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.

The Effect of Synovial Fluid from Degenerated Facet on Hypertrophy and Ossification of the Ligamentum Flavum / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study OBJECTIVES: To examine the effect of a synovial supernatant on the cell viability, osteogenic phenotype, mRNA expression of the types collagen and various transcriptional factors on osteogenesis in ligamentum flavum (LF) cells stimulated with synovial fluid from a degenerated facet joint. LITERATURE REVIEW: In degenerative lumbar spinal stenosis, hypertrophied LF or osteoarthritic hypertrophy of a facet joint often causes neurogenic claudication. The facet joint is a synovial joint with hyaline cartilage on each side. Therefore, osteoarthritis of a facet joint eventually occurs with aging and other degenerative conditions of the spine. In lumbar spinal degeneration, inflammatory mediators or cytokines are released from the facet joint tissue, which consequently affects the adjacent LF because the LF covers posterolateral aspect of the spinal canal near facet joints. However, there are no reports on the relationship between a degenerated facet joint fluid and the LF in the lumbar spine. MATERIALS AND METHODS: LF surgical specimens were obtained from patients with a lumbar spine stenosis, and the cells were isolated by enzymatic digestion. Each of the synovium tissues were weighed and recorded. Each tissue was cut into small pieces with a pair of scissors and then washed 3 times with PBS. The washed tissue pieces were then cultured for 96 hr at 37degrees C, 5% CO2 in DMEM/F-12-0.1% FBS with a density of 200 mg/ml medium. The supernatant was collected after 96 hr. In order to measure quantitatively the proliferation of cells, the AlamarBlue assay was used. The total cellular RNA was extracted from the cells and amplification reactions specific to the following types of cDNA were performed: the osteogenic master transcription factors, Dlx5, Runx2, osterix, and types collagen and osteocalcin. Alkaline phosphatase staining for the biochemical assay and western blotting for osteocalcin protein expression were performed. RESULTS: Human LF cells cultured with the supernatant from the facet synovium showed a slightly stronger AlamarBlue staining than the intensity of the control culture. RT-PCR revealed the upregulation of the osteogenic master transcription factors, Dlx5, Runx2, and osterix in the synovium supernatant group from one hour to 72 hours, and an increase in osteocalcin, types collagen I, III, V, XI levels from one hour to one week. LF cells cultured with the supernatant from the facet synovium showed positive staining for alkaline phosphatase. The level of the osteocalcin protein in the LF cells cultured with the supernatant from the facet synovium was higher than the control group. Conclusions: The supernatant of the facet joint from patients with degenerative spinal stenosis affects LF cells by increasing the level of cellular proliferation, upregulating the mRNA expression of osteocalcin, types of collagen, osteogenic transcription factors, positive alkaline phosphatase staining, and osteocalcin protein expression. Therefore, degenerated synovial fluid from the facet joint is an important mechanism of LF hypertrophy and ossification.

Comparison of the Predictive Value of Myelography, Computed Tomography and MRI on the Treadmill Test in Lumbar Spinal Stenosis

To date, there have been no prospective, objective studies comparing the accuracy of the MRI, myelo-CT and myelography. The purpose of this study is to compare the diagnostic and predictive values of MRIs, myelo-CTs, and myelographies. Myelographies with dynamic motion views, myelo-CTs, MRIs and exercise treadmill tests were performed in 35 cases. The narrowest AP diameter of the dural sac was measured by myelography. At the pathologic level, dural cross-sectional area (D-CSA) was calculated in the MRI and Myelo-CT. The time to the first symptoms (TAF) and the total ambulation time (TAT) were measured during the exercise treadmill test and used as the standard in the comparison of correlation between radiographic parameters and walking capacity. The mean D-CSA by CT was 58.3 mm2 and 47.6 mm2 by MRI. All radiographic parameters such as AP diameters and D-CSA have no correlation to TAF or TAT (p > 0.05). Our data showed no statistically significant differences in the correlation of the patients' walking capacity to the severity of stenosis as assessed by myelography, myelo-CT and MRI.

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study. OBJECTIVES: To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF-beta1 and BMP-2. SUMMARY OF LITERATURE REVIEW: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor. MATERIAL AND METHOD: Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5 mm), frozen at -70 degrees C, and then lyophilized at -50 degrees C. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF-beta1 (10 ng/ml) or BMP-2 (100 ng/ml) or both. After 1 and 2 week culture periods, the DNA synthesis was measured by [3H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35S] sulphate. Reverse transcription-polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM). RESULTS: The IVD cultures in collagen type II with TGF-beta1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF-beta1 and BMP-2 in the matrix synthesis or for the mRNA expression of the matrix components. CONCLUSIONS: Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study. OBJECTIVES: To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF-beta1 and BMP-2. SUMMARY OF LITERATURE REVIEW: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor. MATERIAL AND METHOD: Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5 mm), frozen at -70 degrees C, and then lyophilized at -50 degrees C. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF-beta1 (10 ng/ml) or BMP-2 (100 ng/ml) or both. After 1 and 2 week culture periods, the DNA synthesis was measured by [3H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35S] sulphate. Reverse transcription-polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM). RESULTS: The IVD cultures in collagen type II with TGF-beta1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF-beta1 and BMP-2 in the matrix synthesis or for the mRNA expression of the matrix components. CONCLUSIONS: Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.

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.