인테그라 피부 재생 템플릿은 친절 인테그라 생명 과학 회사에 의해 제공되었다. 작업을 지원하는 자금의 출처는 : (. 수 (Nr) 15,090,007)이 작품은 부분적으로 CIRM-BMBF 초기 번역 상 II 상 및 게놈 규제 FONDAP 센터 JTE에게 모두에 의해 자금을 조달했다.

비계의 1 준비 <ol><li> 12mm 생검 펀치를 사용하여 지지체의 샘플을 생성한다. </li><li> , 비계에 생리 활성 분자 또는 세포를 소개 부드럽게 멸균 거즈로 압박하여 발판을 배출합니다. 이어서 생체 활성 분자 또는 관심 세포를 포함하는 용액 160 μl를 첨가하여 지지체를 재수. 두 번 확인 등 MTT 분석 실험 등의 대사 분석을 통해 세포 생리 활성의 성공을. </li><li> 필요한 경우 단계 120에서 설명한 바와 같이, 재수 통해 다시 피브린 트롬빈 용액 또는 하이드로 겔, 예를 들면, 그들에 의해 전달 된 화합물 또는 세포가 지지체 내부 테스트 할 고정한다. 참고 : 화합물 또는 세포가 기계적으로 재료에 부착하지 않는 경우이 단계가 도움이 될 수 있습니다. 하나는 화합물의 방출 동역학을 제어 할 수있다 .. </li><li> cuttin에 의해, 모든 실험 및 제어 그룹에서 각 발판 아래에 배치됩니다 titanized 메쉬를 준비g 밖으로 둥근 모양의 조각, 직경 약 14mm. 참고 : titanized 메쉬 상처 침대에서 재생 조직을 구분하는 데 필요한 물리적 테두리로 작동합니다. </li></ol> 2 동물 <ol><li> 제시된 모델의 구현에 앞서, 해당 동물 보호법을 협의하고 지방 자치 단체의 허가를 얻을 수 있습니다. 이 작품에서 모든 실험은 현재 독일어 동물 복지 행위에 따라, 마우스로 수행하고 어퍼 바바리아의 지방 정부 (Regierung 폰 Oberbayern)에 의해 승인했다. </li><li> 조심스럽게 동물과 변형을 선택합니다. 생쥐 모델이 확립되었다 아니지만, 예컨대 래트, 토끼, 돼지 또는 다른 동물에 공통으로 번역 될 수있다. </li><li> 당신이 모피 동물로 작업하는 경우, 비계의 주입하기 전에 동물의 뒷면을 면도. 참고 :이 작품은 20g과 25g, 그러나 다른 연령대와 b 사이의 체중과 나이 6~8주의 마우스를 사용로디 가중치가 또한 사용될 수있다. </li></ol> 3 마취 <ol><li> 멸균 조건 하에서 작업을 수행. 동물의 정상 체온을 유지하기 위해, 온난화 매트를 사용한다. </li><li> 마취를 inhalative에 앞서 동물 / kg 체중 0.05-0.1 ㎎, 피하마다 8 시간의 용량으로 Buprenorphin을받을 수 있습니다. </li><li> inhalative 마취 (아이소 플루 란)에서 동물을 배치 또는 이에 상응하는 표준 절차를 적용 할 수 있습니다. 