Annulus Fibrosus Defects Research Articles

Backbone breakthroughs: How rodent models are shaping intervertebral disc disease treatment.

Intervertebral disc degeneration (IVDD) is a widespread, disabling condition that significantly contributes to the global burden of musculoskeletal disorders. To better understand its underlying mechanisms and explore potential therapeutic strategies, animal models serve as valuable tools for simulating the complicated pathophysiology of IVDD. Rodent models are extensively used due to their genetic similarities to humans, cost-effectiveness, and rapid attainment of maturity. These models enable the study of specific molecular pathways involved in IVDD, such as inflammation, matrix degradation, tissue repair, and disc microenvironment homeostasis. This review provides a comprehensive overview of the current status of rodent models used in IVDD research, highlighting their advantages, limitations, and contributions to our understanding of the disease. Specifically, we discussed various rodent models, including traumatic (such as needle puncture in the lumbar and coccygeal region, nucleotomy, and annulus fibrosus defect), non-traumatic (including compression models, lumbar spine instability, and bipedalism), chemically induced models (chymopapain, chondroitinase ABC), and genetically modified models. These models offer insights into the severity of IVDD under different conditions, such as trauma, aging, and genetics. In conclusion, rodent models remain indispensable tools for advancing our understanding of IVDD mechanisms and therapeutic interventions. Carefully selecting animal species and models can provide valuable insights that guide future clinical research and treatment approaches. Our review aims to leverage these models to identify therapeutic targets and strategies that may ultimately reduce the impact of IVDD on human health. PERSPECTIVE: This review describes the role of rodent models in IVDD, highlighting their utility in unraveling disease mechanisms and evaluating therapeutics. By replicating the complex molecular pathways and conditions of disc disease, like trauma, aging, and genetics, these models aid in identifying future advancements in managing lower back pain.

Design and Ex Vivo Evaluation of a PCLA Degradable Device To Improve Annulus Fibrosus Repair.

With a prevalence of over 90% in people over 50, intervertebral disc degeneration (IVDD) is a major health concern. This weakening of the intervertebral discs can lead to herniation, where the nucleus pulpus (NP) leaks through the surrounding Annulus Fibrosus (AF). Considering the limited self-healing capacity of AF tissue, an implant is needed to restore its architecture and function. Here, we developed a biomimetic electrospun nanofibrous biodegradable scaffold that could be potentially used to repair AF defects. To that aim, we synthesized copolymers and blends of ε-caprolactone and lactide to create poly(ε-caprolactone-co-lactide) (PCLA) and PCL/PLA scaffolds with 10, 20, or 30% PLA. Properties of the initial nanofibrous scaffolds and the impact of gamma irradiation sterilization on the mechanical, thermal, and in vitro degradation properties are assessed and discussed with respect to the AF application. It was shown that ovine AF cells colonize the nanofibrous layers with increased metabolic activity over time. As an outcome of these studies, two copolymers were chosen to design a device composed of a 3D nanofibrous stacked scaffold associated with a degradable anchoring system to maintain the scaffold in an AF defect. The implantability of this device was tested in a cadaveric sheep lumbar IVD.

Polyphenol-Mediated Adhesive and Anti-Inflammatory Double-Network Hydrogels for Repairing Postoperative Intervertebral Disc Defects.

Hydrogels have garnered tremendous attention for their applications in the repair of intervertebral disk (IVD) degeneration and postoperative IVD defects. However, it is still challenging to develop a hydrogel fulfilling the requirements for high mechanical properties, adhesive capability, biocompatibility, antibacterial properties, and anti-inflammatory performance. Herein, we report a multifunctional double-network (DN) hydrogel composed of physically cross-linked carboxymethyl chitosan (CMCS) and tannic acid (TA) networks as well as chemically cross-linked acrylamide (AM) networks, which integrates the properties of high strength, adhesion, biocompatibility, antimicrobial activity, and anti-inflammation for the repair of postoperative IVD defects. The treatment with CMCS/TA/PAM DN hydrogels can significantly decrease the levels of inflammatory cytokines and degeneration-related factors and upregulated collagen type II alpha 1. In addition, the hydrogels can effectively seal the annulus fibrosus defect, prevent nucleus pulposus degeneration, retain IVD height, and restore the biomechanical properties of the disc to some extent. This polyphenol-mediated DN hydrogel is promising for sealing IVD defects and preventing herniation after lumbar discectomy.

