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

Journal of Tissue Engineering and Regenerative MedicineVolume 14, Issue 3 p. 497-509 RESEARCH ARTICLEOpen Access Genipin-crosslinked decellularized annulus fibrosus hydrogels induces tissue-specific differentiation of bone mesenchymal stem cells and intervertebral disc regeneration Yizhong Peng, Yizhong Peng Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaContributed equally to this work and shall share the first authorship.Search for more papers by this authorDonghua Huang, Donghua Huang Musculoskeletal Tumor Center, Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, ChinaContributed equally to this work and shall share the first authorship.Search for more papers by this authorJinye Li, Jinye Li Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaContributed equally to this work and shall share the first authorship.Search for more papers by this authorSheng Liu, Sheng Liu Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaSearch for more papers by this authorXiangcheng Qing, Corresponding Author Xiangcheng Qing 353220817@qq.com Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Correspondence Zengwu Shao and Xiangcheng Qing, Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Email: szwpro@163.com; 353220817@qq.comSearch for more papers by this authorZengwu Shao, Corresponding Author Zengwu Shao szwpro@163.com orcid.org/0000-0003-1616-8118 Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Correspondence Zengwu Shao and Xiangcheng Qing, Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Email: szwpro@163.com; 353220817@qq.comSearch for more papers by this author Yizhong Peng, Yizhong Peng Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaContributed equally to this work and shall share the first authorship.Search for more papers by this authorDonghua Huang, Donghua Huang Musculoskeletal Tumor Center, Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, ChinaContributed equally to this work and shall share the first authorship.Search for more papers by this authorJinye Li, Jinye Li Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaContributed equally to this work and shall share the first authorship.Search for more papers by this authorSheng Liu, Sheng Liu Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaSearch for more papers by this authorXiangcheng Qing, Corresponding Author Xiangcheng Qing 353220817@qq.com Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Correspondence Zengwu Shao and Xiangcheng Qing, Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Email: szwpro@163.com; 353220817@qq.comSearch for more papers by this authorZengwu Shao, Corresponding Author Zengwu Shao szwpro@163.com orcid.org/0000-0003-1616-8118 Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Correspondence Zengwu Shao and Xiangcheng Qing, Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Email: szwpro@163.com; 353220817@qq.comSearch for more papers by this author First published: 03 February 2020 https://doi.org/10.1002/term.3014Citations: 19 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 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. 1 INTRODUCTION Intervertebral disc degeneration (IVDD), which is a major cause of low back pain (Luoma et al., 2000), has exerted a large socioeconomic burden worldwide. It could be characterized by progressive degenerative damage of intervertebral discs (IVDs) in company with metabolic alterations of other vertebral parts (Ruiz-Fernandez et al., 2019). Unfortunately, the complicated aetiology/pathogenesis of IVDD largely limits the understanding of this disabling disease and restricts the progression of effective therapies. Annulus fibrosus (AF) defect is a well-known cause for the initiation and progression of IVDD (Buser, Chung, Abedi, & Wang, 2019). Consistent AF defect after discectomy would lead to disc re-herniation (Battie et al., 2014) and persistent low back pain. Operative treatment is the only approach that is clinically applied and mostly preferred for AF defect and disc herniation. However, surgical approaches, especially open discectomy, often require vertebral fusion to avoid spinal instability, which decreases spine mobility in a great extent. Also, postoperative complication, including infection, bleeding, unintentional dural puncture/laceration, and so forth, intends to lengthen the hospital duration and even lead to reoperation (Fjeld et al., 