The storage condition, composition and application of platelet-rich plasma for cartilage regeneration

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의학과, 2020. 8. 이명철.Purpose: The purpose of this study was to investigate the practical use of platelet‐rich plasma (PRP) for cartilage regeneration. For this purpose, the author evaluated 1) the concentrations of growth factors in PRP, depending on the storage conditions; 2) the effects of leukocyte concentrations in PRP on the proliferation and chondrogenesis of synovial membrane-derived mesenchymal stem cells (SDSCs) and chondrocytes; and 3) in vivo effectiveness of PRP with a hyaluronic acid (HA) hydrogel for cartilage regeneration. Materials and Methods: To evaluate growth factor concentration in PRP based on storage conditions, PRP samples were stored at 24℃ (room temperature group), 4℃ (refrigerator group), and −70℃ (deep‐freezer group). In each temperature, four aliquots were prepared based on the time of analysis (immediately, 1, 3, 7 days after preparation). After storage, concentrations of platelet‐derived growth factor‐AA (PDGF‐AA), transforming growth factor‐β (TGF‐β), vascular endothelial growth factor (VEGF), insulin‐like growth factor‐1 (IGF‐1), and fibroblast growth factor‐basic (FGF‐B) were assessed with/without activation using Quantikine colorimetric sandwich immunoassay kits. PRP was activated with 10% Triton‐X for PDGF‐AA, VEGF, FGF‐B, IGF‐1 measurement and sonication for TGF‐β1 measurement. To evaluate the effect of leukocyte concentrations in PRP on the proliferation and chondrogenesis of SDSCs and chondrocytes, we prepared two PRP formulations: (1) leukocyte-poor PRP (P-PRP) and (2) leukocyte-rich PRP (L-PRP). SDSCs and chondrocytes were obtained by enzymatic digestion of synovial tissues and the cartilage from a knee joint undergoing total knee arthroplasty. The primary cells were expanded in Dulbeccos modified Eagles medium supplemented with fetal bovine serum (FBS), L-PRP, or P-PRP. Cell proliferation was measured using the MTT assay. SDSCs and chondrocytes were cultured with chondrogenic medium (CM) only, CM with L-PRP, or CM with P-PRP using a high-density pellet culture system for 3 weeks. The expression of chondrogenesis-related genes (type II collagen, type X collagen, aggrecan, and SOX-9) were analyzed by RT-qPCR. Pellets were stained with safranin-O for proteoglycan detection. The expression of type II collagen was detected by immunohistochemical staining. To evaluate the effect of PRP in combination with the HA hydrogel on in vivo cartilage regeneration, eighteen rabbit osteochondral defect models (round shape defect in the femoral trochlear groove with 4 mm in radius and 3 mm in depth) were made and divided into 3 groups: control group, in which the defect was left untreated; HA group, in which the defect was filled with the HA hydrogel; and HA-PRP group, in which the defect was filled with HA hydrogel and PRP. After 12 weeks, tissue specimens were assessed by macroscopic examination, histological evaluation, and by measuring the glycosaminoglycan (GAG) content. Results: Regarding growth factor concentration in PRP based on storage conditions, PDGF‐AA concentration was highest on day 7 in the room temperature group without activation. With activation, the concentration of PDGF‐AA was constant over the observation period at all temperatures. Without activation, the TGF‐β1 concentration remained negligible over the observation period at all temperatures. However, with activation, TGF‐β1 concentration was highest on day 7 at all temperatures. Over the observation period, VEGF and IGF‐1 concentration were constant with and without activation at all temperatures. Without activation, FGF‐B concentration was highest on day 7 in the deep‐freezer group. With activation, FGF‐B concentration decreased after day 1 in the room temperature group. Regarding the effect of the leukocyte concentration in PRP on the proliferation and chondrogenesis of SDSCs and chondrocytes, L-PRP