M2 macrophage-derived exosomal miR-26b-5p regulates macrophage polarization and chondrocyte hypertrophy by targeting TLR3 and COL10A1 to alleviate osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent chronic musculoskeletal diseases among the elderly population. In this study, macrophage-derived exosomes were isolated and identified. Exosomes were subjected to microRNA (miRNA) sequencing and bioinformatic analysis, and differentially expressed miRNAs were verified. miR-26b-5p target genes were confirmed through target-site mutation combined with a dual-luciferase reporter assay. The effects of miR-26b-5p on macrophage polarization and chondrocyte hypertrophy were assessed in vitro. miR-26b-5p agomir was applied to mice with OA induced by anterior cruciate ligament transection (ACLT). The therapeutic effects of miR-26b-5p were evaluated via pain behavior experiments and histological observations. In vitro, miR-26b-5p repolarized M1 macrophages to an anti-inflammatory M2 type by targeting the TLR3 signaling pathway. miR-26b-5p could target COL10A1, further inhibiting chondrocyte hypertrophy induced by M1 macrophage-conditioned medium (M1-CM). In vivo, miR-26b-5p agomir ameliorated gait abnormalities and mechanical allodynia in OA mice. miR-26b-5p treatment attenuated synovitis and cartilage degeneration, thereby delaying OA progression. In conclusion, M2 macrophage-derived exosomal miR-26b-5p could protect articular cartilage and ameliorate gait abnormalities in OA mice by targeting TLR3 and COL10A1. miR-26b-5p further affected macrophage polarization and chondrocyte hypertrophy. Thus, this exosomal miR-26b-5p-based strategy might be a potential method for OA treatment.

Risk factor analysis of refracture in the same cemented vertebra after percutaneous kyphoplasty for Kümmell's disease.

OBJECTIVE: The objective of this study was to evaluate the factors that affect refracture in the same cemented vertebra after percutaneous kyphoplasty (PKP) for Kümmell's disease (KD) and establish a risk prediction score. METHODS: A total of 2932 patients who were treated with PKP for KD between January 2019 and December 2021 were retrospectively reviewed. After inclusion and exclusion criteria were applied, 191 patients were included in the study. According to the criteria for refracture, there were 50 patients in the refracture group and 141 patients in the no-refracture group. Twenty-five factors were analyzed. Patient demographics, medical history, imaging data, surgical data, and postoperative management were reviewed. Multivariate logistic regression modeling was used to identify the independent risk factors for refracture. Receiver operating characteristic (ROC) curve analysis was used to assess and establish a risk score system and further predict the risk of refracture. RESULTS: In this study, 50 (26.2%) patients developed a refracture. Through univariate analysis, bone mineral density (BMD) (p < 0.001), compression rate (p = 0.007), classification (i.e., the stages determined by the compression ratios) (p < 0.001), bone cement volume (p < 0.001), volume fraction (p < 0.001), distribution pattern (p = 0.007), non-PMMA endplate contact (p < 0.001), and anti-osteoporosis therapy (p < 0.001) were found to be significant factors for post-cement vertebral refracture after PKP in patients with KD. Three independent risk factors were found to be significant for refracture: small volume fraction, low BMD, and no anti-osteoporosis therapy. One point was assigned for each factor. The incidence rates of refracture in patients with scores of 0, 1, 2, and 3 were 3.7%, 4.4%, 42.0%, and 100%, respectively. The area under the ROC curve for this risk prediction score was 0.888 (p < 0.001), indicating moderate accuracy. CONCLUSIONS: Volume fraction, BMD, and osteoporosis therapy are the main factors influencing the refracture of the same cemented vertebra in KD. On the basis of these factors, the risk prediction score developed in this paper can be used to forecast the incidence of refracture.

The role of ferroptosis in intervertebral disc degeneration.

