A gene related study with a review of current osteoporosis medications and a comparison between two kinds of BMP-2

학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2019. 8. 이재협.Osteoporosis is caused by an imbalance between bone formation and bone resorption that results in low bone mass and deteriorated bone microstructure and finally elevates the risk of low-trauma fracture. For developing new therapies for managing osteoporosis, this study compromised 3 stages as follows: 1, the bone formation efficacy of recombinant human bone morphogenetic protein 2 (rhBMP-2) was investigated since bone substitute is necessary for osteoporosis patients; 2, the efficacy of currently available medications was analyzed via meta-analysis; 3, the impact brought by the mutation in Drg2 in bone homeostasis was researched. Their methods and results were as follow. In the first part, we compared the osteoinductivity of Escherichia coli rhBMP-2 (ErhBMP-2) with Chinese hamster ovary cell-derived rhBMP-2 (CrhBMP-2) with human mesenchymal stem cells and rat calvarial defect. In the second part, we systematically reviewed the effect of current osteoporosis medications on preventing secondary osteoporotic vertebral and non-vertebral fractures from randomized controlled studies and synthesized their result via meta-analysis. In the third part, we compared the transcription level of DRG2 in osteoporosis and non-osteoporosis subjects, and furtherly fabricated Drg2 knockout mice and analyzed the difference in bone phenotype of wild type and Drg2 knockout mice. At the end of this part, we investigated the possible mechanisms and signals Drg2 involved in osteoblastic differentiation. The results from the first part showed ErhBMP-2 could have comparable osteoinductivity with Chinese hamster ovary cell-derived BMP-2 while using the demineralized bone matrix as the carrier. In the second part, we found the medications could have a consistent effect on osteoporosis patients, regardless of their fracture history. And in the third part, we found osteoporosis patients had higher expression level of Drg2 and knocking out of Drg2 in mice significantly improved bone mass and mineral density even if mice were ovariectomized. The bone marrow-derived macrophage in Drg2 knockout mice showed lower osteoclastogenesis while the bone marrow mesenchymal stem cell concurrently showed higher osteoblastogenesis than wild type mice. Furtherly, inhibition of Drg2 expression in mouse MC3T3-E1 cells elevated its osteogenicity via canonical and non-canonical BMP pathway. In summary, we found the ErhBMP-2 might have the potential of being used as an anabolic agent for osteoporosis fracture; currently available medications could have a significant effect on preventing secondary osteoporotic fracture; and Drg2 as an important regulator in bone remodeling, which suggested Drg2 inhibitor could be a potential anabolic for treating osteoporosis.골다공증은 골량 감소와 골 미세구조 이상을 야기하는 질환으로 골형성과 골흡수간 불균형에 의해 발생하며, 저손상 골절의 위험을 증가시키는 질환이다. 새로운 골다공증 치료법을 개발하기 위하여 다음 3단계의 연구를 진행하였다. 골다공증 환자는 골대체제가 필요하기 때문에 재조합 골형성단백질 제2형(rhBMP-2) 의 골형성 효능 연구, 현재 사용되는 골다공증 치료제의 2차 골절예방 효능에 관한 메타 분석 연구와 함께, developmentally regulated GTP binding protein 2 (Drg2) 의 골 항상성에 미치는 영향을 연구하였다. 첫번째 연구에서는, 대장균 유래 골형성 단백질 제2형(ErhBMP-2)과 동물세포 유래 골 형성 단백질 제2형(CrhBMP-2)의 골유도성을 인간 간엽줄기세포 및 랫드 두개골 결손모델에서 비교하였고 두번째 연구에서는, 기존 골다공증 치료제가 골다공증성 척추 및 비척추 골절을 예방하는 효과에 대하여 메타분석을 시행하였으며 세번째 연구에서는, 골다공증이 있는 환자군과 정상 대조군의 골수 유래 간엽줄기세포 에서 DRG2의 발현을 비교하였고, Drg2 결손 마우스를 제작하여 대조군과 골 표현형의 차이를 분석하였다. 또한 Drg2 가 조골세포의 분화에 관여하는 기전과 신호전달 연구를 수행하였다. 첫번째 연구에서 ErhBMP-2가 탈회골기질을 담체로 사용할 때 CrhBMP-2와 유사한 골 유도능력을 가질 수도 있음을 확인하였으며 두번째 연구에서는 골다공증 환자의 골절 병력과 무관하게 약물치료가 지속적인 영향이 있을 수 있다는 것을 알 수 있었습니다. 세번째 연구에서 골다공증 환자는 Drg2 mRNA 발현 정도가 정상 대조군보다 더 높았고, Drg2 결손마우스는 난소절제술을 시행하였을 때 골량 보호 효과가 관찰되었다. Drg2 결손 마우스에서 얻은 골수유래 대식세포를 대조군과 비교했을 때, 파골세포 분화력이 낮았으며 골수유래 줄기세포의 조골세포 분화력은 높았다. 또한 마우스 MC3T3-E1 세포에서의 Drg2 발현을 억제시키면 정식 및 비정식 BMP 경로를 통하여 조골세포의 분화가 증가하였다. 결론적으로, 본 연구에서는 ErhBMP-2가 골다공증성 골절에서 동화제제로서 사용 될 수 있는 가능성과 Drg2 유전자가 골 재형성에 있어 중요한 조절 인자임을 확인하였다.Abstract I Table of Contents: IV List of Tables VI List of Figures VII Introduction 1 Methods 5 Osteoinductive treatment of human mesenchymal stem cells 5 ALP staining and and ALP activity assay 5 Calcium staining and assay 6 Real-time PCR 7 Rat calvarial defect model 7 Micro-CT evaluation 8 Hematoxylin and eosin staining 9 Search for studies 10 Selection of studies 10 Data extraction and risk of bias 11 Data analysis and quality of evidence 12 Extraction of mesenchymal stem cells from human 13 Fabrication of DRG2 knock out mouse 13 Genomic typing and gender determination 14 Primary culture of BMMCs and bone marrow MSC 14 TRAP staining 15 shRNA transfection of MC3T3-E1 cell 15 Inducing osteoblastic differentiation in MC3T3-E1 cells 16 Western blot 16 Semiquantitative RT-PCR 17 Feeding and maintaining 17 Serum P1NP and CTX measurement 18 Ovariectomy 18 Calcein labeling 19 Statistics 19 Results 20 ALP assay 20 ALP staining 20 Calcium assay 20 Alizarin red staining 21 Real-time PCR 21 Animal experiments 21 Characteristics of included studies and risk of bias 22 Comparison with control group 24 Comparison between interventions 29 Higher DRG2 expression correlates with lower BMD 30 Knocking out of Drg2 affects mice postnatal bone formation 30 Inhibition of DRG2 improves bone architecture and BMD even in ovariectomized mice 31 Results of the GO enrichment and KEGG pathway analysis 33 Inhibiting the expression of DRG2 inhibits the osteoclastic differentiation of BMMCs and elevates osteoblastic differentiation of bone marrow MSCs 33 Inhibiting the expression of DRG2 elevates osteogenicity of MC3T3-E1 cells 34 Inhibition of DRG2 elevates OB differentiation via canonical and non-canonical BMP signaling 35 Discussion 37 The first section 38 The second section 41 The third section 46 References 50 Figures 61 Table 90 Supplementary material 104 논 문 초 록 123 Acknowledgments 126Docto