부드럽게 동물의 발가락을 압박하거나 피부 핀치를 통해 하나 마취를 확인합니다. </li><li> , exsiccosis 방지 동작의 시작 부분에 0.5 ㎖의 생리 식염수를 피하 주사한다. </li></ol> 피부의 절제 (4) <ol><li> 발생하기 쉬운 위치에 동물을 배치하고 앞쪽 끝이가는 영구 펜 (그림 1A)와 마우스의 뒷면의 중간 선을 표시합니다. </li><li> 절제 영역을 정의합니다. Figu에 표시된 영역에서 결함을 배치1C를 다시. 결함이 너무 하방 배치하는 경우, 동물 드레싱 및 비계를 제거하는 것이 더 경향이 있습니다. </li><li> 10mm 생검 펀치 (그림 1B)를 사용하여 양자의 결함 라운드 만듭니다. 펀치는 신중하게 피부에 대해 강제해야하며, 피부를 통해 완전히 잘라하지만 절제 영역 (그림 1C 및 1E)를 묘사하는 데 사용할 수 없습니다. </li><li> 부드럽게 집게로 표시된 피부를 들어 올려 미세 수술 가위 (그림 1 층)를 사용하여 표시된 원을 따라 절개. 출혈의 경우, 신중하게 멸균 거즈로 압축합니다. 절제의 단계별 설명은 그림 1에 나타나있다. NOTE : 결함 인해 피부 탄력에 결함의 확대를 보상하기 위해, 발판을 생성 한 것보다 더 작은 펀치로 생성된다. </li></ol> 5 비계 주입 <ol><li> TITA위한 공간을 만들기 위해을 인정했다가, 메쉬 2-4밀리미터 (그림 2A)에 대한 상처 국경에서 피부와 하부 조직의 분리를 확장 할 수 있습니다. </li><li> 직접 상처 침대에 상처 가장자리 (그림 2A &amp; B)에서 결함에 메쉬 titanized를 놓습니다. </li><li> 장소는 직접 메쉬를 통해 비계. </li><li> 약간 비계 (그림 2C)를 통해 가장자리를 떠나, 4-6 단일 노트와 인접한 상처 가장자리에 발판을 봉합. 참고 : 생분해 성 봉합사의 사용을 피하십시오. </li><li> 상처 면적 (도 2d)의 모니터링을 허용하는 동시에 발판을 보호하기 위해 상기 결함 드레싱 투명 상처 봉합. </li></ol> (6) 수술 후 케어 <ol><li> 드레싱과 비계의 상호 조작을 막아 케이지 당 하나의 마우스를 보관하십시오. </li><li> 관찰 모터 활동, 체중, 고통의 징후, 공차에 의해 매일 동물의 상태를 평가하기 일반드레싱 및 자동 절단한다. 또한 출혈, 로컬 및 감염의 전신 징후와 드레싱 위치 대 상처 영역을 감시한다. </li><li> 동물의 통증을 최소화하기 위해, 0.05-0.1 ㎎ / ㎏ (체중) 또는 동등한 진통제 약물의 투여 량은 매일 Buprenorphin 주입. </li><li> 마우스가 상처 드레싱을 제거하거나 손상되면 교체하거나 마취를 다시 연결합니다. </li></ol> 비계의 7 안락사 및 적출 <ol><li> 원하는 시점에서, 펜토 바르 비탈 (150 ㎎ / ㎏)에 의해 과량의 동물을 희생. </li><li> 비계 가능 (그림 3A)로 다시 마우스 피부의 정도를 포함해야 영구 마커, 피부 절제의 사이트를 표시합니다. </li><li> 가위 또는 메스 쌍 표시된 선을 따라 피부를 절개. (무딘 준비를 통해 하부 조직에서 인터넷을 발판과 titanized 메시를 포함하여 전체 피부를 분리gure 3B). </li><li> 페트리 접시에 펼쳐 외식 조직을 배치 (그림 3C와 D) </li></ol> 혈관 네트워크의 8 시각화 및 정량화 <ol><li> Transillumination : <ol><li> 흰색 빛 (100 와트 전구 표준)의 강한 소스 위의 페트리 접시를 장착하여, transillumination 장치를 설정합니다. </li><li> 디지털 사진을 얻기 위해 트랜스 일루미네이터 위의 디지털 카메라를 고정합니다. </li><li> 거꾸로 transillumination 장치를 통해 페트리 접시에 샘플을 놓습니다. </li><li> 정상적인 피부의 동일한 크기의 영역뿐만 아니라 매크로 모드에서 완전한 비계의 사진을 촬영합니다. 또한 디지털 분석을 위해 TIFF 형식으로 이미지를 저장합니다. </li><li> 또한 생화학 적 또는 조직 학적 분석을위한 조직을 저장합니다. </li></ol></li><li> 디지털 분할 및 정량화 : <ol><li> FR을 얻을 수있다 VesSeg - 도구 소프트웨어를 다운로드하여 설치:에서 충전 EE <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> . </li><li> VesSeg - 도구 소프트웨어를 사용하여 사진을 엽니 다. </li><li> 디지털 분석 (선택 → 이미지)를위한 발판이 적용 이미지의 영역을 선택합니다. </li><li> 옵션 &quot;히스테리시스 임계 값&quot;(계산 용기 향상 → 탑 햇 변환 → 이미지 → 선박 향상 필터 → 히스테리시스 임계 값)를 사용하여 선박의 명암을 증가합니다. </li><li> 용기지도 (이미지 → 선박 향상 필터 → 히스테리시스 임계 값의 → 임계 테두리)의 분할을 진행합니다. 제 1 임계 값 ( &quot;선박 보험&quot;), 그래서 심지어 원격으로 혈관처럼 모든 픽셀이, 용기가 부착되어 있습니다를 선택합니다. 특히 용기와 같은 용기가 부착되어 있습니다들만 픽셀을 초 임계 ( &quot;배경의 범위를&quot;)를 선택합니다. </li><li> 비 캡처 한 선박을 추가하고 일반적으로 머리카락이나 봉합 길고 얇은 혈관과 같은 구조 인 일반적인 위양성 구조를 제거하기 위해 수동으로 자동 분할 제안을 확인합니다. </li><li> 선박의 길이와 (이진 이미지 통계 → 이미지 → 이미지 통계) 선박에 의해 커버되는 전체 영역을 계산합니다. NOTE : 혈관 길이의 계산을 위해, 생성 된 혈관의 혈관 맵 1 화소 (20)의 폭과 라인 박형화된다. </li></ol></li><li> 기본 조직에 100 %의 값을 할당하는 발판과 같은 파라미터에 따라 고유의 피부 영역을 분석하여 그 값을 지지체에 관한 것이다. 흰색 픽셀의 비율이 정상 조직에서 60 %와 30 % 지지체의 경우 예를 들어, 상기 지지체의 50 %의 혈관 신생을 나타낼 것이다. 참고 : 흰색 용기 적용 범위가 발판 주위에 사각형의 영역에 할당됩니다. 흰색 픽셀 커버리지 consi의 수를 조정원의 면적은 평방 (4 ×의 R 2)보다 훨씬 작은 (π의 X의 R 2)입니다 디링. </li></ol>

