In the present study, we prepared two types of DAFM/chitosan hybrid hydrogels by altering the ratio of DAFM to chitosan. Rat AFSCs were able to adhere, proliferate, and secrete annulus fibrosus-related extracellular matrix on both types of DAFM/chitosan hybrid hydrogels. In vivo experiments further demonstrated the reparative effects of both of the DAFM/chitosan hybrid hydrogels on defects in the rat tail annulus fibrosus.
The AFM images revealed the presence of collagen fibers on the surface of the hybrid hydrogels. SEM analysis revealed that both the DAFM/chitosan-1 hybrid hydrogel and the DAFM/chitosan-2 hybrid hydrogel exhibited a porous structure. Following cell implantation, the rat AFSCs were observed to grow, not only on the hydrogel surface, but also within the internal structure, facilitated by interconnected micropores that enhanced the nutrient exchange. Cell cytoskeleton staining showed that the AFSCs showed superior proliferation on the DAFM/chitosan-1 hybrid hydrogel, compared with the DAFM/chitosan-2 hybrid hydrogel, which was potentially attributable to the higher DAFM content, which was also indicated by AFM analysis. In another study, human dermal fibroblasts and human epithelial keratinocytes have been shown to be able to attach and proliferate on decellularized, dehydrated human amniotic membrane. In contrast, dehydrated human amnion/chorion membrane did not support cell attachment and proliferation under the same conditions[28]. Li et al.[29] have fabricated a decellularized matrix hydrogel using human dental pulp (hDDPM-G) and observed that surfaces coated with hDDPM-G promoted adhesion, migration, and proliferation of human dental pulp stem cells.
According to the gene expression results, AFSCs exhibited higher expression levels of annulus fibrous extracellular matrix-related metabolic genes, including Col-I, Col-II, and aggrecan genes, in DAFM/chitosan-1 hybrid hydrogels compared with the expression in DAFM/chitosan-2 hybrid hydrogels. In contrast, the expression of the degradation-related gene MMP-9 was found to be lower in DAFM/chitosan-1 hybrid hydrogels compared with DAFM/chitosan-2 hybrid hydrogels. These findings suggested that DAFM enhanced the expression of metabolic genes but suppressed the expression of a degradation-related gene associated with the annulus fibrosus extracellular matrix. Bioscaffolds originating from the decellularized extracellular matrix of tissues have the capacity to replicate the complex extracellular microenvironment by preserving the specific composition, biomechanics, and structure of the native extracellular matrix. Previous studies have demonstrated that decellularized matrix can enhance the secretion of decellularized-derived extracellular matrix. For instance, Fu et al. have developed a blood vessel matrix (BVM) hydrogel and demonstrated that coating a plate with the BVM hydrogel enhanced the tube formation of human umbilical vein endothelial cells in vitro. Furthermore, when injected into skin flaps, the BVM hydrogel improved the flap survival rate and increased blood perfusion and capillary density[30]. Bahrami et al.[31] have created a scaffold using decellularized sheep knee cartilage for osteochondral tissue engineering. This scaffold not only facilitated EnSC osteogenic differentiation, but also induced the chondrogenic differentiation of stem cells. In vivo, acellularized scaffolds have demonstrated a good ability to support cell adhesion and proliferation, leading to improved regeneration of articular lesions in rats after 4 weeks. Huang et al.[32] have prepared human periodontal ligament cell decellularized matrix (PDLC-DCM), dental pulp cell decellularized matrix (DPC-DCM), and gingival fibroblast decellularized matrix (GF-DCM) and found that PDLC-DCM facilitated osteogenic differentiation and periodontal ligament differentiation of PDLSCs, while DPC-DCM and GF-DCM promoted osteogenic differentiation.. In our previous study, we also observed increased gene expression and extracellular matrix secretion of col-I, col-II, and aggrecan in annulus fibrosus-derived stem cells cultured on DAFM/PECUU electrospun scaffolds, compared with cells cultured on PECUU fibrous scaffolds[33]. It is of interest that DAFM can downregulate the expression of MMP-9. It is widely recognized that disc degeneration is characterized by the accelerated degradation of elastin, which is caused by an increase in the expression of MMP-9[34], MMP-9 degrades denatured collagen molecules and basement membrane collagens, which has been shown to be correlated with the lumbar intervertebral disc disease grade[35]. Kao et al.[36] have observed that the expression of the Lipocalin-2 gene and protein in rat AF cells was upregulated upon Nerve Growth Factor stimulation, resulting in increased MMP-9 activity and glycosaminoglycan release, which are likely to potentiate the degeneration of AF tissue in vivo. A similar result was found in another study in which AF cells were cultured on a plastic surface coated with fibronectin, vitronectin, col-I, gelatin, and a cell-free matrix deposited by human nucleus pulposus cells. The results showed that fibronectin, collagen, and gelatin downregulated the expression of MMP-2 and MMP-9 in the culture media[37].
