Nucleus Pulposus Tissue Engineering Research Articles

Research Progress in Nucleus Pulposus Tissue Engineering in Lumbar Intervertebral Disc

Lumbar intervertebral disc degeneration is a common pathological process in the spine,with the main clinical symptoms of low back pain,numbness of lower limbs,and defecation dysfunction.The occurrence and development of lumbar intervertebral disc degeneration are determined by multiple factors,and the pathophysiological and cellular mechanisms remain to be fully understood.Nucleus pulposus tissue engineering is a new biotherapy that combines biological histology with material science to treat diseases including lumbar intervertebral disc degeneration.Clinicians should fully learn the complex relationship between nucleus pulposus tissue engineering and lumbar intervertebral disc degeneration,which will facilitate the clinical treatment of lumbar intervertebral disc degeneration,the rehabilitation of lumbar intervertebral disc after treatment,and the prevention of this disease in the population.

Silk-hydrogel functionalized with human decellularized Wharton's jelly extracellular matrix as a minimally invasive injectable hydrogel system for potential nucleus pulposus tissue replacement therapy

Intervertebral disc degeneration is a primary cause for chronic low back pain, a common health problem with high incidence and the leading cause of disability globally. The early stages of disc degeneration in terms of functional and anatomical abnormalities start from the central nucleus pulposus tissue of the intervertebral disc; hence its regeneration has become a prime concern. A plethora of hydrogel systems have been investigated as nucleus pulposus tissue substitute over the years, with limited clinical translation. In the present study, we formulated a minimally invasive injectable cross-linker-free bioactive silk-based hybrid hydrogel system functionalized with decellularized human Wharton's jelly extracellular matrix as an ampule of bioactive cues. The centrifugation based decellularization method removed >92 % of cellular components and preserved >83 % of extracellular matrix composition. The hydrogels were investigated for secondary structure and surface properties through infrared spectroscopy and electron micrographs, respectively. Notably, the developed hydrogels were found to mimic the rheological and mechanical properties of native nucleus pulposus tissue when decellularized Wharton's jelly extracellular matrix content was 0.5 % (w/v) in the base silk hydrogel. Finally, the hydrogels were found to support cell viability, proliferation, and tissue maturation offering great potential for future applications related to nucleus pulposus tissue engineering.

Codelivery of TGF-β1 and anti-miR-141 by PLGA microspheres inhibits progression of intervertebral disc degeneration

BackgroundCervical and lumbar pain is usually caused by degeneration of the nucleus pulposus (NP). As a powerful therapeutic strategy, tissue engineering can effectively restore the normal biological properties of the spinal unit. Previous studies suggested that poly(lactic-co-glycolic acid) (PLGA) microspheres are effective carriers of cells and biomolecules in NP tissue engineering. This study aims to explore the therapeutic effect of PLGA microspheres coloaded with transforming growth factor-β1 (TGF-β1) and anti-miR-141 on intervertebral disc degeneration (IDD).MethodsPLGA microspheres were characterized by scanning electron microscopy, a laser particle size analyzer, and laser confocal microscopy. The in vitro release rate of biomolecules from the microspheres was analyzed by reversed-phase high-performance liquid chromatography and agarose gel electrophoresis. The rat NP cells (NPCs) treated with the solutions released from microspheres for different lengths of time were assigned to a control group (Ctrl), an empty PLGA microsphere group (Mock microsphere, MS), a TGF-β1-loaded PLGA microsphere group (TMS), an anti-miR-141-loaded PLGA microsphere group (AMS), and an anti-miR-141 + TGF-β1-loaded PLGA microsphere group (ATMS). The proliferation and apoptosis of NPCs were observed by alamar blue and flow cytometry. The gene and protein expression of cartilage markers COL2A1 and ACAN were observed by RT-qPCR and Western blot. The rat model of IDD was established by tail puncture. Rats were divided into a control group (Ctrl), a mock operation group (Mock), a TGF-β1 microsphere group (TMS), an anti-miR-141 microsphere group (AMS), and an anti-miR-141 + TGF-β1 microsphere group (ATMS). The degree of rat tail IDD was assessed in each group through magnetic resonance imaging (MRI), safranin O-fast green staining, immunohistochemistry, and Western blotting.ResultsPLGA microspheres were stably coloaded and could sustainably release TGF-β1 and anti-miR-141. The results of in vitro cell experiments showed that the release solution of PLGA microspheres significantly enhanced the proliferation of NPCs without inducing their apoptosis and significantly upregulated cartilage markers in NPCs. The effect of microspheres was greater in the ATMS group than that in the TMS group and AMS group. In vivo experiments showed that IDD could be effectively inhibited and reversed by adding microspheres coloaded with TGF-β1 and/or anti-miR-141, and the effect was greatest in the ATMS group.ConclusionPLGA microspheres coloaded with TGF-β1 and anti-miR-141 can reverse IDD by inhibiting the degeneration of NPCs.

