Tissue Engineering of the Intervertebral Disc with Cultured Nucleus Pulposus Cells Using Atelocollagen Scaffold and Gene Therapy

STUDY DESIGN: This is an in-vitro experiment using rabbit intervertebral disc (IVD) cells and growth factors. OBJECTIVES: We wanted to determine the effect of types I, and II atelocollagen and growth factor gene therapy for matrix regeneration of rabbit IVD cells. SUMMARY OF THE LITERATURE REVIEW: Adenovirus-medicated growth factor gene therapy is efficient for matrix regeneration of the IVD. Atellocollagen has provided a favorable environment for matrix synthesis. However, a combined approach using gene and cell therapy in an atelocollagen scaffold has not yet been attempted. MATERIALS AND METHODS: Rabbit IVD cells were transduced with Ad/TGF-beta1 and Ad/BMP-2. The cells were then implanted to the atelocollagen scaffold. The [methyl-3H]thymidine incorporation for DNA synthesis and the [35S]sulfur incorporation for proteoglycan synthesis were measured. RT-PCR was performed for assessing the aggrecan, collagen type I, collagen type II and osteocalcin mRNA expressions. RESULTS: The rabbit IVD cells with Ad/TGF-beta1 and that were cultured in type I atelocollagen showed a 130% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/TGF-beta1 and that were cultured in type II atelocollagen showed a 180% increase in new proteoglycan synthesis (p<0.05). The rabbit IVD cells with Ad/BMP-2 and that were cultured in type I atelocollagen showed a 70% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/BMP-2 and that were cultured in type II atelocollagen showed a 95% increase (p<0.05). Rabbit IVD cells with Ad/TGF-beta1 and Ad/BMP-2 and that were cultured in type I and II atelocollagen demonstrated increased collagen type I and II mRNA expressions without an osteocalcin mRNA expression (p<0.05). CONCLUSION: Cell and gene therapy in an atelocollagen scaffold provided a efficient mechanism for chondrogenic matrix regeneration of rabbit IVD cells.

Pulsed Electromagnetic Field Stimulates Cellular Proliferation in Human Intervertebral Disc Cells

PURPOSE: The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. MATERIALS AND METHODS: Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650Omega, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [3H]-thymidine, and [35S]-sulfate incorporation. To detect phenotypical expression, reverse transcription-polymerase chain reactions (RT-PCR) were performed for aggrecan, collagen type I, and type II mRNA expression. To assess action mechanism of EMF, IVD cells were exposed to EMF with NG-Monomethyl-L-arginine (NMMA) and acetylsalicylic acid (ASA). RESULTS: There was no cytotoxicity in IVD cells with the EMF group in MTT assay. Cellular proliferation was observed in the EMF group (p < 0.05). There was no difference in newly synthesized proteoglycan normalized by DNA synthesis between the EMF group and the control. Cultures with EMF showed no significant change in the expression of aggrecan, type I, and type II collagen mRNA compared to the control group. Cultures with NMMA (blocker of nitric oxide) or ASA (blocker of prostaglandin E2) exposed to EMF demonstrated decreased DNA synthesis compared to control cultures without NMMA or ASA (p < 0.05). CONCLUSION: EMF stimulated DNA synthesis in human IVD cells while no significant effect on proteoglycan synthesis and chondrogenic phenotype expressions. DNA synthesis was partially mediated by nitric oxide and prostaglandin E2. EMF can be utilized to stimulate proliferation of IVD cells, which may provide efficient cell amplification in cell therapy to degenerative disc disease.