<ol>
	<li>Rahaman, M. N., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+composite+tissue+constructs+for+regenerative+medicine.">Stem cell-based composite tissue constructs for regenerative medicine.</a> Biotechnology and Bioengineering. 91 (3), 261-284 (2005).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature Biotechnology. 23, 47-55 (2005).</li><li>Machens, H. G., Berger, A. C., Mailaender, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioartificial+skin.">Bioartificial skin.</a> Cells Tissues Organs. 167, 88-94 (2000).</li><li>Priya, S. G., Jungvid, H., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+tissue+engineering+for+tissue+repair+and+regeneration.">Skin tissue engineering for tissue repair and regeneration.</a> Tissue Engineering Part B: Reviews. 14, 105-118 (2008).</li><li>Papavasiliou, G., Cheng, M. H., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+vascularization+of+polymer+scaffolds.">Strategies for vascularization of polymer scaffolds.</a> Journal of Investigative Medicine. 58 (7), 838-844 (2010).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+tissue+engineering:+breathing+life+into+constructed+tissue+substitutes.">Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes.</a> Tissue Engineering. 12, 2093-2104 (2006).</li><li>Zhong, S. P., Zhang, Y. Z., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+scaffolds+for+skin+wound+healing+and+dermal+reconstruction.">Tissue scaffolds for skin wound healing and dermal reconstruction.</a> Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology. 2 (5), 510-525 (2010).</li><li>Liu, G., Zhang, Y., Liu, B., Sun, J., Li, W., Cui, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+regeneration+in+a+canine+cranial+model+using+allogeneic+adipose+derived+stem+cells+and+coral+scaffold.">Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold.</a> Biomaterials. 34 (11), 2655-2664 (2013).</li><li>Hansson, A., Di Francesco, T., Falson, F., Rousselle, P., Jordan, O., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+nanoparticles+for+directed+tissue+engineering.">Preparation and evaluation of nanoparticles for directed tissue engineering.</a> International Journal of Pharmaceutics. 439 (1-2), 73-80 (2012).</li><li>Sarkar, S. D., Farrugia, B. L., Dargaville, T. R., Dhara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan-collagen+scaffolds+with+nano/microfibrous+architecture+for+skin+tissue+engineering.">Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering.</a> Journal of Biomedical Materials Research Part A. 18, (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+knitted+mesh-reinforced+collagen-chitosan+hybrid+scaffold+in+the+one-step+repair+of+full-thickness+skin+defects+in+rats.">The roles of knitted mesh-reinforced collagen-chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats.</a> Acta Biomaterials. 9 (8), 7822-7832 (2013).</li><li>Rizzi, S. C., Upton, Z., Bott, K., Dargaville, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+dermal+wound+healing:+biomedical+device+approaches.">Recent advances in dermal wound healing: biomedical device approaches.</a> Expert Review of Medical Devices. 1, 143-154 (2010).</li><li>des Rieux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+systems+delivering+VEGF+to+promote+angiogenesis+for+tissue+engineering.">3D systems delivering VEGF to promote angiogenesis for tissue engineering.</a> Journal of Controlled Release. 150, 272-278 (2011).</li><li>Reckhenrich, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactivation+of+dermal+scaffolds+with+a+non-viral+copolymer-protected+gene+vector.">Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector.</a> Biomaterials. 32, 1996-2003 (2011).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Key+Regulatory+Roles+of+the+PI3K/Akt+Signaling+Pathway+in+the+Functionalities+of+Mesenchymal+Stem+Cells+and+Applications+in+Tissue+Regeneration.">The Key Regulatory Roles of the PI3K/Akt Signaling Pathway in the Functionalities of Mesenchymal Stem Cells and Applications in Tissue Regeneration.</a> Tissue Engineering Part B Rev. 19, 516-528 (2013).</li><li>Fedorovich, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+progenitor+cells+in+prevascularized+bone+tissue+engineering:+development+of+heterogenous+constructs.">The role of endothelial progenitor cells in prevascularized bone tissue engineering: development of heterogenous constructs.</a> Tissue Engineering Part A. 16 (7), 2355-2367 (2010).