Gait cycle comparions of cruciate sacrifice for total knee design.-explicit finite element

Gait cycle comparions of cruciate sacrifice for total knee design.-explicit finite elementope

Phenotypical Stability and Matrix Synthesis of Human Intervertebral Disc Cells in Response to Dexamethasone and Transforming Growth Factor-β1

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-β1 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-β1) 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-β1 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-β1 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-β1. Conclusions: DEX demonstrated a proliferative effect on human IVD cells, with the combination of DEX and TGF-β1 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.ope

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors

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-β1 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℃, and then lyophilized at -50℃. 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-β1 (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-β1 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-β1 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.ope

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.ope

아텔로 콜라겐 지지체와 성장 인자로 배양된 수핵세포를 이용한 조직 공

Dept. of Medical Science/석사[한글]연구계획 : 실험자 연구 연구목적 : 제 1형, 2형 아텔로콜라겐 지지체에 TGF-β1, BMP-2를 투여하여 이식된 추간판 세포의 세포 증식, 기질 생성, 표현형 발현을 알아보기 위함.연구배경 : 최근, 조직공학은 퇴행성 추간판 질환의 생물학적 치료를 위한 미래의 새로운 연구 기술로 언급되고 있으며, 이는 신체의 조직과 기관의 재생을 가능케 하는 전도유망한 기술로 각광받고 있다. 인공 지지체에 세포를 이식하는 연구는 세포의 생존 및 증식, 재생되려는 조직의 고유한 표현형 유지, 그리고 성장인자와 같은 생물학적인 자극 등의 조건들이 모두 조화를 이루어야만 한다.대상 및 방법 : 뉴질랜드 흰 토끼 30마리로부터 추간판 조직내의 수핵 부위를 분리하고 순차적 효소처리에 의해 수핵 세포를 배양한다. 각각 1%의 제1형, 2형 아텔로콜라겐을 96-well plate에 넣고, -70℃에서 냉동시킨 후 다시 -50℃에서 냉동건조 처리를 한다. 이렇게 제작된 다공성의 교원질 물질은 교차 결합 방식에 의해 지지체로 완성된다. 표면 장력을 이용하여 추간판 세포들이 아텔로콜라겐 지지체로 이식되고, 세포가 이식된 지지체에는 각각10ng/ml의 TGF-β1, 100ng/ml의 BMP-2, 그리고 두 성장인자들이 1:1로 혼합된 용액을 첨가한다. 배양한 지 각각 1, 2, 4주 째 되는 날, [3H]-thymidine incorporation을 통해 DNA의 양을 측정하고, [35S]-sulfate incorporation을 통해서 기질 생성량을 측정한다. 또한 RT-PCR을 통해 기질성분인 aggrecan, 제1형, 2형 교원질 그리고 골성인자인 osteocalcin의 mRNA 발현 정도를 알아보고, 주사 전자 현미경 관찰을 통해 세포가 이식된 아텔로콜라겐 지지체의 내부 형태를 관찰한다.결과 : TGF-β1이 투여된 제2형 아텔로콜라겐 지지체 배양군은 세포의 기질 생성이 증가하였고, 기질 성분인 aggrecan, 제1형, 2형 교원질의 mRNA 발현도 대조군에 비해 유의하게 증가하였다. 한편, 제1형 아텔로콜라겐 지지체 배양군은 세포의 증식이 매우 활발하였으며 제 1형, 2형 교원질의 mRNA 발현도 증가하는 양상을 보였다. 그렇지만 어떤 지지체의 배양군에서도 추간판 연골 세포의 골성인자인 osteocalcin의 mRNA 발현은 나타나지 않았으며, 기질 생성과 기질 성분의 mRNA 발현에 있어서 TGF-β1과 BMP-2간의 상호 상승작용은 나타나지 않았다. 한편 주사전자현미경 상으로 이식된 세포가 각각의 지지체에 안정적으로 부착된 상태이면서 세포 표면에 기질이 방출된 것을 확인하였다.결론 : 제1형, 2형 아텔로콜라겐 지지체에 이식 배양된 토끼의 추간판 수핵 세포는 생존 가능하였고, 제1형 아텔로콜라겐 지지체는 추간판 세포의 증식이 활발하였으며, 제2형 아텔로콜라겐 지지체는 세포외 기질 생성이 제1형 아텔로콜라겐 지지체에 비해 탁월하였다. 또한 두 지지체의 배양군들은 TGF-β1 및 BMP-2 성장인자에 생물학적으로 반응하였다. 따라서 성장인자가 처리된 아텔로콜라겐 지지체에서 자란 추간판 세포는 조직공학적 추간판의 재생에 적합하였다. [영문]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-β1 and BMP-2.Summary of literature review: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment about degenerative IVD diseases and has been highlighted as a promising technique for the regeneration of tissues and organs in human body. Research on cell transplantation in artificial scaffolds should be validated in terms of cell viability and proliferation, maintenance of characteristicphenotype, and biologically active growth factor.Materials and Methods: Lumbar IVD were harvested from 10 New Zealand white rabbits, with the nucleus pulposus (NP) cells were isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5mm), frozen at -70℃, and then lyophilized at -50℃. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were then treated with TGF-β1 (10ng/ml) or BMP-2 (100ng/ml) or both. After 1, 2, and 4 week culture periods, the DNA synthesis was measured by [3H]-thymidine incorporation and newly synthesized proteoglycan was measured by incorporation of [35S]-sulfate. Reverse transcription-polymerase chain reactions for the mRNA expressions of aggrecan, types I, II collagens, and osteocalcin were performed. The inner morphology of cell seeded scaffolds was determined by scanning electron microscopy (SEM).Results: The NP cell cultures in atelocollagen type II with TGF-β1 demonstrated increase in proteoglycan synthesis and upregulation of aggrecan, types I and II collagen mRNA expressions, compared to control. IVD cultures in type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of types I, II collagen mRNA expressions. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no upregulation of the osteocalcin mRNA expression. Furthermore there was no synergistic effect of TGF-β1 and BMP-2 in matrix synthesis and mRNA expression of matrix components. The SEM images showed stable cell adhesion on each matrix and releasing of extracellular matrices on cell surfaces.Conclusion: NP cells from rabbit were viable in types I and type II atelocollagen scaffolds. Type I atelocollagen scaffold was suitable for cell proliferation, but type II atelocollagen scaffold was suitable for extracellular matrix synthesis. The NP cells in both scaffolds were biologically responsive to growth factors. Taken together, NP cells in atelocollagen scaffolds, with anabolic growth factors provide a mechanism for tissue engineering of IVD.ope