The expression of IL-6, an inflammatory mediator, was lower in DAFM/chitosan-1 hydrogel than in DAFM/chitosan-2 hydrogel, indicating that DAFM could downregulate the expression of IL-6. Similar results have also been found in the study of Ahmed et al. in which an injectable hydrogel enriched with liver extracellular matrix and silver nanoparticles was developed for the treatment of acute liver failure, and significant reductions in the expression of IL-6 and transforming growth factor-beta were observed[38]. This decreased expression of pro-inflammatory factors resulted in a weaker immune response of the cells toward the material.
In the present study, we employed microsurgical instruments to generate defects in the annulus fibrosus of rat caudal vertebrae. After 3 months, MRI was conducted, which revealed that both the DAFM/chitosan hybrid hydrogel-1 and DAFM/chitosan hybrid hydrogel-2 groups had experienced some loss of intervertebral space height and darkening of disc signals, although a discernible intervertebral space was still present. The annulus fibrosus degeneration group exhibited a significant loss of intervertebral space height and indications of fusion. The sham group did not show any significant abnormalities in the intervertebral space height, and the disc signal remained normal. The results of HE staining and Safranine and Fast Green staining demonstrated similar findings to the MRI results. Furthermore, Safranine and Fast Green staining resulted in distinct visualization of the annulus fibrosus and nucleus pulposus tissue in the sham group, showing normal endplate morphology. The DAFM/chitosan-1 hybrid hydrogel and the DAFM/chitosan-2 hybrid hydrogel groups showed partial endplate morphology, while no obvious endplate morphology was observed in the annulus fibrosus degeneration group. Degeneration of the annulus fibrosus results in altered spinal stress distribution, sustained irregular stress, and aggravated intervertebral disc and spinal degeneration, ultimately leading to loss of intervertebral space height[39]. The results of MRI, HE staining, and Safranine and Fast Green staining all demonstrated that the DAFM/chitosan hybrid hydrogels were incapable of sustaining the signal of the intervertebral disc; nevertheless, the hydrogels could efficiently mend defects in the annulus fibrosus, retard intervertebral disc degeneration, and forestall additional loss of intervertebral disc height. Our previous study also demonstrated that the implantation of a DAFM/PECUU blended fiber scaffold at the site of an annulus fibrosus defect in rats could partially maintain disc height in degenerated intervertebral space and potentially contributed to the reversal of disc degeneration. Studies have also demonstrated that the implantation of biomaterials can help maintain the height of degenerated intervertebral space. In a study by Li et al, the in vivo repair efficacy of hydrogels was assessed using a rat AF defect model. The findings indicated that the implantation of PEGDA/DAFM/TGF-β1 hydrogels successfully sealed the AF defect, prevented nucleus pulposus atrophy, preserved the disc height, and partially restored the biomechanical properties of the intervertebral disc[40].
There are some limitations with the present study. Annulus fibrosus is a heterogeneous tissue with considerable variations in the cell type, extracellular matrix composition, and mechanical properties along the radial regions. On the basis of the differences in mechanical properties and biochemical composition, previous studies have divided the annulus fibrosus into inner and outer regions. The radial heterogeneity of the actual annulus fibrosus was overlooked in the present study. In future studies, we will extract the inner and outer regions of the annulus fibrosus separately and prepare decellularized extracellular matrix. These matrices will be crosslinked with chitosan to create hydrogels. We will observe the proliferation of rat annulus fibrosus stem cells on these two hydrogels and monitor the secretion of extracellular matrix by annulus fibrosus cells.