A bio-inspired nano-material recapitulating the composition, ultra-structure, and function of the glycosaminoglycan-rich extracellular matrix of nucleus pulposus

The nucleus pulposus (NP) of intervertebral disc represents a soft gel consisting of glycosaminoglycans (GAGs)-rich extracellular matrix (ECM). Significant loss of GAGs and normal functions are the most prevalent changes in degenerated disc. Attempts targeted to incorporate GAGs into collagen fibrous matrices have been made but the efficiency is very low, and the resulting structures showed no similarity with native NP. Inspired by the characteristic composition and structures of the ECM of native NP, here, we hypothesize that by chemically modifying the collagen (Col) and hyaluronic acid (HA) and co-precipitating with GAGs, a bio-inspired nano-material recapitulating the composition, ultra-structure and function of the GAG-rich ECM will be fabricated. Compositionally, the bio-inspired nano-material namely Aminated Collagen-Aminated Hyaluronic Acid-GAG (aCol-aHA-GAG) shows a record high GAG/hydroxyproline ratio up to 39.1:1 in a controllable manner, out-performing that of the native NP. Ultra-structurally, the nano-material recapitulates the characteristic ‘nano-beads’ (25 nm) and ‘bottle-brushes’ (133 nm) features as those found in native NP. Functionally, the nano-material supports the viability and maintains the morphological and phenotypic markers of bovine NP cells, and shows comparable mechanical properties of native NP. This work contributes to the development of a compositionally, structurally, and functionally biomimetic nano-material for NP tissue engineering.

The combinatory effect of scaffold topography and culture condition: an approach to nucleus pulposus tissue engineering.

Scaffold topography and culture medium conditions for human wharton's jelly mesenchymal stem cells (hWJ-MSC) are critical components of the approach to nucleus pulposus (NP) tissue engineering.Aim:To evaluate the silk fibroin (SF) scaffold topography analysis (optimal thickness and pore diameter) and to determine culture medium conditions for the growth and differentiation of hWJ-MSC.Method:hWJ-MSCs were seeded into different thicknesses and pore size diameters and grown in different concentrations of glucose, platelet rich plasma (PRP) and oxygen. The cell-seeded scaffold was evaluated for cell attachment, growth and differentiation potency.Results & discussion:The results indicated that SF scaffold with a minimum thickness 3.5 mm and pore diameter of 500 μm with cells cultured under low glucose, 10% PRP and normoxia conditions induced the growth and differentiation of hWJ-MSCs, indicated by the accumulation of glycosaminoglycans content and the presence of type II collagen, as markers of NP-like cells.