Repair of annulus fibrosus defects using decellularized annulus fibrosus matrix/chitosan hybrid hydrogels

Degenerative disc disease is the leading cause of lower back and leg pain, considerably impacting daily life and incurring substantial medical expenses for those affected. The development of annulus fibrosus tissue engineering offers hope for treating this condition. However, the current annulus fibrosus tissue engineering scaffolds fail to accurately mimic the natural biological environment of the annulus fibrosus, resulting in limited secretion of extracellular matrix produced by the seeded cells and poor biomechanical properties of the constructed biomimetic annulus fibrosus tissue. This inability to match the biomechanical performance of the natural annulus fibrosus hinders the successful treatment of annulus fibrosus defects. In this study, we fabricated decellularized annulus fibrosus matrix (DAFM)/chitosan hydrogel-1 (DAFM: Chitosan 6:2) and DAFM/chitosan hydrogel-2 (DAFM: Chitosan 4:4) by varying the ratio of DAFM to chitosan. Rat annulus fibrosus (AF)-derived stem cells were cultured on these hydrogel scaffolds, and the cell morphology, AF-related gene expression, and Interleukin-6 (IL-6) levels were investigated. Additionally, magnetic resonance imaging, Hematoxylin and eosin staining, and Safranine and Fast Green staining were performed to evaluate the repair effect of the DAFM/chitosan hydrogels in vivo. The gene expression results showed that the expression of Collagen type I (Col-I), Collagen type I (Col-II), and aggrecan by annulus fibrosus stem cells (AFSCs) cultured on the DAFM/chitosan-1 hydrogel was higher compared with the DAFM/chitosan-2 hydrogel. Conversely, the expression of metalloproteinase-9 (MMP-9) and IL-6 was lower on the DAFM/chitosan-1 hydrogel compared with the DAFM/chitosan-2 hydrogel. In vivo, both the DAFM/chitosan-1 and DAFM/chitosan-2 hydrogels could partially repair large defects of the annulus fibrosus in rat tail vertebrae. In conclusion, the DAFM/chitosan-1 hydrogel could be regarded as a candidate scaffold material for the repair of annulus fibrosus defects, offering the potential for improved treatment outcomes.

Innovative hydrogel-patch combination for large annulus fibrosus defects: a prospective approach to address herniation recurrence

BACKGROUND CONTEXTLarge annulus fibrosus (AF) defects often lead to a high rate of reherniation, particularly in the medial AF region, which has limited self-healing capabilities. The increasing prevalence of herniated discs underscores the need for effective repair strategies. PURPOSEThe objectives of this study were to design an AF repair technique to reduce solve the current problems of insufficient mechanical properties and poor sealing capacity. STUDY DESIGNIn vitro biomechanical experiments and finite element analysis. METHODSThe materials used in this study were patches and hydrogels with good biocompatibility and sufficient mechanical properties to withstand loading in the lumbar spine. Five repair techniques were assessed in this study: hydrogel filler (HF), AF patch medial barrier (MB), AF patch medial barrier and hydrogel filler (MB&HF), AF patch medial-lateral barrier (MLB), and AF patch medial-lateral barrier and hydrogel filler (MLB&HF). The repair techniques were subjected to in vitro testing (400 N axial compression and 0–500 N fatigue loading at 5Hz) and finite element analysis (400 N axial compression) to evaluate the effectiveness at repairing large AF defects. The evaluation included repair tightness, spinal stability, and fatigue resistance. RESULTSFrom the in vitro testing, the failure load of the repair techniques was in the following order HF <MB <MB&HF <MLB <MLB&HF. Both HF and MB groups failed to effectively increase intervertebral disc (IVD) stiffness, resulting in a reduction in spatial stability. The MLB, MB&HF, and MLB&HF groups partially restored IVD stiffness, with MLB&HF showing the most effective recovery (−24.13% ± 3.59%). From the finite element models, incorporating a hydrogel filler was best able to maintain the IVD height. Patch repair alone could not adequately reduce the high AF stress due to AF injury, but with hydrogel support, stress was substantially low and more uniformly distributed. All repair techniques demonstrated reduced stress around the damaged area on the AF, in comparison to the unrepaired model. The NP pressure in the HF group was closest to the intact group, and the patch repair reduced the NP pressure. The maximum patch deformation and suture stress were ranked as MB >MLB >MB&HF >MLB&HF. CONCLUSIONSThe combined use of patches and hydrogels exhibited promising mechanical properties postdiscectomy, providing a promising solution for addressing large AF defects and improving disc stability. CLINICAL SIGNIFICANCEThis study introduces a promising method for repairing large annular fissure (AF) defects after disc herniation, combining patch repair with a hydrogel filler. These techniques hold potential for developing clinical AF repair products to address this challenging issue.

