Annulus Fibrosus Tissue Engineering Research Articles

From Mechanobiology to Mechanical Repair Strategies: A Bibliometric Analysis of Biomechanical Studies of Intervertebral Discs.

Neck pain and low back pain are major challenges in public health, and intervertebral disc (IVD) biomechanics is an important multidisciplinary field. To date, no bibliometric literature review of the relevant literature has been performed, so we explored the emerging trends, landmark studies, and major contributors to IVD biomechanics research. We searched the Web of Science core collection (1900–2022) using keywords mainly composed of “biomechanics” and “intervertebral disc” to conduct a bibliometric analysis of original papers and their references, focusing on citations, authors, journals, and countries/regions. A co-citation analysis and clustering of the references were also completed. A total of 3189 records met the inclusion criteria. In the co-citation network, cluster #0, labeled as “annulus fibrosus tissue engineering”, and cluster #1, labeled as “micromechanical environment”, were the biggest clusters. References by MacLean et al and Holzapfel et al were positioned exactly between them and had high betweenness centrality. There existed a research topic evolution between mechanobiology and mechanical repair strategies of IVDs, and the latter had been identified as an emerging trend in IVD biomechanics. Numerous landmark studies had contributed to several fields, including mechanical testing of normal and pathological IVDs, mechanical evaluation of new repair strategies and development of finite element model. Adams MA was the author most cited by IVD biomechanics papers. Spine, the European Spine Journal, and the Journal of Biomechanics were the three journals where the most original articles and their references have been published. The United States has contributed most to the literature (n = 1277 papers); however, the research output of China is increasing. In conclusion, the present study suggests that IVD repair is an emerging trend in IVD biomechanics.

Fabrication and characterization of an acellular annulus fibrosus scaffold with aligned porous construct for tissue engineering.

Scaffolds mimicking the native annulus fibrosus (AF) extracellular matrix (ECM) structure are crucial to guide the seeding cells to regenerate aligned tissue, while fabricating such a scaffold by synthetic material is challengeable. Native acellular scaffolds derived from AF tissue certainly possess the advantages of natural structure and composition. Based on previous studies, we modified decellularization procedure and especially compared two drying methods, including gradient dehydration and freeze-drying. The decellularization process can effectively remove the host cells and antigens such as α-Gal, while maintaining the original ECM including GAG and collagen I. Compared with gradient dehydration, freeze-drying not only rendered the decellularized scaffold in dry state for storage but also gave the scaffold more aligned porous structure and hydrophilicity. And, the acellular porous scaffold manifested better capacity of supporting cell ingrowth when seeded human bone marrow mesenchymal stem cells (hBMSCs) or implanted in vivo. Furthermore, this optimized freeze-dried scaffold showed similar mechanical elastic modulus as native AF and demonstrated rare inflammatory granuloma and immune rejection as observed in HE staining and immunohistochemistry staining (IHC) of CD8 and MAC387 epitopes when implanted subcutaneously in vivo. To sum up, through our decellularization and freeze-drying procedure, an aligned porous three-dimensional scaffold derived from the natural AF ECM was successfully fabricated with good retention of ECM components and benign biocompatibility. It will be a promising scaffold for AF tissue engineering.

The Functional Role of Interface Tissue Engineering in Annulus Fibrosus Repair: Bridging Mechanisms of Hydrogel Integration with Regenerative Outcomes.

