The project was supported by CTSA (UL1TR000058) from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University. The contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.

모든 혈액 그리기 절차는 허가 및 인증 전문가에 의해 수행되어야합니다. 연구를위한 인체의 사용은 임상 시험 심사위원회 또는 기타 적절한 권한 승인을 포함한다. 동의서에 관한 및 참가자 식별을 보호하는 특별주의 사항을 따라야합니다. 이 프로토콜에 나와있는 모든 실험은 인간의 혈액 및 / 또는 혈액 제품과 적절한 개인 보호 장비의 처리는 항상 착용해야합니다 포함한다. 폐기물은 생물로 간주 규정에 따라 처리해야한다. 1. 정맥 천자 <ol><li> 환자 / 참가자를 확인하고 기존의 기록을 확인합니다. 세부 연구를 설명하고 동의를 얻을. </li><li> 평평하고 안정된 표면에 표시 튜브와 개별 환자에 대한 설정 트레이가 있습니다. </li><li> 절차를 설명하고 지원 팔을 편안하게 환자 좌석을 확인합니다. 환자 자신 또는 SH 알리전자는 작은 핀치를 느낄 것이다 및 절차 전반에 걸쳐 여전히 남아 있어야한다. </li><li> 어댑터에 바늘을 연결합니다. </li><li> 손을 씻고 장갑을 착용하십시오. </li><li> 바깥쪽으로 중심에서 동심원 70 % 이소 프로필 알코올로 청소하여 정맥 천자에 대한 전주 포사를 준비합니다. 30 초 동안 자연 건조 사이트를 허용합니다. </li><li> 촉진하여 적절한 정맥을 확인합니다. </li><li> (아직 반경 펄스를 느끼면서) 너무 꽉 있는지 만드는 천자 사이트 위의 지혈대 3-4을 적용합니다. </li><li> 하나의 부드러운 동작으로 피부에 바늘 15 ~ 30 도의 경사를 삽입하여 정맥 천자를 수행합니다. 바늘을 통해 혈관을 누르고 전체 혈액의 9 ml의를 수집합니다. </li><li> 지혈대를 제거합니다. 바늘에서 튜브를 제거합니다. </li><li> 바늘을 철회하고 바늘을 제거하기 전에 천자 부위에 거즈 광장을 적용합니다. 적절한 생물 학적 용기에 바늘을 폐기​​하십시오. </li><li> 홍보 유지하기 위해 환자에게 문의펑크 사이트에서 essure. </li><li> 튜브 레이블 </li><li> 출혈이 중지 확인하기 위해 천자 사이트를 확인하십시오. </li><li> 환자가 (통증이 팽창하거나 빛 머리가) 확실히 느끼고 있는지 물어, 거즈 광장을 통해 접착 붕대 나 테이프를 적용 </li><li> 배출하기 전에 환자 / 참가자 감사합니다. </li></ol> 2. L-PRF 준비 <ol><li> 바로 붉은 얹어 건조 유리 튜브에 정맥 채혈 한 후, 원심 분리기에 넣습니다. </li><li> 적절한 카운터 밸런스를 배치 한 후 실온에서 12 분 동안 400 XG에 원심 분리기. </li><li> 주기의 끝에서 튜브를 제거한다. 혈소판 가난한 플라즈마 (PPP), 혈소판 풍부 피브린 (L-PRF) 및 적혈구베이스 (그림 1) : 세 개의 층을 확인할 수 있습니다. </li><li> 피펫을 사용하여 PPP를 대기음. 사용 핀셋 부드럽게 L-PRF를 당겨 멸균, 천공 금속 메쉬에 넣습니다. </li><li> 외과 용 메스를 사용하여 조심스럽게 버프 떠나는 RBC 층의 벌크를 긁어Y 코트 그대로. </li><li> 부드럽게 30 초 동안 (멸균 금속 플레이트, 대략적인 무게 225g 사용) L-PRF 응고를 압축. 혈소판 가난한 플라즈마는 압착된다. </li><li> 플레이트를 제거하고 부드럽게 L-PRF 막을 올립니다. L-PRF 막은 실험 12에서 사용하기위한 준비가되어있다. </li></ol> 3. 일축 인장 시험 <ol><li> 취급의 용이성을 위해 여과지에 놓고 L-PRF 막 (N = 6)과 (7.5 mm의 게이지 길이 자신의 좁은 지점에 폭 2.75 mm)를 주문 제작 금속이 죽었을 사용하여 &quot;개 뼈&quot;에 펀치. </li><li> 세 지점에서 각 샘플의 두께를 측정하고 평균을 취. </li><li> 신중 축성 테스트 시스템의 턱 그립의 중앙에 L-PRF 막 결합. </li><li> 조심스럽게 L-PRF 막을 노출 파일러 용지 지원을 찢어. </li><li> 프로그램 장비 가동 헤드가 일정한 속도 (10.0 mm / 분)로 작동되도록 할 때 L- 실험을 시작할PRF는 아직 젖어있다. </li><li> 단축 테스트 시스템과 함께 제공되는 소프트웨어의 휴식, 휴식 에너지​​ 및 변형을 탄성 계수를 기록합니다. 이 값은 자동으로 계산하며 사용자 정의 입력이 필요하지 않습니다. 그림 3을 참조하십시오. </li></ol> 4. 