2019). For these reasons, an increasing interest has been attracted in biological strategies for IVD regeneration. Among those, biomaterial-based therapy is a promising approach that can achieve early intervention or postoperative AF repair after AF injuries and prevent further operation. As it has long been identified that cellular physiology of disc cells is largely affected by the mechanical environment in AF tissues (Vergroesen et al., 2015), many engineered scaffolds have been fabricated for AF biomechanical restoration and pain relief, including natural scaffolds (Zitnay et al., 2018), synthetic polymers (Christiani, Baroncini, Stanzione, & Vernengo, 2019), and their combination (Yang et al., 2017). Among various types of scaffolds, in situ tissue-derived biomaterials, which closely mimic the prominent features of the native tissue microarchitecture and support an appropriate, tissue-specific cell phenotype, have their innate advantages in native tissue repair (Bejleri & Davis, 2019). Decellularized matrices are biomaterials produced by removing cellular components of the tissues while retaining most of extracellular matrix (ECM). The process of decellularization not only maintains the original structure and biological function of the native tissues that are essential for cell adhesion, proliferation, and differentiation, but also significantly reduces autoimmunity (Bejleri & Davis, 2019; Xu et al., 2014). Thus, the native tissue-derived decellularized matrices could provide the innate tissue architecture as well as vital biochemical signaling molecules for the resident cells in various types of tissue regeneration. Unfortunately, despite the several advantages, the IVD-derived decellularized matrix is limited in direct application of IVD repair due to its nonplasticity. ECM hydrogels from decellularized IVD tissues are considered to be reliable materials for IVD repair as the injectable property that could fill irregularly shaped defects as well as the matrix structure and biological signals retained from the native source tissue (Mercuri, Gill, & Simionescu, 2011). However, the capacity of decellular IVD-derived hydrogel in directing cell behavior and promoting in situ tissue repair is poorly understood, especially for decellularized AF (DAF)-derived hydrogel. Thus, we produced a novel decellularized AF-derived hydrogel and explored its biocompatibility, cellular fate decision, and ability in IVD defects restoration in vitro/in vivo. Due to the relatively low mechanical strength of the AF-derived hydrogel compared with the native AF tissues, genipin, which was extracted from the natural compound geniposide with low toxicity and high biocompatibility, was chosen as a crosslinking agent for the AF-derived hydrogel. The genipin-crosslinked DAF hydrogels (g-DAF-G) could mimic the mechanical property of native AF tissues better than DAF-only hydrogel (DAF-G). Moreover, it has been reported that micromechanical properties determined by the microstructure of materials were effective on cell differentiation induction (Wen et al., 2014). Larger Young modulus (13.4 MPa) was reported to better induce collagen I (Col I) expression than the smaller one (2.5 MPa) in AF-derived stem/progenitor cells (Guo et al., 2015), suggesting that larger Young modulus is potentially more likely to induce AF-specific phenotype. Thus, the scaffolds with a relative high stiffness might increase the tendency of resident stem cells into AF-like differentiation and benefit the AF tissues defects better. In this study, we developed a genipin-crosslinked DAF-G that were injectable and could manifest better in situ formability to overcome the limitations of noncrosslinked DAF-G. The microstructure, mechanical properties, and biocompatibility of the produced hydrogel in the present study were thoroughly assessed. The differentiation of human bone mesenchymal stem cells (hBMSCs) on the hydrogel and in vivo repair of AF defects rat model was also evaluated. The general schematic design of our study was shown in Figure S1. We hypothesized that the produced hydrogen could effectively direct specific differentiation of hBMSCs towards AF cells and exhibit high efficiency of in vivo AF tissue repair. We hope that our research could offer new insights into the regeneration of IVD. 2 MATERIALS AND METHODS 2.1 Bovine AF specimen All animal experiments were managed with the protocol approved by the Animal Experimentation Committee of Huazhong University of Science and Technology. Twenty disc specimens were harvested from four bovine tails that were obtained from a local abattoir. In details, five disc specimens were harvested from each tail by cutting transversely, while surrounding soft tissues and vertebral body were cautiously removed. Then, the outer AF tissues were obtained from each disc and used in the following experiment. Throughout the whole preparation process, the extracted AF samples were regularly sprayed with distilled water to keep them moist. 