочко ПВ, Аляев ЮГ, Шехтер АБ, Винаров АЗ, Истранов ЛП, Истранова ЕВ и др. Экспериментальное обоснование создания гибридной матрицы и тканеинженерной конструкции на основе сетки из полигликолида и реконструированного коллагена с целью последующей заместительной уретропластики. Урология. 2015; 6: 5–13. Glybochko PV, Alyaev YuG, Shekhter AB, Vinarov AZ, Istranov LP, Istranova EV i dr. Ehksperimental’noe obosnovanie sozdaniya gibridnoj matricy i tkaneinzhenernoj konstrukcii na osnove setki iz poliglikolida i rekonstruirovannogo kollagena s cel’yu posleduyushchej zamestitel’noj uretroplastiki. Urologiya. 2015; 6: 5–13.

9. Kufelt O, El-Tamer A, Sehring C, Schlie-Wolter S, Chichkov BN. Hyaluronic acid based materials for scaffolding via two-photon polymerization. Biomacromolecules. 2014; 15 (2): 650–659. doi: 10.1021/bm401712q.

10. Wang C, Lau TT, Loh WL, Su K, Wang DA. Cytocompatibility study of a natural biomaterial crosslinker – Genipin with therapeutic model cells. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2011; 97 (1): 58–65. doi: 10.1002/jbm.b.31786.

11. Поляков М, Баграташвили ВН. Сверхкритические среды: растворители для экологически чистой химии. Журнал Рос. хим. об-ва им. Д.И. Менделеева. 1999; 43 (2): 93–99. Poljakov M, Bagratashvili VN. Sverhkriticheskie sredy: rastvoriteli dlja jekologicheski chistoj himii. Zhurnal Ros. him. ob-va im. D.I. Mendeleeva. 1999; 43 (2): 93–99.

12. Zhang J, Davis TA, Matthews MA, Drews MJ, LaBerge M, An YH. Sterilization using high-pressure carbon dioxide. The Journal of Supercritical Fluids. 2006; 38 (3): 354–372. doi: 10.1016/j.supfl u.2005.05.005.

13. Tayton E, Purcell M, Aarvold A, Smith JO, Kalra S, Briscoe A et al. Supercritical CO2 fl uid-foaming of polymers to increase porosity: A method to improve the mechanical and biocompatibility characteristics for use as a potential alternative to allografts in impaction bone grafting? Acta biomaterialia. 2012; 8 (5): 1918–1927. doi: 10.1016/j.actbio.2012.01.024. Epub 2012 Jan 24.

14. Akopova TA, Timashev PS, Demina TS, Bardakova KN, Minaev NV, Burdukovskii VF et al. Solid-state synthesis of unsaturated chitosan derivatives to design 3D structures through two-photon-induced polymerization. Mendeleev Communications. 2015; 25 (4): 280–282. doi: 10.1016/j.mencom.2015.07.017.

15. Timashev PS, Demina TS, Minaev NV, Bardakova KN, Koroleva AV, Kufelt OA et al. Fabrication of microstructured materials based on chitosan and its derivatives using two-photon polymerization. High Energy Chemistry. 2015; 49 (4): 300–303. doi: 10.1016/j.mencom.2015.07.017.

16. Тимашев ПС, Бардакова КН, Демина ТС, Пудовкина ГИ, Новиков ММ, Марков МА и др. Новый биосовместимый материал на основе модифицированного твердофазным методом хитозана для лазерной стереолитографии. Современные технологии в медицине. 2015; 7 (3). doi: 10.7868/S0023119315040178. Timashev PS, Bardakova KN, Demina TS, Pudovkina GI, Novikov MM, Markov MA i dr. Novyj biosovmestimyj material na osnove modifi cirovannogo tverdofaznym metodom hitozana dlya lazernoj stereolitografi i. Sovremennye tekhnologii v medicine. 2015; 7 (3).

17. Евсеев АВ, Марков МА, Панченко ВЯ, Якунин ВП. Способ отверждения фотополимеризующейся композиции на основе акрилового олигомера путем инициирования полимеризации в установках радиационного отверждения покрытий. Патент РФ № 2148060. 2000 Апр 27. Evseev AV, Markov MA, Panchenko VYa, Yakunin VP. Sposob otverzhdeniya fotopolimerizuyushchejsya kompozicii na osnove akrilovogo oligomera putem iniciirovaniya polimerizacii v ustanovkah radiaci￾onnogo otverzhdeniya pokrytij. Patent RF № 2148060. 2000 Apr 27.

18. Timashev PS, Kotova SL, Glagolev NN, Aksenova NA, Solovieva AB, Bagratashvili VN. Cleaning of cantilevers for atomic force microscopy in supercritical carbon di oxide. Russian Journal of Physical Chemistry B. 2014; 8 (8): 1081–1086. ISSN 1990–7931.

19. Ernst Breel. Characterizing the micro-mechanical properties of immersed hydrogels by nanoindentation. Technical Report. January 2015. doi: 10.13140/2.1.3580.9606.

20. Yuehua Yuan, T. Randall Lee. Contact Angle and Wetting Properties. Surface Science Techniques. 2013; 51: 3–34.

21. ГОСТ ISO 10993-5-2011 «Изделия медицинские. Оценка биологического действия медицинских изделий. Часть 5. Исследования на цитотоксичность: методы in vitro». GOST ISO 10993-5-2011 «Izdeliya medicinskie. Ocenka biologicheskogo dejstviya medicinskih izdelij. Chast’ 5. Issledovaniya na citotoksichnost’: metody in vitro». International Standard ISO 10993. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity.

22. Шишацкая ЕИ. Клеточные матриксы из резорбируемых полигидроксиалканоатов. Гены и клетки. 2007; 2 (2). Shishackaja EI. Kletochnye matriksy iz rezorbiruemyh poligidroksialkanoatov. Geny i kletki. 2007; 2 (2).

23. Popov VK, Evseev AV, Ivanov AL, Roginski VV, Volozhin AI, Howdle SM. Laser stereolithography and supercritical fl uid processing for custom-designed implant fabrication. J. Materials Science: Materials in Medicine. 2004; 15 (2): 123–128.