Ex-vivo biomechanical evaluation of the application of a novel annulus closure device to closure of annulus fibrosus.

To investigate the technical feasibility of applying a simple suture guide device to close the annulus fibrosus (AF) of the intervertebral discs (IVD). 30 sheep functional discal units (FDUs) were obtained and subjected to mock discectomy. Mock sutures were performed using 3-0 non-absorbable sutures under a novel AF suture device following a suture procedure. The FDUs were compressed under axial loading at 1.8mm/min and evaluated for Failure load (N). The failure loads of the hand stitching group (Group H) and suture device stitching group (Group S) were significantly higher than those of the control group (Group C) (p = 0.033; p < 0.001). This study provides reasonable reasons to believe that the simple suture guide device described here is technically feasible for AF defect closure. It thus constitutes an encouraging proof of concept for the proposed device; however, it does not constitute a complete demonstration of the device's feasibility in the clinical setting considering that the annulus closure operation is performed ex vivo on functional spinal units, as opposed to within an environment that mimics the clinical setting. To this end, confirmatory experiments will be conducted such as more multiaxial or dynamic mechanical testing, and notably performing the surgery on sheep models instead of on ex vivo functional spinal units.

Extracellular Vesicles Functional “Brick‐Cement” Bio‐Integrated System for Annulus Fibrosus Repair

AbstractDue to the deficiency of mechanical supporting after discectomy and weak proliferative capacity of annulus fibrosus (AF) cells, the AF defect repair remains a clinical challenge. Herein, a myofibroblasts derived extracellular vesicles (M‐EVs) functional “brick‐cement” bio‐integrated system (M‐EVs@PGBgel) is developed to repair AF defect. The modified Poly(glycerol‐sebacate) (PGBS), “bio‐brick” layer, exhibited excellent support features on account of its elastomeric mechanical properties. The loaded M‐EVs in the “bio‐cement” layer activated ITGA6/PI3K/AKT pathway, regulated M2 macrophage polarization, thus synergistically promoting AF cell proliferation and migration. The “bio‐cement” layer integrated PGBS and remnant tissue at the defect through the Schiff base reaction and aided M‐EVs’ sustained release. This study demonstrated that M‐EVs@PGBgel significantly improved the disc's biological and mechanical properties in the AF defect microenvironments and promoted AF regeneration in vivo. The M‐EVs@PGBgel shows promise as an effective strategy to simultaneously address the mechanical imbalance and biological disruptions resulting from AF defect.