Nucleus pulposus, annulus fibrosus, and cartilage endplate constitute an avascular intervertebral disc (IVD), which is crucial for spinal and intervertebral joint mobility. As one of the most widespread health issues worldwide, intervertebral disc degeneration (IVDD) is recognized as a key contributor to back and neck discomfort. A number of degenerative disorders have a strong correlation with ferroptosis, a recently identified novel regulated cell death (RCD) characterized by an iron-dependent mechanism and a buildup of lipid reactive oxygen species (ROS). There is growing interest in the part ferroptosis plays in IVDD pathophysiology. Inhibiting ferroptosis has been shown to control IVDD development. Several studies have demonstrated that in TBHP-induced oxidative stress models, changes in ferroptosis marker protein levels and increased lipid peroxidation lead to the degeneration of intervertebral disc cells, which subsequently aggravates IVDD. Similarly, IVDD is significantly relieved with the use of ferroptosis inhibitors. The purpose of this review was threefold: 1) to discuss the occurrence of ferroptosis in IVDD; 2) to understand the mechanism of ferroptosis and its role in IVDD pathophysiology; and 3) to investigate the feasibility and prospect of ferroptosis in IVDD treatment.

Biomechanical study of two-level oblique lumbar interbody fusion with different types of lateral instrumentation: a finite element analysis.

Objective: The aim of this study was to verify the biomechanical properties of a newly designed angulated lateral plate (mini-LP) suited for two-level oblique lumbar interbody fusion (OLIF). The mini-LP is placed through the lateral ante-psoas surgical corridor, which reduces the operative time and complications associated with prolonged anesthesia and placement in the prone position. Methods: A three-dimensional nonlinear finite element (FE) model of an intact L1-L5 lumbar spine was constructed and validated. The intact model was modified to generate a two-level OLIF surgery model augmented with three types of lateral fixation (stand-alone, SA; lateral rod screw, LRS; miniature lateral plate, mini-LP); the operative segments were L2-L3 and L3-L4. By applying a 500 N follower load and 7.5 Nm directional moment (flexion-extension, lateral bending, and axial rotation), all models were used to simulate human spine movement. Then, we extracted the range of motion (ROM), peak contact force of the bony endplate (PCFBE), peak equivalent stress of the cage (PESC), peak equivalent stress of fixation (PESF), and stress contour plots. Results: When compared with the intact model, the SA model achieved the least reduction in ROM to surgical segments in all motions. The ROM of the mini-LP model was slightly smaller than that of the LRS model. There were no significant differences in surgical segments (L1-L2, L4-L5) between all surgical models and the intact model. The PCFBE and PESC of the LRS and the mini-LP fixation models were lower than those of the SA model. However, the differences in PCFBE or PESC between the LRS- and mini-LP-based models were not significant. The fixation stress of the LRS- and mini-LP-based models was significantly lower than the yield strength under all loading conditions. In addition, the variances in the PESF in the LRS- and mini-LP-based models were not obvious. Conclusion: Our biomechanical FE analysis indicated that LRS or mini-LP fixation can both provide adequate biomechanical stability for two-level OLIF through a single incision. The newly designed mini-LP model seemed to be superior in installation convenience, and equally good outcomes were achieved with both LRS and mini-LP for two-level OLIF.

Minimally invasive percutaneous new designed transpedicular lag-screw fixation for the management of Hangman fracture using O-arm-based navigation: a clinical study.

BACKGROUND: To investigate the outcomes and safety of using minimally invasive percutaneous new transpedicular lag-screw fixation with intraoperative, full rotation, three-dimensional image (O-arm)-based navigation for the management of Hangman fracture. METHODS: Twenty-two patients with Hangman fracture were treated with minimally invasive percutaneous new transpedicular lag-screws using intraoperative, full rotation, and three-dimensional image (O-arm)-based navigation. The preoperative and postoperative conditions of the patients were evaluated according to the ASIA (American Spinal Injury Association) scale. The patient's VAS (visual analog scale) scores before and after surgery, operation time, cervical vertebral activity, intervertebral angle and bone healing were recorded and collected, and repeated measures analysis of variance was used for statistical analysis. RESULTS: All patients were satisfactorily repositioned after surgery, and the VAS scores for neck pain were significantly lower than those before surgery on the first day and at 1 month, 3 months and the last follow-up (P < 0.001). According to the ASIA scale, four patients recovered from preoperative grade D to postoperative grade E. Bony fusion was achieved for all cases, and the range of neck rotation was restored to normal at the last follow-up. The post-surgery angular displacement (AD) demonstrated the stability of C2-3 after our new screw fixation for the treatment of Hangman fracture. CONCLUSIONS: Minimally invasive percutaneous new transpedicular lag-screw fixation using intraoperative, full rotation, three-dimensional image (O-arm)-based navigation achieved satisfactory clinical results with the advantages of immediate stability, safety and effectivity. We suggest that it is a reliable and advanced technique for the management of Hangman fracture.

