Annulus Fibrosus-derived Stem Cells Research Articles

The most influential articles on stem cells in intervertebral disc degeneration

BackgroundStem cell-related studies have been increasingly conducted to facilitate the regeneration of degenerative discs. However, analyses of high-impact articles focused on this topic are rare. This study aimed to determine and summarize the most-cited studies examining stem cells in the context of intervertebral disc degeneration (IDD).MethodsWe searched the Web of Science (WoS) database for stem cell-related articles in IDD, and the 50 highest-cited papers were summarized. A correlation analysis was conducted to determine the relationship among WoS citations, Altmetric Attention Score (AAS), and Dimensions.ResultsThe number of citations of the top 50 manuscripts ranged from 92 to 370. The top three countries were the United States (14), China (10), and Japan (9). Spine (12) was the most prevalent journal, and this was followed by Biomaterials (6). Bone marrow-derived stem cells were the most common subject (38), and they were followed by nucleus pulposus-derived stem cells (4) and annulus fibrosus-derived stem cells (4). Humans were the most studied species (31), and the next most studied were rabbits (9) and rats (7). There was a very high correlation between WoS and Dimension citations (p < 0.001, r = 0.937).ConclusionsFor the first time, the highest impact articles examining stem cells in the context of IDD were assessed together. The current study provides a deepened understanding of historical studies focused on stem cells in IDD and is beneficial for future studies in this field.

Leukoreduced PRP enhanced proliferation and ECM production yet inhibited senescence, inflammation, and multi-differentiation potential of AFSCs by downregulating HMGB1

BackgroundThis study assessed the role and potential mechanism of platelet-rich plasma (PRP) in the progression of intervertebral disk degeneration (IVDD).MethodsAnnulus fibrosus (AF)-derived stem cells (AFSCs) from New Zealand white rabbits received the transfection with high mobility group box 1 (HMGB1) plasmids and the subsequent treatment with bleomycin, 10% leukoreduced PRP or leukoconcentrated PRP. Dying cells were indicated by immunocytochemistry analysis for senescence-associated β-galactosidase (SA-β-gal) staining. The proliferation of these cells was evaluated based on the population doubling time (PDT). The expressions of HMGB1, pro-aging and anti-aging molecules, extracellular matrix (ECM)-related catabolic/anabolic factors, and inflammatory genes at the molecular or transcriptional levels were quantified via Western blot or reverse transcription-quantitative PCR (RT-qPCR). Besides, the adipocytes, osteocytes, and chondrocytes were separately dyed by Oil Red O, Alizarin Red S, and Safranin O staining.ResultsBleomycin enhanced the senescent morphological changes and increased the PDT and the expressions of SA-β-gal, pro-aging molecules, ECM-related catabolic factors, inflammatory genes, and HMGB1 while suppressing the expressions of anti-aging and anabolic molecules. Leukoreduced PRP reversed the effects of bleomycin and inhibited the differentiation of AFSCs into adipocytes, osteocytes, and chondrocytes. Besides, HMGB1 overexpression offset the roles of leukoreduced PRP in AFSCs.ConclusionLeukoreduced PRP promotes cell proliferation and ECM production of AFSCs, while inhibiting their senescence, inflammation, and multi-differentiation potentials via downregulating HMGB1 expression.

Graded-Three-Dimensional Cell-Encapsulating Hydrogel as a Potential Biologic Scaffold for Disc Tissue Engineering.

Intervertebral disk (IVD) degeneration, which can cause lower back pain, is a major predisposing factor for disability and can be managed through multiple approaches. However, there is no satisfactory strategy currently available to reconstruct and recover the natural properties of IVDs after degeneration. As tissue engineering develops, scaffolds with embedded cell cultures have proved critical for the successful regeneration of IVDs. In this study, an integrated scaffold for IVD replacement was developed. Through scanning electron microscopy and other mechanical measurements, we characterized the physical properties of different hydrogels. In addition, we simulated the physiological structure of natural IVDs. Nucleus pulposus (NP) cells and annulus fibrosus-derived stem cells (AFSCs) were seeded in gelatin methacrylate (GelMA) hydrogel at different concentrations to evaluate cell viability and matrix expression. It was found that different concentrations of GelMA hydrogel can provide a suitable environment for cell survival. However, hydrogels with different mechanical properties influence cell adhesion and extracellular matrix component type I collagen, type II collagen, and aggrecan expression. This tissue-engineered IVD implant had a similar structure and function as the native IVD, with the inner area mimicking the NP tissue and the outer area mimicking the stratified annulus fibrosus tissue. The new integrated scaffold demonstrated a good simulation of disc structure. The preparation of efficient and regeneration-promoting tissue-engineered scaffolds is an important issue that needs to be explored in the future. It is hoped that this work will provide new ideas and methods for the further construction of functional tissue replacement discs.