봉합 유지 강도 <ol><li> 장소 L-PRF 처리의 용이성을 위해 여과지에 멤브레인 (N = 3)과 수술 메스를 사용하여 측정 시료의 직사각형 (10mm X 25mm)로 잘라. </li><li> 각 시료 ((3)의 평균)의 두께를 측정한다. </li><li> 스테인리스 교정 결찰 와이어 (지름 220 μm의)을 사용하여 시료의 중심에 핀홀을 만든다. </li><li> 루프를 형성하고, 인장 시험기에 그것을 해결하기 위해 핀홀을 통해 합자 와이어를 전달합니다. 아래턱 그립 (13)에 L-PRF 멤브레인의 가장자리를 놓습니다. </li><li> 이동 헤드가 일정한 속도 (10mm / 분) 시작에서 작동되도록 기기를 프로그래밍실험. </li><li> 단축 테스트 시스템과 함께 제공되는 소프트웨어의 휴식, 휴식 에너지​​ 및 변형을 탄성 계수를 기록합니다. 이 값은 자동으로 계산하며 사용자 정의 입력이 필요하지 않습니다. 그림 3을 참조하십시오. </li></ol> 5. 형태 학적 검사 <ol><li> 10mm 피부 생검 펀치를 사용하여 현미경 검사의 L-PRF 샘플을 준비하고 24 웰 플레이트에 배치합니다. </li><li> PBS로 샘플을 씻고 20 분 (PBS)에 2.5 % 글루 타르 알데하이드로 고정한다. </li><li> 순차적으로 5 분마다의 에탄올 농도 (50 %, 70 %, 80 %, 90 % 및 100 %) 증가에 침지하여 시험편 탈수. </li><li> 5 분 동안 100 % HMDS (헥사 메틸 디 실라 잔) 0.5 ㎖로 처리한다. HMDS (14) 초과 제거 O / N을 공급하십시오. </li><li> 양면 테이프를 이용하여 스터브에 마운트 샘플은 70 초 동안 백금 스퍼터 코팅 및 가속 작동 주사 전자 현미경으로 검사또한 알고리즘 20 kV의 전압 (또는 적절한 설정). </li></ol> L-PRF, 트립신 감수성과 닌히 드린 분석 6. Genipin 가교 <ol><li> genipin 가교 L-PRF를 준비하려면, PBS로 세포막을 씻어 48 시간 동안 (70 % 에탄올) 1 % genipin 용액 4 mL에 흠뻑 젖어. 초과 genipin (15), (16)를 제거하기 전에 실험에 PBS로 씻어. </li><li> 트립신 분해에의 저항에 의해 L-PRF의 genipin 가교의 안정성을 평가합니다. 장소 L-PRF 막 (N = 3), 0.01 % 트립신 500 μL에 가교 L-PRF genipin 트립신의 일일 변화로 3 일 동안 37 ℃에서 배양 하였다. <ol><li> 시작에 노출 하루 3에서 차이를 효소와 무게는 효소 분해 (17)를 나타냅니다 종료 1 일 이전에 샘플의 무게를 측정. </li></ol></li><li> 닌히 드린 시험을하여 처리 genipin L-PRF (G-PRF)로 가교 결합의 양을 정량화. 먼저 표준 CURV를 준비E 글리신을 사용하여 (1 ㎜의 0.031 mM)을 곡선은 유리 아미노산 농도 (FAA)와 흡광도 사이의 관계를 수립한다. <ol><li> 2 % 1 ㎖와 샘플 열 PRF (w / v)의 100 ℃에서 15 분 동안 닌히 드린. </li><li> 용액을 실온으로 냉각하고 50 % 에탄올 1.5 mL를 추가 할 수 있습니다. </li><li> 적합한 분광 광도계를 사용하여 570 nm에서 흡광도를 분석한다. </li><li> (15) 아래의 식을 이용하여 가교 결합 백분율을 결정 </li></ol></li></ol><img alt="식 (1)" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. MTS 세포 증식 분석 <ol><li> 80 %의 합류 단층을 얻을 때까지 T-75 플라스크에 최소 필수 중간 - 알파 수정 (αMEM)에서 MC3T3 (마우스 두개골 preosteoblasts)을 성장. </li><li> 신선한 멸균 L-PRF 막 (세포 배양 후드 안쪽 L-PRF 튜브를 열)를 준비하고 새로운 세포 배양 접시에 막 전송. </li><li> 대기음 플라스크에서 미디어와, PBS와 단층 린스 5 ml의 0.05 % 트립신을 추가 5 분 동안 37 오 C 배양기에 플라스크를 놓고, 5, 400 XG에서 원심 분리 튜브에서 원심 분리에 플라스크의 내용물을 피펫 분. </li><li> 부드럽게, 뜨는을 가만히 따르다 세포 펠렛을 깰 튜브를 누르고 신선한 α의 (MEM)의 4 ㎖로 다시 중단, 혈구에 세포 현탁액 (50 μL)와 트리 판 블루 (50 μL)의 혼합물을 분배하고의 수를 계산 세포 </li><li> 시드 막 내에 세포를 유지하기 위해 L-PRF 막 위에 배치 10mm 유리 클로닝 고리 내에 4 × 5 셀 (링 24 시간 후에 제거 될 수있다). </li><li> 4 일에, 10 분 동안 PBS의 세 번에 구조를 씻어. </li><li> 각 웰에 혈청이없는 배지 200 ㎕를 MTS 시약 1 ML을 추가하고 37 ℃에서 2 시간 동안 품어. </li><li> 490 nm에서 200 μL 씩에서 흡광도를 측정한다. </li></ol>