Moderate mechanical stimulation antagonizes inflammation of annulus fibrosus cells through YAP-mediated suppression of NF-κB signaling.

Intervertebral disc degeneration (IDD) is a leading cause of low back pain. The inflammatory responses caused by aberrant mechanical loading are one of the major factors leading to annulus fibrosus (AF) degeneration and IDD. Previous studies have suggested that moderate cyclic tensile strain (CTS) can regulate anti-inflammatory activities of AF cells (AFCs), and Yes-associated protein (YAP) as a mechanosensitive coactivator senses diverse types of biomechanical stimuli and translates them into biochemical signals controlling cell behaviors. However, it remains poorly understood whether and how YAP mediates the effect of mechanical stimuli on AFCs. In this study, we aimed to investigate the exact effects of different CTS on AFCs as well as the role of YAP signaling involving in it. Our results found that 5% CTS inhibited the inflammatory response and promoted cell growth through inhibiting the phosphorylation of YAP and nuclear localization of NF-κB, while 12% CTS had a significant proinflammatory effect with the inactivation of YAP activity and the activation of NF-κB signaling in AFCs. Furthermore, moderate mechanical stimulation may alleviate the inflammatory reaction of intervertebral discs through YAP-mediated suppression of NF-κB signaling in vivo. Therefore, moderate mechanical stimulation may serve as a promising therapeutic approach for the prevention and treatment of IDD.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration.

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades. However, efficient harvest and handling of cell sheets remain challenging, including insufficient extracellular matrix content and poor mechanical strength. Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types. However, currently, there are no effective ways to apply mechanical loading to cell sheets. In this study, we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide) (PNIPAAm) to poly(dimethylsiloxane) (PDMS) surfaces. The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting. Subsequently, MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates. Upon maturation, the cell sheets were harvested by lowering the temperature. We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning. Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated. After implantation into the critical-sized calvarial defects of mice, the mechanically conditioned cell sheets significantly promoted new bone formation. Findings from this study reveal that thermo-responsive elastomer, together with mechanical conditioning, can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.

Bone Cement Reperfusion Revision Surgery for Symptomatic Recurrence of Kümmell's Disease After Percutaneous Kyphoplasty.

STUDY DESIGN: Retrospective study. OBJECTIVES: To demonstrate that repeat Percutaneous vertebroplasty (PVP) performed for the same cemented vertebrae in Kümmell's disease can offer therapeutic benefit for patients with recurrent symptoms after initial percutaneous kyphoplasty (PKP) treatment. METHODS: From January 2019 to December 2021, we investigated 2932 patients with PKP. Among them, 191 patients were diagnosed Kümmell's disease. 33 patients upon presentation of recurrent symptoms underwent repeat PVP procedure. Radiologic outcomes and clinic indices were investigated. RESULTS: Bone cement reperfusion surgery was successfully completed in 33 patients. The average age was 73.5 ± 8.2 years old. The kyphosis angle showed significant correction from pre-operation to the final follow-up, descending from pre-operation (20.6 ± 11.1°) to final follow-up (15.4 ± 7.9°). The vertebral heights at different follow-up appointments were significantly higher than the pre-operative appointments. The VAS and ODI scores at final follow-up were respectively 1.2 ± .8 and 27.3 ± 5.4%, which were both significantly lower than those before operation. No complications such as cement leakage into the spinal canal or cement displacement occurred during follow-up. CONCLUSIONS: Bone cement reperfusion surgery can ameliorate kyphosis and restore vertebral height to some extent. Repeat PVP is a minimally invasive surgery that provides superior long-term results in clinical and radiological outcomes but is technically more difficult to perform.

TGF-ß1-supplemented decellularized annulus fibrosus matrix hydrogels promote annulus fibrosus repair.