Biodegradable devices for annulus fibrosus defect closure after lumbar discectomy

To study the properties of materials and select the most suitable one for appropriate device covering annulus fibrosus defect after lumbar discectomy. Prototype devices made of poly(L-)lactide, polycaprolactone (PLC) and copolymers of(L-)lactide and ε-caprolactone (LCL) without prior purification were used. Sponge materials were prepared using 1.4-dioxane without pretreatment. Physical and mechanical tests in compression were performed on Instron 5982 and Instron 5965 machines at a temperature of 37 °C in thermal chamber. Strain ranged from 0 to 80%, displacement rate - 50% of specimen height per a minute. Specimens were cylindrical in shape with a length of 10-15 mm and diameter of 8 mm. All experiments were performed in at least three repetitions. The ram lumbar spine was used as laboratory model. Prototypes #2 made of PLC and LCL copolymers were the most perspective, but require partial or complete intraoperative removal of nucleus pulposus. For all devices, we estimated their physical and mechanical characteristics, namely modulus and compressive strength, and compared these values with mechanics of native annulus fibrosus. A method for implant delivery to annulus fibrosus defect site was proposed for the developed devices, and bench tests of devices were carried out on the lumbar spine of the ram.

Extracellular vesicles loaded dual-network bioactive sealant via immunoregulation and annulus fibrosus repair for intervertebral disc herniation

Intervertebral disc herniation (IVDH) is a common manifestation of intervertebral disc degeneration (IVDD) characterized by inflammation that results in the rupture of the annulus fibrosus (AF) and herniation of the nucleus pulposus (NP). While current clinical research primarily focuses on regulating the degenerative NP, the crucial role of the AF in maintaining the mechanical stability and metabolic balance of the intervertebral disc (IVD) has been overlooked. Resolving immunoregulation and AF repair is imperative to effectively prevent recurrent herniation. Therefore, this study introduces a bioactive sealant (OD/GM/QCS-sEVs), which combines gelatin methacryloyl (GM) and oxidized dextran (OD) with quaternized chitosan (QCS) and incorporates small extracellular vesicles (sEVs). The developed sealant possesses injectability, self-healing capabilities, tissue adhesiveness, and mechanical stability, with an average adhesive strength of 109.63 kPa. In vitro experiments demonstrate that OD/GM/QCS-sEVs effectively seal AF defects while preserving mechanical properties comparable to those of a normal IVD. Additionally, the sealant releases sEVs through a pH-responsive mechanism, thereby modulating macrophage polarization to the M2 phenotype via the NF-κB signaling pathway. This mechanism facilitates immunoregulation and anti-inflammatory effects, and promotes stem cell differentiation into fibrocartilage. Animal experiments confirm the ability of OD/GM/QCS-sEVs to seal defects, prevent proteoglycan loss, inhibit IVDD development, and promote AF regeneration. Overall, OD/GM/QCS-sEVs hold promise as an innovative bioactive sealant for recurrent herniation by resolving immunoregulation and AF regeneration.

A novel rat model of annulus fibrosus injury for intervertebral disc degeneration

BACKGROUND CONTEXTIn clinical practice, acute trauma and chronic degeneration of the annulus fibrosus (AF) can promote further degeneration of the intervertebral disc (IVD). Therefore, it is critical to understand the AF repair process and its consequences on IVD. However, the lack of cost-effective and reproducible in vivo animal models of AF injury has limited research development in this field. PURPOSESThe purpose of this study was to establish and evaluate the utility of a novel animal model for full-thickness AF injury. Three foci were proposed: (1) whether this new modeling method can cause full-layer AF damage; (2) the repair processes and pathological changes in the damaged area after AF injury, and (3) the morphological and histological changes in the IVD are after AF injury. STUDY DESIGN/SETTINGIn vivo rat AF injury model with characterization of AF damage repair, IVD degeneration. METHODSA total of 72,300 g male rats were randomly assigned to one of the two groups: experimental or sham. Annulus fibrosus was separated layer by layer under the microscope with a #11 blade up to the AF- nucleus pulpous (NP) junction. The repair process of the horizontal AF and morphological changes in the sagittal IVD were evaluated with HE staining. Sirius red staining under polarized light. Immunofluorescence was conducted to analyze changes in the expression of COL1 and COL3 in the AF injury area and 8-OHdg, IL-6, MMP13, FSP1, and ACAN in the IVD. The disc height and structural changes after AF injury were measured using X-ray and contrast-enhanced micro-CT. Additionally, the resistance of the AF to stretching was analyzed using three-point bending. RESULTSAnnulus fibrosus-nucleus pulpous border was identified to stably induce the full-thickness AF injury without causing immediate NP injury. The AF repair process after injury was slow and expressed inflammation factors continuously, with abundant amounts of type III collagen appearing in the inner part of the AF. The scar at the AF lesion had decreased resistance to small molecule penetration and weakened tensile strength. Full-thickness AF injury induced disc degeneration with loss of disc height, progressive unilateral vertebral collapse, and ossification of the subchondral bone. Inflammatory-induced degeneration and extracellular matrix catabolism gradually appeared in the NP and cartilage endplate (CEP). CONCLUSIONSWe established a low-cost and reproducible small animal model of AF injury which accurately replicated the pathological state of the limited AF self-repair ability and demonstrated that injury to the AF alone could cause further degeneration of the IVD. CLINICAL RELEVANCEThis in vivo rat model can be used to study the repair process of the AF defect and pathological changes in the gradual degeneration of IVD after AF damage. In addition, the model provides an experimental platform for in vivo experimental research of potential clinical therapeutics.