2.2 Preparation of genipin-crosslinked DAF matrix hydrogels The dissected AF tissues were cut into 5-cm pieces for a chemical decellularization process. Briefly, cellular components in AF were sequentially extracted by Triton X-100 (2%) and sterile water washes at 4°C. After the freeze-drying processes for five cycles, the AF tissues with Triton X-100 (2%) were gently shaken on open air shaker for 72 hr at 18°C. Then, the supernatant liquid was removed after 1,200 rpm for 5 min and 1% sodium dodecyl sulfate was applied to wash the AF tissues on a shaker for 72 hr at 18°C. Next, the AF tissues were washed by distilled water for three times (30 min for each time) in order to fully remove the residual sodium dodecyl sulfate solution. For sterilization, the resulting DAF matrix was lyophilized, smashed into powder by a micromill (Thomas Wiley® Mini-Mill, USA), fumigated in a sealed box by 95% alcohol for 2 hr at 37°C, and irradiated under ultraviolet overnight with ventilation, generating DAF powder (DAF-P). The obtained DAF-P (1.5% w/v) was weighed and solubilized in 0.01 mol/L HCl containing pepsin (concentration of pepsin is 1.5 mg/ml) with moderate agitation for 48 hr. The pH of the digested solution (DAF in pepsin–HCl solution) was adjusted to 7.4 using 0.1 mol/L HCL and 0.1 mol/L NaOH, and the salt concentration was adjusted with 10 times phosphate-buffered saline (PBS; 1/10 of final neutralized volume) to form DAF-Gs. Genipin (Aladdin, China) dissolved in 75% EtOH was applied to crosslink DAF-G, with various genipin concentrations (0.01%, 0.02%, and 0.04% w/v), forming g-DAF-Gs. Commercialized hydrogel products, Col I (Corning, USA), was utilized in further studies. The final concentration of Col I, DAF, and g-DAF-P in the hydrogel was 1.5% w/v. The solution-to-gelatin transition was induced by adjusting the temperature to the physiological range (37°C, about 30 min). All procedures were carried out under sterile conditions. 2.3 Characterization of fresh AF and DAF-P To identify the efficiency of AF decellularization, residual DNA was detected, and histological structures were evaluated by hematoxylin and eosin (HE) staining. To analyze the abundance of ECM components, the concentrations of glycosaminoglycans (GAGs) in fresh AF (FAF) and DAF-P were detected by tissue GAGs alcian blue colorimetry assay kit (GenMed Scientifics, GMS 19236.2, USA), after lyophilization of FAF and DAF-P. A standard curve was performed to estimate the concentration of GAGs in each sample. The DNA of FAF and DAF-P was extracted using the Animal Tissues/Cells Genomic DNA Extraction Kit (Solarbio, Cat#D1700, Beijing, China) and analyzed by NanoDrop microvolume sample retention system (Thermo Scientific). For histological evaluation, FAF and DAF-P were fixed in 4% formalin, washed three times with distilled water, embedded in paraffin, and sectioned using an ultrathin slicer (Leica EM UC7, Germany). The sections were stained with HE, 4',6-diamidino-2-phenylindole, picrosirius red, improved special Masson trichrome and immunohistochemical staining of collagen 1A1 (COL1A1, A1352, Abclonal) and collagen 2A1 (COL2A1, 15943-1-AP, Proteintech), and photographed under a microscope (Olympus, Japan). 2.4 Rheological property of hydrogels To monitor the process of gelation at 37°C (physiological temperature), the pH and ion-balanced solution was placed on a 20-mm parallel plate rheometer (Thermo Scientifc, HAAKE MARS III) at 4°C to ensure homogeneous distribution and liquidity of the prehydrogel solutions between the plates. Parameters in the test were set to 1% strain and 1 Hz. To observe the gelation process at 37°C, a dynamic time sweep (10 min) was performed with a fast heating from 4°C to 37°C (200°C/min) at the beginning stage. Storage modulus of each sample was recorded to assess the mechanical characteristics of hydrogels. 