PREPARATION AND BIOCOMPATIBILITY OF IN SITU CROSSLINKING HYALURONIC ACID HYDROGEL_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIANGJiabi ^1,2 , LIJun ² , WANGTing ² , LIANGYuhong ² , ZOUXuenong ^3,4 , ZHOUGuangqian ² ,  ZHOUZhiyu ^2,4

1. Pharmaceutical Department, the Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai Guangdong, 519000, P.R.China;
2. Shenzhen Key Laboratory for Anti-ageing and Regenerative Medicine, Center for Anti-ageing and Regenerative Medicine, School of Medicine, Shenzhen University;
3. Institute of Orthopaedics, the First Affiliated Hospital Department of Spinal Surgery;
4. Institute of Orthopaedics, the First Affiliated Hospital, Sun Yat-sen University;

Corresponding author：

ZHOUZhiyu, Email: zzy990802@126.com

Keywords：

Hyaluronic acid; Hydrogel; In situ crosslinking; Biocompatibility; Tissue engineered scaffold

DOI：

10.7507/1002-1892.20160156

Video：

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Objective To fabricate in situ crosslinking hyaluronic acid hydrogel and evaluate its biocompatibility in vitro. Methods The acrylic acid chloride and polyethylene glycol were added to prepare crosslinking agent polyethylene glycol acrylate (PEGDA), and the molecular structure of PEGDA was analyzed by Flourier transformation infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. Hyaluronic acid hydrogel was chemically modified to prepare hyaluronic acid thiolation (HA-SH). And the degree of HA-SH was analyzed qualitatively and quantitatively by Ellman method. HA-SH solution in concentrations (W/V) of 0.5%, 1.0%, and 1.5% and PEGDA solution in concentrations (W/V) of 2%, 4%, and 6% were prepared with PBS. The two solutions were mixed in different ratios, and in situ crosslinking hyaluronic acid hydrogel was obtained; the crosslinking time was recorded. The cellular toxicity of in situ crosslinking hyaluronic acid hydrogel (1.5% HA-SH and 4% PEGDA mixed) was tested by L929 cells. Meanwhile, the biocompatibility of hydrogel was tested by co-cultured with human bone mesenchymal stem cells (hBMSCs). Results Flourier transformation infrared spectroscopy showed that most hydroxyl groups were replaced by acrylate groups; 1H nuclear magnetic resonance spectroscopy showed 3 characteristic peaks of hydrogen representing acrylate and olefinic bond at 5-7 ppm. The thiolation yield of HA-SH was 65.4%. In situ crosslinking time of hyaluronic acid hydrogel was 2 to 70 minutes in the PEGDA concentrations of 2%-6% and HA-SH concentrations of 0.5%-1.5%. The hyaluronic acid hydrogel appeared to be transparent. The toxicity grade of leaching solution of hydrogel was grade 1. hBMSCs grew well and distributed evenly in hydrogel with a very high viability. Conclusion In situ crosslinking hyaluronic acid hydrogel has low cytotoxicity, good biocompatibility, and controllable crosslinking time, so it could be used as a potential tissue engineered scaffold or repairing material for tissue regeneration.

Citation： LIANGJiabi, LIJun, WANGTing, LIANGYuhong, ZOUXuenong, ZHOUGuangqian, ZHOUZhiyu. PREPARATION AND BIOCOMPATIBILITY OF IN SITU CROSSLINKING HYALURONIC ACID HYDROGEL. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(6): 767-771. doi: 10.7507/1002-1892.20160156 Copy

