• Polymer(Korea)
• /
• v.39 no.1
• /
• pp.144-150
• /
• 2015

Gellan gum as a natural polysaccharide has good heat resistance, acid resistance and enzymes resistance. However, one of the drawbacks of gellan gum might be the lower mechanical strength. In this work, gellan gum scaffolds were mixed with poly(lactic-co-glycolic acid) (PLGA) microsphere in order to improve mechanical properties. The gellan gum scaffolds with various contents of PLGA microsphere were prepared for the regeneration of disc tissues. To evaluate the mechanical strength of hybrid structure of gellan gum and PLGA microsphere, compression strength of the fabricated scaffolds was measured. MTT analysis, SEM observation, histological evaluation and RT-PCR were performed to confirm the effect on the cell growth and extracellular matrix secretion. As a result, it showed the best cell proliferation and extracellular matrix secretion in gellan gum sponge containing 50% PLGA microspheres. In conclusion, this study confirmed that the hybrid structure of gellan gum and PLGA microspheres was found suitable in regeneration of the intervertebral disc.

• Journal of Korean Neurosurgical Society
• /
• v.44 no.5
• /
• pp.327-333
• /
• 2008

Objective: The purpose of this study is to verify the usefulness of the rabbit model for disc degeneration study. Materials: The L1-L2, L2-L3, L3-L4. or L4-L5 lumbar intervertebral disc (IVD) of 9 mature male New Zealand White rabbits were injured by inserting a 16-gauge needle to a depth of 5 mm in the left anterolateral annulus fibrosus while leaving L5-L6 IVD uninjured. Three other rabbits also received intradiscal injections of rabbit disc cells transfected with adenovirus and bone morphogenetic protein-2 (ad-BMP-2) at L4-L5 in addition to injury by 16-gauge needle at the L1-L2 level. Using digitized radiographs, measurements of IVD height were made and analyzed by using the disc height index (DHI). Magnetic resonance imaging (MRI) scans of the injured discs, injected discs, and uninjured L5-L6 discs were performed at 15 weeks post surgery and compared with preoperative MRI scans. Results: All twelve rabbits showed consistent results of disc degeneration within 15 weeks following annular puncture. DHIs of injured discs were significantly lower than that of the uninjured L5-L6 discs (p<0.05). The mean value of disc degeneration grade of injured discs was significantly higher than that of uninjured discs (p<0.05). The injection of disc cell transfected with ad-BMP-2 did not induce disc regeneration at 15 weeks after injection. Conclusion: This study showed that the injured disc had a significant change in DHI on simple lateral radiograph and disc degeneration grade on MRI scans within 15 weeks in all rabbits. Rabbit annular puncture model can be useful as a disc degeneration model in vivo.

• Journal of Biomedical Engineering Research
• /
• v.28 no.4
• /
• pp.512-519
• /
• 2007

The purpose of this study is to investigate the potential of a novel tissue engineering approach to regenerate intervertebral disc. In this study, thermosensitive scaffold (chitosan-Pluronic hydrogel) and nanofiber were used to replace the nucleus pulposus (NP) and annulus fibrosus of a degenerated intervertebral disc, leading to an eventual regeneration of the disc using the minimally invasive surgical procedure and organ culture. In preliminary study, disc cells were seeded into the scaffolds and cellular responses were assessed by MTT assay and scanning electron microscopy (SEM). Based on these results, we could know that tissue engineered scaffolds might provide favorable environments for the regeneration of tissues. Organ culture was performed in fresh porcine spinal motion segments with endplates on both sides. These spinal motion segments were classified into three groups: control (Intact), injured NP (Defect), and inserting tissue engineered scaffolds (Insert). The specimens were cultivated for 7 days, subsequently structural stability, cell proliferation and morphological changes were evaluated by the relaxation time, quantity of DNA, GAG and histological examination. In these results, inserting group showed higher relaxation time, reduced decrement of DNA contents, and accumulated GAG amount. Consequently, the tissue engineered scaffolds used in this study seen to be a promising base scaffolds for regenerative intervertebral disc due to its capacity to absorb external dynamic loading and the possible ideal environment provided for disc cell growing.