The experimental study of regeneration of annulus fibrosus using decellularized annulus fibrosus matrix/poly(ether carbonate urethane)urea-blended fibrous scaffolds with varying elastic moduli.

Although tissue engineering has attracted increasing attention for the treatment of degenerative intervertebral disc disease, the biochemical properties, structural organization, and mechanical characteristics of annulus fibrosus tissue have restricted progress. Differentiation of annulus fibrosus-derived stem cells (AFSCs) can be regulated by the elasticity of substrates such as poly(ether carbonate urethane)urea (PECUU). Decellularized annulus fibrosus matrix (DAFM) has good biocompatibility and biodegradability, making it suitable for cell adhesion, proliferation, and differentiation. In this study, we used a coaxial electrospinning method to synthesize DAFM/PECUU-blended fibrous scaffolds with elasticities approximating that of native inner and outer annulus fibrosus tissue. AFSCs cultured on DAFM/PECUU-blended fibrous scaffolds exhibited increased collagen type I gene expression with increasing elasticity of the scaffold material; notably, collagen type II and aggrecan gene expression exhibited the opposite trend. Regarding extracellular matrix secretion, collagen type I content gradually increased with substrate elasticity, while collagen type II and aggrecan contents decreased. In vivo evaluations employing magnetic resonance imaging, hematoxylin and eosin staining, and immunohistochemistry indicated that DAFM/PECUU-blended fibrous scaffolds could effectively repair defects of annulus fibrosus tissue. Our findings provide a theoretical and practical basis for the development of bionic annulus fibrosus tissue that closely mimics the biological properties, mechanical function, and matrix composition of native tissue.

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.

BackgroundTissue 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.MethodsPoly-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.ResultsThe 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.ConclusionFindings 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 articleEffective 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.

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.

Mechanical and Topographical Guidance of Stem Cell Differentiation &lt;i&gt;in Vitro&lt;/i&gt; Via YAP-Dependent Mechanotransduction: A Biomimetic Approach Toward Annulus Fibrosus Regeneration

The current engineered bionic disc tissue is considered as a potential way for treating intervertebral disc degeneration disease, which yet still remains challenging due to the complex radial gradient of natural annulus fibrosus (AF) tissue in cell phenotype, biochemical composition, topographical features, and mechanical properties. Previously, we have found that the differentiation of annulus fibrosus-derived stem cells (AFSCs) could be regulated by the elasticity of scaffold. In this study, we attempted to examine the combined effect of both mechanical and topographical features on the gene expression of AFSCs. Cells were cultured on four types of poly(ether carbonate urethane) urea (PECUU) scaffolds with controlled elasticity and fiber size (soft, small size; stiff, small size; soft, large size and stiff, large size). Results showed that when the fiber size of scaffold was kept constant, the expression of collagen-I (Col-I) in AFSCs increased with scaffold elasticity, while the expression of collagen-II (Col-II) and aggrecan genes showed an opposite trend. Moreover, when scaffold elasticity was controlled, the gene expression of Col-I in AFSCs increased with fiber size. In contrast, the expression of Col-II and aggrecan decreased. Such substrate elasticity and topography dependent changes of AFSC were similar to the gradient characteristics of native AF tissue. In addition, increasing elasticity and fiber size of scaffolds promoted YAP activation and its nuclear translocation. The results illustrate unambiguously that the mechanical property is a potent regulator of AFSC differentiation. Moreover, we revealed that fiber size of scaffold significantly affects spreading area, focal adhesion and differentiation of AFSCs. Therefore, both mechanical property and topography features of scaffolds regulate AFSC differentiation, possibly through a YAP-dependent mechanotransduction mechanism.

Identification and Characterizations of Annulus Fibrosus-Derived Stem Cells.