Annulus fibrosus (AF) repair remains a challenge because of its limited self-healing ability. Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair. Although the unique and beneficial characteristics of decellularized extracellular matrix (ECM) in tissue repair have been demonstrated, the poor mechanical property of ECM hydrogels largely hinders their applications in tissue regeneration. In the present study, we combined polyethylene glycol diacrylate (PEGDA) and decellularized annulus fibrosus matrix (DAFM) to develop an injectable, photocurable hydrogel for AF repair. We found that the addition of PEGDA markedly improved the mechanical strength of DAFM hydrogels while maintaining their porous structure. Transforming growth factor-ß1 (TGF-ß1) was further incorporated into PEGDA/DAFM hydrogels, and it could be continuously released from the hydrogel. The in vitro experiments showed that TGF-ß1 facilitated the migration of AF cells. Furthermore, PEGDA/DAFM/TGF-ß1 hydrogels supported the adhesion, proliferation, and increased ECM production of AF cells. In vivo repair performance of the hydrogels was assessed using a rat AF defect model. The results showed that the implantation of PEGDA/DAFM/TGF-ß1 hydrogels effectively sealed the AF defect, prevented nucleus pulposus atrophy, retained disc height, and partially restored the biomechanical properties of disc. In addition, the implanted hydrogel was infiltrated by cells resembling AF cells and well integrated with adjacent AF tissue. In summary, findings from this study indicate that TGF-ß1-supplemented DAFM hydrogels hold promise for AF repair.

The role of microenvironment in stem cell-based regeneration of intervertebral disc.

Regenerative medicine for intervertebral disc (IVD) disease, by utilizing chondrocytes, IVD cells, and stem cells, has progressed to clinical trials in the treatment of back pain, and has been studied in various animal models of disc degeneration in the past decade. Stem cells exist in their natural microenvironment, which provides vital dynamic physical and chemical signals for their survival, proliferation and function. Long-term survival, function and fate of mesenchymal stem cells (MSCs) depend on the microenvironment in which they are transplanted. However, the transplanted MSCs and the endogenous disc cells were influenced by the complicated microenvironment in the degenerating disc with the changes of biochemical and biophysical components. It is important to understand how the MSCs and endogenous disc cells survive and thrive in the harsh microenvironment of the degenerative disc. Furthermore, materials containing stem cells and their natural microenvironment have good clinical effects. However, the implantation of tissue engineering IVD (TE-IVD) cannot provide a complete and dynamic microenvironment for MSCs. IVD graft substitutes may need further improvement to provide the best engineered MSC microenvironment. Additionally, the IVD progenitor cells inside the stem cell niches have been regarded as popular graft cells for IVD regeneration. However, it is still unclear whether actual IVD progenitor cells exist in degenerative spinal conditions. Therefore, the purpose of this review is fourfold: to discuss the presence of endogenous stem cells; to review and summarize the effects of the microenvironment in biological characteristics of MSC, especially those from IVD; to explore the feasibility and prospects of IVD graft substitutes and to elaborate state of the art in the use of MSC transplantation for IVD degeneration in vivo as well as their clinical application.

Multifunctional Nanofibrous Scaffolds with Angle-Ply Microstructure and Co-Delivery Capacity Promote Partial Repair and Total Replacement of Intervertebral Disc.

There is an urgent clinical need for the treatment of annulus fibrosus (AF) impairment caused by intervertebral disc (IVD) degeneration or surgical injury. Although repairing injured AF through tissue engineering is promising, the approach is limited by the complicated angle-ply microstructure, inflammatory microenvironment, poor self-repairing ability of AF cells and deficient matrix production. In this study, electrospinning technology is used to construct aligned core-shell nanofibrous scaffolds loaded with transforming growth factor-ß3 (TGFß3) and ibuprofen (IBU), respectively. The results confirm that the rapid IBU release improves the inflammatory microenvironment, while sustained TGFß3 release enhances nascent extracellular matrix (ECM) formation. Biomaterials for clinical applications must repair local AF defects during herniectomy and enable AF regeneration during disc replacement, so a box defect model and total IVD replacement model in rat tail are constructed. The dual-drug delivering electrospun scaffolds are assembled into angle-ply structure to form a highly biomimetic AF that is implanted into the box defect or used to replace the disc. In two animal models, it is found that biomimetic scaffolds with good anti-inflammatory ability enhance ECM formation and maintain the mechanical properties of IVD. Findings from this study demonstrate that the multifunctional nanofibrous scaffolds provide inspirations for IVD repair.

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway.