Construction of tissue-engineered nucleus pulposus by stimulation with periodic mechanical stress and BMP-2

SummaryIntervertebral disc (IVD) degeneration, which is common among elderly individuals, mainly manifests as low back pain and is caused by structural deterioration of the nucleus pulposus (NP) due to physiological mechanical stress. NP mesenchymal stem cells (NPMSCs) around the IVD endplate have multidirectional differentiation potential and can be used for tissue repair. To define favorable conditions for NPMSC proliferation and differentiation into chondroid cells for NP repair, the present study simulated periodic mechanical stress (PMS) of the NP under physiological conditions using MSC chondrogenic differentiation medium and recombinant human BMP-2 (rhBMP-2). rhBMP-2 effectively promoted NPMSC proliferation and differentiation. To clarify the mechanism of action of rhBMP-2, integrin alpha 1 (ITG A1) and BMP-2 were inhibited. PMS regulated the BMP-2/Smad1/RUNX2 pathway through ITG A1 and promoted NPMSC proliferation and differentiation. During tissue-engineered NP construction, PMS can effectively reduce osteogenic differentiation and promote extracellular matrix protein synthesis to enhance structural NP recovery.

Sa12b-Modified Functional Self-Assembling Peptide Hydrogel Enhances the Biological Activity of Nucleus Pulposus Mesenchymal Stem Cells by Inhibiting Acid-Sensing Ion Channels

Various hydrogels have been studied for nucleus pulposus regeneration. However, they failed to overcome the changes in the acidic environment during intervertebral disc degeneration. Therefore, a new functionalized peptide RAD/SA1 was designed by conjugating Sa12b, an inhibitor of acid-sensing ion channels, onto the C-terminus of RADA16-I. Then, the material characteristics and biocompatibility of RAD/SA1, and the bioactivities and mechanisms of degenerated human nucleus pulposus mesenchymal stem cells (hNPMSCs) were evaluated. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that RAD/SA1 self-assembling into three-dimensional (3D) nanofiber hydrogel scaffolds under acidic conditions. Analysis of the hNPMSCs cultured in the 3D scaffolds revealed that both RADA16-I and RAD/SA1 exhibited reliable attachment and extremely low cytotoxicity, which were verified by SEM and cytotoxicity assays, respectively. The results also showed that RAD/SA1 increased the proliferation of hNPMSCs compared to that in culture plates and pure RADA16-I. Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting demonstrated that the expression of collagen I was downregulated, while collagen II, aggrecan, and SOX-9 were upregulated. Furthermore, Ca2+ concentration measurement and western blotting showed that RAD/SA1 inhibited the expression of p-ERK through Ca2+-dependent p-ERK signaling pathways. Therefore, the functional self-assembling peptide nanofiber hydrogel designed with the short motif of Sa12b could be used as an excellent scaffold for nucleus pulposus tissue engineering. Moreover, RAD/SA1 exhibits great potential applications in the regeneration of mildly degenerated nucleus pulposus.

Bardoxolone Methyl Ameliorates Compression-Induced Oxidative Stress Damage of Nucleus Pulposus Cells and Intervertebral Disc Degeneration Ex Vivo.

Intervertebral disc degeneration (IDD) is the main cause of low back pain, and little is known about its molecular and pathological mechanisms. According to reports, excessive compression is a high-risk factor for IDD; compressive stress can induce oxidative stress in nucleus pulposus (NP) cells during IDD progression that, in turn, promotes cell apoptosis and extracellular matrix (ECM) degradation. Currently, NP tissue engineering is considered a potential method for IDD treatment. However, after transplantation, NP cells may experience oxidative stress and induce apoptosis and ECM degradation due to compressive stress. Therefore, the development of strategies to protect NP cells under excessive compressive stress, including pretreatment of NP cells with antioxidants, has important clinical significance. Among the various antioxidants, bardoxolone methyl (BARD) is used to protect NP cells from damage caused by compressive stress. Our results showed that BARD can protect the viability of NP cells under compression. BARD inhibits compression-induced oxidative stress in NP cells by reducing compression-induced overproduction of reactive oxygen species (ROS) and malondialdehyde. Thus, BARD has a protective effect on the compression-induced apoptosis of NP cells. This is also supported by changes in the expression levels of proteins related to the mitochondrial apoptosis pathway. In addition, BARD can inhibit ECM catabolism and promote ECM anabolism in NP cells. Finally, the experimental results of the mechanism show that the activation of the Nrf2 signaling pathway participates in the protection induced by BARD in compressed NP cells. Therefore, to improve the viability and biological functions of NP cells under compression, BARD should be used during transplantation.