2.5 SEM observation FAF, DAF-P, lyophilized DAF-G and g-DAF-G were fixed in 2.5% glutaraldehyde for 8 h, rinsed in deionized water for 30 min, and then gradiently dehydrated in a series of alcohol solutions (30%, 50%, 75%, 100% ethanol) for half an hour of each rinse. Finally samples were lyophilized and torn to generate the fracture surface. After coating with platinum, the surface of each sample was photographed by scanning electron microscope (SEM; HITACHI S-4800) at 10 kV. The structural parameters of samples were measured by image analysis software (Image J; National Institutes of Health). More specifically, three SEM pictures from three independent samples for each group were collected for quantitative analysis. Three squares (1/10 width and length of the whole picture) were randomly placed on the picture and the area within the squares were identified as region of interest (ROI). Then, diameters of the fibers included in ROIs were measured, and the largest distance between adjacent fibers included in ROI was measured and defined as fibers distance. 2.6 Protein distribution analysis by sodium dodecyl sulfate–polyacrylamide gel To compare the difference of retained ECM-related components among DAF-P, DAF-G, and g-DAF-G, they were lyophilized and ground into powder, weighed, and the protein components were extracted using radioimmunoprecipitation assay lysis buffer (Nanjing Jiancheng Bioengineering institute) containing a protease inhibitor cocktail (Nanjing Jiancheng Bioengineering institute) and a protein phosphatase inhibitor mixture (Nanjing Jiancheng Bioengineering institute). Next, the samples were centrifuged for 25 min at 13,000 rpm and 4°C, and total protein concentration was measured by a BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein in each sample (30 μg) were electrophoretically separated by a 12% sodium dodecyl sulfate–polyacrylamide gel, which was stained with Coomassie Brilliant Blue R250 protein fast stain kit (Nanjing Jiancheng Bioengineering institute) for 1 hr according to the instructions. After image, total protein in the gel was quantified using Image Lab 4.0 software (Bio-Rad Laboratories, Hercules, CA, USA). 2.7 ATR-FTIR analysis A Fourier transform infrared (FTIR) Spectrometer (Spectrum 100, Perkin Elmer, USA) equipped with a germanium single bounce micro attenuated total reflection (ATR) objective was applied to measure the infrared radiation (IR) spectrum of each sample. All IR spectra were collected from 75 scans between 500 and 4,000 cm−1 at a wavenumber resolution of 5 cm−1. The exhibited IR absorbance spectrum for each sample is the mean of spectra collected randomly from three independent locations. 2.8 Human bone marrow samples All experimental protocols involving human tissue and cells were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, China. Human bone marrow specimens were obtained during the femoral head arthroplasty surgery of six volunteer donors (age from 30 to 50 years). 2.9 Cell extraction hBMSCs were isolated from bone marrow aspirates and cultured in vitro as follows: 10 ml of bone marrow was diluted (1:1) with human MSC complete medium (Cyagen, USA) and loaded over Percoll (Sigma, USA) for density gradient centrifugation. Mononucleated cells were obtained from the interface after centrifugation (900g, 25 min) and washed by PBS (Bosterbio, USA) twice. Then, cells were resuspended in human MSC complete medium and seeded in T25 flasks (Corning, USA) at a density of 1.0 × 105/cm2 at 37°C and 5% CO2. After 48 hr, medium change was conducted to remove nonadherent cells. The adherent cells were cultured for about 2 weeks until cell clones reached about 75–85% confluence, then digested with 0.25% trypsin-0.02% ethylenediaminetetraacetic acid (Sigma, USA) and finally subcultured at a density of 1.0 × 105 cells/cm2 in new T25 flasks. The third generation of hBMSCs was used throughout the whole experiments. The neutralized hydrogels were uniformly spread in a six-well culture plate for 100 μl per well and placed in cell incubator (37°C) for 30 min for gelation. A thin hydrogel layer coating was created after gelation, and the suspension cells in the third generation were counted and plated onto the surface of hydrogel scaffolds with Dulbecco's modified Eagle medium/ha F-12 (Gibco, USA) containing 1% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Beyotime, China) at 37°C and 5% CO2. The cells cultured on coating hydrogels were used for cell viability assessment and AF specific gene detection. 