Cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution

Abstract

Chondroprogenitor cells encapsulated in a chondrogenically supportive, three-dimensional hydrogel scaffold represents a promising, regenerative approach to articular cartilage repair. In this study, we have developed an injectable, biodegradable methacrylated gelatin (mGL)-based hydrogel capable of rapid gelation via visible light (VL)-activated crosslinking in air or aqueous solution. The mild photocrosslinking conditions permitted the incorporation of cells during the gelation process. Encapsulated human-bone-marrow-derived mesenchymal stem cells (hBMSCs) showed high, long-term viability (up to 90 days) throughout the scaffold. To assess the applicability of the mGL hydrogel for cartilage tissue engineering, we have evaluated the efficacy of chondrogenesis of the encapsulated hBMSCs, using hBMSCs seeded in agarose as control. The ability of hBMSC-laden mGL constructs to integrate with host tissues after implantation was further investigated utilizing an in vitro cartilage repair model. The results showed that the mGL hydrogel, which could be photopolymerized in air and aqueous solution, supports hBMSC growth and TGF-β3-induced chondrogenesis. Compared with agarose, mGL constructs laden with hBMSCs are mechanically stronger with time, and integrate well with native cartilage tissue upon implantation based on push-out mechanical testing. VL-photocrosslinked mGL scaffold thus represents a promising scaffold for cell-based repair and resurfacing of articular cartilage defects.

FIG. 1.

(A) Visible light (VL)–activated gelation using illumination from a dental lamp (wavelength 430–490 nm, power 1400 mw/cm²). (B) Live/dead analysis of human-bone-marrow-derived mesenchymal stem cells (hBMSCs) encapsulated into methacrylated gelatin (mGL) hydrogel at different depths (1/3 and 2/3 from surface), using commonly used ultraviolet (UV)/I2959 or VL/lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Green, live cells; red or orange, dead cells as indicated by arrows. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 2.

(A) Compressive Young's moduli of mGL hydrogels prepared by different exposure times up to 8 min. (B) Collagenase digestion behavior of mGL hydrogels prepared by 2-, 4-, and 8-min light exposure. Wet weight loss of the scaffolds in collagenase solution was estimated at different times up to 180 min.

FIG. 3.

Live/Dead assay of hBMSCs cultured in agarose at day 1 (A) or 90 (C), or in mGL hydrogel at day 1 (B) or 90 (D). Green, live cells; red or orange, dead cells as indicated by arrows. Scale bar=100 μm. (E) MTS assay of mGL or agarose constructs at day 0 and 90 (**p<0.01). Color images available online at www.liebertpub.com/tea

FIG. 4.

Real-time reverse transcription–polymerase chain reaction analysis of chondrogenic gene expression in hBMSC-laden agarose or mGL hydrogels at day 90 (*p<0.05, **p<0.01). Genes analyzed include Sox 9, collagen type II, and aggrecan. hBMSCs cultured on tissue culture plastic without chondrogenic induction served as control. All values are normalized to control.

FIG. 5.

Quantitation of glycosaminoglycan (GAG) content deposited in hBMSC-laden constructs after 90 days of chondrogenic culture. (A) Total GAG in a construct. Blank agarose and mGL without cells served as control. (B) GAG production normalized to DNA content (**p<0.01).

FIG. 6.

Alcian blue staining of sGAG in histological sections of the hBMSC-laden mGL and agarose constructs after 90 days of chondrogenic culture with Fast Red as nuclear counterstaining. Scale bar=1 mm (A, C) or 100 μm (B, D). Color images available online at www.liebertpub.com/tea

FIG. 7.

Mechanical properties of hBMSC-laden mGL and agarose constructs at day 0 and 90 culture in chondrogenic medium (*p<0.05).

FIG. 8.

In vitro assessment of integration of hBMSC-laden mGL and agarose constructs into native cartilage. (A) Native bovine cartilage rings with 8-mm outer diameter and 4-mm inner diameter. (B) The inner gap in the cartilage explants was filled with hBMSC-laden mGL or agarose. (C) The composite constructs were cultured in chondrogenic medium for 6 weeks, and were then subjected to push-out mechanical testing. (D–G) Push-out test device. The composite constructs were placed in a custom-designed chamber (4), which held the cartilage in position, allowing push-out access to the inner-implanted component. The motor (1) controlled the movement of plunger (3), and the sensor (2) recorded the real-time force. (H) Typical stress–displacement curves of the agarose/hBMSC and mGL/hBMSC constructs from the push-out test. (I) Failure stress values for the different groups (*p<0.05, **p<0.01). Color images available online at www.liebertpub.com/tea