| IntroductionThe intervertebral disc (IVD) is composed of the nucleus pulposus (NP), the annulus fibrosus (AF), and the endplates (EP). Among them, NP contains a large amount of disc-matrix proteins which are negatively charged proteoglycans (PG) within a collagen type Ⅱ network1. Consequently, a swelling pressure is present due to the high concentration of water in the NP2. Notably, IVD has poor self-repair capacity and its degeneration often progresses to an irreversible change. This process usually starts in the NP. In this condition, there is an increase of collagen type Ⅰ and denatured collagen type Ⅱ in extracellular matrix (ECM), whereas proteoglycans are reduced because of the activation of matrix-degrading enzyme which degrades the components of ECM3,4.Growth factor therapy is one of the promising modalities to regenerate IVD. It has been shown that transforming growth factor β1(TGFβ1), bone morphogenetic protein (BMP)-7, growth differentiation factor-5(GDF-5), basic fibroblast growth factor (bFGF) and transforming growth factor β3(TGFβ3) could stimulate IVD cells to proliferate and produce ECM in vitro5,6. Moreover, TGFβ3and GDF-5could inhibit IVD degeneration in animal models7,8. However, the short half-life of growth factors represents a problem for their application in vivo. For example, Walsh et al. reported that the anular fibrochondrocytes in degenerated IVDs are responsive to TGF-β1only after multiple injections in vivo9.Several groups have investigated whether authentic NP tissue could be engineered in vitro and in vivo by transplanting allogenic donor NP cells and mesenchymal stem cells (MSCs) seeded in3D scaffolds10-12. Among the scaffolds evaluated, mainly solid and preformed scaffolds comprising porous foams and fibrous meshes have been used. However, these types of scaffolds have the major drawback of requiring a surgical incision to position the scaffolds into desired sites in the body. Recently, a wide variety of biodegradable PLG A spherical particles have been utilized for the sustained delivery of bioactive molecules as an injectable depot formulation13,14. Moreover, PLGA is known to be biocompatible that has a broad range of applications in tissue engineering and clinic application15,16. Therefore, PLGA microspheres may be a suitable material for the preparation of injectable scaffolds for NP tissue engineering.Microparticulate systems have attracted increasing interest over the past few years as carriers for the delivery of cells and proteins due to minimal invasive procedures in tissue engineering16-18. Growth factors and drugs have been co-incorporated in PLGA microspheres using microparticulate systems for NP tissue differentiation19-21. Especially, anti-inflammatory agents such as dexamethasone can minimize implantation-associated inflammation and promote cell differentiation22,23. It is important to administer both anti-inflammatory corticosteroids and growth factors in a localized environment at low doses, so that both agents can function symbiotically without interference.This study contains five parts:(1) Isolation and identification of SD ADSCs; Construction and characterized the overexpressed GFP/ADSCs and TGF-P3/ADSCs cell lines;(2) Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc;(3) Dual delivery for stem cell differentiation using dexamethasone and TGF-β3in/on polymeric microspheres as a cell carrier for nucleus pulposus regeneration;(4) Dual delivery for stem cell matrix accumulation using dexamethasone and TGF-β3in/on a layered polymeric microspheres in a rat disc degeneration model. Part I Isolation and identification of SD ADSCs; Construction and characterized the overexpressed GFP/ADSCs and TGF-p3/ADSCs cell linesAim:The purpose of this study was to construct a lentiviral vector containing TGF-β3/GFP gene and transfect into adipose derived stem cells in vitro. Then we use drugs to select GFP/ADSCs and TGF-β3/ADSCs cell lines, which are always characterized as stem cells. It will facilitated the following development of gene and cell therapy in treating diseases of intervertebral disc degeneration.Methods and results:Firstly, we cultured SD rat ADSCs through cutting adipose into pieces, digesting using enzyme and isolating by adherent method. Separated ADSCs were identified by studying stem cell morphology, growth curve, surface antigen marker expression profiles and three-line differentiation capacity. Gateway technology was used to construct lentiviral vectors carrying the TGF-β3, and/or GFP gene, and which were transfected into ADSCs later. Antibiotics were used to obtain a stable expression of TGF-β3and/or GFP gene in cell lines TGF-β3/ADSCs and/or GFP/ADSCs. Finally, in accordance with the morphology, surface antigen marker expression profiles and three-line differentiation capacity of stem cells, TGF-β3/ADSCs and GFP/ADSCs were identified. The expression of TGF-β3gene was detected by RT-PCR and Western Blot methods in all three cell lines. SD rats ADSCs were successfully established, cultured and expanded in vitro, and they were also successfully induced into osteogenic, adipogenic and cartilage cells. The hematopoietic stem cell phenotypic markers of ADSCs were negative, while the expression of mesenchymal stem cell phenotypic markers was positive. After48h transfection, strong green fluorescence could be seen, and the transfection rate of Lenti-GFP-TGF-β3and Lenti-GFP were above90%. Compared with GFP/ADSCs and ADSCs cells, the expression of TGF-β3gene is overexpression in TGF-β3/ADSCs cells by RT-PCR and Western blot detection (P <0.05). In addition, the characteristics of stem cells could still be identified in GFP/ADSCs and TGF-β3/ADSCs cell lines.Conclusions:SD rats ADSCs were easily isolated and cultured in vitro, which have multiple differentiation potential abilities and mesenchymal stem cell characteristics. Lentiviral vectors containing the TGF-β3/GFP gene were successfully transfected into ADSCs,then GFP/ADSCs and TGF-β3/ADSCs cell lines were established.Part Ⅱ Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral discAim:Human adipose-derived mesenchymal stem cells (ADMSCs) may be ideal source of cells for intervertebral disc (IVD) regeneration, but the harsh chemical microenvironment of IVD may significantly influence the biological and metabolic vitality of ADMSCs and impair their repair potential. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of ADMSCs in the chemical microenvironment of IVD under normal and degeneration conditions.Methods and results:ADMSCs were harvested from young (aged8-12years, n=6) and mature (aged33-42years, n=6) male donors and cultured under standard condition and IVD-like conditions (low glucose, acidity, high osmolarity, and combined conditions) for2weeks. Cell viability was measured by annexin V-FITC and PI (AV-PI) staining and cell proliferation was measured by MTT assay. The expression of aggrecan, collagen-Ⅰ, and collagen-Ⅱ was detected by real-time quantitative polymerase chain reaction and Western blot analysis. IVD-like glucose condition slightly inhibited cell viability, but increased the expression of aggrecan. In contrast, IVD-like osmolarity, acidity and the combined conditions inhibited cell viability and proliferation and the expression of aggrecan and collagen-Ⅰ. ADMSCs from young and mature donors exhibited similar responses to the chemical microenvironments of IVD.Conclusions:IVD-like low glucose is a positive factor but IVD-like high osmolarity and low pH are deleterious factors that affect the survival and biological behaviors of ADMSCs. These findings may promote the translational research of ADMSCs in IVD regeneration for the treatment of low back pain. Part Ⅲ Dual delivery for stem cell differentiation using dexamethasone and TGF-β3in/on polymeric microspheres as a cell carrier for nucleus pulposus regenerationAim:This study aimed to investigate the feasibility of the nanostructured3D poly(lactide-co-glycolide)(PLGA) constructs, which are loaded with dexamethasone (DEX) and growth factor embeded heparin/poly(L-lysine) nanoparticles via a layer-by-layer system, to serve as an effective scaffold for nucleus pulposus (NP) tissue engineering.Methods and results:Our results demonstrated that the microsphere constructs were capable of simultaneously releasing TGF-β3and DEX with approximately zero order kinetics. The dual bead microspheres showed no cytotoxicity, and promoted the proliferation of the rat adipose-derived mesenchymal stem cells (ADSCs) by lactate dehydrogenase assay and CCK-8assay. After4weeks of cultivation in vitro, the ADSCs-scaffold hybrids contained significantly higher levels of sulfated GAG/DNA and collagen type Ⅱ than the control samples. Moreover, quantitative real time PCR analysis revealed that the expression of disc-matrix proteins including collagen type Ⅱ, aggrecan, and versican in the ADSCs-scaffold hybrids was significantly higher than that in the control group, whereas the expression of osteogenic differentiation marker (collagen type I) was decreased.Conclusions:Taken together, these data indicate that Dex/TGF-β3/PLGA microspheres could be used as a scaffold to improve the ADSCs growth and differentiating into NP like cells, and reduce the inflammatory response for IVD tissue engineering.Part Ⅳ Dual delivery for stem cell matrix accumulation using dexamethasone and TGF-β3in/on a layered polymeric microspheres in a rat disc degeneration modelAim:Low back pain is frequently caused by nucleus pulposus (NP) degeneration. Tissue engineering is a powerful therapeutic strategy which could restore the normal biomechanical motion of the human spine. Previously we reported a new nanostructured3D poly(lactide-co-glycolide)(PLGA) microsphere, which is loaded with dexamethasone and growth factor embedded heparin/poly(L-lysine) nanoparticles via a layer-by-layer system, was an effective cell carrier in vitro for NP tissue engineering. This study aimed to investigate whether the implantation of adipose-derived stem cell (ADSCs) seeded PLGA microspheres into the rat intervertebral disc degeneration model could regenerate the degenerated disc.Methods and results:Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix associated gene expressions were evaluated in normal controls (NC)(without operations), degeneration control (DC) group (with needle puncture, only DMEM injection); PLGA microspheres (PM) treatment group (with needle puncture, PLGA microspheres only injection); and PLGA microspheres loaded with ADSCs treatment (PMA) group (with needle puncture, PLGA microspheres loaded with ADSCs injection) for a24-week period. The results showed that24weeks post-transplantation, PM and PMA groups regained a disc height value of about63%and76%, MRI signal intensity of about47%and76%, respectively, compared to NC group. Biochemistry, immunohistochemistry and gene expression analysis indicated the restoration of proteoglycan accumulation in the discs of PM and PMA groups.Conclusions:Taken together, these data suggest that ADSCs-seeded PLGA microspheres could partly regenerate the degenerated disc in vivo after implantation into the rat intervertebral disc degeneration model. PLGA microsphere may serve as a promising carrier in cell transplantation therapy for degenerative disc disease. |
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