The nucleus pulposus microenvironment in the intervertebral disc: the fountain of youth?

The intervertebral disc (IVD) is a complex tissue, and its degeneration remains a problem for patients, without significant improvement in treatment strategies. This mostly age-related disease predominantly affects the nucleus pulposus (NP), the central region of the IVD. The NP tissue, and especially its microenvironment, exhibit changes that may be involved at the outset or affect the progression of IVD pathology. The NP tissue microenvironment is unique and can be defined by a variety of specific factors and components characteristic of its physiology and function. NP progenitor cell interactions with their surrounding microenvironment may be a key factor for the regulation of cellular metabolism, phenotype, and stemness. Recently, celltransplantation approaches have been investigated for the treatment of degenerative disc disease, highlighting the need to better understand if and how transplanted cells can give rise to healthy NP tissue. Hence, understanding all the components of the NP microenvironment seems to be critical to better gauge the success and outcomes of approaches for tissue engineering and future clinical applications. Knowledge about the components of the NP microenvironment, how NP progenitor cells interact with them, and how changes in their surroundings can alter their function is summarised. Recent discoveries in NP tissue engineering linked to the microenvironment are also reviewed, meaning how crosstalk within the microenvironment can be adjusted to promote NP regeneration. Associated clinical problems are also considered, connecting bench-to-bedside in the context of IVD degeneration.

The application of decellularized nucleus pulposus matrix/chitosan with transforming growth factor β3 for nucleus pulposus tissue engineering.

Low back pain caused by intervertebral disc degeneration has become a global problem that seriously affects public health. The application of nucleus pulposus tissue engineering to disc degeneration has attracted increasing attention. A scaffold is important for nucleus pulposus tissue engineering, which provides a three-dimensional growth space with an appropriate biomechanical and biochemical microenvironment for seed cell differentiation and proliferation. In this study, a decellularized nucleus pulposus matrix/chitosan (DNPM/chitosan) hydrogel scaffold was prepared with crosslinker genipin. Nucleus pulposus stem cells (NPSCs) were cultured in hybrid hydrogels with or without transforming growth factor-β3 (TGF-β3) and then cell morphology, proliferation, and nucleus pulposus-related gene expression were analyzed. TGF-β3 was successfully incorporated into the DNPM/chitosan hydrogel and NPSCs grew well on both kinds of hydrogel. Moreover, gene expression of collagen-I, collagen-II, and aggrecan was enhanced in the DNPM/chitosan hydrogel with TGF-β3. These results indicate that the DNPM/chitosan hybrid hydrogel is a promising candidate scaffold for nucleus pulposus tissue engineering.

Stiffness of photocrosslinkable gelatin hydrogel influences nucleus pulposus cell propertiesin vitro.

A key early sign of degenerative disc disease (DDD) is the loss of nucleus pulposus (NP) cells (NPCs). Accordingly, NPC transplantation is a treatment strategy for intervertebral disc (IVD) degeneration. However, in advanced DDD, due to structural damage of the IVD and scaffold mechanical properties, the transplanted cells are less viable and secrete less extracellular matrix, and thus, are unable to efficiently promote NP regeneration. In this study, we evaluated the encapsulation of NPCs in a photosensitive hydrogel made of collagen hydrolysate gelatin and methacrylate (GelMA) to improve NP regeneration. By adjusting the concentration of GelMA, we prepared hydrogels with different mechanical properties. After examining the mechanical properties, cell compatibility and tissue engineering indices of the GelMA‐based hydrogels, we determined the optimal hydrogel concentration of the NPC‐encapsulating GelMA hydrogel for NP regeneration as 5%. NPCs effectively combined with GelMA and proliferated. As the concentration of the GelMA hydrogel increased, the survival, proliferation and matrix deposition of the encapsulated NPCs gradually decreased, which is the opposite of NPCs grown on the surface of the hydrogel. The controllability of the GelMA hydrogels suggests that these NPC‐encapsulating hydrogels are promising candidates to aid in NP tissue engineering and repairing endogenous NPCs.