Prediction of Ebolavirus Genomes Encoded MicroRNA-Like Small RNAs Using Bioinformatics Approaches

Recent findings revealed that certain viruses encoded microRNA-like small RNAs using the RNA interference machinery in the host cells. However, the function of these virus-encoded microRNA-like small RNAs remained unclear, and whether these microRNA-like small RNAs were involved in the replication of the virus and viral infection was still disputable. In this chapter, the negative-sense RNA genome of Ebola virus (EBOV) was scanned using bioinformatics tools to predict the EBOV-encoded microRNA-like small RNAs. Then, the potential influence of viral microRNA-like small RNAs on the viral immune evasion, host cellular signaling pathway, and epigenetic regulation of antiviral defense mechanism were also detected by the reconstructed regulatory network of target genes associated with viral encoded microRNA-like small RNAs. In this analysis, EBOV-encoded microRNA-like small RNAs were proposed to inhibit the host immune response factors, probably facilitating the evasion of EBOV from the host defense mechanisms. In conclusion, systematic investigation of microRNA-like small RNAs in EBOV genome may shed light on the underlying molecular mechanisms of the pathological process of Ebola virus disease (EVD)

Effect of medications on prevention of secondary osteoporotic vertebral compression fracture, non-vertebral fracture, and discontinuation due to adverse events: a meta-analysis of randomized controlled trials