Annulus fibrosus (AF) injuries are common in degenerative disc disease (DDD) and can lead to substantial deterioration of the intervertebral disc. However, repair or regeneration of AF remains challenging. Recently, we have found that there exists a subpopulation of cells, which form colonies in vitro and could self-renew, in AF tissue. These cells express typical surface antigen molecules of mesenchymal stem cells, including CD29, CD44, and CD166. They also express common stem cell markers such as Oct-4, nucleostemin, and SSEA-4. In addition, they can be induced to differentiate into adipocytes, osteocytes, and chondrocytes. Being AF tissue-specific, such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using stem cell-based cell therapies or tissue engineering approaches.

The characteristics of stem cells in human degenerative intervertebral disc.

Background:The aim of this study is to identify which possessed the best stem-cell-like characteristics in 3 kinds of cell in human degenerative intervertebral disc: NPSCs (nucleus pulposus-derived stem cells), AFSCs (annulus fibrosus-derived stem cells), or CESCs (cartilage endplate-derived stem cells).Methods:We separated the disc samples obtained from 15 surgically treated patients with disc degenerative diseases into nucleus pulposus, annulus fibrosus, and cartilage endplate. After cultivating, we used the cell counting kit-8 to analysis the cell activity of 3 kinds of disc tissue-derived stem cell separately; different stem cells were defined with multilineage (osteogenic, chondrogenic, and adipogenic) differentiation. We extracted the total RNA and detected the expression of different lineage differentiation-related genes with the real-time polymerase chain reaction (RT-PCR).Results:Cell morphology of NPSCs, AFSCs, and CESCs did not show significant difference. Cell proliferation capacity of NPSCs and AFSCs was stronger than that of CESCs. The differentiation outcomes showed that osteocyte-like cells were stained red by Alizarin red S, chondrocyte-like cells blue by toluidine blue, and adipocyte-like red by oil red O. The RT-PCR reflected that the expression of different lineage differentiation-related genes of AFSCs was stronger than NPSCs and CESCs.Conclusion:In conclusion, we found that the cell morphology was not significantly different among NPSCs, AFSCs, and CESCs. Both differentiation and RT-PCR tests demonstrated that AFSCs had the best stem-cell-like characteristics in the human degenerative intervertebral disc.

The comparison of biological characteristics of three kinds of stem cells in human degenerative intervertebral disc

Objective To compare the biological characteristics of nucleus pulposus-derived stem cells (NPSCs), annulus fibrosus-derived stem cells (AFSCs) and cartilage endplate-derived stem cells (CESCs) from the same patient who had degenerative disc. Methods The human discs isolated from 7 patients with intervertebral disc herniation were separated carefully and digested and filtered before getting the original NPSCs, AFSCs and CESCs. When cultivated to third passage, the cell activity of stem cells was analyzed by using cell counting kit-8 (CCK-8); multilineage (adipogenic, osteogenic and chondrogenic) differentiation potential was analyzed to define different stem cells. The total RNA of third-passage-cells was extracted, and then reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the relative expression of adipogenic, osteogenic and chondrogenic differentiation-related genes. Results Cell morphology of NPSCs, AFSCs and CESCs from the same patient showed no significant difference. The growth curve showed that proliferative ability of CESCs (A value at day 1, 3, 5, 7, 9, 11, 13, 15 was 0.35±0.01, 0.52±0.02, 0.64±0.08, 0.80±0.19, 0.79±0.2, 1.06±0.11, 1.37±0.09, 1.31±0.41) and AFSCs (0.38±0.01, 0.50±0.03, 0.72±0.06, 0.82±0.13, 0.87±0.08, 0.87±0.09, 1.20±0.05, 1.43±0.05)was more active than that of NPSCs (0.32±0.02, 0.42±0.04, 0.59±0.01, 0.64±0.17, 0.65±0.19, 0.68±0.21, 0.67±0.12, 0.59±0.12). Multilineage differentiation results showed adipocyte-like red by oil red O, osteocyte-like cells were stained red by Alizarin red, and chondrocyte-like cells blue by toluidine blue. The RT-PCR results showed that AFSCs was better than NPSCs and CESCs in the aspect of the expression of adipogenic [the relative expression of peroxisome proliferator-activated receptor gamma (PPARγ) gene was 6.85±0.32 vs. 6.63±0.50 and 7.21±0.21; the relative expression of lipoprotein lipase (LPL) gene was 13.72±0.37 vs. 8.41±0.42 and 10.62±0.37] and chondrogenic [the relative expression of type 2 collagen (COL-2) gene was 10.45±1.10 vs. 3.53±0.62 and 8.26±0.49; the relative expression of sex determining region Y-box 9(SOX-9) gene was 14.39±1.22 vs. 7.66±0.68 and 13.70±1.10] differentiation-related genes, whereas the expression of osteogenic differentiation-related genes was best in CESCs[the relative expression of runt related transcription factor-2(RUNX-2) gene was 7.30±1.1 vs. 2.61±0.06 and 6.20±0.92; the relative expression of COL-2 gene was 8.22±0.84 vs. 7.05±0.82 and 4.94±0.90] and the expression of adipogentic differentiation-related genes, particularly LPL gene was worse in NPSCs. Conclusion The cell morphology was similar among NPSCs, AFSCs and CESCs in vitro, but biological characteristics of those were different. Induced differentiation and RT-PCR results both showed that AFSCs had more advantages than NPSCs and CESCs in the field of intervertebral disc tissue engineering. Key words: Intervertebral disc degeneration; Nucleus pulposus stem cells; Annulus fibrosus stem cells; Cartilaginous endplate stem cells; Biological characteristics