A possible injectable tissue engineered nucleus pulposus constructed with platelet-rich plasma and ADSCs in vitro

BackgroundInjectable tissue engineered nucleus pulposus is a new idea for minimally invasive repair of degenerative intervertebral disc. The platelet-rich plasma (PRP) and adipose-derived stromal cells (ADSCs) could be harvested from autologous tissue easily. PRP contains numerous autologous growth factors and has reticulate fibrous structure which may have the potential to make ADSCs differentiate into nucleus pulposus-like cells. The goal of this study was to explore the feasibility of constructing a possible injectable tissue engineered nucleus pulposus with PRP gel scaffold and ADSCs.MethodsAfter identification with flow cytometry, the rabbit ADSCs were seeded into PRP gel and cultured in vitro. At the 2nd, 4th, and 8th week, the PRP gel/ADSCs complex was observed by macroscopy, histological staining, BrdU immunofluorescence, and scanning electron microscopy. The glycosaminoglycans (GAG) in the PRP gel/ADSCs complex were measured by safranin O staining with spectrophotometry. In PRP gel/ADSCs complex, gene expression of HIF-1α, aggrecan, type II collagen were tested by RT-PCR. The injectability of this complex was evaluated.ResultsMacroscopically, the complex was solidified into gel with smooth surface and good elasticity. The safranin O dye was almost no positive staining at 2nd week; however, the positive staining of extracellular matrix was enhanced obviously at 4th and 8th week. The HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immunofluorescence showed that ADSCs can survive and proliferate in PRP gel at each time points. The level of GAG at 4th week was higher than those at 2nd week (P < 0.05), and significant difference was also noted between 4th and 8th week (P < 0.05). HIF-1α, aggrecan, type II collagen gene expression at 4th week were much more than those at 2nd week (P < 0.05), and significant differences were also noted between 4th and 8th week (P < 0.05). The flow rate of complex was 0.287 mL/min when passed through the 19-gauge needle with the 100 mmHg injection pressure.ConclusionsOur preliminary findings suggest that the PRP gel make it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after coculture in vitro. According to the results, it is a better feasible method for construction of autologous injectable tissue engineered nucleus pulposus.

Exploring Gelation and Physicochemical Behavior of in Situ Bioresponsive Silk Hydrogels for Disc Degeneration Therapy.

Hydrogels have received considerable attention in the field of tissue engineering because of their unique structural and compositional resemblance to the highly hydrated human tissues. In addition, controlled fabrication processes benefit them with desirable physicochemical features for injectability in minimally invasive manner and cell survival within hydrogels. Formulation of biologically active hydrogels with desirable characteristics is one of the prerequisites for successful applications like nucleus pulposus (NP) tissue engineering to address disc degeneration. To achieve such a benchmark, in this study, two naturally derived silk fibroin proteins (Bombyx mori, BM SF; and Antheraea assamensis, AA SF) were blended together to allow self-assembly and transformation to hydrogels in absence of any cross-linker or external stimuli. A comprehensive study on sol-gel transition of fabricated hydrogels in physiological fluid microenvironment (pH, temperature, and ionic strength) was conducted using optical and fluorescence analysis. Tunable gelation time (∼8-40 min) was achieved depending on combinations. The developed hydrogels were validated by extensive physicochemical characterizations which include confirmation of secondary structure, surface morphology, swelling and degradation. Mechanical behavior of the hydrogels was further analyzed in various in vitro-physiological-like conditions with varying pH, ionic strength, diameter, storage time, and strain values to determine their suitability in native physiological environments. Rheological study, cytocompatibility using primary porcine NP cells and ex vivo biomechanics of hydrogels were explored to validate their in situ applicability in minimally invasive manner toward potential disc regeneration therapy.