• Journal of Korean Neurosurgical Society
• /
• v.53 no.4
• /
• pp.207-212
• /
• 2013

Objective : Recent studies have shown encouraging progress toward the use of autogenic and allogenic mesenchymal stem cells (MSCs) to arrest, or even lead to partial regeneration in, intervertebral disc (IVD) degeneration. However, this technology is still in its infancy, and further development is required. The aim of this study was to analyze whether rat adipose-derived mesenchymal stem cells (ADMSC) can differentiate towards IVD-like cells after treatment with transforming growth factor ${\beta}3$ (TGF-${\beta}3$) in vitro. We also performed quantitative analysis of gene expression for ADMSC only, ADMSCs treated with TGF-${\beta}3$, and co-cultured ADMSCs treated with TGF-${\beta}3$. Methods : ADMSCs were sub-cultured to homogeneity and used in fluorocytometry assays for CD11, CD45, and CD90/Thy1. ADMSCs were differentiated in spheroid culture towards the chondrogenic lineage by the presence of TGF-${\beta}3$, dexamethasone, and ascorbate. We also co-cultured pure ADMSCs and nucleus pulposus cells in 24-well plates, and performed immunohistochemical staining, western blotting, and RT-PCR for quantitative analysis of gene expression. Results : Results of fluorocytometry were positive for CD90/Thy1 and negative for CD11 and CD45. TGF-${\beta}3$-mediated induction of ADMSCs led to the expression of the differentiation markers of intervertebral disc-like cells, such as aggrecan, collagen II, and sox-9. Co-cultured ADMSCs treated with TGF-${\beta}3$ showed higher expression of differentiation markers and greater extracellular matrix production compared with ADMSCs treated with TGF-${\beta}3$ alone. Conclusion : ADMSC treated with TGF-${\beta}3$ may be an attractive source for regeneration therapy in degenerative IVD. These findings may also help elucidate the pathologic mechanism of MSC therapy in the degeneration of IVD in vivo.

• Polymer(Korea)
• /
• v.37 no.6
• /
• pp.669-676
• /
• 2013

Demineralized bone particle (DBP) is a biomaterial used widely in the field of tissue engineering. In this study, in order to study the effect of DBP/poly(lactic-co-glycolic acid) (PLGA) scaffold on disc regeneration in vivo environment, we prepared the porous DBP/PLGA hybrid scaffold. Disc defect was induced by removing the nucleus pulposus tissue after incision the annulus fibrosus tissue in half and scaffolds were transplanted. After 1, 2 and 3 months later, the extracted discs were confirmed by collagen synthesis and glycosaminoglycan (sGAG). We conducted histology (H&E, Safranin-O, Alcian blue, Type I Collagen, Type II Collagen). From the results, it was confirmed that collagen and sGAG content were high in DBP/PLGA scaffold, and the regeneration of intervertebral disc was possible.

• Journal of Haehwa Medicine
• /
• v.25 no.1
• /
• pp.37-44
• /
• 2016

Objectives : This study was designed to investigate the effect of Gamishinchubogun-tang (JiaweiShenzhuibujian-tang; GSB) on regeneration of PC12 cells. Methods : PC12 cells have been used extensively as a model for studying the cellular and molecular effects of neuronal cells. In order to check the effect of GSB on the regeneration of PC12 cells, the morphological change of PC12 cells were observed comparatively in GSB group and control group. Results : The significant changes in neurite length of PC12 cells have been observed on GSB group. In proportion to the concentration of GSB it was observed an increase in neurite outgrowth. Conclusions : This study confirmed that GSB made a significant influence on regeneration of PC12 cells.