Hydrogels are extraordinarily versatile by design and can enhance repair in diseased and injured musculoskeletal tissues. Biological fixation of these constructs is a significant determinant factor that is critical to the clinical success and functionality of regenerative technologies for musculoskeletal repair. In the context of an intervertebral disc (IVD) herniation, nucleus pulposus tissue protrudes through the ruptured annulus fibrosus (AF), consequentially impinging on spinal nerve roots and causing debilitating pain. Discectomy is the surgical standard of care to treat symptomatic herniation; however these procedures do not repair AF defects, and these lesions are a significant risk factor for recurrent herniation. Advances in tissue engineering utilize adhesive hydrogels as AF sealants; however these repair strategies have yet to progress beyond preclinical animal models because these biomaterials are often plagued by poor integration with AF tissue and lead to large variability in repair outcomes. These critical barriers to translation motivate this article to review the material composition of hydrogels that have been evaluated in situ for AF repair, proposed mechanisms of how these biomaterials interface with AF tissue, and their functional outcomes after treatment in order to inform the development of new hydrogels for AF repair. In this systematic review, we identify 18 hydrogel formulations evaluated for AF repair, all of which demonstrate large heterogeneity in their interfacing mechanisms and reported outcome measures to assess the effectiveness of repair. Hydrogels that covalently bond to AF tissue were found to be the most successful in improving IVD biomechanical properties from the injured state, but none were able to restore properties to the intact state suggesting that new repair strategies with innovative surface chemistries are an important future direction. We additionally review biomechanical evaluation methods and recommend standardization in the field of AF tissue engineering to establish mechanical benchmarks for translation and ensure clinical feasibility.

Regeneration of annulus fibrosus tissue using a DAFM/PECUU-blended electrospun scaffold

Low back pain and disc degeneration affect quality of life and imposes an enormous financial burden. Although annulus fibrosus (AF) tissue engineering provides an alternative therapeutic possibility in the treatment of degenerative intervertebral disc disease, it is restricted by the biochemical properties, organizational structure, and mechanical characteristics of the scaffold. The ideal scaffold should closely mimic the natural extracellular matrix (ECM) in structure and function for long-term stability and survival. Poly(ether carbonate urethane) urea (PECUU) can be electrospun into nanofibrous scaffolds to mimic ECM architecture with the appropriate mechanical properties. However, PECUU scaffolds lack the bioactivity of natural ECM. On the other hand, a decellularized annulus fibrosus matrix (DAFM) has good biocompatibility and biodegradability and has been shown to promote secretion of AF-related ECM. Herein, DAFM/PECUU-blended electrospun scaffolds were fabricated with the help of coaxial electrospinning technology for the first time. AF-derived stem cells were cultured on DAFM/PECUU electrospun scaffolds, and cellular metabolic activity, morphology, and gene expression assays as well as AF-related ECM synthesis were performed. The results showed that AF-derived stem cells proliferated well on the scaffolds. Gene expression and ECM secretion of collagen type I and II and aggrecan from AF-derived stem cells cultured on DAFM/PECUU electrospun scaffolds were higher than from those on PECUU fibrous scaffolds. Thus, DAFM/PECUU electrospun scaffolds are a potential candidate for AF tissue engineering applications.

Biomimetic angle-ply multi-lamellar scaffold for annulus fibrosus tissue engineering.

Constructing a biomimetic scaffold that replicates the complex architecture of intervertebral disc annulus fibrosus (AF) remains a major goal in AF tissue engineering. In this study, a biomimetic angle-ply multi-lamellar polycaprolactone/silk fibroin (PCL/SF) AF scaffold was fabricated. Wet-spinning was used to obtain aligned PCL/SF microfiber sheets, and these were excised into strips with microfibers aligned at +30° or -30° relative to the strip long axis. This was followed by stacking two strips with opposing fiber alignment and wrapping them concentrically around a mandrel. Our results demonstrated that the scaffold possessed spatial structure and mechanical properties comparable to natural AF. The scaffold supported rabbit AF cells adhesion, proliferation, infiltration and guided oriented growth and extracellular matrix deposition. In conclusion, our angle-ply multi-lamellar scaffold offers a potential solution for AF replacement therapy and warrants further attention in future investigations.

