Thermosensitive injectable hyaluronic acid hydrogel for adipose tissue engineering

Abstract

A series of thermosensitive copolymer hydrogels, aminated hyaluronic acid-g-poly(N-isopropylacrylamide) (AHA-g-PNIPAAm), were synthesized by coupling carboxylic end-capped PNIPAAm (PNIPAAm-COOH) to AHA through amide bond linkages. AHA was prepared by grafting adipic dihydrazide to the HA backbone and PNIPAAm-COOH copolymer was synthesized via a facile thermo-radical polymerization technique by polymerization of NIPAAm using 4,4'-azobis(4-cyanovaleric acid) as an initiator, respectively. The structure of AHA and AHA-g-PNIPAAm copolymer was determined by (1)H NMR. Two AHA-g-PNIPAAm copolymers with different weight ratios of PNIPAAm on the applicability of injectable hydrogels were characterized. The lower critical solution temperature (LCST) of AHA-g-PNIPAAm copolymers in PBS were measured as approximately 30 degrees C by rheological analysis, regardless of the grafting degrees. Enzymatic resistance of AHA-g-PNIPAAm hydrogels with 28% and 53% of PNIPAAm in 100U/mL hyaluronidase/PBS at 37 degrees C was 12.3% and 37.6% over 28 days, respectively. Equilibrium swelling ratios of AHA-g-PNIPAAm hydrogels with 28% of PNIPAAm were 21.5, and significantly decreased to 13.3 with 53% of PNIPAAm in PBS at 37 degrees C. Results from SEM observations confirm a porous 3D AHA-g-PNIPAAm hydrogel structure with interconnected pores after freeze-drying and the pore diameter depends on the weight ratios of PNIPAAm. Encapsulation of human adipose-derived stem cells (ASCs) within hydrogels showed the AHA-g-PNIPAAm copolymers were noncytotoxic and preserved the viability of the entrapped cells. A preliminary in vivo study demonstrated the usefulness of the AHA-g-PNIPAAm copolymer as an injectable hydrogel for adipose tissue engineering. This newly described thermoresponsive AHA-g-PNIPAAm copolymer demonstrated attractive properties to serve as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications.

Fig. 1

¹H NMR spectra of (a) AHA and (b) AHA-g-PNIPAAm in D₂O.

Fig. 2

Viscosity (a) and storage modulus G′ (b) of AHA-g-PNIPAAm copolymer with 5 wt % concentration in PBS as a function of temperature. Insets are in situ gelation of AHA-g-PNIPAAm hydrogel in PBS.

Fig. 3

Degradation of AHA-g-PNIPAAm copolymer hydrogels in PBS and 100U/ml hyaluronidase/PBS at 37°C with respect to weight loss. Values reported are an average n=5, ± standard deviation.

Fig. 4

SEM images to show the internal structures of AHA-g-PNIPAAm-28 (a–c) and AHA-g-PNIPAAm-53 (d–f) hydrogels before and after incubation in PBS at 37°C for different times. (b, e) and (c, f) are 7 and 21d, respectively.

Fig. 5

SEM images to show the internal structures of AHA-g-PNIPAAm-28 (a–b) and AHA-g-PNIPAAm-53 (c–d) hydrogels after degradation at 37 °C in 100U/ml hyaluronidase/PBS for 7d (a, c) and 21d (b, d).

Fig. 6

Equilibrium swelling ratio of AHA-g-PNIPAAm copolymer hydrogels as a function of incubation time in PBS and DMEM/F12/10%FBS at 37°C. Values reported are an average n=5, ± standard deviation.

Fig. 7

(a) DNA contents of encapsulated ASCs in AHA-g-PNIPAAm hydrogels as a function of culture time. Values reported are an average n=3, ± standard deviation. (b) Macroscopic morphologies of cultured ASCs/hydrogels matrices after 21d culture. Left: AHA-g-PNIPAAm-28; Right: AHA-g-PNIPAAm-53.

Fig. 8

CLSM image showing encapsulated ASCs in AHA-g-PNIPAAm-28 (a–b) and AHA-g-PNIPAAm-53 (c–d) hydrogel after 7d (a, c) and 21d (b, d) culture. Cell seeding density: 5×10⁶/mL. The live cells were stained with FDA (Green) and the dead cell nuclei were stained with PI (Red).

Fig. 9

SEM image to show the morphology of encapsulated ASCs in AHA-g-PNIPAAm-28 (a) and AHA-g-PNIPAAm-53 (b) hydrogels after 21d culture. Cell seeding density: 5×10⁶/mL.

Fig. 10

Subcutaneous injection of 5 wt% AHA-g-PNIPAAm-53 copolymer hydrogels implants in athymic nude mice after 2h. Yellow arrows denote hydrogel bumps after injection.

Fig. 11

H & E staining of AHA-g-PNIPAAm-53 copolymer hydrogels after 5d of implantation. T: tissue; H: hydrogel.

Scheme 1

Synthetic route of (a) AHA, (b) PNIPAAm-COOH, and (c) AHA-g-PNIPAAm.