| 摘要: | Background: The anterior cruciate ligament (ACL) partial tears constitute a poor healing capacity due to its intra-articular location and inadequate blood supply. Clinically, ACL partial tear treatments are limited by immobilization and rehabilitation. Therefore, different tissue engineering approaches have been emerged. Among them, cell-derived (CD) extracellular matrix (ECM) is related to a low possibility of pathogen transmission, owns an ideal microenvironment with complex fibrillar proteins and growth factors for cell adhesion and proliferation. Nonetheless, CD-ECM exhibited weak biomechanical properties, and processing of CD-ECM into three-dimensional (3D) scaffolds, including implantable sponge and injectable gels, have been studied scarcely. The gel and sponge scaffolds provide suitable platforms for 3D cell culture and tissue regeneration.
Aim: To develop genipin cross-linked adipose-derived stem cell (ADSC) ECM-graphene oxide (GO) composite sponge and genipin cross-linked ADSC-ECM gel as well as to investigate efficacy of the fabricated scaffolds on rabbit ACL partial tear.
Materials and Methods: ADSC-ECM sponges were fabricated by several steps, including isolation and expansion of rabbit ADSCs, preparation of ADSCs sheets, decellularization, lyophilization, adding graphene oxide, and cross-linking, while the genipin cross-linked ADSC-ECM gel was produced by additional enzymatic digestion and pH neutralization along with the above steps. The physicochemical properties of the scaffolds were assessed by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), mass spectrometry, western blotting, rheological, and degradation analysis. Cytocompatibility of the scaffolds was assessed on mouse skin fibroblast (L929), rabbit ADSCs and anterior cruciate ligament fibroblasts (ACLFs). Biocompatibility and biodegradation of the sponge were evaluated after implanting rat subcutaneous tissue using histological analysis, blood biochemical test, and micro–CT. Local efficacy the fabricated 3D scaffolds were analyzed after implantation by using MRI and histological analysis.
Results: SEM analyses of the cross-linked ADSC-ECM sponges and gels demonstrated a highly porous microstructure and the ADSC-ECM sponges had 71.22 ± 19.52 µm of pore sizes. The in vitro degradation rate was found to be significantly lower in both cross-linked ADSC-ECM sponges and gels in comparison with non-cross-linked ECM sponges and gels. Successful cross-linking of ADSC-ECM sponges and gels as well as conjugation of graphene oxide into the sponges were depicted by characteristic peaks in FTIR analysis. Moreover, mass spectrometry and western blotting analyses showed that ADSC-ECM was containing collagen, biglycan, decorin, fibronectin, vimentin, vitronectin, and transforming growth factor beta 1 (TGF-β1). Furthermore, MTT assay showed that the developed sponges and gels could maintain L929, ADSCs, and ACLFs viability. After subcutaneous implantation, ADSC-ECM sponges containing a medium amount of GO showed appropriate biodegradation with a lower inflammatory reaction. Results of histology analysis demonstrated that both genipin cross-linked ADSC-ECM-GO composite sponge and genipin cross-linked ADSC-ECM gel could assist in improving ACL partial tear healing in the rabbit model.
Conclusion: Genipin cross-linked ADSC-ECM-GO composite sponge and genipin cross-linked ADSC-ECM gel were successfully developed along with improved mechanical properties. Besides, the ADSC-ECM contained collagen, fibronectin, biglycan, and TGF-β1. Fabricated natural 3D scaffolds could maintain ACLFs, ADSCs, and L929 cells viability and morphology. Finally, both genipin cross-linked ADSC-ECM-GO composite sponge and genipin cross-linked ADSC-ECM gel could improve rabbit ACL partial tear healing. These natural 3D scaffolds might be suitable for orthopedic tissue engineering application in the future. |