Development of annulus fibrosus tissue construct with hydrogel coils containing pre-conditioned mesenchymal stem cell

Low back pain associated with degenerative disc diseases has been a major health concern that brings suffering to the patients physically and economically. Many existing therapeutic strategies provide short-term relief of symptoms rather than treatment of the underlying cause. Development of an engineered tissue for disc regeneration is still in its infancy due to the limited autologous healthy disc cell source from the patients. It is also challenging to mimic the complexity of micro-architecture in the native disc tissue that determine their unique structural properties. To date, simple tissue models that mimic the annulus fibrosus (AF) micro-environment for understanding the potential of mesenchymal stem cells (MSCs) in AF tissue engineering are still lacking. In this study, the assembly of a coiled hydrogel microfiber has shown its capability to encapsulate MSCs and create an engineered tissue model that mimics the multiple lamellae of native AF. Using this model, we investigated the potential of MSCs that were previously induced by ascorbic acid (AA). Compared to non-induced MSCs, AA-induced MSCs exhibited significant increase in AF-associated biomarkers during later development in the engineered AF tissue model and also encouraged collagen accumulation through the down-regulated catabolic gene MMP1 and upregulated anti-catabolic gene TIMP1. Furthermore, AA-induced MSCs exhibited a Col2/Col1 ratio closer to that of a native AF tissue. These results suggested that AA-induced MSCs could be a potential cell source for AF tissue engineering and this established tissue model may provide a simple tool for successful AF tissue engineering strategies in the future.

Scaffold-Free tissue engineering with aligned bone marrow stromal cell sheets to recapitulate the microstructural and biochemical composition of annulus fibrosus

Current tissue engineering strategies through scaffold-based approaches fail to recapitulate the complex three-dimensional microarchitecture and biochemical composition of the native Annulus Fibrosus tissue. Considering limited access to healthy annulus fibrosus cells from patients, this study explored the potential of bone marrow stromal cells (BMSC) to fabricate a scaffold-free multilamellar annulus fibrosus-like tissue by integrating micropatterning technologies into multi-layered BMSC engineering. BMSC sheet with cells and collagen fibres aligned at ~30° with respect to their longitudinal dimension were developed on a microgroove-patterned PDMS substrate. Two sheets were then stacked together in alternating directions to form an angle-ply bilayer tissue, which was rolled up, sliced to form a multi-lamellar angle-ply tissue and cultured in a customized medium. The development of the annulus fibrosus-like tissue was further characterized by histological, gene expression and microscopic and mechanical analysis. We demonstrated that the engineered annulus fibrosus-like tissue with aligned BMSC sheet showed parallel collagen fibrils, biochemical composition and microstructures that resemble the native disk. Furthermore, aligned cell sheet showed enhanced expression of annulus fibrosus associated extracellular matrix markers and higher mechanical strength than that of the non-aligned cell sheet. The present study provides a new strategy in annulus fibrosus tissue engineering methodology to develop a scaffold-free annulus fibrosus-like tissue that resembles the microarchitecture and biochemical attributes of a native tissue. This can potentially lead to a promising avenue for advancing BMSC-mediated annulus fibrosus regeneration towards future clinical applications.

Tissue Engineering the Annulus Fibrosus Using 3D Rings of Electrospun PCL:PLLA Angle-Ply Nanofiber Sheets

Treatments to alleviate chronic lower back pain, caused by intervertebral disc herniation as a consequence of degenerate annulus fibrosus (AF) tissue, fail to provide long-term relief and do not restore tissue structure or function. The future of AF tissue engineering relies on the production of its complex structure assisted by the many cells that are resident in the tissue. As such, this study aims to mimic the architecture and mechanical environment of outer AF tissue using electrospun fiber scaffolds made from a synthetic biopolymer blend of poly(ε-caprolactone) (PCL) and poly(L-lactic) acid (PLLA). Initially, an aligned bilayer PCL:PLLA scaffold was manually assembled at ±30° fibers direction to resemble the native AF lamellar layers; and bovine AF cells were used to investigate the effect of construct architecture on cell alignment and orientation. Bilayer scaffolds supported cell adhesion and influenced their orientation. Furthermore, significant improvements in tensile stiffness and strength were achieved, which were within the reported range for human AF tissue. Electrospun bilayer scaffolds are, however, essentially two-dimensional and fabrication of a complete three-dimensional (3D) circular construct to better replicate the AF's anatomical structure is yet to be achieved. For the first time, a custom-built Cell Sheet Rolling System (CSRS) was utilized to create a 3D circular lamellae construct that mimics the complex AF tissue and which overcomes this translational limitation. The CSRS equipment is a quick, automated process that allows the creation of multilayered, tube-like structures (with or without cells), which is ideal for mimicking human cervical AF tissue in term of tissue architecture and geometry. Tube-like structures (6 layers) were successfully created by rolling ±30° bilayer PCL:PLLA scaffolds seeded with bovine AF cells and subsequently cultured for 3 weeks. Cells remained viable, purposefully oriented with evidence of collagen type I deposition, which is the main structural component of AF tissue. This is the first study focused on applying CSRS technology for the fabrication of a more clinically-relevant, 3D tissue engineered scaffold for AF tissue regeneration.