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1ß stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin ß1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin ß1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.

Substrate Topography Regulates Differentiation of Annulus Fibrosus-Derived Stem Cells via CAV1-YAP-Mediated Mechanotransduction.

Regeneration of annulus fibrosus (AF) through tissue engineering techniques shows promise as a treatment for patients with degenerative disc disease (DDD). Yet, it remains challenging because of the intrinsic heterogeneity of AF tissue and shortage of in-depth knowledge of its structure-function correlation. In the current study, we fabricated fibrous poly(ether carbonate urethane)urea (PECUU) scaffolds with various fiber sizes to mimic the microstructural feature of native AF and aimed to regulate the differentiation of AF-derived stem cells (AFSCs) by controlling the topographical cues of the scaffold. We found that the morphology of AFSCs varied significantly on scaffolds with various fiber sizes. Meanwhile, the expression of the phenotypic marker genes of outer AF was up-regulated on scaffolds with large fibers. Meanwhile, enhanced expression of the phenotypic marker genes of inner AF was seen on scaffolds with small fibers. Such topography-dependent gene expression in AFSCs approximated the biochemical profile of AF tissue in various zones. Moreover, cell spreading and nucleus translocation of Yes-associated protein (YAP) were facilitated with increased fiber size. Formation and maturation of focal adhesions of AFSCs were also promoted. We also found that Caveolin-1 (CAV1) positively modulated the mechano-responses of YAP in response to substrate topography. In conclusion, depending on the activation of the CAV1-YAP mechanotransduction axis, tuning the fiber size of scaffolds can effectively induce changes in cell shape, adhesions, and extracellular matrix expression. This work may therefore provide new insights in the design of novel materials toward AF tissue regeneration.

Regulation of differentiation of annulus fibrosus-derived stem cells using heterogeneous electrospun fibrous scaffolds.

BACKGROUND: Tissue engineering of the annulus fibrosus (AF) shows promise as a treatment for patients with degenerative disc disease (DDD). However, it remains challenging due to the intrinsic heterogeneity of AF tissue. Fabrication of scaffolds recapitulating the specific cellular, componential, and microstructural features of AF, therefore, is critical to successful AF tissue regeneration. METHODS: Poly-L-lactic acid (PLLA) fibrous scaffolds with various fiber diameters and orientation were prepared to mimic the microstructural characteristics of AF tissue using electrospinning technique. AF-derived stem cells (AFSCs) were cultured on the PLLA fibrous scaffolds for 7 days. RESULTS: The morphology of AFSCs significantly varied when cultured on the scaffolds with various fiber diameters and orientation. AFSCs were nearly round on scaffolds with small fibers. However, they became spindle-shaped on scaffolds with large fibers. Meanwhile, upregulated expression of collagen-I gene happened in cells cultured on scaffolds with large fibers, while enhanced expression of collagen-II and aggrecan genes was seen on scaffolds with small fibers. The production of related proteins also showed similar trends. Further, culturing AFSCs on a heterogeneous scaffold by overlaying membranes with different fiber sizes led to the formation of a hierarchical structure approximating native AF tissue. CONCLUSION: Findings from this study demonstrate that fibrous scaffolds with different fiber sizes effectively promoted the differentiation of AFSCs into specific cells similar to the types of cells at various AF zones. It also provides a valuable reference for regulation of cell differentiation and fabrication of engineered tissues with complex hierarchical structures using the physical cues of scaffolds. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Effective AF repair is an essential need for treating degenerative disc disease. Tissue engineering is a promising approach to achieving tissue regeneration and restoring normal functions of tissues. By mimicking the key structural features of native AF tissue, including fiber size and alignment, this study deciphered the effect of scaffold materials on the cell differentiation and extracellular matrix deposition, which provides a solid basis for designing new strategies toward more effective AF repair and regeneration.

Tissue Engineering and Regenerative Medicine: Achievements, Future, and Sustainability in Asia.