Melatonin-loaded self-healing hydrogel targets mitochondrial energy metabolism and promotes annulus fibrosus regeneration

Intervertebral disc (IVD) herniation is a major cause of chronic low back pain and disability. The current nucleus pulposus (NP) discectomy effectively relieves pain symptoms, but the annulus fibrosus (AF) defects are left unrepaired. Tissue engineering approaches show promise in treating AF injury and IVD degeneration; however, the presence of an inflammatory milieu at the injury site hinders the mitochondrial energy metabolism of AF cells, resulting in a lack of AF regeneration. In this study, we fabricated a dynamic self-healing hydrogel loaded with melatonin (an endocrine hormone well-known for its antioxidant and anti-inflammatory properties) and investigate whether melatonin-loaded hydrogel could promote AF defect repair by rescuing the matrix synthesis and energy metabolism of AF cells. The protective effects of melatonin on matrix components (e.g. type I and II collagen and aggrecan) in AF cells were observed in the presence of interleukin (IL)-1β. Additionally, melatonin was found to activate the nuclear factor erythroid 2-related factor signaling pathway, thereby safeguarding the mitochondrial function of AF cells from IL-1β, as evidenced by the increased level of adenosine triphosphate, mitochondrial membrane potential, and respiratory chain factor expression. The incorporation of melatonin into a self-healing hydrogel based on thiolated gelatin and β-cyclodextrin was proposed as a means of promoting AF regeneration. The successful implantation of melatonin-loaded hydrogel has been shown to facilitate in situ regeneration of AF tissue, thereby impeding IVD degeneration by preserving the hydration of nucleus pulposus in a rat box-cut IVD defect model. These findings offer compelling evidence that the development of a melatonin-loaded dynamic self-healing hydrogel can promote the mitochondrial functions of AF cells and represents a promising strategy for IVD regeneration.

Sustained release of basic fibroblast growth factor in micro/nanofibrous scaffolds promotes annulus fibrosus regeneration.