<ol>
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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=Cross-linking+methods+of+electrospun+fibrinogen+scaffolds+for+tissue+engineering+applications.">Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.</a> Biomed Mater. 3 (4), (2008).</li><li>Gorczyca, G., 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=Preparation+and+characterization+of+genipin+cross-linked+porous+chitosan-collagen-gelatin+scaffolds+using+chitosan-CO2+solution.">Preparation and characterization of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution.</a> Carbohydr Poly. 102, 901-911 (2014).</li><li>Rozman, P., Semenic, D., Wislet-Gendebien, S. a. b. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.+The+Role+of+Platelet+Gel+in+Regenerative+Medicine.">Chapter 15. The Role of Platelet Gel in Regenerative Medicine.</a> Advances In Regenerative Medicine. , 319-349 (2011).</li><li>Dohan Ehrenfest, ., Lemo, D. M., Jimbo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+a+relevant+animal+model+for+testing+the+in+vivo+effects+of+Choukroun&#8217;s+platelet-rich+fibrin+(PRF):+Rabbit+tricks+and+traps.">Selecting a relevant animal model for testing the in vivo effects of Choukroun&#8217;s platelet-rich fibrin (PRF): Rabbit tricks and traps.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 110 (4), 413-416 (2010).</li><li>Guilak, F., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering:+Ten+more+years+of+progress.">Functional tissue engineering: Ten more years of progress.</a> J Biomec. 47 (9), 1931-1932 (2014).</li><li>Guilak, F., Butler, D. L., Goldstein, S. A., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+mechanobiology+in+functional+tissue+engineering.">Biomechanics and mechanobiology in functional tissue engineering.</a> J Biomec. 47 (9), 1933-1940 (2014).</li><li>Collet, J. P., Shuman, H., Ledger, R. E., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+an+individual+fibrin+fiber+in+a+clot.">The elasticity of an individual fibrin fiber in a clot.</a> Proc Natl Acad Sci. 102 (26), 9133-9137 (2005).</li><li>Liu, 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=Fibrin+fibers+have+extraordinary+extensibility+and+elasticity.">Fibrin fibers have extraordinary extensibility and elasticity.</a> Science. 313 (5787), 634 (2006).</li><li>American National Standard. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8:+Test+methods+for+vascular+prostheses.">Chapter 8: Test methods for vascular prostheses.</a> Association for the Advancement of Medica lnstrumentation Guidance document: Cardiovascular Implants - Tubular vascular prostheses. , 33-34 (2001).</li></ol>