Injectable, redox-polymerized carboxymethylcellulose hydrogels promote nucleus pulposus-like extracellular matrix elaboration by human MSCs in a cell density-dependent manner.

Low back pain is a major cause for disability and is closely linked to intervertebral disc degeneration. Mechanical and biological dysfunction of the nucleus pulposus in the disc has been found to initiate intradiscal degenerative processes. Replacing or enriching the diseased nucleus pulposus with an injectable, stem cell-laden biomaterial that mimics its material properties can provide a minimally invasive strategy for biological and structural repair of the tissue. In this study, injectable, in situ-gelling carboxymethylcellulose hydrogels were developed for nucleus pulposus tissue engineering using encapsulated human marrow-derived mesenchymal stromal cells (hMSCs). With the goal of obtaining robust extracellular matrix deposition and faster construct maturation, two cell-seeding densities, 20 × 106 cells/ml and 40 × 106 cells/ml, were examined. The constructs were fabricated using a redox initiation system to yield covalently crosslinked, cell-seeded hydrogels via radical polymerization. Chondrogenic culture of the hydrogels over 35 days exhibited high cell viability along with deposition of proteoglycan and collagen-rich extracellular matrix, and mechanical and swelling properties similar to native human nucleus pulposus. Further, the matrix production and distribution in the carboxymethylcellulose hydrogels was found to be strongly influenced by hMSC-seeding density, with the lower cell-seeding density yielding a more favorable nucleus pulposus-specific matrix phenotype, while the rate of construct maturation was less dependent on the cell-seeding density. These findings are the first to demonstrate the utility of redox-polymerized carboxymethylcellulose hydrogels as hMSC carriers for potential minimally invasive treatment strategies for nucleus pulposus replacement.

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

Functional self-assembled peptide scaffold inhibits tumor necrosis factor-alpha-induced inflammation and apoptosis in nucleus pulposus cells by suppressing nuclear factor-κB signaling.

Although nucleus pulposus (NP) tissue engineering has achieved tremendous success, researches still face the huge obstacles in maintaining cell survival and function. A novel functional self-assembled peptide RADA-KPSS was constructed by conjugating BMP-7 short active fragment (KPSS) to the C-terminus of RADA16-I that displays anti-inflammatory and anti-apoptosis effects. However, whether this functional self-assembled RADA-KPSS peptide can alleviate inflammation and NPC apoptosis induced by tumor necrosis factor-alpha (TNF-α) has not been studied. Therefore, we cultured NPCs treated with TNF-α for 48 h with the RADA-KPSS peptide, and compared the results to those with RADA16-I peptide. The cell apoptosis rate, inflammatory mediator secretion, expression of matrix-degrading enzymes, and extracellular matrix (ECM) protein levels were evaluated. The expression of nuclear factor-κB-p65 (NF-κB-p65) protein was also tested. TNF-α-treated NPCs cultured with the RADA16-I peptide showed up-regulated gene expression for matrix-degrading enzymes, such as matrix metalloproteinases-3 (MMP-3), MMP-9, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4), and down-regulated gene expression for ECM proteins such as aggrecan, collagen II, and Sox-9. The RADA-KPSS peptide could attenuate the expression of MMP-3, MMP-9, and ADAMTS-4, promote accumulation of ECM proteins, and increase secretion of glycosaminoglycan as compared with the RADA16-I peptide. Moreover, the TNF-α-damaged NPCs was further demonstrated to inhibit NF-κB-p65, IL-1, IL-6, and prostaglandin E-2 proteins and decrease cell apoptosis in RADA-KPSS peptide. In conclusion, the functional self-assembled RADA-KPSS peptides have anti-inflammatory and anti-apoptotic effects by promoting anabolic processes and inhibiting catabolic processes in intervertebral disk degeneration. These peptides may be feasible for clinical applications in NP tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1082-1091, 2018.