Decellularized Annulus Fibrosus Matrix/Chitosan Hybrid Hydrogels with Basic Fibroblast Growth Factor for Annulus Fibrosus Tissue Engineering.

Low back pain caused by degenerative disc disease affects many people worldwide and brings huge economical burden. Thus, attentions have focused on annulus fibrosus (AF) tissue engineering for treatment of intervertebral disc degeneration. To engineer a functional replacement for the AF, it is important to fabricate scaffolds that mimic the structural and mechanical properties of native tissue. AF-derived stem cells are promising seed cells for AF tissue engineering due to their tissue specificity. In the present study, decellularized AF matrix (DAFM)/chitosan hybrid hydrogels were fabricated using genipin as a crosslinker. AF stem cells were cultured on hydrogel scaffolds with or without basic fibroblast growth factor (bFGF), and cell proliferation, morphology, gene expression, and AF tissue synthesis were examined. Overall, more collagen-I, collagen-II, and aggrecan were secreted by AF stem cells grown on hydrogels with bFGF compared to those without. These results support the application of DAFM/chitosan hybrid hydrogels as an appropriate candidate for AF tissue engineering. Furthermore, incorporation of bFGF into hydrogels promoted AF-related tissue synthesis.Impact StatementThe investigation of annulus fibrosus (AF)-related tissue secretion and gene expression in extracellular matrix (ECM) of AF-derived stem cells (AFSCs) provided theoretical and practical basis for the choice of scaffold materials and growth factors for AF tissue engineering. The innovations of the present work are obvious. First, AFSCs were used because they are more easily differentiated into AF cells, thereby producing more AF-related ECM. Second, the decellularized AF matrix (DAFM) was derived from native AF tissue, but had reduced immunogenicity after decellularization. Furthermore, the DAFM structure mimicked the fibrous network of actual AF tissue, which was advantageous to AFSC adhesion and growth. Third, basic fibroblast growth factor was successfully incorporated into the DAFM, showed gradual sustained release, and effectively promoted production of AF tissue ECM factors collagen-I, collagen-II, aggrecan, and glycosaminoglycan.

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 SignificancePhysical 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.

In vitro evaluation of 3D printed polycaprolactone scaffolds with angle-ply architecture for annulus fibrosus tissue engineering.

Tissue engineering of the annulus fibrosus (AF) is currently being investigated as a treatment for intervertebral disc degeneration, a condition frequently associated with low back pain. The objective of this work was to use 3D printing to generate a novel scaffold for AF repair that mimics the structural and biomechanical properties of the native tissue. Multi-layer scaffolds were fabricated by depositing polycaprolactone struts in opposing angular orientations, replicating the angle-ply arrangement of the native AF tissue. Scaffolds were printed with varied strut diameter and spacing. The constructs were characterized morphologically and by static and dynamic mechanical analyses. Scaffold surfaces were etched with unidirectional grooves and the influence on bovine AF cell metabolic activity, alignment, morphology and protein expression was studied in vitro. Overall, the axial compressive and circumferential tensile properties of the scaffolds were found to be in a similar range to the native AF tissue. Confocal microscopy images indicated that cells were able to attach and spread on the smooth polycaprolactone scaffolds, but the surface texture induced cellular alignment and proliferation. Furthermore, immunofluorescence analysis demonstrated the aligned deposition of collagen type I, aggrecan and the AF-specific protein marker tenomodulin on the etched scaffolds. Overall, results demonstrated the potential for using the scaffolds as a template for AF regeneration.