2.10 Assessment of cell viability Cell compatibility assays were conducted using Cell Counting Kit-8 (CCK-8, KeyGEN BioTECH, China). Cells were plated on 96-well plates (control) or the Col I, DAF-G, or g-DAF-G precoated 96-well plates (2,000 cells per well). After 1, 7, 14, and 21 days, 100-μl CCK-8 working solution (CCK-8 reagent: serum-free medium = 1:10) was added into each well of the plate and incubated for 2 hr. Then, to avoid the influence of the bottom hydrogel scaffolds on the absorbency, the CCK-8 working solution of each well was successively transferred to a new blank 96-well plate. Finally, absorbency, indicating cell viability, was detected at 450 nm using a spectrophotometer (ELx808 Absorbance Microplate Reader, Bio-Tek, USA). In addition, Live-Dead cell staining kit (KeyGEN BioTECH, China) was carried out by treating cells with calcein AM and PI for 10 min, after hBMSCs were cultured on 96-well plates (control) or the Col I, DAF-G, or g-DAF-G precoated 96-well plates for 14 or 21 days, and then observing the image by a fluorescence microscope (Olympus, IX73, Japan). 2.11 LDH assay The 96-well plate was precoated with none (control), 20-μl DAF-G and 20-μl g-DAF-G of various genipin concentrations (0.01%, 0.02%, and 0.04%). hBMSCs were seeded at a density of 2,000 cells per well, and lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Beyotime, Shanghai, China) was utilized to evaluate the cytotoxicity of the various genipin concentration on hBMSCs at Day 21 according to the protocol. LDH release activity was calculated from optical density value measured by spectrophotometer (ELx808 Absorbance Microplate Reader, Bio-Tek, USA). 2.12 Quantitative real-time RT-PCR analysis Gene expression was measured by reverse transcription polymerase chain reaction (RT-PCR) after 7 or 21 days of hydrogel culturing. Total RNA from hBMSCs was harvested using the TRIZOL reagent (Invitrogen, USA). Complementary DNA was synthesized using the RevertAid™ First Strand cDNA Synthesis Kit (K1622; Fermentas) and oligo (dT) primers (15 min at 37°C and 5 s at 85°C) on a RT-PCR system (Eastwin Life Science, Beijing, China). RT-PCR was carried out with a Bio-Rad CFX96™ Real-Time System using the SsoFast™ EvaGreen Supermix Kit (Bio-Rad). Primer sequences for collagen-1A1, collagen-5A1, integrin-binding sialoprotein (IBSP), fibulin-1 (FBLN1), tenomodulin (TNMD), and glyceraldehyde-3-phosphate dehydrogenase (the control) were listed in Table 1. The relative expression levels were analyzed by the 2−ΔΔCt method and normalized to the control. Table 1. Summary of primers used in quantitative reverse transcription polymerase chain reaction Gene Sequence (5'-3') COL1A1 Forward primer GATTGACCCCAACCAAGGC Reverse primer GAATCCATCGGTCATGCTCT COL5A1 Forward primer CCGATTGGCTACCCAGGTC Reverse primer CACCCTTGATGCCCATGTCT TNMD Forward primer TTGGTATCCTGGCCCTAA Reverse primer CAGTGCCATTTCCGCTTC IBSP Forward primer AGGGCAGTAGTGACTCATCCG Reverse primer AGCCCAGTGTTGTAGCAGAAAG FBLN1 Forward primer TCTCTGTGGATGGCAGGTCA Reverse primer ACACTGGTAGGAGCCGTAGA GAPDH Forward primer AATCCCATCACCATCTTCCAG Reverse primer GAGCCCCAGCCTTCTCCAT Abbreviations: FBLN1, fibulin-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IBSP, integrin-binding sialoprotein; TNMD, tenomodulin. 2.13 Rat tail acupuncture degenerative model Sixteen male Sprague Dawley rats around 300 g (8–12 weeks old) were used for the in vivo experiments. The surgical procedures were described as follows: After anaesthetized by 1% pentobarbital sodium (ml/g) via intraperitoneal injection. The coccygeal vertebrae Co4/Co5 and Co5/Co6 were located by manual palpation and counting and confirmed by a trial radiograph. The IVDs were punctured by a 18-G sterile needle with an outer diameter of 1.27 mm at a depth from the skin to near the border of the inner AF and nucleus pulposus (NP) tissues with NP intact. Then, the needle was rotated 360° and held for 30 s. Next, 10-μl physiological saline or the neutralized prehydrogel solution was injected into the predisposed defect of AF tissues by microsyringes (Hamilton injection needle) attached to 20-G needles. The concentration of genipin in g-DAF-G was 0.02% (w/v). The injection of hydrogels was delayed for approximately 20 min, to allow for pregelation (forming Col I, DAF-G, and g-DAF-G) and avoid diffusion or leakage. The rats were randomly divided into four groups for different treatment (four rats in each group): the degeneration group (with needle puncture and physiological saline injection); the Col I group (with needle puncture and Col I injection); the DAF-G group (with needle puncture and DAF-G injection); and the g-DAF-G group (with needle puncture and g-DAF-G injection). The process of gelation could be observed in situ after injection for 30 min. A semitransparent gel was formed in situ for the Col I and DAF-G groups, whereas a blue gel was coagulated for the g-DAF-G group. Magnetic resonance imaging (MRI), and histological analysis were performed to assess the repair effect of hydrogel on AF defects in each group. 