Enzymes in ginkgolide biosynthetic pathway : cloning and genomic structure

Thesis (master`s)--서울대학교 대학원 :농생명공학부,2004.Part 1 The Ginkgo biloba is a perennial woody plant growing throughout the whole region of Korea. It accumulates various ginkgolides, whose oxidative modification step of terpenoid intermediates in biosynthesis is monooxygenation by cytochrome P450 monooxygenases. The partial cDNAs from G. biloba root using homology-based PCR yielded five distinct cytochrome P450 families (GbCYP&#8729;A, GbCYP&#8729;B, GbCYP&#8729;C, GbCYP&#8729;D, GbCYP&#8729;E). The GbCYP&#8729;E sequence information derived from rapid amplification of cDNA ends (RACE) was used to produce 1655 bp of a full-length cDNA sequence including 1455 bp of an open reading frame for 485 amino acids with a deduced size of 55.172 kDa. The deduced amino acid sequence displayed 29% identity with taxane 14β-hydroxylase of Taxus cuspidata. Also, a cytochrome P450 cDNA which was 2038 bplong and partially deficient of the 5 -UTR (untranslated region) has been isolated from the cDNA library of G. biloba root, naming it as GbCYP&#8729;F. This showed to have an open reading frame 1761 bp-long and a 586 amino acid sequence with its size 66.196 kDa. The deduced amino acid sequences of GbCYP&#8729;F displayed 75% identity with CYP97B2 monooxygenase of Glycine max. This is the first report on cDNA isolation of the cytochrome P450 genes from G. biloba.Maste