showed a stronger proliferative effect on both types of cells than P-PRP and FBS. Meanwhile, RT-qPCR revealed higher cartilage-related gene expression in SDSCs and chondrocytes in the P-PRP group compared with that in the L-PRP and CM groups. However, SDSCs and chondrocytes in both PRP groups showed weaker safranin-O staining than those in the CM group. In immunohistochemical analysis, positive staining of SDSCs were not observed in either of the PRP groups and staining of chondrocytes in both PRP groups were weaker than those in the CM group. Regarding the effect of PRP combined with the HA hydrogel on in vivo cartilage regeneration, the macroscopic ICRS scores were not different among the three groups. The HA and HA-PRP groups showed significantly higher microscopic ODriscoll scores and a significantly higher GAG content than the control group, although the values were not different between the HA and HA-PRP groups. Conclusion: The growth factor concentrations significantly differed in PRP, depending on the storage temperature, duration of storage, and activation. Regardless of the leukocyte concentration, PRP showed a negative effect on chondrogenesis of SDSCs and chondrocytes. In addition, a combined use of the HA hydrogel and PRP did not show better chondrogenic effects in vivo compared with those of the HA hydrogel alone. Considering the results of our study, a further study is necessary to clarify the ideal composition and application of PRP for cartilage regeneration.목 적: 본 연구에서는 혈소판 풍부 혈장을 이용한 연골재생에 대해 알아보고자 한다. 이를 위해 먼저 보관조건에 따른 혈소판 풍부 혈장 내 성장인자 농도를 조사하고, 혈소판 풍부 혈장 내 백혈구 농도 차이가 인체활막세포와 연골세포에 미치는 영향을 알아본 후, 마지막으로 혈소판 풍부 혈장과 지지체의 복합체 형성이 연골재생에 미치는 영향을 알아보았다. 대상 및 방법: 보관조건에 따른 혈소판 풍부 혈장 내 성장인자 농도를 조사하기 위해, 혈소판 풍부 혈장을 24도, 4도, -70도에서 7일 동안 보관한 후 활성화 전후로 PDGF‐AA, TGF‐β, VEGF, IGF‐1, FGF‐B 농도를 ELISA를 이용하여 측정하였다. 또한 혈소판 풍부 혈장 내 백혈구 농도 차이가 인체활막세포와 연골세포에 미치는 영향을 알아보기 위해, 백혈구 풍부 (Leukocyte-rich)와 순수 (Leukocyte-poor) 혈소판 풍부 혈장을 제조하고 이들을 각각 인체활막세포, 연골세포와 같이 3주간 pellet 배양하였다. 이후 세포 증식을 MTT assay로 평가하고, 연골형성능은 RT-PCR로 연골형성에 관련된 유전자 (Type II collagen, Type X collagen, SOX-9, aggrecan) 발현을 측정한 후 조직학/면역학적 염색을 시행하였다. 마지막으로 혈소판 풍부 혈장과 지지체의 복합체 형성이 연골재생에 미치는 영향을 조사하기 위해, 토끼 대퇴 활차에 골연골 결손을 만들고 대조군, 지지체군, 복합체군 (혈소판 풍부 혈장+지지체)으로 나눈 후 12주에 ICRS macroscopic score와 Modified ODriscoll score를 얻고 DMB assay를 통해 GAG 합성을 평가하였다. 결 과: 보관조건에 따른 혈소판 풍부 혈장 내 성장인자 농도의 경우, 활성화 전 PDGF‐AA 농도는 24도에서 7일째 가장 높았고, TGF‐β는 모든 온도에서 7일 동안 잘 검출되지 않았으며, VEGF와 IGF‐1 농도는 모든 온도에서 7일 동안 일정하게 유지되고, FGF‐B 농도는 -4도에서 7일째 가장 높았다. 활성화 후 PDGF‐AA, VEGF, IGF‐1 농도가 모든 온도에서 7일 동안 일정하게 유지된 것에 반해, TGF‐β 농도는 모든 온도에서 7일에 가장 높았고, FGF‐B 농도는 24도에서 1일 후 급격히 감소하는 양상을 보였다. 혈소판 풍부 혈장 내 백혈구 농도 차이가 인체활막세포와 연골세포에 미치는 영향의 경우, 백혈구 풍부 혈소판 풍부 혈장에서 인체활막세포와 연골세포 모두 보다 큰 증식능력을 보였으나, RT-PCR의 경우 두 세포 모두 백혈구 순수 혈소판 풍부 혈장에서 보다 많은 유전자 발현이 관찰되었다. 하지만 인체활막세포와 연골세포의 조직학/면역학적 염색상, 백혈구 농도에 관계없이 두 혈소판 풍부 혈장 모두에서 연골 분화가 억제되는 양상이 관찰되었다. 혈소판 풍부 혈장과 지지체의 복합체 형성이 연골재생에 미치는 영향의 경우, 세군 모두에서 ICRS macroscopic score에 차이가 없었다. Modified ODriscoll score와 GAG 농도의 경우, 지지체군과 복합체군 모두 대조군보다 높았으나, 두 군 간에는 차이가 관찰되지 않았다. 결 론: 혈소판 풍부 혈장에서 방출되는 성장인자들의 농도는 보관온도, 보관기간, 활성화 여부에 따라 큰 차이가 있었다. 또한 혈소판 풍부 혈장 내 백혈구 농도에 관계없이 혈소판 풍부 혈장은 인체활막세포와 연골세포의 연골형성에 부정적인 영향을 보였으며, 혈소판 풍부 혈장과 지지체의 복합체 사용은 지지체 단독 사용에 비해 향상된 연골형성으로 이어지지 못했다. 이번 연구결과들을 기반으로 연골재생을 위한 최적의 혈소판 풍부 혈장 사용에 대한 추가연구가 필요 할 것으로 생각된다.Introduction 1 Chapter I. Effect of storage conditions and activation on growth factor concentration in platelet‐rich plasma 6 I-1. Materials and Methods 7 I-2. Results 8 I-3. Discussion 17 Chapter II. The effect of leukocyte concentration in platelet‐rich plasma on the proliferation and chondrogenesis of synovium-derived mesenchymal stem cell and chondrocytes 22 II-1. Materials and Methods 23 II-2. Results 28 II-3. Discussion 35 Chapter III. The effect of platelet‐rich plasma with hyaluronic acid hydrogel on in vivo chondrogenesis in a rabbit osteochondral defect model 40 III-1. Materials and Methods 41 III-2. Results 47 III-3. Discussion 53 Conclusion 56 References 57 국문 초록 69Docto