Tissue engineering has promising applications in the treatment of intervertebral disc degeneration (IDD). The annulus fibrosus (AF) is critical for maintaining the physiological function of the intervertebral disc (IVD), but the lack of vessels and nutrition in AF makes it difficult to repair. In this study, we used hyaluronan (HA) micro-sol electrospinning and collagen type I (Col-I) self-assembly techniques to fabricate layered biomimetic micro/nanofibrous scaffolds, which released basic fibroblast growth factor (bFGF) to promote AF repair and regeneration after discectomy and endoscopic transforaminal discectomy. The bFGF enveloped in the core of the poly-L-lactic-acid (PLLA) core-shell structure was released in a sustained manner and promoted the adhesion and proliferation of AF cells (AFCs). Col-I could self-assemble on the shell of the PLLA core-shell scaffold to mimic the extracellular matrix (ECM) microenvironment, providing structural and biochemical cues for the regeneration of AF tissue. The in vivo studies showed that the micro/nanofibrous scaffolds promoted the repair of AF defects by simulating the microstructure of native AF tissue and inducing endogenous regeneration mechanism. Taken together, the biomimetic micro/nanofibrous scaffolds have clinical potential for the treatment of AF defects caused by IDD. STATEMENT OF SIGNIFICANCE: The annulus fibrosus (AF) is essential for the intervertebral disc(IVD) physiological function, yet it lacks vascularity and nutrition, making repair difficult. Micro-sol electrospinning technology and collagen type I (Col-I) self-assemblytechniquewere combined in this study to create a layered biomimetic micro/nanofibrous scaffold that releases basic fibroblast growth factor (bFGF) to promote AF repair and regeneration. Col-I could mimic the extracellular matrix (ECM) microenvironment,in vivo, offering structural and biochemical cues for AF tissue regeneration. This research indicates that micro/nanofibrous scaffolds have clinical potential for treating AF deficits induced by IDD.

Targeting Endogenous Reactive Oxygen Species Removal and Regulating Regenerative Microenvironment at Annulus Fibrosus Defects Promote Tissue Repair

The excessive reactive oxygen species (ROS) level, inflammation, and weak tissue regeneration ability after annulus fibrosus (AF) injury constitute an unfavorable microenvironment for AF repair. AF integrity is crucial for preventing disc herniation after discectomy; however, there is no effective way to repair the AF. Herein, a composite hydrogel integrating properties of antioxidant, anti-inflammation, and recruitment of AF cells is developed through adding mesoporous silica nanoparticles modified by ceria and transforming growth factor β3 (TGF-β3) to the hydrogels. The nanoparticle loaded gelatin methacrylate/hyaluronic acid methacrylate composite hydrogels eliminate ROS and induce anti-inflammatory M2 type macrophage polarization. The released TGF-β3 not only plays a role in recruiting AF cells but is also responsible for promoting extracellular matrix secretion. The composite hydrogels can be solidified in situ in the defect area to effectively repair AF in rats. The strategies targeting endogenous ROS removal and improving the regenerative microenvironment by the nanoparticle-loaded composite hydrogels have potential applications in AF repair and intervertebral disc herniation prevention.

Core-shell oxygen-releasing fibers for annulus fibrosus repair in the intervertebral disc of rats.

The repair of annulus fibrosus (AF) defect after discectomy in the intervertebral disc (IVD) has presented a challenge over the past decade. Hostile microenvironments in the IVD, including, compression and hypoxia, are critical issues that require special attention. Till date, little information is available on potential strategies to cope with the hypoxia dilemma in AF defect sites. In this study, perfluorotributylamine (PFTBA) core-shell fibers were fabricated by coaxial electrospinning to construct oxygen-releasing scaffold for promoting endogenous repair in the AF after discectomy. We demonstrated that PFTBA fibers (10% chitosan, chitosan: PCL, 1:6) could release oxygen for up to 144​h. The oxygen released from PFTBA fibers was found to protect annulus fibrosus stem cells (AFSCs) from hypoxia-induced apoptosis. In addition, the PFTBA fibers were able to promote proliferation, migration and extracellular matrix (ECM) production in AFSCs under hypoxia, highlighting their therapeutic potential in AF defect repair. Subsequent in vivo studies demonstrated that oxygen-supplying fibers were capable of ameliorating disc degeneration after discectomy, which was evidenced by improved disc height and morphological integrity in rats with the oxygen-releasing scaffolds. Further transcriptome analysis indicated that differential expression genes (DEGs) were enriched in "oxygen transport" and "angiogenesis", which likely contributed to their beneficial effect on endogenous AF regeneration. In summary, the oxygen-releasing scaffold provides novel insights into the oxygen regulation by bioactive materials and raises the therapeutic possibility of oxygen supply strategies for defect repair in AF, as well as other aerobic tissues.