Background Bone loss with aging and menopause increases the risk of fragile vertebral fracture, osteoporotic vertebral compression fracture (OVCF). The fracture causes severe pain, impedes respiratory function, lower the quality of life, and increases the risk of new fractures and deaths. Various medications have been prescribed to prevent a secondary fracture, but few study summarized their effects. Therefore, we investigated their effects on preventing subsequent OVCF via meta-analyses of randomized controlled trials. Methods Electronic databases, including MEDLINE, EMBASE, CENTRAL, and Web of Science were searched for published randomized controlled trials from June 2015 to June 2019. The trials that recruited participants with at least one OVCF were included. We assessed the risk of bias of every study, estimated relative risk ratio of secondary OVCF, non-vertebral fracture, gastrointestinal complaints and discontinuation due to adverse events. Finally, we evaluated the quality of evidence. Results Forty-one articles were included. Moderate to high quality evidence proved the effectiveness of zoledronate (Relative Risk, RR: 0.34; 95% CI, 0.17–0.69, p = 0.003), alendronate (RR: 0.54; 95% CI: 0.43–0.68; p < 0.0001), risedronate (RR: 0.61; 95% CI: 0.51–0.73; p < 0.0001), etidronate (RR, 0.50; 95% CI, 0.29–0.87, p < 0.01), ibandronate (RR: 0.52; 95% CI: 0.38–0.71; p < 0.0001), parathyroid hormone (RR: 0.31; 95% CI: 0.23–0.41; p < 0.0001), denosumab (RR, 0.41; 95% CI, 0.29–0.57; p < 0.0001) and selective estrogen receptor modulators (Raloxifene, RR: 0.58; 95% CI: 0.44–0.76; p < 0.0001; Bazedoxifene, RR: 0.66; 95% CI: 0.53–0.82; p = 0.0002) in preventing secondary fractures. Moderate quality evidence proved romosozumab had better effect than alendronate (Romosozumab vs. alendronate, RR: 0.64; 95% CI: 0.49–0.84; p = 0.001) and high quality evidence proved that teriparatide had better effect than risedronate (risedronate vs. teriparatide, RR: 1.98; 95% CI: 1.44–2.70; p < 0.0001). Conclusion Zoledronate, alendronate, risedronate, etidronate, ibandronate, parathyroid hormone, denosumab and selective estrogen receptor modulators had significant secondary prevention effects on OVCF. Moderate quality evidence proved romosozumab had better effect than alendronate. High quality evidence proved PTH had better effect than risedronate, but with higher risk of adverse events.This work was supported by Mid-career Researcher Program through NRF grant (2016R1A2B3015048) funded by the Korea government (MSIP)

Effect of Whitlockite as a new bone substitute for bone formation in spinal fusion and ectopic ossification animal model

Background Bone substrates like hydroxyapatite and tricalcium phosphate have been widely used for promoting spinal fusion and reducing the complications caused by autograft. Whitlockite has been reported to promote better bone formation in rat calvaria models compare with them, but no study investigated its effect on spinal fusion yet. Also, the higher osteoinductivity of whitlockite raised concern of ectopic ossification, which was a complication of spinal fusion surgery that should be avoided. Methods In this study, we compared the osteoinductivity of whitlockite, hydroxyapatite, and tricalcium phosphate porous particles with SD rat spine posterolateral fusion model and investigated whether whitlockite could induce ectopic ossification with SD rat abdominal pouch model. Results The micro-CT result from the posterolateral fusion model showed whitlockite had slightly but significantly higher percent bone volume than tricalcium phosphate, though none of the materials formed successful fusion with surrounding bone tissue. The histology results showed the bone formed on the cortical surface of the transverse process but did not form a bridge between the processes. The result from the abdominal pouch model showed whitlockite did not induce ectopic bone formation. Conclusion Whitlockite had a potential of being a better bone substrate hydroxyapatite and tricalcium phosphate in spinal fusion with low risk of inducing ectopic ossification.This study was supported by clinical research program funded by SMG-SNU Boramae Medical Center (03–2015-1)

Comparison of demineralized bone matrix and hydroxyapatite as carriers of Escherichia coli recombinant human BMP-2

Background Autograft has been widely used in various orthopedic and dental surgery for its superior osteogenicity, osteoinductivity and osteoconductivity. But the available volume of the autograft is limited and the efficacy of it is highly affected by the condition of the patients. Therefore, growth factors such as Escherichia coli bone morphogenetic protein-2 (ErhBMP-2) has been widely used in some countries and regions with various carriers that could affect the effects of the growth factors. Demineralized bone matrix (DBM) has been widely used as a bone graft substitute and growth factor carrier, but its effect as a carrier of ErhBMP-2 was less investigated. Materials and methods Rat calvaria defect model was used in this study. We implanted ErhBMP-2 with DBM or hydroxyapatite (HA) as a carrier in 8 mm calvaria defect and compared their bone regeneration effect in 4th week and 8th week after implantation with micro-CT and histology. The data was analyzed with one-way ANOVA method with Bonferroni post-hoc analysis. Result The group with DBM as the carrier showed significantly higher bone volume and bone thickness than the groups with HA as the carrier in both weeks. And the histology sections showed less adipose tissue formed in the groups with DBM as the carrier. Conclusion DBM could be a better carrier for ErhBMP-2 than HA.This study was supported by a research grant for animal studies from Daewoong Pharmaceutical (Seoul, Korea) (No. 800–20140080)

Enhanced heat transport in thermal convection with suspensions of rod-like expandable particles

Thermal convection of fluid is a more efficient way than diffusion to carry heat from hot sources to cold places. Here, we experimentally study the Rayleigh–Bénard convection of aqueous glycerol solution in a cubic cell with suspensions of rod-like particles made of polydimethylsiloxane. The particles are inertial due to their large thermal expansion coefficient and finite sizes. The thermal expansion coefficient of the particles is three times larger than that of the background fluid. This contrast makes the suspended particles lighter than the local fluid in hot regions and heavier in cold regions. The heat transport is enhanced at relatively large Rayleigh number ( Ra ) but reduced at small Ra . We demonstrate that the increase of Nusselt number arises from the particle–boundary layer interactions: the particles act as ‘active’ mixers of the flow and temperature fields across the boundary layers