A network analysis of ¹⁵O-H₂O PET reveals deep brain stimulation effects on brain network of Parkinson's disease

PURPOSE: As Parkinson's disease (PD) can be considered a network abnormality, the effects of deep brain stimulation (DBS) need to be investigated in the aspect of networks. This study aimed to examine how DBS of the bilateral subthalamic nucleus (STN) affects the motor networks of patients with idiopathic PD during motor performance and to show the feasibility of the network analysis using cross-sectional positron emission tomography (PET) images in DBS studies. MATERIALS AND METHODS: We obtained [¹⁵O]H₂O PET images from ten patients with PD during a sequential finger-to-thumb opposition task and during the resting state, with DBS-On and DBS-Off at STN. To identify the alteration of motor networks in PD and their changes due to STN-DBS, we applied independent component analysis (ICA) to all the cross-sectional PET images. We analysed the strength of each component according to DBS effects, task effects and interaction effects. RESULTS: ICA blindly decomposed components of functionally associated distributed clusters, which were comparable to the results of univariate statistical parametric mapping. ICA further revealed that STN-DBS modifies usage-strengths of components corresponding to the basal ganglia-thalamo-cortical circuits in PD patients by increasing the hypoactive basal ganglia and by suppressing the hyperactive cortical motor areas, ventrolateral thalamus and cerebellum. CONCLUSION: Our results suggest that STN-DBS may affect not only the abnormal local activity, but also alter brain networks in patients with PD. This study also demonstrated the usefulness of ICA for cross-sectional PET data to reveal network modifications due to DBS, which was not observable using the subtraction method.ope

A Study of Expression through Reinterpretation of Images in Terms of Visual Perception: With a Focus on the Researchers Own Works