Mimicking the Annulus Fibrosus Using Electrospun Polyester Blended Scaffolds.

Treatments to alleviate chronic lower back pain, caused by intervertebral disc herniation as a consequence of degenerate annulus fibrosus (AF) tissue, fail to provide long-term relief and do not restore tissue structure or function. This study aims to mimic the architecture and mechanical environment of AF tissue using electrospun fiber scaffolds made from synthetic biopolymers-poly(ε-caprolactone) (PCL) and poly(L-lactic) acid (PLLA). Pure polymer and their blends (PCL%:PLLA%; 80:20, 50:50, and 20:80) are studied and material properties-fiber diameter, alignment, % crystallinity, tensile strength, and water contact angle-characterized. Tensile properties of fibers angled at 0°, 30°, and 60° (single layer scaffolds), and ±0°, ±30°, and ±60° (bilayer scaffolds) yield significant differences, with PCL being significantly stiffer with the addition of PLLA, and bilayer scaffolds considerably stronger. Findings suggest PCL:PLLA 50:50 fibers are similar to human AF properties. Furthermore, in vitro culture of AF cells on 50:50 fibers demonstrates attachment and proliferation over seven days. The optimal polymer composition for production of scaffolds that closely mimic AF tissue both structurally, mechanically, and which also support and guide favorable cell phenotype is identified. This study takes a step closer towards successful AF tissue engineering and a long-term treatment for sufferers of chronic back pain.

Influence of dynamic mechanical stimulation on tissue engineering annulus fibrosus

Objective To investigate the influence of dynamic mechanical stimulation on the annulus fibrosus (AF) cells seeded on silk scaffolds. Methods AF cells were isolated from rabbits and were seeded on the scaffold, then cultured for 3, 7, 14 days with different range of dynamic compression. Stereomicroscope and scanning electron microscope (SEM) was used to observe the surface morphology of tissue engineering annulus fibrosus cells (TE-AFs). After fixation, samples were harvested for histological staining. AF cells related extracellular matrix (ECM) was evaluated by the quantitative analysis of total DNA, proteoglycan and collagen I. The mechanical properties were compared within different groups. Results Stereomicroscope and SEM results showed that the colors of TE-AFs in all groups were deepening with time going. SEM showed cell adhesion on the scaffold and the secretion of extracellular matrix. Histological, immunohistochemical staining, biochemical quantitative analysis and total DNA content showed that the AF cells inside scaffolds could support AF cell attachment, proliferation and secretion. As a result, the compressive properties were enhanced with increasing culture time. Stereomicroscope showed that the colors of TE-AFs in all groups were deepening with time going after dynamic compression. HE staining, Safranin O staining and Type I collagen staining showed that cell proliferation and secretion, GAG secretion and collagen secretion were increased with time going within different groups. Quantitation of GAG achieved maximum in 15% strain group, and quantitation of collagen achieved maximum in 10% strain group. The total DNA content achieved maximum in 5% strain group, and compression elastic modulus achieved maximum in 15% strain group. The height of TE-AFs did not change after mechanical stimulation for 14 days. Conclusion Suitable mechanical stimulation is a positive factor for new AF tissue engineering that will tend to the nature tissue. Excessive compression can accelerate the progress of cell apoptosis. Key words: Tissue engineering; Silk; Tissue scaffolds; Biomechanics

A novel electrospun-aligned nanoyarn/three-dimensional porous nanofibrous hybrid scaffold for annulus fibrosus tissue engineering.