The research progress of the mechanism of endogenous repair failure during IVD degeneration

Intervertebral disc (IVD) degeneration is considered to be the main cause of low back pain (LBP), however, there are lack of long-lasting and effective methods of clinical treatment. Tissue engineering technique based on stem cells be-comes an essential research direction on repair of IVD degeneration at present, and its effectiveness and feasibility have been con-firmed, but it is difficult to maintain the sufficiency and vitality of stem cells in IVD. Previous studies showed that stem cells exist-ed naturally in IVD, and stem cells from stem cell niche could migrate to IVD physiologically to maintain the IVD environment bal-ance under the adverse microenvironment. Unfortunately, these behaviors cannot preclude IVD degeneration. Therefore, theoreti-cal basis for the regeneration of nucleus pulposus (NP) in situ can be obtained from studying the mechanism that the endogenous repair failure during IVD degeneration, the cell death and the migration of stem cells in IVD, and the key regulatory targets to sus-tain the quantity and quality of the stem cells. Although there have been few researches to study the mechanism of the cell death and the migration of stem cells in IVD so far, studies demonstrated that the major inducing factors of IVD degeneration (pressure and hypoxia) could decrease the number of NP cells by autophagy, apoptosis and necroptosis, and chemokines and their receptors played a critical role in the migration of mesenchymal stem cells. These researches provide a clue for studying the mechanism of endogenous repair failure during IVD degeneration. We reviewed the current research situation and progress of the mechanism that endogenous repair failure during IVD degeneration in the following articles. First, we exhibited the potential of IVD stem cells in IVD degeneration repair. Second, the effect of the adverse microenvironment (pressure, hypoxia, etc) on the migration of IVD stem cells was discussed. Third, the mechanism of the stem cell death, autophagy, apoptosis and necroptosis under the adverse (pressure, hypoxia, etc.) microenvironment, and the correlation between the IVD stem cells migration and autophagy, apoptosis and necroptosis was studied. And then tissue engineering of NP was also discussed to achieve the endogenous repair of IVD degenera-tion. These studies will provide an innovative research direction on endogenous repair and a new strategy of early therapy for IVD degeneration.

Advances in the application of nucleus pulposus stem cells in nucleus pulposus tissue engineering

Intervertebral disc degeneration (IVDD) takes great affection on people health all over the world. With the extension of human life and aging, the number of sick people will grow rapidly. At present, both conservative and surgical treatment can only alleviate the symptoms, rather than the fundamental treatment for the process of degeneration. Therefore, the application of cell therapy in the treatment of degenerative disc regeneration is an innovative treatment. However, it's a great bottleneck for exogenous transplanted stem cells to survive in degenerated intervertebral disc with harsh environment containing strong force, acidic pH, hypoxia, hyperosmolarity and limited nutrition. In recent years, tissue endogenous repair has been demonstrated to be present in almost all human tissues, and endogenous stem cell play an important role in the process. Similarly, nucleus pulposus was also confirmed the existence of such a kind of tissue specific stem cells-nucleus pulposus derived stem cells (NPSCs)-a new promising candidate for the treatment of IVDD. NPSCs, as a new kind of stem cells, hold either the characteristic of stem cell and the special potential of themselves, such as the nice adaption of the environment in intervertebral disc and the superior potential of cartilage differentiation. Nonetheless, the isolations of NPSCs were different; cell phenotypes were lack of wildly acceptable standard and the biological characteristics were various among different NP sources. In addition, whether the application of NPSCs could be used in nucleus pulposus tissue engineering still calls for further research. Due to the unclear in degeneration mechanism of IVDD, the study on degeneration mechanism of NPSCs may play a great important role in clarifying this issue.