Tissue-mimetic hybrid bioadhesives for intervertebral disc repair.

Intervertebral disc (IVD) degeneration and herniation often necessitate surgical interventions including a discectomy with or without a nucleotomy, which results in a loss of the normal nucleus pulposus (NP) and a defect in the annulus fibrosus (AF). Due to the limited regenerative capacity of the IVD tissue, the annular tear may remain a persistent defect and result in recurrent herniation post-surgery. Bioadhesives are promising alternatives but show limited adhesion performance, low regenerative capacity, and inability to prevent re-herniation. Here, we report hybrid bioadhesives that combine an injectable glue and a tough sealant to simultaneously repair and regenerate IVD post-nucleotomy. The glue fills the NP cavity while the sealant seals the AF defect. Strong adhesion occurs with the IVD tissues and survives extreme disc loading. Furthermore, the glue can match native NP mechanically, and support the viability and matrix deposition of encapsulated cells, serving as a suitable cell delivery vehicle to promote NP regeneration. Besides, biomechanical tests with bovine IVD motion segments demonstrate the capacity of the hybrid bioadhesives to restore the biomechanics of bovine discs under cyclic loading and to prevent permanent herniation under extreme loading. This work highlights the synergy of bioadhesive and tissue-engineering approaches. Future works are expected to further improve the tissue specificity of bioadhesives and prove their efficacy for tissue repair and regeneration.

Injectable Cell-Laden Nanofibrous Matrix for Treating Annulus Fibrosus Defects in Porcine Model: An Organ Culture Study.

Lower back pain commonly arises from intervertebral disc (IVD) failure, often caused by deteriorating annulus fibrosus (AF) and/or nucleus pulposus (NP) tissue. High socioeconomic cost, quality of life issues, and unsatisfactory surgical options motivate the rapid development of non-invasive, regenerative repair strategies for lower back pain. This study aims to evaluate the AF regenerative capacity of injectable matrix repair strategy in ex vivo porcine organ culturing using collagen type-I and polycaprolactone nanofibers (PNCOL) with encapsulated fibroblast cells. Upon 14 days organ culturing, the porcine IVDs were assessed using gross optical imaging, magnetic resonance imaging (MRI), histological analysis, and Reverse Transcriptase quantitative PCR (RT-qPCR) to determine the regenerative capabilities of the PNCOL matrix at the AF injury. PNCOL-treated AF defects demonstrated a full recovery with increased gene expressions of AF extracellular matrix markers, including Collagen-I, Aggrecan, Scleraxis, and Tenascin, along with anti-inflammatory markers such as CD206 and IL10. The PNCOL treatment effectively regenerates the AF tissue at the injury site contributing to decreased herniation risk and improved surgical outcomes, thus providing effective non-invasive strategies for treating IVD injuries.

Engineered multifunctional Silk fibroin cryogel loaded with exosomes to promote the regeneration of annulus fibrosus

Annulus fibrosus (AF) defect is a common clinical problem caused by lumbar disc herniation. Many AF mechanical closure devices have been developed to prevent recurrent herniation after discectomy. However, the high-inflammatory and high-ROS environment caused by AF defects make it difficult for mechanical devices to effectively reverse the degenerative process and promote regeneration of AF defects. Based on this, in this study, a multifunctional silk fibroin (SF) cryogel scaffold loaded with annulus fibrosus cell-derived exosomes (AEs) was constructed to inhibit intervertebral disc degeneration and promote regeneration of defects by regulating the immune microenvironment and scavenging ROS. SF cryogel has a uniform macroporous structure and good shape memory function, which could effectively seal AF and achieve the sustained release of AEs. In vitro cell experiments showed that AEs/SF cryogel possessed anti-inflammatory, antioxidant stress, cell recruitment, and regulating fibrocartilage differentiation abilities. In vivo animal experiments also further confirmed that AEs/SF cryogel could effectively seal AF defect of the rat caudal vertebra after acupuncture and regulate the IVD immune microenvironment by promoting the polarization of macrophages from M1 to M2 phenotype and reduce oxidative stress, which contributed to reversing the degeneration process and promote the regeneration of AF defects, indicating a potential application for AF defect repair.