2.14 MRI procedures and data processing At 4 and 8 weeks after injection, all rats were anesthetized with 1% pentobarbital sodium (ml/g) and were put on examination plate with their tails straightened. T2-weighted sections in the sagittal plane were imaged by MRI (MAGNETOM Avanto, 1.5 T, Germany) to analyze the water content and the structure of the target disc. ImageJ was used for quantitative analysis of the image slices to measure the relative NP water content by the ratio of punctured disc to adjacent intact disc. The alterations of the NP tissues were also assessed by modified Pfirrmann disc degeneration grading system (Che et al., 2019). Two researchers carried out the assessment independently. 2.15 Histological analysis After injection for 8 weeks, target disc specimens in each group were harvested by euthanizing the rats with 1% pentobarbital sodium (ml/g). The disc samples were rinsed by PBS buffer solution, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5.0 μm per section (Thermo#shandon finesse 325). Next, disc specimens were stained with HE and safranin O–fast green (S&O). The cellularity and morphology of the IVD tissues were assessed by two authors separately according to the scoring criteria described by Han et al. (2008) 2.16 Statistical analysis The outcomes were presented as the mean ± standard deviation (SD), and a value of p < 0.05 was considered statistically significant. The statistical differences between treatment groups were evaluated by one-way analysis of variance, following the Tukey post hoc test. All experiments were repeated at least three times using independent samples. Statistical analyses were performed using the SPSS software package (IBM SPSS software package 18.0). All the statistical chart was drawn by GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA). 3 RESULTS 3.1 FAF and DAF-P characterization After decellularization, the DAF tissue became white and porous, and HE staining showed an absence of nuclei and a loose-layered structure in histological sections (Figure 1a). Also, 4',6-diamidino-2-phenylindole staining suggested obviously less positive staining after decellularization, indicating efficient removal of DNA. (Figure 1b). During gelation, the genipin-crosslinked AF hydrogels gradually turned into blue, and AF hydrogels formed a semitransparent gel (37°C, pH 7.3–7.5; Figure 1c). Residual DNA content for FAF and DAF-P were 360 ± 31 and 40 ± 4 ng/mg, respectively. DNA derived from DAF-P was lower than the internationally recognized criterion of 50 ng/mg (Crapo et al., 2012; Figure 1d). The outcomes of the GAGs content for FAF and DAF-P showed 2.27 ± 0.56 and 1.16 ± 0.13 μg/mg, respectively (Figure 1e). Also, as what COL1A1, COL2A1, picrosirius red, and masson staining suggested, the arrangement of ECM collagens became a little bit loose, and the staining intensity, indicating collagen content, did not get altered significantly after decellularization (Figure S2). Therefore, our protocol for removing cellular component while preserving ECM is reliable and efficient. Figure 1Open in figure viewerPowerPoint Fabrication of decellularized annulus fibrosus (AF) hydrogel and evaluation of decellularization efficiency. (a) General appearance and hematoxylin and eosin staining of fresh AF (FAF) and decellularized AF powder (DAF-P). (b) 4',6-Diamidino-2-phenylindole staining revealed significant removal of cellular components after decellularization. (c) Decellularized AF hydrogels (DAF-G) appealed to be semitransparent and oyster milk, and genipin-crosslinked decellularized AF hydrogels (g-DAF-G) turned blue after gelation and was more stable. Black bar = 200 μm, white bar = 500 μm. Quantitative analysis of (d) DNA and (e) glycosaminoglycans suggested high efficiency of decellula