저자는 공개 아무것도 없어 그리고이 책과 관련된 관심의 알려진 충돌이 없는지 확인합니다.

자가 혈소판 농축 때문에 성장 인자의 풍부 (18)의 재생 의료 분야에서 유망. 그러나, 이러한 제제는 종종 외과 적 조작이 매우 곤란하게 정의 구조 부족. 여러 번, 현탁액 젤은 예측할 수없는 결과를 초래, 배달의 현장에서 효과적으로 유지되지 않습니다. L-PRF는 혈소판의 발전에 큰 사전이 본질적으로 내포 된 혈소판과 회사 섬유소 막 인 것을 집중 나타냅니다. 이 고체 막은 우수한 핸들링...

The use of blood and blood-derived products to seal wounds and improve healing in different clinical situations started with fibrin glues, which are mainly fibrinogen concentrates. Addition of platelets to fibrin glue not only improved their strength but also promoted neoangiogenesis and regeneration. These benefits are attributed to the release of a variety of peptide growth factors from the alpha-granules of platelets upon activation1. Platelet concentrates (PC) were seen as a practical way to deliver growth factors2 and its use was driven by commercial interests rather than research characterization3. In fact, PCs are difficult to characterize unlike homogenous and defined pharmacological preparations, they are a potpourri of signaling molecules and blood cells (platelet and leukocytes) entrapped within a fibrin matrix. Different commercial and proprietary preparations yield a variety of PC that are different in cellular composition, growth factor recovery and kinetics of release4. 
It is important to realize that in most oral surgeries, platelet-rich plasma (PRP) preparations are used as a gel in open surgical wounds and not as platelet suspensions. In these situations, the gelation is induced by the addition of thrombin, calcium chloride, batroxobin or other agents and directly placed in the site of injury5. Due to rapid activation, fibrinogen polymerization is often incomplete and results in friable fibrin gels with very little mechanical strength. In addition, injectable PRP gels undergo rapid fibrinolysis6,7.
In contrast, the processes of blood coagulation (fibrinogen polymerization), platelet enrichment and activation occur simultaneously in the preparation of L-PRF8. The coagulation cascade is triggered when whole blood contacts the walls of a dry glass tube and continues throughout the centrifugation process. This results in the formation of a mechanically-strong blood clot (L-PRF) that can be surgically handled and used. 
Even though L-PRF has been investigated in terms of optimal methods of preparation, growth factor release and cell distribution9-11, detailed mechanical characterization of these membranes are lacking. This is significant gap in knowledge, given the popularity of these membranes in clinical practice as well as its potential to be used as a biomaterial. Current study focusses on the protocol for deriving L-PRF as well as methods that can be employed to study its mechanical properties. This data is intended to serve as baseline for ongoing studies investigating the viscoelastic properties of this interesting natural biomaterial.

