• Journal of Korean Neurosurgical Society
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• v.48 no.1
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• pp.1-7
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• 2010

Objective : Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. Methods : Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). Results : AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-$1{\beta}$, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-$1{\beta}$, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. Conclusion : We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

• Polymer(Korea)
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• v.34 no.3
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• pp.282-288
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• 2010

The porcine small intestinal submucosa (SIS) has been widely used as a biomaterial without immuno rejection responses and hyalunonic acid (HA) can be used as biocompatible materials to regenerate tissue. We developed the SIS sponge and HA loaded SIS sponges (SIS/HA) for the possibility of the application of the tissue engineering using annulus fibrosus (AF). SEM observation shows that SIS and SIS/HA sponges have interconnected and open pores. We demonstrated the presence of HA in SIS/HA sponge from C-O functional group observed by the FTIR analysis. In conclusion, we suggest that SIS/HA sponge may be useful to tissue engineering using AF cell. This may be due to the enhanced biocompatibility and higher water retention capacity of HA.

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

• Journal of Biomedical Engineering Research
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• v.39 no.1
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• pp.22-29
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• 2018

Intervertebral disc(IVD) mainly consists of Annulus fibrosus(AF) and Nucleus pulposus(NP), playing a role of distributing a mechanical load on vertebral body. IVD tissue engineering has been developed the methods to achieve anatomic morphology and restoration of biological function. The goal of present study is to identify the possibilities for creating a substitute of IVD the morphology and biological functions are the same as undamaged complete IVD. To fabricate the AF and NP combine biphasic IVD tissue, AF tissue scaffolds have been printed by 3D bio-printing system with natural biomaterials and NP tissues have been prepared by scaffold-free culture system. We evaluated whether the combined structure of 3D printed AF scaffold and scaffold-free NP tissue construct could support the architecture and cell functions as IVD tissue. 3D printed AF scaffolds were printed with 60 degree angle stripe patterned lamella structure(the inner-diameter is 5mm, outer-diameter is 10 mm and height is 3 mm). In the cytotoxicity test, the 3D printed AF scaffold showed good cell compatibility. The results of histological and immunohistochemical staining also showed the newly synthesized collagens and glycosaminoglycans, which are specific makers of AF tissue. And scaffold-free NP tissue actively synthesized glycosaminoglycans and type 2 collagen, which are the major components of NP tissue. When we combined two engineered tissues to realize the IVD, combined biphasic tissues showed a good integration between the two tissues. In conclusion, this study describes the fabrication of Engineered biphasic IVD tissue by using enable techniques of tissue engineering. This fabricated biphasic tissue would be used as a model system for the study of the native IVD tissue. In the future, it may have the potential to replace the damaged IVD in the future.

• Polymer(Korea)
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• v.32 no.6
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• pp.516-522
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• 2008

Biodegradable polymers have been used extensively as scaffolding materials to regenerate new tissues and the ingrowth of tissue have been reported to be dependent directly of the porosity, pore diameter, pore shape, and porous structure of the scaffold. In this study, porous poly (L-lactide-co-glycolide) (PLGA) scaffolds with five different pore sizes were fabricated to investigate the effect of pore sizes for AF tissue regeneration. Cellular viability and proliferation were assayed by MTT test. Hydroxyproline/DNA content of AF cells on each scaffold was measured. sGAG analyses were performed at each time point of 2 and 6 weeks. Scaffold seeded AF cells were implanted into the back of athymic nude mouse to observe the difference of formation of disc-like tissue depending on pore size in vivo. We confirmed that scaffold with $180{\sim}250{\mu}m$ pores displayed high cell viability in vitro and produced higher ECM than scaffold with other pore sizes in vivo.

• Polymer(Korea)
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• v.31 no.1
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• pp.14-19
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• 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)
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• v.32 no.2
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• pp.109-115
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• 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.

• Journal of Korean Neurosurgical Society
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• v.44 no.5
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• pp.327-333
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• 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
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• v.28 no.4
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• pp.512-519
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• 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.