조직생체배양기 내에서 배양된 인대 및 건 조직 재건용 줄기세포 담지 생체유래 지지체의 분석

Dept. of Medical Science/박사Since many years, the injury of anterior cruciate ligament (ACL) has been cured by the biological grafting material such as autograft and allograft. These natural scaffolds derived from human tissues have many advantages, including eliminated donor site morbidity, shortened operation time, large-sized bone grafts, minimized surgical incisions, and reduced incidence of arthrofibrosis. However there are potential disadvantages such as some risk of viral and bacterial disease transmission, potential for immune rejection, and longer incorporation time, which can require longer protection from potentially injurious effects. However, donor screening, high pressure saline washing, antibiotics, and sterilization may safely preserve them from those disadvantages. Besides, it became known that natural graft can affect patient’s immune rejection by its own cellular components remaining in the tendon as antigens. Thus, the important of decellularization is increased for the safety of tendon graft. Decellularized tissues are successfully used in tissue engineering and regenerative medical field for the purpose of removing tissue antigens from a cellular component. However, chemical decellularization which is processed by ionic solution or enzymatic treatment that may decrease the mechanical property of tendon graft and deform the extracellular matrix (ECM). In addition, some cleaning detergents can be harmful and dangerous when it remains in the patient’s body. Even though the decellularization technique is considered as effective and safe method for tissue sterilization, most of soft tissues are processed restrictively by antibiotics or high pressure saline washing. While decellularization technique is used in tissue sterilization to remove immunoreactive material, the property of tendon graft must be preserved of mechanical strength decline by decellularization must be recovered. For this study, we designed a tissue bioreactor to give biaxial cyclic loading to the tissue. The advanced bioreactor is expected to provide ideal conditions for the cultivation of functional tissue grafts. The system of tissue bioreactor is composed of motion part that gives mechanical stimulation such as translational and rotational strain, control part that regulates time, frequency, temperature, and gases such as CO2, and O2 and chamber part that receives physical stimulation conditionally. The purpose of this study is to confirm ECM synthesis and mechanical efficiency of cell-natural scaffold construct that is cultured under cyclic loading in the tissue bioreactor. During decellularization process, porcine anterior tibialis tendons were treated with enzymatic solution and ultrasonic cleaning. In the final step, hypertonic and hypotonic solutions were rinsed to remove residue during cell lysis. Decellularized tendons were recellularized by bone marrow derived stem cell-collagen mixture, and cultivated under cyclic loading in the bioreactor. In this study, the ultimate tensile load of the decellularized tendons was lowered by 20% than the fresh tendon. However, after cultivation in the tissue bioreactor with cyclic loading, which was conditioned as 10% of tension, 90° of torsion and 1Hz of frequency, the ultimate tensile load was further increased as compared with the fresh tendon. This study shows that the natural graft is decellularized as biologically safe and the cyclic loading in the bioreactor system increases the mechanical property of engineered cell-natural scaffold construct compared with the fresh natural graft. The recellularized natural scaffold composite, which is cultivated under cyclic loading in the bioreactor, will be essential for the ligament and tendon reconstruction.ope

Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using atelocollagen scaffold and growth factors

STUDY DESIGN: In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors. OBJECTIVE: To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II. SUMMARY OF BACKGROUND DATA: Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before. METHODS: Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-β1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed. RESULTS: Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-β1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-β1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-β1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05). CONCLUSION: NP cells in atelocollagens under the stimulation of TGF-β1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.ope

Herniated Intervertebral Disk Induces Hypertrophy and Ossification of Ligamentum Flavum

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). OBJECTIVES: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. METHODS: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. RESULTS: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. CONCLUSIONS: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.ope