학위논문 (석사)-- 서울대학교 대학원 : 동양화과, 2012. 8. 정형민.시각예술은 보는 것을 근간으로 한다. 우리는 보는 것으로부터 가장 많은 정보를 외부로부터 받아들이고, 이에 의존하며 살아가고 있기 때문에 , 본다는 행위가 가지는 주관적인 측면을 쉽게 잊어버린다. 눈을 통해 본다는 것은, 우리가 인간이기 때문에 인간의 방식으로만 볼 수 있다. 눈이 없는 생물은 빛이 없는 환경에서도 다른 정보들을 이용해 살아간다. 인간으로서 무엇을 본다는 행위는 객관적이며 수동적인 것이 아니라, 상대적이며 능동적인 행위이다. 본다는 행위의 상대적인 특성은 더 나아가 각 개인에게 자신을 둘러싼 환경에 의한 자신만의 고유한 시각적 상징체계, 즉 보는 법을 가지게 한다. 보는 법은 인간이 성장함에 따라 둘러싼 환경에 적응하는 과정에서 수정되고 발전된다. 보는 법은 철로와 같다. 우리는 어떤 철로를 달릴 것인지 결정할 수 있는 기관사일 수 있지만 철로가 없이는 아예 기차가 달릴 수 없다. 무언가를 알아본다는 것은 이미 그것에 대해 철로가 놓여있는 것이다. 새로 생기는 지점이나 사라지는 길들은 기차들의 흐름과 앞서 존재하는 철로로부터 자유로울 수 없다. 앞서 존재하는 사회문화적 현상을 보기 위해 우리는 매체를 학습해야 한다. 여기서 매체란 사람과 사람 사이를, 어제와 오늘을 이어 주는 광범위한 사회적 연결망이다. 이 매체는 시대, 사회마다 다르다. 과거에는 이러한 차이들이 자연환경과 지리적 거리에 기인했지만, 오늘날에는 빠르게 변화하고 있다. 이 변화의 방향은 무엇보다도 빠른 정보의 전달속도에 의해 결정된다. 빠르게 정보가 교환되어 질수록 매체는 단순히 사람과 사회를 이어주는 기능을 넘어 제2의 세계, 가상세계가 되었다. 새로운 매체의 변화는 예술가에게 매체에 대한 이해를 새로운 과제로서 요구한다. 이 논문은 보는 법에 대한 위의 내용들을 크게 두 장으로 나누어 본인의 작업과 관련하여 분석하려 하였다. 먼저, 눈의 빛을 받아들이는 시각구조상의 특징을 이용하여 착시를 유도한 작업을 진행하였고, 더 나아가 감각적 착시만을 이용하는 것을 넘어 문화적으로 고정된 보는 법에 개입하여 지각적 착시를 유도하였다. 둘째, 기존의 유명한 도상들을 차용하여 현재 일어나고 있는 매체의 변화를 은유적으로 표현하는 작업을 진행하기위해, 차용한 각 도상의 상징을 현 시점에 통용되고 있는 의미로 분석하고, 이를 응용해 새로운 상징을 도상결합을 통해 만들어내었다. 위와 같은 일련의 작업들은 석사과정 동안 해왔던 여러 가지 생각들을 시각적으로 구체화한 것이다. 작업들의 이미지 자체는 사실 아무것도 아니다. 이는 내 생각을 전달하는 수단이지 목적이 아니다. 또한 이 논문의 목적 역시 내 작업을 설명하기 위한 것이 아니라 이와 같은 시대적 상황을 이해하고 그 속에서 나의 역할을 정의하는 것에 있다. 즉, 예술가는 상징의 교환을 통하여 실재와 허구를 뛰어넘어 지각적 차원에서 관람자에게 이상적인 꿈을 경험할 수 있도록 해 주는 배역이다. 주요어 : 상징, 이미지, 시각, 복제, 매체, 도식, 착시 학 번 : 2010-21265The visual arts are based on seeing. Because we receive the greatest amount of information from the outside through and live by depending on seeing, we easily forget the subjective aspect of the act of seeing. Because we are human beings, even seeing with the eyes can occur only in a human way. Living beings without eyes live even in environments without light by using other types of information. The act of seeing as a human being is not an objective and passive one but a relative and active one. Furthermore, the relative characteristics of the act of seeing prompt each individual to possess a unique visual symbol system according to the surrounding environment, or a way of seeing. Ways of seeing are modified and developed in the process through which humans adapt to the surrounding environment as they grow. They are like railroads. While we may be engineers who can decide which railroad to travel, trains cannot travel at all without railroads. Recognizing something means that there already is a railroad to that object. Nor