<table border="0" cellpadding="0" cellspacing="0">
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			<td>Needle 19G</td>
			<td>BD</td>
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			<td>Needle Disposal Container</td>
			<td>Fisherbrand</td>
			<td>14-827-122</td>
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			<td>BD</td>
			<td>8020129</td>
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			<td>Tyco</td>
			<td>5750</td>
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			<td>Johnson &#38; Johnson</td>
			<td>5005989</td>
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			<td>Surshield</td>
			<td>Terumo</td>
			<td>SV*S19BL</td>
			<td>Safety winged infusion set</td>
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			<td>Blood Collection Assembly</td>
			<td>BD</td>
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			<td>BD</td>
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			<td>Intra-Spin System&#160;</td>
			<td>Intra-Lock International</td>
			<td>ISS110</td>
			<td>Centrifuge and Xpression L-PRF FabricationKit&#160;</td>
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			<td>Pipettes (Serological &#38; Micro)</td>
			<td>Corning</td>
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			<td>Scalpel</td>
			<td>Exelint</td>
			<td>29552</td>
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			<td>MTS Bionix 200</td>
			<td>MTS Systems Corporation</td>
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			<td>Material testing systems</td>
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			<td>MTS Test Works 4</td>
			<td>MTS Systems Corporation</td>
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			<td>628-4228</td>
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			<td>Koptec</td>
			<td>V1001</td>
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			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Aldrich</td>
			<td>440191</td>
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			<td>Aluminium Mounting Stubs</td>
			<td>Ted Pella</td>
			<td>16324</td>
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			<td>Double Sided Carbon Tape</td>
			<td>PELCO Tabs</td>
			<td>16084-1</td>
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			<td>Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>LV 5610</td>
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			<td>Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>51026282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimicotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
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		<tr>
			<td>Genipin</td>
			<td>Wako</td>
			<td>078-03021</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Media</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>Minimum Essential Medium-Alpha</td>
		</tr>
		<tr>
			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G1118</td>
			<td></td>
		</tr>
		<tr>
			<td>PMS Reagent</td>
			<td>Sigma</td>
			<td>P9625</td>
			<td></td>
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		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td></td>
			<td>Epoch Spectrophotometer</td>
		</tr>
		<tr>
			<td>10mm Glass Cloning Rings</td>
			<td>Corning</td>
			<td>3166-10</td>
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			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
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			<td>DPBS</td>
			<td>Corning</td>
			<td>55-031-PB</td>
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			<td>Ninhydrin 98%</td>
			<td>Aldrich</td>
			<td>454044</td>
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			<td>24 Well Plate</td>
			<td>Corning</td>
			<td>3987</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>Acu Punch</td>
			<td>P1025</td>
			<td></td>
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		<tr>
			<td>Digital Micrometer</td>
			<td>Pittsburgh</td>
			<td>68305</td>
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			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
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		<tr>
			<td>12 Well Plate</td>
			<td>Corning</td>
			<td>3336</td>
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		<tr>
			<td>96 Well Plate</td>
			<td>Corning</td>
			<td>3596</td>
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</table>

characterization of leukocyte-platelet rich fibrin, a novel biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

(상부, 중간 및 하부) 층이도 2에 도시 된 다른 섹션에서 L-PRF 응고의 주사 전자 현미경 이미지. 알 수있는 바와 같이, 상기 상부는 더 세포 주로 섬유소 네트워크로 구성된다. 중간 층은 자신의 활성화 및 탈과립의 증거와 혈소판 풍부하다. 하층은 백혈구 및 피브린 매트릭스 내에 포획 적혈구의 혼합물을 갖는다. 단축 인장 시험 및 봉합 고정 강도 시험 : 기계적 ?...

Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

백혈구 - 혈소판 풍부한 섬유소, 소설 생체 재료의 특성

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

characterization-of-leukocyte-platelet-rich-fibrin-a-novel-biomaterial

Research

JoVE Journal

Bioengineering

12.8K 조회수.  School of Dentistry, Virginia Commonwealth University. Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing. 

비디오: Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

심장 조직 공학을위한 섬유소 히드로겔에 Cardiomyocytes의 캡슐

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

연구

생체공학

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

위한 실크 영화 문화 시스템 체외에서 분석 및 Biomaterial 디자인

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

섬유아세포 매개 피브린 겔 다짐 관찰 및 정량화

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

소설 보컬 폴드 생물 반응기의 구조 및 특성

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

신경 성장 인자 인 캡슐 레이션을위한 새로운 HDL 모방 나노 입자의 제조 및 특성 규명

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Heparinized StarPEG Nanocoating와 췌 장 독도의 표면 공학

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

제조 및 Macrostructure를 포함 하는 광학 조직 팬텀의 특성화

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

서 주 신장 조직 세그먼트와 소설 비 계 없는 건설 기술 관

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

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 ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.