IntroductionHerniation of the nucleus pulposus (NP) because of defects in the annulus fibrosus (AF) is a well-known cause of low back pain. Defects in the AF thus remain a surgical challenge, and efforts have been made to develop new techniques for closure and repair. In this study, we developed an electrospun aligned nanoyarn scaffold (AYS) and nanoyarn/three-dimensional porous nanofibrous hybrid scaffold (HS) for AF tissue engineering.MethodsThe AYS was fabricated via conjugated electrospinning, while the aligned nanofibrous scaffold (AFS) was prepared by traditional electrospinning as a baseline scaffold. The HS was constructed by freeze-drying and cross-linking methods. Scanning electron microscopy and mechanical measurement were used to characterize the properties of these scaffolds. Bone marrow derived mesenchymal stem cells (BMSCs) were seeded on scaffolds, and cell proliferation was determined by CCK-8 assay, while cell infiltration and differentiation were assessed by histological measurement and quantitative real-time polymerase chain reaction, respectively.ResultsMorphological measurements showed that AYS presented a relatively better three-dimensional structure with larger pore sizes, higher porosity, and better fibers’ alignment compared to AFS. Mechanical testing demonstrated that the tensile property of AFS and AYS was qualitatively similar to the native AF tissue, albeit to a lesser extent. When BMSCs were seeded and cultured on these scaffolds, the number of cells cultured on HS and AYS was found to be significantly higher than that on AFS and culture plate after 7 days of culture (P<0.05). In addition, cell infiltration was significantly higher in HS when compared with AFS and AYS (P<0.05). A part of BMSCs ingressed into the inner part of AYS upon long-term in vitro culture. No significant difference was observed between AFS and AYS in terms of the median infiltration depth (P>0.05). BMSCs seeded on AYS demonstrated an increased expression of COL1A1, while the expression levels of SOX-9, COL2A1, and Aggrecan were higher in HS compared to other scaffolds (P<0.05).ConclusionThese findings indicate that HS makes a proper scaffold for the AF tissue engineering as it replicates the axial compression and tensile property of AF, thereby providing a better platform for cell infiltration and cell–scaffold interaction.

Tissue Engineering of the Intervertebral Disc's Annulus Fibrosus: A Scaffold-Based Review Study.

Tissue engineering as a high technology solution for treating disc's problem has been the focus of some researches recently; however, the upcoming successful results in this area depends on understanding the complexities of biology and engineering interface. Whereas the major responsibility of the nucleus pulposus is to provide a sustainable hydrated environment within the disc, the function of the annulus fibrosus (AF) is more mechanical, facilitating joint mobility and preventing radial bulging by confining of the central part, which makes the AF reconstruction important. Although the body of knowledge regarding the AF tissue engineering has grown rapidly, the opportunities to improve current understanding of how artificial scaffolds are able to mimic the AF concentric structure-including inter-lamellar matrix and cross-bridges-addressed unresolved research questions. The aim of this literature review was to collect and discuss, from the international scientific literature, information about tissue engineering of the AF based on scaffold fabrication and material properties, useful for developing new strategies in disc tissue engineering. The key parameter of this research was understanding if role of cross-bridges and inter-lamellar matrix has been considered on tissue engineering of the AF.

The fabrication and characterization of a multi-laminate, angle-ply collagen patch for annulus fibrosus repair.

One major limitation of intervertebral disc (IVD) repair is that no ideal biomaterial has been developed that effectively mimics the angle-ply collagen architecture and mechanical properties of the native annulus fibrosus (AF). Furthermore, it would be beneficial to devise a simple, scalable process by which to manufacture a biomimetic biomaterial that could function as a mechanical repair patch to be secured over a large defect in the outer AF that will support AF tissue regeneration. Such a biomaterial would: (1) enable the employment of early-stage interventional strategies to treat IVD degeneration (i.e. nucleus pulposus arthroplasty); (2) prevent IVD re-herniation in patients with large AF defects; and (3) serve as a platform to develop full-thickness AF and whole IVD tissue engineering strategies. Due to the innate collagen fibre alignment and mechanical strength of pericardium, a procedure was developed to assemble multi-laminate angle-ply AF patches derived from decellularized pericardial tissue. Patches were subsequently assessed histologically to confirm angle-ply microarchitecture, and mechanically assessed for biaxial burst strength and tensile properties. Additionally, patch cytocompatibility was evaluated following seeding with bovine AF cells. This study demonstrated the effective removal of porcine cell remnants from the pericardium, and the ability to reliably produce multi-laminate patches with angle-ply architecture using a simple assembly technique. Resultant patches demonstrated their inherent ability to resist biaxial burst pressures reminiscent of intradiscal pressures commonly borne by the AF, and exhibited tensile strength and modulus values reported for native human AF. Furthermore, the biomaterial supported AF cell viability, infiltration and proliferation. Copyright © 2016 John Wiley & Sons, Ltd.