can newly generated points and disappearing roads be free from the flow of trains and preexisting railroads. To see preexisting sociocultural phenomena, we must study media. Here, media are broad social network that connect one person to another and yesterday to today. These media differ for each era and society. In the past, such differences arose from the natural environment and geographical distancetoday, they are changing swiftly. The direction of such changes is determined above all by the rapid rates at which information is transmitted. With the increasingly rapid exchange of information, media have become a second world, a virtual world, transcending the role simply of connecting people and society. Changes in new media make a demand, as a new task, that artists understand them. The present study analyzed the above contents regarding ways of seeing in two parts, with respect to the present researchers artistic works. First, it created optical illusions by using the characteristics of the optical structure that receives light and, moreover, created cognitive optical illusions by intervening in culturally fixed ways of seeing, transcending merely sensory optical illusions. Second, to express metaphorically current changes in media by borrowing famous existing symbol images, the symbolism of each of the borrowed images was analyzed in terms of currently common significance and, by applying this, new symbolism was created through combination with the symbols. The series of tasks above are visual concretization of diverse ideas that the present researcher held throughout the masters program. In fact, however, the images from these tasks are nothing. They are but the means to deliver my thoughts, not the ends. In addition, the purpose of the present study likewise lies not in explaining my artistic works but in understanding the situation of the present age and to define the role of the present researcher in it. In other words, the artists role is to allow the viewer to experience ideal dreams on a cognitive level by transcending actuality and fiction and through the exchange of symbols. Keywords: Symbols, images, vision, replication, media, schemata, optical illusions Student ID: 2010-21265차 례 국문초록 ·ⅰ 차 례 ·ⅲ 도판목록 ·ⅴ Ⅰ. 서론 1. 연구목적·1 2. 연구내용·1 3. 연구방법·3 Ⅱ. 시지각적 개입을 통한 착시표현 1. 인간의 시감각적 특징을 이용한 착시표현·9 2. 매체로의 개입을 통한 지각적 착시표현·21 3. 가치체계 개입을 통한 지각적 착시표현·30 4. 이미지의 지각적 재해석을 통한 표현·51 Ⅲ. 도상의 재해석을 통한 매체변화의 은유적 표현 1. 반가사유상을 차용한 표현·60 2. Apple사(社)의 로고를 차용한 표현·72 Ⅳ. 맺음말 1. 결론·84 2. 후속연구·86 참고문헌 ·88 부록 ·92 영문초록 ·95Maste