1.	Cheung HK, Han TT, Marecak DM, et al. Composite hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with adipose-derived stem cells. Biomaterials, 2014, 35(6): 1914-1923.
2.	Bi X, Liang A, Tan Y, et al. Thiolene crosslinking polyamidoamine dendrimer-hyaluronic acid hydrogel system for biomedical applications. J Biomater Sci Polym Ed, 2016, 27(8): 743-757.
3.	Bian S, He M, Sui J, et al. The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cells culture. Colloids and Surface B: Biointerfaces, 2016, 140(1): 392-402.
4.	Koshy ST, Ferrante TC, Lewin SA, et al. Injectable, porous, and cell-responsive gelatin cryogels. Biomaterials, 2014, 35(8): 2477-2487.
5.	Palumbo FS, Fiorica C, Di Stefano M, et al. In situ forming hydrogels of hyaluronic acid and inulin derivatives for cartilage regeneration. Carbohydrate Polymers, 2015, 122: 408-416.
6.	Kablik J, Monheit GD, Yu L, et al. Comparative physical properties of hyaluronic acid dermal fillers. Dermatol Surg, 2009, 35 Suppl 1: 302-312.
7.	Kaderli S, Boulocher C, Pillet E, et al. A novel oxido-viscosifying hyaluronic acid-antioxidant conjugate for osteoarthritis therapy: biocompatibility assessments. Eur J Pharm Biopharm, 2015, 90: 70-79.
8.	Mayol L, De Stefano D, De Falco F, et al. Effect of hyaluronic acid on the thermogelation and biocompatibility of its blends with methyl cellulose. Carbohydrate Polymers, 2014, 112: 480-485.
9.	Kaderli S, Boulocher C, Pillet E, et al. A novel biocompatible hyaluronic acid-chitosan hybrid hydrogel for osteoarthrosis therapy. Int J Pharm, 2015, 483(1-2): 158-168.
10.	Lima SV, de Oliveira Rangel AE, de Melo Lira MM, et al. The biocompatibility of a cellulose exopolysaccharide implant in the rabbit bladder when compared with dextranomer microspheres plus hyaluronic acid. Urology, 2015, 85(6): 1520.e1-6.
11.	Prestwich GD, Marecak DM, Marecek JF, et al. Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives. J Control Release, 1998, 53(1-3): 93-103.
12.	Xu X, Jha AK, Harrington DA, et al. Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter, 2012, 8(12): 3280-3294.
13.	Burdick JA, Presteich GD. Hyaluronic acid hydrogels for biomedical applications. Adv Mat, 2011, 23(12): H41-56.
14.	Bencherif SA, Srinivasan A, Horkay F, et al. Influence of the degree of methacrylation on hyaluronic acid hydrogels properties. Biomaterials, 2008, 29(12): 1739-1749.
15.	Tomihata K, Ikada Y. Crosslinking of hyaluronic acid with water-soluble carbodiimide. J Biomed Mater Res, 1997, 37(2): 243-251.
16.	Butterworth PH, Baum H, Porter JW. A modification of the Ellman procedure for the estimation of protein sulfhydryl groups. Arch Biochem Biophys, 1967, 118(3): 716-723.
17.	Wang W, Huang X, Yin H, et al. Polyethylene glycol acrylate-grafted polysulphone membrane for artificial lungs: plasma modification and haemocompatibility improvement. Biomed Mater, 2015,10(6): 65-78.
18.	Chong JY, Mulet X, Postma A, et al. Novel RAFT amphiphilic brush copolymer steric stabilisers for cubosomes: Poly (octadecyl acrylate)-block-poly (polyethylene glycol methyl ether acrylate). Soft Matter, 2014, 10(35): 6666-6676.
19.	Munoz-Pinto DJ, Jimenez-Vergara AC, Gharat TP, et al. Characterization of sequential collagen-poly (ethylene glycol) diacrylate interpenetrating networks and initial assessment of their potential for vascular tissue engineering. Biomaterials, 2015, 40: 32-42.
20.	Ali S, Cuchiara ML, West JL. Micropatterning of poly(ethylene glycol) diacrylate hydrogels. Methods Cell Biol, 2014, 121: 105-119.