Exploring innovative solutions to improve the healthcare of the aging and diseased population continues to be a global challenge. Among a number of strategies toward this goal, tissue engineering and regenerative medicine (TERM) has gradually evolved into a promising approach to meet future needs of patients. TERM has recently received increasing attention in Asia, as evidenced by the markedly increased number of researchers, publications, clinical trials, and translational products. This review aims to give a brief overview of TERM development in Asia over the last decade by highlighting some of the important advances in this field and featuring major achievements of representative research groups. The development of novel biomaterials and enabling technologies, identification of new cell sources, and applications of TERM in various tissues are briefly introduced. Finally, the achievement of TERM in Asia, including important publications, representative discoveries, clinical trials, and examples of commercial products will be introduced. Discussion on current limitations and future directions in this hot topic will also be provided.

Effects of Matrix Stiffness on the Differentiation of Multipotent Stem Cells.

Differentiation of stem cells, a crucial step in the process of tissue development, repair and regeneration, can be regulated by a variety of mechanical factors such as the stiffness of extracellular matrix. In this review article, the effects of stiffness on the differentiation of stem cells, including bone marrow-derived stem cells, adipose-derived stem cells and neural stem cells, are briefly summarized. Compared to two-dimensional (2D) surfaces, three-dimensional (3D) hydrogel systems better resemble the native environment in the body. Hence, the studies which explore the effects of stiffness on stem cell differentiation in 3D environments are specifically introduced. Integrin is a well-known transmembrane molecule, which plays an important role in the mechanotransduction process. In this review, several integrin-associated signaling molecules, including caveolin, piezo and Yes-associated protein (YAP), are also introduced. In addition, as stiffness-mediated cell differentiation may be affected by other factors, the combined effects of matrix stiffness and viscoelasticity, surface topography, chemical composition, and external mechanical stimuli on cell differentiation are also summarized.

Substrate stiffness- and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated protein.

Annulus fibrosus (AF) tissue engineering has attracted increasing attention as a promising therapy for degenerative disc disease (DDD). However, regeneration of AF still faces many challenges due to the tremendous complexity of this tissue and lack of in-depth understanding of the structure-function relationship at cellular level within AF is highly required. In light of the fact that AF is composed of various types of cells and has gradient mechanical, topographical and biochemical features along the radial direction. In this study, we aimed to achieve directed differentiation of AF-derived stem cells (AFSCs) by mimicking the mechanical and topographical features of native AF tissue. AFSCs were cultured on four types of electrospun poly(ether carbonate urethane)urea (PECUU) scaffolds with various stiffness and fiber size (soft, small size; stiff, small size; soft, large size and stiff, large size). The results show that with constant fiber size, the expression level of the outer AF (oAF) phenotypic marker genes in AFSCs increased with the scaffold stiffness, while that of inner AF (iAF) phenotypic marker genes showed an opposite trend. When scaffold stiffness was fixed, the expression of oAF phenotypic marker genes in AFSCs increased with fiber size. While the expression of iAF phenotypic marker genes decreased. Such substrate stiffness- and topography-dependent changes of AFSCs was in accordance with the genetic and biochemical distribution of AF tissue from the inner to outer regions. Further, we found that the Yes-associated protein (YAP) was translocated to the nucleus in AFSCs cultured with increasing stiffness and fiber size of scaffolds, yet it remained mostly phosphorylated and cytosolic in cells on soft scaffolds with small fiber size. Inhibition of YAP down-regulated the expression of tendon/ligament-related genes, whereas expression of the cartilage-related genes was upregulated. The results illustrate that matrix stiffness is a potent regulator of AFSC differentiation. Moreover, we reveal that fiber size of scaffolds induced changes in cell adhesions and determined cell shape, spreading area, and extracellular matrix expression. In all, both mechanical property and topography features of scaffolds regulate AFSC differentiation, possibly through a YAP-dependent mechanotransduction mechanism. STATEMENT OF SIGNIFICANCE: Physical cues such as mechanical properties, topographical and geometrical features were shown to profoundly impact the growth and differentiation of cultured stem cells. Previously, we have found that the differentiation of annulus fibrosus-derived stem cells (AFSCs) could be regulated by the stiffness of scaffold. In this study, we fabricated four types of poly(ether carbonate urethane)urea (PECUU) scaffolds with controlled stiffness and fiber size to explore the potential of induced differentiation of AFSCs. We found that AFSCs are able to present different gene expression patterns simply as a result of the stiffness and fiber size of scaffold material. This work has, for the first time, demonstrated that larger-sized and higher-stiffness substrates increase the amount of vinculin assembly and activate YAP signaling in pre-differentiated AFSCs. The present study affords an in-depth comprehension of materiobiology, and be helpful for explain the mechanism of YAP mechanosensing in AF in response to biophysical effects of materials.