Progress and challenges in tissue engineering of intervertebral disc annulus fibrosus

Degenerative disc disease (DDD) is a leading cause of low back pain, which severely affects the quality of life and incurs significant medical cost. Annulus fibrosus(AF) injuries can lead to substantial deterioration of intervertebral disc degeneration. However, the AF repair/regeneration remains a challenge due to the intrinsic cellular, biochemical and biomechanical heterogeneity of AF tissue. Tissue engineering would be a promising approach for AF regeneration. This article aims to provide a brief overview of the fundamental aspects of AF, the current achievements and future challenges of AF tissue engineering. A multidisciplinary approach is proposed for future studies to fully mimic the native AF tissue and its microenvironment, including choosing adequate cell source, preparing scaffolds with hierarchical microstructures, supplementing appropriate growth factors, and enforcing suitable mechanical stimulation. Hopefully, the engineered AF tissues would be effectively used to facilitate the treatment of DDD in the future.

Matrix elasticity-dependent differentiation of annulus fibrous-derived stem cells

Matrix elasticity-dependent differentiation of annulus fibrous-derived stem cells

Modulation of the gene expression of annulus fibrosus-derived stem cells using poly(ether carbonate urethane)urea scaffolds of tunable elasticity

Annulus fibrosus (AF) injuries commonly lead to substantial deterioration of the intervertebral disc (IVD). While tissue engineering has recently evolved into a promising approach for AF regeneration, it remains challenging due to the cellular, biochemical, and mechanical heterogeneity of AF tissue. In this study, we explored the use of AF-derived stem cells (AFSCs) to achieve diversified differentiation of cells for AF tissue engineering. Since the differentiation of stem cells relies significantly on the elasticity of the substrate, we synthesized a series of biodegradable poly(ether carbonate urethane)urea (PECUU) materials whose elasticity approximated that of native AF tissue. When AFSCs were cultured on electrospun PECUU fibrous scaffolds, the gene expression of collagen-I in the cells increased with the elasticity of scaffold material, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. At the protein level, the content of collagen-I gradually increased with substrate elasticity, while collagen-II and GAG contents decreased. In addition, the cell traction forces (CTFs) of AFSCs gradually decreased with scaffold elasticity. Such substrate elasticity-dependent changes of AFSCs were similar to the gradual transition in the genetic, biochemical, and biomechanical characteristics of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that AFSCs, depending on the substrate elasticity, have strong tendencies to differentiate into various types of AF-like cells, thereby providing a solid foundation for the tissue engineering applications of AFSCs. Statement of significanceRepairing the annulus fibrosus (AF) of intervertebral disc (IVD) is critical for the treatment of disc degeneration disease, but remains challenging due to the significant heterogeneity of AF tissue. Previously, we have identified rabbit AF-derived stem cells (AFSCs), which are AF tissue-specific and hold promise for AF regeneration. In this study, we synthesized a series of poly(ether carbonate urethane)ureas of various elasticity (or stiffness) and explored the potential of induced differentiation of AFSCs using electrospun PECUU scaffolds. This work has, for the first time, found that AFSCs are able to present different gene expression patterns simply as a result of the elasticity of scaffold material. Therefore, our findings will help supplement current knowledge of AF tissue regeneration and may benefit a diversified readership from scientific, engineering, and clinical settings whose work involves the biology and tissue engineering of IVD.

The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells.

Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.