Multilayer Electrospun-Aligned Fibroin/Gelatin Implant for Annulus Fibrosus Repair: An In Vitro and In Vivo Evaluation.

Annulus fibrosus (AF) damage is proven to prompt intervertebral disc (IVD) degeneration, and unrepaired AF lesions after surgical discectomy may boost herniation of the nucleus pulposus (NP) which may lead to further compression of neural structures. Moreover, vascular and neural ingrowth may occur within the defect which is known as a possible reason for discogenic pain. Due to a limited healing capacity, an effective strategy to repair and close the AF defect is necessary. In this study, using electrospinning technology, two nature polymers, silk fibroin and gelatin, were linked to imitate the unique lamellae structure of native AF. Our findings revealed that a multilayer electrospun-aligned fibroin/gelatin scaffold with mechanical and morphological properties mimicking those of native AF lamellae have been developed. The average diameter of the nanofiber is 162.9 ± 38.8 nm. The young’s modulus is around 6.70 MPa with an ultimate tensile strength of around 1.81 MP along preferred orientation. The in vitro test confirmed its biocompatibility and ability to maintain cell viability and colonization. Using a porcine model, we demonstrated that the multilayer-aligned scaffold offered a crucial microenvironment to induce collagen fibrous tissue production within native AF defect. In the implant-repaired AF, H&E staining showed homogeneous fibroblast-like cell infiltration at the repaired defect with very little vascular ingrowth, which was confirmed by magnetic resonance imaging findings. Picrosirius red staining and immunohistochemical staining against type I collagen revealed positively stained fibrous tissue in an aligned pattern within the implant-integrated site. Relative to the intact control group, the disc height index of the serial X-ray decreased significantly in both the injury control and implant group at 4 weeks and 8 weeks (p < 0.05) which indicated this scaffold may not reverse the degenerative process. However, the results of the discography showed that the effectiveness of annulus repair of the implant group is much superior to that of the untreated group. The scaffold, composed with nature fibroin/gelatin polymers, could potentially enhance AF healing that could prevent IVD recurrent herniation, as well as neural and neovascular ingrowth after discectomy surgeries.

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-β3 (TGFβ3) and ibuprofen (IBU), respectively. The results confirm that the rapid IBU release improves the inflammatory microenvironment, while sustained TGFβ3 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.

A Biodegradable Polymeric Matrix for the Repair of Annulus Fibrosus Defects in Intervertebral Discs.

Tissue defects in the annulus fibrosus (AF) due to intervertebral disc (IVD) degeneration or after nucleodiscectomy have little self-healing capacity. To prevent progressive degeneration of the IVD, the AF must be repaired. Biological closure has not yet been achieved and is a challenge for the research community. In this study, a scaffold made of absorbable poly (glycolic acid) (PGA) and hyaluronan (HA) that exhibit excellent biocompatibility and cell colonization properties was used to repair AF defects in an ovine model. A partial resection was performed in AF in L3/4 or L4/5 of 10 sheep and PGA-HA scaffolds were implanted on the defects (n = 5), while defects in the control group were left untreated (n = 5). Three months post-operation, the lumbar discs were sectioned and stained with hematoxylin and eosin and safranin-O/fast-green. Histological features including proteoglycan content, annular structure, cellular morphology, blood vessel ingrowth and tear/cleft formation were scored using a modified scoring scheme by 3 investigators and evaluated by a pathologist independently. The treated AF exhibited significantly enhanced repair tissue structure with signs of proteoglycan formation compared to the untreated group. The median scores were 4.3 for the treated and 9.8 for the untreated group. Cystic degeneration, perivascular infiltration, inflammation and necrosis were only present in the untreated group. Blood vessel ingrowth and tear/cleft formation were increased, though not significant, in the untreated group while cell morphology was comparable in both groups. PGA-HA scaffolds used for AF closure support repair tissue formation in an ovine lumbar disc defect model.