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+and+angiogenesis+of+tissue-engineered+bone+constructed+by+prevascularized+&#946;-tricalcium+phosphate+scaffold+and+mesenchymal+stem+cells.">Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized &#946;-tricalcium phosphate scaffold and mesenchymal stem cells.</a> Biomaterials. 36, 9452-9461 (2010).</li><li>Cuadra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+results+of+burned+hands+treated+with+Integra.">Functional results of burned hands treated with Integra.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 65 (2), 228-234 (2012).</li><li>Wilcke, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF(165)+and+bFGF+protein-based+therapy+in+a+slow+release+system+to+improve+angiogenesis+in+a+bioartificial+dermal+substitute+in+vitro+and+in+vivo.">VEGF(165) and bFGF protein-based therapy in a slow release system to improve angiogenesis in a bioartificial dermal substitute in vitro and in vivo.</a> Langenbecks Arch Surg. 392 (3), 305-314 (2007).</li><li>Condurache, A., Aach, T., Grzybowsky, S., Machens, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+and+analysis+in+laboratory+skin+transplant+micro-angiograms.">Vessel segmentation and analysis in laboratory skin transplant micro-angiograms.</a> Proceedings of the Eighteenth IEEE Symposium on Computer-Based Medical Systems. , 21-26 (2005).</li><li>Danner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+human+sweat+gland-derived+stem+cells+for+enhancing+vascularization+during+dermal+regeneration.">The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration.</a> Journal of Investigative Dermatology. 132 (6), 1707-1716 (2012).</li><li>Shaterian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+Time+Analysis+of+the+Kinetics+of+Angiogenesis+and+Vascular+Permeability+in+an+Animal+Model+of+Wound+Healing.">Real Time Analysis of the Kinetics of Angiogenesis and Vascular Permeability in an Animal Model of Wound Healing.</a> Burns. 35 (6), 811-817 (2009).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nature Medicine. 9 (6), 713-725 (2003).</li><li>Bergeron, L., Tang, M., Morris, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+vascular+injection+techniques+for+the+study+of+perforator+flaps.">A review of vascular injection techniques for the study of perforator flaps.</a> Plastic and Reconstructive Surgery. 117, 2050-2057 (2006).</li><li>Schlatter, P., K&#246;nig, M. F., Karlsson, L. M., Burri, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+intussusceptive+capillary+growth+in+the+chorioallantoic+membrane+(CAM)+of+the+chicken+embryo.">Quantitative study of intussusceptive capillary growth in the chorioallantoic membrane (CAM) of the chicken embryo.</a> Microvascular Research. 54 (1), 65-73 (1997).</li><li>Lehr, H. A., Leunig, M., Menger, M. D., Nolte, D., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+skinfold+chamber+technique+for+intravital+microscopy+in+nude+mice.">Dorsal skinfold chamber technique for intravital microscopy in nude mice.</a> American Journal of Pathology. 143 (4), 1055-1062 (1993).</li><li>Menger, M. D., J&#228;ger, S., Walter, P., Hammersen, F., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+studies+on+the+microvasculature+of+transplanted+islets+of+Langerhans+in+vivo.">A novel technique for studies on the microvasculature of transplanted islets of Langerhans in vivo.</a> International journal of microcirculation, clinical and experimental. 9 (1), 103-117 (1990).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+spheroids+of+adipose-derived+mesenchymal+stem+cells+are+potent+initiators+of+blood+vessel+formation+in+porous+polyurethane+scaffolds.">Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds.</a> Acta Biomaterials. 9 (6), 6876-6884 (2013).</li><li>Ega&#241;a, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+mesenchymal+cells+to+improve+vascularization+in+a+mouse+model+for+scaffold-based+dermal+regeneration.">Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration.</a> Tissue Eng Part A. 15 (5), 1191-1200 (2009).</li><li>Condurache, A., Aach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+in+angiograms+using+hysteresis+thresholding.">Vessel segmentation in angiograms using hysteresis thresholding.</a> Proceedings of the Ninth IAPR Conference on Machine Vision Applications. , 269-272 (2005).</li><li>Ega&#241;a, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+method+to+visualize+and+quantify+vascular+networks+in+native+and+tissue+engineered+skin.">Ex vivo method to visualize and quantify vascular networks in native and tissue engineered skin.</a> Langenbecks Archives of Surgery. 394, 349-356 (2009).</li></ol>