• Polymer(Korea)
• /
• v.31 no.1
• /
• pp.14-19
• /
• 2007

Demineralized bone particle (DBP) has been used as one of the powerful inducers of bone and cartilage tissue specialization. In this study, we fabricated DBP/PLGA scaffold for tissue engineered disc regeneration. We manufactured dual-structured scaffold to compose inner cylinder and outer doughnut similar to nature disc tissue. The DBP/PLGA scaffold was characterized by porosity, wettability, and water uptake ability. We isolated and cultured nucleus pulposus (NP) and annulus fibrosus (AF) cells from rabbit intervertebral disc. We seeded NP cells into the inner core of the hybrid scaffold and AF cells into the outer portion of it. Cellular viability and proliferation were assayed by 3-(4,5-dimethylthiazole-2-yl) -2,5- diphenyltetrazolium -bromide (MTT) test. PLGA and PLGA/DBP scaffolds were implanted in subcutaneous of athymic nude mouse to observe the formation of disc-like tissue in vivo. And then we observed change of morphology and hematoxylin and eosin (H&E). Formation of disc-like tissue was better DBP/PLGA hybrid scaffold than control. Specially, we confirmed that scaffold impregnated 20 and 40% DBP affected to proliferation of disc cell and formation of disc-like tissue.

• Polymer(Korea)
• /
• v.32 no.2
• /
• pp.109-115
• /
• 2008

Because demineralized bone particle (DBP) contains various bioactive molecules such as cytokines, it is widely used biomaterials in the field of tissue engineering. In this study, we investigated the effect of 2-dimensional DBP/PLGA hybrid films on adhesion, proliferation and phenotype maintenance of intervertebral disc cells. PLGA films incorporated with different amount (0, 10, 20, 40 and 80 wt%) of DBP were prepared by the solvent evaporation method and characterized by scanning election microscopy (SEM). PLGA film has a flat and smooth surface. According to the increase of content of DBP, the surface of DBP/PLGA film exhibited few agglomerates and increased the roughness of the surface. Annulus fibrosus (AF) and nucleus pulposus (NP) cells were cultured on PLGA and DBP/PLGA film surface, and then examined the cell adhesion and proliferation by the cell count and SEM observation. The result of cell count and SEM observation revealed that 10 and 20% DBP in DBP/PLGA films were superior to adhesion and proliferation of both AF and NP cells. We confirmed that specific gene expression of disc cells on DBP/PLGA film based on the cell count result. Disc cells seeded on 20% DBP/PLGA film expressed the gene of type I and II collagen continuously. Therefore, pertinent content of biomaterials could provide more appropriate condition on adhesion and proliferation of cell. And this results may be used as a basic data for the intervertebral disc regeneration using tissue engineering.

• Polymer(Korea)
• /
• v.32 no.1
• /
• pp.26-30
• /
• 2008

This study was designed to investigate the effect of hybridization of synthetic/natural materials for annulus fibrosus (AF) tissue regeneration in vitro and in vivo. The synthetic/natural hybrid scaffolds were prepared using PLGA (poly (lactic-co-glycolic) acid), SIS (small intestinal submucosa) and DBP (demineralized bone particles). PLGA, PLGA/SIS(20%), PLGA/DBP(20%) and PLGA/SIS (10%)/DBP (10%) scaffold were manufactured by solvent casting/salt leaching method. Compressive strength was measured. Rabbit AF cells were isolated, cultured and seeded into experimental groups. Hydroxyproline production and DNA quantity of AP cells on each scaffold was measured at 2, 4 and 6 weeks after in vitro culture. Cell-scaffold composites were implanted subcutaneously into athymic mice. After 1,4 and 6 weeks postoperatively, specimens were taken and H&E, Safranin-O and type I collagen staining were carried out concerning formation of cartilagenous tissue. In vitro PLGA/SIS scaffold was evaluated for total collagen content (bydroryproline/DNA content) and PLGA scaffold was evaluated for compressive strength.