뇌파와 기능적 자기공명영상의 동시 측정 기법을 이용한 수면과 각성시 기능적 뇌 네트워크의 탐색 연구

Dept. of Medical Science/박사Sleep is regulated by modulation of neural interaction between particular brain regions. Brain neural activity can be estimated by various neuroimaging methods such as electroencephalography (EEG), functional magnetic resonance imaging (fMRI) and so on. Further analysis of the acquired data based on graph theoretical approaches aims to identify the functional brain networks. Moreover, in the recent neuroscience field, many research studies have defined the brain as a large complex system that is organized by the reciprocal interactions between brain regions.To investigate the changes of functional brain network during sleep and wakefulness (awake), we acquired the data from 11 healthy participants using simultaneous EEG and fMRI. The sleep data were obtained approximately 7 hours while participants are sleeping. Unfortunately, the artifacts caused by movements of participants as well as characteristics of instruments had occurred inevitably during the process of data acquisition. Thus we removed these artifacts using the average artifact subtraction for EEG data and the sub-volume utilization for fMRI data. Then, the preprocessed sleep data were visually classified and scored for every 30 sec epochs by sleep S1, S2, S3 and REM according to the international standard of AASM (American Academy of Sleep Medicine) guideline. In network analysis, to construct the functional brain network, we defined nodes depicting 95 regions of interest using brain anatomical atlas from the Automated Anatomical Labeling (AAL) and Freesurfer. For the construction of functional connectivity we applied the Pearson’s correlation coefficient between pairs of node time series.The ventral diencephalon (vDC) seeded functional connectivity has dense connections in the sleep associated regions with subcortex during sleep stages rather than wakefulness. Comparing the network topology of whole brain, we observed the main effect of sleep in the mean clustering coefficient that was significantly increased during sleep S2, S3 and REM compared with wakefulness. The global / local efficiency showed significant differences between the pairs of only sleep S1, the transitional state from wake to sleep. This functional brain network could be decomposed with independent functional subnetworks revealed by graph ICA (independent component analysis). Our results support the assumption that the functional brain network is organized by locally distributed unit functions and it is functionally segregated during sleep.In this study, we observed that the sleep data could be acquired by concordant EEG and fMRI during very long time. Our result demonstrated that the functional brain network could be reorganized by the dynamic changes of spontaneous brain activity depending on sleep stages.restrictio

SI 엔진의 흡기관 내에서의 유량 측정에 관한 연구

학위논문(석사)--서울대학교 대학원 :기계공학과,1995.Maste

Transient air fuel ratio control of a gasoline engine using an integration type ultrasonic flow meter

학위논문(박사)--서울대학교 대학원 :기계공학과,2000.Docto

Activation of the Occipital Cortex and Deactivation of the Default Mode Network During Working Memory in the Early Blind.

Although blind people heavily depend on working memory to manage daily life without visual information, it is not clear yet whether their working memory processing involves functional reorganization of the memory-related cortical network. To explore functional reorganization of the cortical network that supports various types of working memory processes in the early blind, we investigated activation differences between 2-back tasks and 0-back tasks using fMRI in 10 congenitally blind subjects and 10 sighted subjects. We used three types of stimulus sequences: words for a verbal task, pitches for a non-verbal task, and sound locations for a spatial task. When compared to the sighted, the blind showed additional activations in the occipital lobe for all types of stimulus sequences for working memory and more significant deactivation in the posterior cingulate cortex of the default mode network. The blind had increased effective connectivity from the default mode network to the left parieto-frontal network and from the occipital cortex to the right parieto-frontal network during the 2-back tasks than the 0-back tasks. These findings suggest not only cortical plasticity of the occipital cortex but also reorganization of the cortical network for the executive control of working memory. (JINS, 2011, 17, 1-16).ope

Are brain networks stable during a 24-hour period?

Despite the widespread view of the brain as a large complex network, the dynamicity of the brain network over the course of a day has yet to be explored. To investigate whether the spontaneous human brain network maintains long-term stability throughout a day, we evaluated the intra-class correlation coefficient (ICC) of results from an independent component analysis (ICA), seed correlation analysis, and graph-theoretical analysis of resting state functional MRI, acquired from 12 young adults at three-hour intervals over 24 consecutive hours. According to the ICC of the usage strength of the independent network component defined by the root mean square of the temporal weights of the network components, the default mode network centered at the posterior cingulate cortex and precuneus, the superior parietal, and secondary motor networks showed a high temporal stability throughout the day (ICC>0.5). However, high intra-individual dynamicity was observed in the default mode network, including the anterior cingulate cortex and medial prefrontal cortex or posterior-anterior cingulate cortex, the hippocampal network, and the parietal and temporal networks. Seed correlation analysis showed a highly stable (ICC>0.5) extent of functionally connected regions from the posterior cingulate cortex, but poor stability from the hippocampus throughout the day. Graph-theoretical analysis using local and global network efficiency suggested that local brain networks are temporally stable but that long-range integration behaves dynamically in the course of a day. These results imply that dynamic network properties are a nature of the resting state brain network, which remains to be further researched.ope