1. Cheung HK, Han TT, Marecak DM, et al. Composite hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with adipose-derived stem cells. Biomaterials, 2014, 35(6): 1914-1923.
2. Bi X, Liang A, Tan Y, et al. Thiolene crosslinking polyamidoamine dendrimer-hyaluronic acid hydrogel system for biomedical applications. J Biomater Sci Polym Ed, 2016, 27(8): 743-757.
3. Bian S, He M, Sui J, et al. The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cells culture. Colloids and Surface B: Biointerfaces, 2016, 140(1): 392-402.
4. Koshy ST, Ferrante TC, Lewin SA, et al. Injectable, porous, and cell-responsive gelatin cryogels. Biomaterials, 2014, 35(8): 2477-2487.
5. Palumbo FS, Fiorica C, Di Stefano M, et al. In situ forming hydrogels of hyaluronic acid and inulin derivatives for cartilage regeneration. Carbohydrate Polymers, 2015, 122: 408-416.
6. Kablik J, Monheit GD, Yu L, et al. Comparative physical properties of hyaluronic acid dermal fillers. Dermatol Surg, 2009, 35 Suppl 1: 302-312.
7. Kaderli S, Boulocher C, Pillet E, et al. A novel oxido-viscosifying hyaluronic acid-antioxidant conjugate for osteoarthritis therapy: biocompatibility assessments. Eur J Pharm Biopharm, 2015, 90: 70-79.
8. Mayol L, De Stefano D, De Falco F, et al. Effect of hyaluronic acid on the thermogelation and biocompatibility of its blends with methyl cellulose. Carbohydrate Polymers, 2014, 112: 480-485.
9. Kaderli S, Boulocher C, Pillet E, et al. A novel biocompatible hyaluronic acid-chitosan hybrid hydrogel for osteoarthrosis therapy. Int J Pharm, 2015, 483(1-2): 158-168.
10. Lima SV, de Oliveira Rangel AE, de Melo Lira MM, et al. The biocompatibility of a cellulose exopolysaccharide implant in the rabbit bladder when compared with dextranomer microspheres plus hyaluronic acid. Urology, 2015, 85(6): 1520.e1-6.
11. Prestwich GD, Marecak DM, Marecek JF, et al. Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives. J Control Release, 1998, 53(1-3): 93-103.
12. Xu X, Jha AK, Harrington DA, et al. Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter, 2012, 8(12): 3280-3294.
13. Burdick JA, Presteich GD. Hyaluronic acid hydrogels for biomedical applications. Adv Mat, 2011, 23(12): H41-56.
14. Bencherif SA, Srinivasan A, Horkay F, et al. Influence of the degree of methacrylation on hyaluronic acid hydrogels properties. Biomaterials, 2008, 29(12): 1739-1749.
15. Tomihata K, Ikada Y. Crosslinking of hyaluronic acid with water-soluble carbodiimide. J Biomed Mater Res, 1997, 37(2): 243-251.
16. Butterworth PH, Baum H, Porter JW. A modification of the Ellman procedure for the estimation of protein sulfhydryl groups. Arch Biochem Biophys, 1967, 118(3): 716-723.
17. Wang W, Huang X, Yin H, et al. Polyethylene glycol acrylate-grafted polysulphone membrane for artificial lungs: plasma modification and haemocompatibility improvement. Biomed Mater, 2015,10(6): 65-78.
18. Chong JY, Mulet X, Postma A, et al. Novel RAFT amphiphilic brush copolymer steric stabilisers for cubosomes: Poly (octadecyl acrylate)-block-poly (polyethylene glycol methyl ether acrylate). Soft Matter, 2014, 10(35): 6666-6676.
19. Munoz-Pinto DJ, Jimenez-Vergara AC, Gharat TP, et al. Characterization of sequential collagen-poly (ethylene glycol) diacrylate interpenetrating networks and initial assessment of their potential for vascular tissue engineering. Biomaterials, 2015, 40: 32-42.
20. Ali S, Cuchiara ML, West JL. Micropatterning of poly(ethylene glycol) diacrylate hydrogels. Methods Cell Biol, 2014, 121: 105-119.

Chinese Journal of Reparative and Reconstructive Surgery

PREPARATION AND BIOCOMPATIBILITY OF IN SITU CROSSLINKING HYALURONIC ACID HYDROGEL

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