Phenotypical Stability and Matrix Synthesis of Human Intervertebral Disc Cells in Response to Dexamethasone and Transforming Growth Factor-beta1 / 대한척추외과학회지

STUDY DESIGN: An in vitro experiment. OBJECTIVES: To evaluate the mRNA expressions of matrix components, and analyze the cellular proliferation and proteoglycan synthesis of human intervertebral disc cells in response to dexamethasone and TGF-beta1 SUMMARY OF LITERATURE REVIEW: Corticosteroids are responsible for the regulation of a diverse range of biological processes through modulation of the expression of target genes. The direct injection of methylprednisolone to the intervertebral disc (IVD) has been shown to cause degeneration and calcification of the disc in rabbits. Systemic administration of hydrocortisone induced degeneration of notochordal cells, which accelerated the aging process of the disc in mice. Transforming growth factor beta-1 (TGF-beta1) is known as a potent agent for the proliferation, differentiation and matrix synthesis of IVD. MATERIALS AND METHODS: IVD cells were isolated from ten patients, and subsequently cultured. Various doses of dexamethasone (DEX) and/or TGF-beta1 were administered to the IVD cultures. DNA and proteoglycan syntheses were measured by the incorporation of [3H]-thymidine and [35S]-sulfate, respectively. RT-PCRs were performed for the expressions of aggrecan, collagen types I and II, and osteocalcin mRNA. RESULTS: Cultures with DEX showed increased cellular proliferation and decreased proteoglycan synthesis (p<0.05). TGF-beta1 potentiated the proliferative effect of DEX, but failed to stimulate proteoglycan synthesis in the cultures containing DEX. There were no recognizable changes in the mRNA expressions of aggrecan, collagen types I and II, and osteocalcin in response to DEX and TGF-beta1. CONCLUSIONS: DEX demonstrated a proliferative effect on human IVD cells, with the combination of DEX and TGF-beta1 showing potentiation of the proliferative effect, while at high doses(100 and 1000nM, the DEX was shown to down-regulate the proteoglycan synthesis. Caution should be exercised in the use of corticosteroid in the therapeutic approaches for the treatment of disc disease or in the regenerative matrix of the IVD.

The Effect of Pulsed Electromagnetic Field in Human Intervertebral Disc Cell / 대한척추외과학회지

STUDY DESIGN: An in vitro experiment. OBJECTIVES: To assess the effect of pulsed sinusoidal EMF on human intervertebral disc (IVD) cells. LITERATURE REVIEW SUMMARY: Electromagnetic field (EMF) is known to modify some relevant physiological parameters of cells cultured in vitro, such as proliferation, synthesis, secretion of growth factors and transcription. EMF induces bone formation in delayed, non union and spinal fusion models. Also, the exposure of EMF has been shown to protect against the hazardous effect of smoking in the rabbit IVD. MATERIALS AND METHODS: Human IVD cells were three-dimensionally cultured in alginate beads and exposed to a 650 omega, 1.8millitesla magnetic flux density, 60Hz sinusoidal wave of EMF. The cultures were divided into the control and EMF groups, with various exposure times. The cytotoxicity, and DNA and proteoglycan syntheses were measured by the MTT assay, and [3H]-thymidine and [35S]-sulfate incorporation, respectively. RT-PCRs were performed for aggrecan, and collagen types I and II mRNA expressions. RESULTS: There was no recognizable cytotoxicity in the EMF group, but cellular proliferation was stimulated (p<0.05). Newly synthesized proteoglycan, normalized by DNA synthesis, was decreased in the EMF group (p<0.05) as were the expressions of aggrecan (48hour exposure) and type II collagen (72 hours exposure) mRNA compared to the control group. CONCLUSIONS: EMF seems to be hazardous in the synthesis of the chondrogenic matrix, while marginally beneficial in the cellular proliferation of human IVD cells.

The Effect of Pulsed Electromagnetic Field in Human Intervertebral Disc Cell / 대한척추외과학회지

STUDY DESIGN: An in vitro experiment. OBJECTIVES: To assess the effect of pulsed sinusoidal EMF on human intervertebral disc (IVD) cells. LITERATURE REVIEW SUMMARY: Electromagnetic field (EMF) is known to modify some relevant physiological parameters of cells cultured in vitro, such as proliferation, synthesis, secretion of growth factors and transcription. EMF induces bone formation in delayed, non union and spinal fusion models. Also, the exposure of EMF has been shown to protect against the hazardous effect of smoking in the rabbit IVD. MATERIALS AND METHODS: Human IVD cells were three-dimensionally cultured in alginate beads and exposed to a 650 omega, 1.8millitesla magnetic flux density, 60Hz sinusoidal wave of EMF. The cultures were divided into the control and EMF groups, with various exposure times. The cytotoxicity, and DNA and proteoglycan syntheses were measured by the MTT assay, and [3H]-thymidine and [35S]-sulfate incorporation, respectively. RT-PCRs were performed for aggrecan, and collagen types I and II mRNA expressions. RESULTS: There was no recognizable cytotoxicity in the EMF group, but cellular proliferation was stimulated (p<0.05). Newly synthesized proteoglycan, normalized by DNA synthesis, was decreased in the EMF group (p<0.05) as were the expressions of aggrecan (48hour exposure) and type II collagen (72 hours exposure) mRNA compared to the control group. CONCLUSIONS: EMF seems to be hazardous in the synthesis of the chondrogenic matrix, while marginally beneficial in the cellular proliferation of human IVD cells.

Mesenchymanl Stem Cell Based Intradiscal Gene Therapy: Therapeutic Implication in Degenerative Disc Disease / 대한척추외과학회지

STUDY DESIGN: In-vitro experiments using human mesenchymal stem cells (MSCs), intervertebral disc (IVD) cells and type 5 adenovirus/transforming growth factor-beta1 construct (Ad/TGF-beta1). OBJECTIVES: To determine the effect of MSC-based gene therapy for matrix regeneration of IVD cells. SUMMARY OF LITERATURE REVIEW: MSCs are known to be multipotent in tissue regeneration. In degeneration of IVD, cellular replacement with genetic modification other than that of IVD cells may prove an enhanced mechanism for the regeneration of MATERIALS AND METHODS: MSCs and IVD cells were cultured and an adenovirus construct containing TGF-beta1 cDNA (Ad/TGF-beta1) was also produced. In the first step, the MSCs were transduced with Ad/TGF-beta1, then mixed with IVD cells in various proportions and three dimensionally cultured. [methyl-(3)H]Thymidine and [(35)S]Sulfur incorporation for DNA and proteoglycan synthesis, respectively, were measured. RT-PCR was performed to assess the aggrecan and collagen types I and II mRNA RESULTS: Mixed cultures of MSC and IVD cells showed relatively similar amounts of newly synthesized proteoglycan compared with cultures of IVD cells only. In mixed cultures transduced with Ad/TGF-beta1, there were significant decreases in newly synthesized proteoglycan with increasing the proportions of MSCs, which was also found with the aggrecan and collagen type II mRNA expressions. However, the collagen type I mRNA expression increased with increased proportions of MSCs transduced with Ad/TGF-beta1. CONCLUSION: Cell therapy with MSCs and IVD cells provided a mechanism for cellular augmentation. However, MSC-based gene therapy coupled with IVD cells did not maintain a chondrogenic phenotype.

Molecular Biological and Pathological Aspects of Intercostal Muscles and Intervertebral Discs in Adolescent Idiopathic Scoliosis in Korea / 대한척추외과학회지

STUDY DESIGN: A molecular biological study of intercostal muscles and intervertebral disc cells of Korean scoliosis patients. OBJECTIVES: To study the pathological results of intercostal muscles and molecular biological activity of intervertebral disc cells of the scoliotic major curve in Korean patients. SUMMARY OF LITERATURE REVIEW : The cause of idiopathic scoliosis has been investigated in terms of many parameters. Although, molecular biological studies of intercostal muscles and intervertebral disc cells have been performed in foreign countries, few studies have been conducted in Korea. MATERIALS AND METHODS: Ten patients, one male and nine female, who underwent thoracoscopic surgery were reviewed. The age range was 13 to 23 years old. Intercostal muscles were taken from the portal site of the major curve (1x1 cm sized). Ten tissues were stained with H/E and ATPase immunohistochemical staining. An appropriate amount of intervertebral disc was taken from the major curve of three scoliotic patients and each concentration of collagen type I, II, GAG gene and proteoglycan synthesis activity was measured. The results were compared with those of grade 0 and grade II degenerative change on each MRI. RESULTS: The intercostal muscle of scoliotic patients showed 60.4+/-8.4% in type I muscle fiber and 39.6+/-8.8% in type II-A. These results were not different from those of previous studies. The size of muscle fiber was 48-65 microns, which was slightly smaller than the absolute value, but the difference was not statistically significant. The amount of produced proteoglycans was slightly higher in the intervertebral disc cells of scoliotic patients, the total amount of collagen was significantly lower and there was a difference in the production of type II collagen. CONCLUSIONS: The intercostal muscles were not affected by the muscle of scoliotic patients and there was no molecular biological significant difference between control and scoliotic patients. We can assume that scoliosis was not caused by problems of intervertebral disc or intercostal muscles.

Dual Roles of Ligamentum Flavum for Spinal Fusion: As an Osteoinductive Agent and Carrier for Ex-vivo Gene Transfer / 대한척추외과학회지

STUDY DESIGN: An in-vitro experiment using human ligamentum flavum (LF) and the adnovirus-BMP-2 construct, Ad/BMP-2. OBJECTIVES: To determine the dual roles of LF as an osteoinductive agent and carrier for ex-vivo gene transfer. SUMMARY OF LITERATURE REVIEW: LF is known to have osteogenic potential. Pathologically, ossified LF may cause myelopathy and radiculopathy. BMP-2 is known as an important factor in the differentiation, and maintenance, of osteoblast phenotypes. Ex-vivo gene transfer, using human LF for spinal fusion, has never been attempted before. MATERIALS AND METHODS: The LF cells were cultured from the degenerated LF of spinal stenosis patients. An adenovirus construct, containing BMP-2 cDNA (Ad/BMP-2), was also produced. The LF cell cultures were exposed to the adenoviral construct. The Osteocalcin expression was analysed by Western blot analysis. The osteocalcin and BMP-2 mRNA expressions were analysed by RT-PCR. Bone formation was assessed by alkaline phosphatase and Von Kossa stains. RESULTS: The LF cell cultures, with Ad/BMP-2, showed transgene expression in the Western blot analysis. Also, the cultures exhibited the mRNA expressions of both osteocalcin and BMP-2, in a dose-dependent manner. The LF cultures, with Ad/BMP-2, demonstrated alkaline phosphatase expression and bone nodule formations from the Von Kossa staining. CONCLUSION: The genetically modified LF strongly induced osteogenesis, which can be used during a spinal fusion, as an osteoinductive agent and carrier, for ex-vivo gene transfer.

Osteogenesis by Transfer of Bone Morphogenetic Protein-2 cDNA in Ligamentum Flavum Cells: Approach Toward Tissue Engineering / 대한척추외과학회지

STUDY DESIGN: In-vitro experiment. OBJECTIVES: To determine the effect of bone morphogenetic protein-2 in the osteogenesis of human ligamentum flavum cells and test the feasibility of gene transfer to these cells. SUMMARY OF LITERATURE REVIEW: Bone morphogenetic protein-2 (BMP-2) is known to be an important factor in the differentiation and maintenance of the osteoblastic phenotype. Tissue engineering for osteogenesis in ligamentum flavum by BMP-2 and gene transfer has not been previously studied. MATERIALS AND METHODS: Ligmentum flavum cells were harvested and cultured from surgical patients with spinal stenosis. BMP-2 was produced by transfecting pcDNA3.1/Hygro/BMP-2 into CHO cells using Lipofectamine 2000. Adenovirus-lacZ (Ad/lacZ) was also produced, and administered with BMP-2 to cell culture. The expression of lacZ was analyzed by X-gal staining. Bone formation was assessed by alkaline phosphatase, von Kossa, and alizarin Red-S staining, and the expression of osteocalcin was determined immunocytochemically. RESULTS: Ligamentum flavum cell cultures with Ad/lacZ showed marker gene expression. BMP-2 induced osteogenesis in ligamentum flavum cells as evidenced by alkaline phosphatase, von Kosa, and alizarin Red-S staining. Also, cell culture with BMP-2 showed strong positivity with osteocalcin by immunocytochemistry. CONCLUSION: BMP-2 more strongly induced the osteogenesis of ligamentum flavum, and also its gene transfer to ligamentum flavum was found to be feasible. These results may open a new era of ligamentum flavum tissue engineering.