Genipin‐crosslinked decellularized annulus fibrosus hydrogels induces tissue‐specific differentiation of bone mesenchymal stem cells and intervertebral disc regeneration

Abstract

Biomaterial‐based therapy that can restore annulus fibrosus (AF) function in early stage and promote endogenous repair of AF tissues is a promising approach for AF tissue repair. In this study, we established a genipin‐crosslinked decellularized AF hydrogels (g‐DAF‐G) that are injectable and could manifest better in situ formability than noncrosslinked decellularized AF hydrogel, while preserving the capacity of directing differentiation of human bone mesenchymal stem cells (hBMSCs) towards AF cells. Hematoxylin and eosin staining, 4',6‐diamidino‐2‐phenylindole staining, and so forth showed that the majority of cellular components were removed, whereas extracellular matrix and microstructure were largely preserved. The storage modulus increased from 465.5 ± 9.4 Pa to 3.29 ± 0.24 MPa after 0.02% genipin crosslinking of decellularized AF hydrogels (DAF‐G) to form g‐DAF‐G. AF‐specific genes (COL1A1, COL5A1, TNMD, IBSP, FBLN1) were significantly higher in DAF‐G and g‐DAF‐G groups than that in control group after 21 days of culturing. g‐DAF‐G significantly restored nucleus pulposus water content and preserved intervertebral structure in vivo. Summarily, we produced a novel AF regeneration biomaterial, g‐DAF‐G, which exhibited well biocompatibility, great bioactivity, and much higher mechanical strength than DAF‐G. This study will provide an easy and fast therapeutic alternative to repair AF injuries or tears.

Decellularized Disc Hydrogels for hBMSCs tissue-specific differentiation and tissue regeneration

“…Freeze-drying processes were then performed and the lyophilized decellularized tissues were homogenized into DNP powder or DAF powder. Decellularized matrix hydrogels were prepared as previously described [ 16 ]. Specifically, a certain amount of sterilized DTM powder was added into 0.01 M hydrochloric acid containing pepsin (concentration of pepsin is 1.5 mg/ml) to adjust its concentration to 10 mg/ml.…”

“…All experimental protocols involving human tissue and cells were carried out following The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and approved by the medical ethics committee of Tongji Medical College of Huazhong University of Science and Technology, and the informed consent of all participating subjects was obtained. As previously described [ 16 ], hBMSCs were extracted from the bone marrow in artificial hip joint replacements. The stemness was evaluated by cellular markers (anti-CD73-APC, anti-CD90-APC, anti-CD105-APC, anti-34-APC, anti-CD45-APC, Biolegend, USA) ( Supplementary Fig.…”

“…Regarding AF, structural repair is a hot spot. Many strategies have been proposed to repair the herniation site, including mesh attachment, void filling, and collagen reconstruction [ [15] , [16] , [17] ]. However, biomaterials designed for the regeneration and integrity of AF defects have not yet observed satisfying clinical outcomes [ 5 ].…”

Post-decellularization techniques ameliorate cartilage decellularization process for tissue engineering applications

“…Furthermore, in a study comparing the ECM derived from human umbilical cord, crosslinked by genipin and N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) for neural tissue application, authors demonstrated that genipin, rather than EDC, improved the bio-stability of injectable ECM hydrogel in biocompatible concentration. 290 In another example, Yizhong Peng et al, 291 established an injectable genipin-crosslinked decellularized annulus fibrosus (dAF) hydrogels and showed that they are better in case of formability, biocompatibility, bioactivity, and mechanical strength in comparison to non-crosslinked dAF.…”

“…In another example, Yizhong Peng et al, 291 established an injectable genipin-crosslinked decellularized annulus fibrosus (dAF) hydrogels and showed that they are better in case of formability, biocompatibility, bioactivity, and mechanical strength in comparison to non-crosslinked dAF.…”