Biomechanics in Annulus Fibrosus Degeneration and Regeneration.

Degenerative disc degeneration (DDD) is the major cause of low back pain, which seriously affects the life of patients. Current surgical and conservative treatments only relieve the pain temporarily, yet fail to restore the normal biomechanics and functions of healthy spine. Indeed, high recurrence of disc herniation commonly happens after discectomy. Degenerative changes in biomechanical and structural properties of the intervertebral disc (IVD), including fissures in annulus fibrosus (AF) and volume loss of nucleus pulposus (NP), mainly contribute to DDD development. AF plays a critical role in the biomechanical properties of IVD as it structural integrity is essential to confine NP and maintain physiological intradiscal pressure under loading. Maintaining the homeostasis of AF and NP, and thereby IVD, requires regulation of their biomechanics, which is also involved in the onset and subsequent development of AF degeneration. Therefore, it is essential to understand the biomechanical changes of AF during degeneration, which can also provide valuable insights into the repair and regeneration of AF. In this review, we focus on the biomechanical properties of AF tissue associated with its homeostasis and degeneration, and discuss the biomechanical stimulus required for regeneration of AF. We also provide an overview of recent strategies to target and modulate cell mechanics toward AF regeneration.

Strategies for Annulus Fibrosus Regeneration: From Biological Therapies to Tissue Engineering.

Intervertebral disc (IVD) is an avascular tissue which contributes to the weight bearing, motion, and flexibility of spine. However, IVD is susceptible to damage and even failure due to injury, pathology, and aging. Annulus fibrosus (AF), the structural and functional integrity of which is critically essential to confine nucleus pulpous (NP) and maintain physiological intradiscal pressure under mechanical loading, plays a critical role in the biomechanical properties of IVD. AF degeneration commonly results in substantial deterioration of IVD. During this process, the biomechanical properties of AF and the balance between anabolism and catabolism in IVD are progressively disrupted, leading to chronic back pain, and even disability of individuals. Therefore, repairing and regenerating AF are effective treatments to degeneration-associated pains. However, they remain highly challenging due to the complexity of natural AF tissue in the aspects of cell phenotype, biochemical composition, microstructure, and mechanical properties. Tissue engineering (TE), by combining biological science and materials engineering, shed lights on AF regeneration. In this article, we review recent advances in the pro-anabolic approaches in the form of cell delivery, bioactive factors delivery, gene therapy, and TE strategies for achieving AF regeneration.

Interleukin-1ß induces apoptosis in annulus fibrosus cells through the extracellular signal-regulated kinase pathway.

PURPOSE: The loss of intervertebral disc (IVD) cells due to excessive apoptosis induced by inflammatory cytokines is a major cause of IVD degeneration. This study aims to explore the mechanism of interleukin-1ß (IL-1ß)-induced apoptosis of annulus fibrosus cells (AFCs). It's hypothesized that IL-1ß induces apoptosis through the extracellular signal-regulated kinase (ERK) pathway in AFCs. METHODS: The mRNA and protein expression levels of apoptosis-associated genes were analyzed by quantitative real-time PCR and Western blotting. The apoptotic rate was measured by flow cytometry. Three experimental groups were established, including Control, IL-1ß, and IL-1ß+U0126 groups, respectively. RESULTS: Increase in the expression of apoptosis-associated genes including B-cell lymphoma-2 associated X (Bax), caspase-3, and caspase-9, and meanwhile, decrease in the expression of B-cell lymphoma-2 (Bcl-2) gene were found in patients with degenerative IVDs. In in vitro tests, both apoptosis and phosphorylated ERK expression in rat AFCs decreased in the IL-1ß+U0126 group compared with the IL-1ß group. The expression levels of Bax, caspase-3, and caspase-9 in AFCs decreased significantly in the IL-1ß+U0126 group compared with those in the IL-1ß group. The expression level of Bcl-2, on the other hand, significantly increased. CONCLUSIONS: Findings from this study suggest that IL-1ß induces apoptosis in AFCs through the ERK pathway, and therefore, ERK inhibition may provide certain protection against the adverse effects of IL-1ß.