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering

Background ContextDisc degeneration is the leading cause of low back pain and is often characterized by a loss of disc height, resulting from cleavage of chondroitin sulfate proteoglycans (CSPGs) present in the nucleus pulposus. Intact CSPGs are critical to water retention and maintenance of the nucleus osmotic pressure. Decellularization of healthy nucleus pulposus tissue has the potential to serve as an ideal matrix for tissue engineering of the disc because of the presence of native disc proteins and CSPGs. Injectable in situ gelling matrices are the most viable therapeutic option to prevent damage to the anulus fibrosus and future disc degeneration. PurposeThe purpose of this research was to create a gentle decellularization method for use on healthy nucleus pulposus tissue explants and to develop an injectable formulation of this matrix to enable therapeutic use without substantial tissue disruption. Study DesignPorcine nuclei pulposi were isolated, decellularized, and solubilized. Samples were assessed to determine the degree of cell removal, matrix maintenance, gelation ability, cytotoxic residuals, and native cell viability. MethodsNuclei pulposi were decellularized using serial detergent, buffer, and enzyme treatments. Decellularized nuclei pulposi were solubilized, neutralized, and buffered. The efficacy of decellularization was assessed by quantifying DNA removal and matrix preservation. An elution study was performed to confirm removal of cytotoxic residuals. Gelation kinetics and injectability were quantified. Long-term in vitro experiments were performed with nucleus pulposus cells to ensure cell viability and native matrix production within the injectable decellularized nucleus pulposus matrices. ResultsThis work resulted in the creation of a robust acellular matrix (>96% DNA removal) with highly preserved sulfated glycosaminoglycans (>47%), and collagen content and microstructure similar to native nucleus pulposus, indicating preservation of disc components. Furthermore, it was possible to create an injectable formulation that gelled in situ within 45 minutes and formed fibrillar collagen with similar diameters to native nucleus pulposus. The processing did not result in any remaining cytotoxic residuals. Solubilized decellularized nucleus pulposus samples seeded with nucleus pulposus cells maintained robust viability (>89%) up to 21 days of culture in vitro, with morphology similar to native nucleus pulposus cells, and exhibited significantly enhanced sulfated glycosaminoglycans production over 21 days. ConclusionsA gentle decellularization of porcine nucleus pulposus followed by solubilization enabled the creation of an injectable tissue-specific matrix that is well tolerated in vitro by nucleus pulposus cells. These matrices have the potential to be used as a minimally invasive nucleus pulposus therapeutic to restore disc height.

An interpenetrating network composite for a regenerative spinal disc application

Severe degeneration of the intervertebral disc has an immensely debilitating effect on quality of life that has become a serious health and economic burden throughout the world. The disc plays an integral role in biomechanical movement and support within the spine. The emergence of tissue engineering endeavours to restore the structural characteristics and functionality of the native tissue. Hydrogels have been widely investigated as a candidate for regeneration of the gelatinous nucleus pulposus due to its architectural resemblance and fluid retention characteristics. However, hydrogels are often limited due to small compressive stiffness and tear resistance, leading to extrusion complications. Reinforcement of the hydrogel network using polymeric scaffolds may address these issues of inadequate mechanical properties and implant instability. This study investigates the potential of a carrageenan gel-infused polycaprolactone scaffold for nucleus pulposus tissue engineering. Mechanical properties were characterised using viscoelastic and poroelastic frameworks via microindentation. The incorporation of polymeric reinforcement within the gels increased material stiffness to that comparable to the native nucleus pulposus, however permeability was significantly greater than native values. A preliminary cell evaluation culturing NIH 3T3s over 21 days suggested the incorporation of polymeric networks also enhanced cellular proliferation compared to gels alone.