관심 문장의 충돌 : 모든 저자 : 없음 재정 공시 : 저자 중에이 원고에서 언급 된 제품, 장치, 또는 약물의에 재무 적 이해 관계가 없습니다.

생체 물질 내에 혈관 과정을 연구하기위한 새로운 방법의 안정적인 발전을 요구 조직 공학적 구조에 혈류를 개선하는 방법을 성공적으로 확립 할 필요가있다. 생체 가시 골격 혈관을 만들기위한 일반적인 방법은 고해상도 도구를 제공 현미경의 사용을 포함한다. 대부분의 경우에, 그러나,이 방법은 작은 조직 영역의 분석에 제한되고 비용과 시간이 걸리는 경향이있다. 또한, 종종 혈액 관?...

최근 수십 년 동안 조직 공학은 신체의 자신의 세포 하나와 조직 결함을 대체하는 새로운 치료 옵션을 열었다. 조직 재생의 생리 학적 과정을 지원하기 위해, 지지체는 상처 침대에서 세포가 성장하고 결함 2,3 복원 할 시나리오를 제공 생분해 구조로 설계된다. 불충분 한 혈관은 bioartificial 지지체 (4)의 임상 돌파구를 다시 보유하고있는 주요 장애물로 간주됩니다. 세포의 증식과 함께, 영양소 및 산소의 증가 및 혈관 신생의 재료에 대한 요구가 중요하게된다. 부족 또는 지연 혈관 따라서 조직 공학 제품 (5)의 중앙 괴사로 이어질 수 있습니다. 또한, 혈관 면역 적격 세포를 제공하고 회생 영역에서 대사 잔류 물을 제거. 높은 감염률과 낮은 재생은 아르목표로 조직 공학에서 관찰 부족한 혈액 관류의 결과의 일부는 발판 6,7의 혈관을 증가시켜 막을 수 있습니다. 생체 재료 자체와 지지체의 미세 혈관 형성에 중요한 역할 초점 향상을 목표로 여러 전략. 8,9을 복원하기 때문에 (재), 재생하여 수리에서 치유 과정 시프트 하나에 가까운 특성을 갖는 생리 조직을 생성하는 새로운 방법을 개발하기 위해 집중적 인 연구 노력들이있다. 자신의 회생 가능성에 관해서 공부하고 평가 하였다 생체 적합 물질은 콜라겐, 섬유소, 키토산, 알긴산 (10, 11)를 포함했다. 이러한 생체 재료를 사용하고 티슈 탈세 포화, 자기 조립, 조형 등 다양한 전략을 사용하는 새로운 발판을 구축하고 12 전기 방사를위한 백본으로서 결합 될 수있다. ENH 위해서는신체의 자신의 재생 능력 ANCE, 비계는 bioactivated 할 수 있습니다. 재조합 혈관 성장의 혼입 등의 요소 (14) 또는 (13)에 대해 코딩하는 유전자 벡터는 지지체의 혈관을 개선 요인을 보여 주었다. 줄기 세포의 사용은 널리 간엽 기질 세포 및 내피 전구 세포가 가장 관심을 얻고있다 15,16 혈관을 개선하기 위해 유망한 전략으로 밝혀졌다. 다른 방법은 이식 17 일 이전 조립식 용기를 포함하는 네트워크 구조를 구축하기 위해 시도한다. 집중 지지체 디자인 노력과 그들의 생체 활성에도 불구하고, 어떤 전략이 대규모 화상 상처의 피부 대체를 제외하고, 임상 적으로 상당한 수준에서 혈관 생성을 향상하지 않고 있으며, 임상 루틴으로 생체 공학 재료의 번역은 주저 18 일어나는 . 그 이유 중 하나는 혈관 이유인공 조직 구성체, 여전히 미해결 문제이며 생체 내 접근법에서 새로운 기술의 성공 여부를 평가하기 곤란하다. 시험 관내 실험은 비계의 혈관 전위의 중요한 식견을 제공 할 수도 있지만, 적절한 동물 모델은 이러한 재료의 생체 적합성, 특히 중요한 안전성 및 효능을 치료하고, 조직의 혈관 형성과 같은 주요 파라미터를 연구해야 구성. 따라서 신뢰할 수있는 도구를 시각화하고 혈관 네트워크를 생체 내에서 필수적인을 정량화한다. 본 연구에서 우리는 외식 지지체 내부의 혈관 네트워크 시각화 및 정량화를 허용 간단하고 신뢰할 수있는 방법을 제시한다. 이 방법은 조직 transillumination 디지털 분할에 기초한다. 이 방법은 비 침습적이기 때문에, 타겟 재료의 상기 분자 및 조직 학적 분석을 허용한다.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:431px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Ethilon P-3 13 mm 3/8 circle 5-0</td>
			<td style="width:223px;">Ethicon, Norderstedt, Germany</td>
			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
			<td style="width:119px;">100196040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
			<td></td>
		</tr>
	</tbody>
</table>