Distinguishing characteristics of stem cells derived from different anatomical regions of human degenerated intervertebral discs.

Several types of stem cells have been successfully demonstrated to exist in the human degenerated intervertebral disc (IVD), which is composed of annulus fibrosus (AF), nucleus pulposus (NP) and cartilage endplate (CEP). However, the differences in the biological characteristics among these and bone marrow derived mesenchymal stem cells (BM-MSCs) remain unclear. To investigate this issue, cells were harvested from human AF, NP, CEP, and bone marrow, respectively; passage 2 cells were selected using the agarose suspension culture system to obtain stem cell clones. Following expansion in vitro, stem cells from different anatomical regions were compared regarding the morphology, proliferation ability, immunophenotypic expression, and multi-lineage differentiation capacity. In addition, stem cell-alginate bead compositions were constructed for the comparison of DNA and sGAG content. There were subtle differences regarding cell morphology, but no significant differences in proliferation ability among the four types of stem cells. For the immunophenotypic analysis, all stem cells basically fulfilled the criteria for mesenchymal stem cells (MSCs), which have been published by the International Society for Cellular Therapy (ISCT), with a significant difference in CD105 expression. A comparison of the osteogenic capacities indicated: cartilage endplate-derived stem cells (CESCs)>annulus fibrosus-derived stem cells (AFSCs)>BM-MSCs>nucleus pulposus-derived stem cells (NPSCs). The chondrogenesis difference was similar to osteogenesis. For adipogenesis: BM-MSCs>NPSCs>CESCs>AFSCs. In the stem cell/alginate composition, the CESCs consistently showed the superior chondrogenic potential among all those cell types. Our data indicated that all the four types of stem cells shared some similar biological properties (regarding shape, proliferation ability and immunophenotypic expression). CESCs, which had the strongest osteogenic and chondrogenic potentials, may serve as excellent seed cells for NP/cartilage or bone tissue engineering.

Effect of scaffold elasticity on the gene expression of annulus fibrosus-derived stem cells.

This article provides more experimental details and findings of the study as to how the elasticity of scaffold material modulates the gene expression of annulus fibrosus-derived stem cells (AFSCs) (Zhu et al., 2015 [1]). The detailed synthetic route and characterizations of four kinds of biodegradable poly(ether carbonate urethane)ureas (PECUUs) are described. After AFSCs were cultured on electrospun PECUU fibrous scaffolds, the cell proliferation and gene expression analyses were performed to explore the effect of substrate elasticity on the growth and differentiation characteristics of AFSCs.

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.

Identification of rabbit annulus fibrosus-derived stem cells.

Annulus fibrosus (AF) injuries can lead to substantial deterioration of intervertebral disc (IVD) which characterizes degenerative disc disease (DDD). However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies in vitro and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs), including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

Introduction In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus, nucleus pulposus (NP), and cartilage endplate. Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. Materials and Methods In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, and so on. Results We found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs ( p&lt; 0.05); in addition, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray ( p&gt;0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. Conclusion Our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases. Disclosure of Interest None declared

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, etc. evaluations, we found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs (p<0.05); additionally, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray (p>0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. All in all, our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases.