Glucocorticoid Mechanism of Inhibition of the Inflammatory Cells in Lumbar Intervertebral Disc Cells Stimulated by TNF-alpha Production of Nuclear Factor-kappaB / 대한정형외과연구학회지

PURPOSE: To analyze the action mechanism of NF-kappaB, IkappaB-alpha and effect of the Dexamethasone (DEXA) in mediating this inflammation, after stimulating cultured herniated intervertebral disc cells with TNF-alpha. MATERIALS AND METHODS: After cultured human intervertebral disc cells passaged three times, they were divided into four groups: A control group (A), DEXA treatment group (B), TNF-alpha treated group (C), TNF-alpha and DEXA were treated at the same time (D). IL-6 and IL-1beta gene expression were measured with semi-quantitative RT-PCR. Western blot analysis was performed to measure protein expression of IkappaB-alpha in the above groups for 10 minutes, 1 hour, 2 hours. In addition, in order to explain the mechanism of NF-kappaB nuclear binding for each group, the nuclear amount of NF-kappaB binding in the nucleus is measured by EMSA. RESULTS: In RT-PCR, expression of IL-6 and IL-1beta was greatest in group C, followed by group D, group A. IkappaB-alpha expression of the group treated with DEXA was not detected in Western blot results within 10 minutes. However, if stimulated by TNF-alpha, the DEXA was not inhibited of IkappaB-alpha concentration. After 1 hour and 2 hours, IkappaB-alpha levels were expressed by cells autonomously (autoregulatory induction). EMSA results expression levels in nuclear protein was maintained in accordance with protein expression. CONCLUSIONS: Our study shows that DEXA inhibits the production of mediators such as inflammatory IL-6 and IL-1beta, however, may not inhibit the transcription of NF-kappaB stimulated by TNF-alpha.

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.

Double Plate Occipitocervical Fusion After Failed Posterior Fusion C 1-2 With Wiring / 대한척추외과학회지

A 31-year-old female complained of neck pain and limitation in neck motion. She had a 3 month history of treatment with Halovest at another hospital for a fracture of the odontoid process due to a car accident. The patient complained of persistent pain and limitation in neck motion following the cessation of Halovest. A dynamic radiograph demonstrated instability on C1-2 and she underwent a posterior cervical fusion with wiring. A wound infection developed, and loosening of the wire and lysis of the posterior arch at C1-2 were seen on a follow up plain radiograph 2 months postoperatively. She was transferred to our hospital where she underwent occipitocervical fusion with a double plate after control of the infection. There were rigid fixations of the plate and bone union on a follow up radiograph 24 months postoperatively.

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.

Change of TypeI and TypeII Collagen Biosynthesis by Growth Factors in Cultured Cells Isolated from Rabbit Intervertebral Disc / 대한정형외과학회잡지

Growth factors influencing the function of chondrocytes are insulin-like growth factor I(IGF-I), basic fibroblast growth factor(bFGF), transforming growth factor-beta1(TGF-beta1), and epidermal growth factor(EGF). To find out the role of four kinds of growth factors in the biosynthesis of type I and II collagen represented as the phenotype of the disc cells, we cultured the disc cells isolated from rabbit intervertebral discs primarily and then checked cell proliferation, the expression of type I and II procollagen mRNA, and the immunohistochemical stains with type I and II collagen antibodies during in vitro culture in the maintenance medium containing low serum concentration with adding four kinds of growth factors. The results are as follows. FBS(10% Fetal bovine serum) group showed the highest cell proliferation potential. EGF and TGF groups showed remarkable cell proliferation, but there was no significant difference in IGF and FGF groups comparing to control group. A partial clone that encodes the rabbit type II procollagen C-propeptide region(RbCo12A1) was successfully isolated by reverse transcription-polymerase chain reaction using total RNA extracted from articular chondrocytes of rabbits. The identity of the cDNA clone was confirmed by DNA sequencing of the polymerase chain reaction products. A comparison of human al(II) cDNA sequence showed high sequence homology(83.6%). Type I procollagen mRNA expressed highly in EGF group. FGF, IGF, and TGF groups showed no significant expression comparing to control group. FBS group showed lower expression than control group. Type II procollagen expression was increased with passage of time, so at Day 10 it was the highest in all groups. Control group showed the highest expression among 6 experimental groups. The expression of type II procollagen in FGF and TGF groups was slightly lower than that of control. EGF and IGF groups showed markedly decreased expression comparing to control group. That in FBS group was the lowest, so it was three times lower than control group. In immunohistochemical stains with type I collagen, there was no difference among control, FBS, and EGF groups. FGF, IGF, and TGF groups showed increased positivity on stain comparing to control group, but the positivity didnt exceed 10%. For type II collagen, EGF and FGF groups showed decreased positivity, but there was no significant difference in FBS, IGF, and TGF groups comparing to control group. On the basis of this study, it may be concluded that TGF-pl showed the possibility of regeneration or delay the degeneration process of the intervertebral disc through the contribution to the stimulatory effects of cell proliferation and the synthesis of type II collagen. For the clinical use of this, more studies about the combination effects with FBS or other kinds of growth factors and finding out the ideal concentration about TGF-pl will be needed.