a full skin defect model to evaluate vascularization of biomaterials in vivo

불충분 한 혈관이 조직 공학적 구조물의 임상 성공을 제한하는 주요 요인들 중 하나로 간주된다. 혈관 생성을 향상을 목표로 새로운 전략을 평가하기 위해, 신뢰할 수있는 방법을 보이는 바이오 인공 지지체에 새로운 혈관의 성장을 확인하고 그 결과를 정량화하기 위해 요구된다. 지난 몇 년 동안, 우리 그룹은 transillumination에 의해 혈관의 직접적인 시각화를 가능하게하고 디지털 분할을 통해 정량의 가능성을 제공하는 전체 피부 결함 모델을 출시했다. 이 모델에서, 하나는 수술 마우스의 뒷면에 전체 피부 결함을 생성하고 테스트를 재료로 대체합니다. 분자 또는 관심의 세포는 또한 잠재적 인 효과를 연구하기 위해 이러한 물질에 통합 할 수 있습니다. 자신의 선택의 관측 시간 후, 재료는 평가를 위해 외식된다. 양자 상처 번째 내부 비교하기의 가능성을 제공한다연구에 필요한 동물의 수를 감소뿐만 아니라 개인간 아티팩트를 최소화한다. 다른 방법에 비해, 우리의 방법은 간단, 신뢰성과 비용 효과 분석을 제공합니다. 우리는 다른 생체 물질과 생체 인증 방식의 혈관 테스트시 고해상도의 스크리닝을 수행하는 루틴 도구로이 모델을 구현 하였다.

신뢰할 수있는 양자 간 전체 피부 결함은 피부 연구 (그림 2)에서 생체 재료로 대체 될 수있다 마우스 (그림 1)에서 생성 할 수 있습니다. 여기에서, 더 큰 합병증은 감염이나 이물 반응,도 거시적 징후 중 또는 수술 시술 후 관찰되지 않습니다. 마우스를 제거 할 때 드문 경우에, 비계가 손실됩니다. 상처 수축 (그림 3) 관찰되지 않았다. 조직 transillumination는 모?...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

혈관은 성공적인 조직 공학의 접근 방법의 핵심입니다. 따라서, 신뢰성있는 기술은 조직 구조에서의 혈관 망의 발달을 평가하기 위해 필요하다. 여기서 우리는 시각화 및 생체 내에서 혈관 생성을 정량화하기 위해 간단하고 비용 효율적인 방법을 제시한다.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

12.1K 조회수.  Technische Universität München.  혈관은 성공적인 조직 공학의 접근 방법의 핵심입니다. 따라서, 신뢰성있는 기술은 조직 구조에서의 혈관 망의 발달을 평가하기 위해 필요하다. 여기서 우리는 시각화 및 생체 내에서 혈관 생성을 정량화하기 위해 간단하고 비용 효율적인 방법을 제시한다. 

비디오: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

3 차원의 인간 피부 모델을 재구성 : 도구는 일반 피부와 흑색증의 진행을 공부하기

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

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Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

에 내장 혈관 신생에 의하여 조직 공학<em&gt; 생체</em&gt; 조직 공학 상공 회의소

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

촉각 자극에 의​​지 모터 응답의 적시성과 정확성을 평가하는 방법

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Zebrafish 태아 모델 Vivo 시각화 및 Intravital 소재 분석 관련 된 황색 포도상구균 에 감염

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

사용자 지정 외부 Fixator를 안정화 하는 쥐에서 안정적이 고 재현 가능한 크기의 중요 한 단편 대 퇴 결함 모델

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

피부에 있는 유전으로 표를 붙인 기자 세포의 Vivo 2 색 2-광자 화상 진찰에서

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...