Name: characterization of leukocyte-platelet rich fibrin, a novel biomaterial
Uploaded: 2015-09-29T15:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

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>

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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><tbody><tr><td>Needle 19G</td><td>BD</td><td>305186</td><td></td></tr><tr><td>Needle Disposal Container</td><td>Fisherbrand</td><td>14-827-122</td><td></td></tr><tr><td>Red-Topped Glass Collection Tube</td><td>BD</td><td>8020129</td><td></td></tr><tr><td>Gauze Pads</td><td>Tyco</td><td>5750</td><td></td></tr><tr><td>Bandage</td><td>Johnson &amp;amp; Johnson</td><td>5005989</td><td></td></tr><tr><td>Surshield</td><td>Terumo</td><td>SV*S19BL</td><td>Safety winged infusion set</td></tr><tr><td>Blood Collection Assembly</td><td>BD</td><td>303380</td><td></td></tr><tr><td>Tourniquets</td><td>BD</td><td>367203</td><td></td></tr><tr><td>Brand Luer Adapter</td><td>Vacutainer</td><td>L42179</td><td></td></tr><tr><td>Intra-Spin System&amp;nbsp;</td><td>Intra-Lock International</td><td>ISS110</td><td>Centrifuge and Xpression L-PRF FabricationKit&amp;nbsp;</td></tr><tr><td>Pipettes (Serological &amp;amp; Micro)</td><td>Corning</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Exelint</td><td>29552</td><td></td></tr><tr><td>MTS Bionix 200</td><td>MTS Systems Corporation</td><td></td><td>Material testing systems</td></tr><tr><td>MTS Test Works 4</td><td>MTS Systems Corporation</td><td></td><td></td></tr><tr><td>Whatman Filter Paper</td><td>Whatman</td><td>1004 070</td><td></td></tr><tr><td>SS Orthodontic ligature wire</td><td>Patterson Dental</td><td>628-4228</td><td></td></tr><tr><td>200 Proof Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Aldrich</td><td>440191</td><td></td></tr><tr><td>Aluminium Mounting Stubs</td><td>Ted Pella</td><td>16324</td><td></td></tr><tr><td>Double Sided Carbon Tape</td><td>PELCO Tabs</td><td>16084-1</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>JEOL</td><td>LV 5610</td><td></td></tr><tr><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><td></td></tr><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></tr><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><td></td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>DPBS</td><td>Corning</td><td>55-031-PB</td><td></td></tr><tr><td>Ninhydrin 98%</td><td>Aldrich</td><td>454044</td><td></td></tr><tr><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></tr><tr><td>Digital Micrometer</td><td>Pittsburgh</td><td>68305</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G6257</td><td></td></tr><tr><td>12 Well Plate</td><td>Corning</td><td>3336</td><td></td></tr><tr><td>96 Well Plate</td><td>Corning</td><td>3596</td><td></td></tr></tbody></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

12.9K 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

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

ASC의 분화를 제어하는​​ Bilayered 히드로겔 꾸민

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

연구

생체공학

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

준비를 한 다음 감염을 방지하기 위해 새로운 접근 방식으로 PRP<em&gt; 체외에서</emPRP의&gt; 항균 속성

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

면역학 및 감염병학

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

미세 유체 흐름 챔버 지혈 및 혈소판 수혈을 모델링하기 위해 재구성 된 혈액을 사용하여<em&gt; 체외</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

향상된 세포 접착 폴리 테트라 플루오로 에틸렌에 젖은 화학 및 펩티드 고정화

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

혈소판 수혈 및 지혈을위한 미세 유체 유량 용기 모델은 실시간으로 혈소판 증착 및 섬유소 형성을 측정합니다

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

교양된 혈관 세포에 부착 혈소판 백혈구 신규 모집 조사 하 층 플로우 기반 분석

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

철 염화물에 의한 쥐 혈전증 모델

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

의학

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

흐름하에있는 혈소판 탈식 및 분비의 라이브 셀 이미징

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

생물학

<meta charset="utf8"></head><body>재생 의학을 위한 Fibrin Hydrogel의 기계적 기질 혈관 분획

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

<meta charset="utf8"></head><body>저자 스포트라이트: 상처 치유를 위한 지방 유래 기질 혈관 분획의 잠재력 탐구

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

기계적으로 분리된 Stromal Vascular Fraction과 Fibrin Hydrogel의 조합: 처리 프로토콜

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

백혈구 - 혈소판 풍부한 섬유소, 소설 생체 재료의 특성

요약

더 많은 비디오 탐색

이 비디오의 챕터

ASC의 분화를 제어하는 Bilayered 히드로겔 꾸민

준비를 한 다음 감염을 방지하기 위해 새로운 접근 방식으로 PRP

미세 유체 흐름 챔버 지혈 및 혈소판 수혈을 모델링하기 위해 재구성 된 혈액을 사용하여

향상된 세포 접착 폴리 테트라 플루오로 에틸렌에 젖은 화학 및 펩티드 고정화

혈소판 수혈 및 지혈을위한 미세 유체 유량 용기 모델은 실시간으로 혈소판 증착 및 섬유소 형성을 측정합니다

교양된 혈관 세포에 부착 혈소판 백혈구 신규 모집 조사 하 층 플로우 기반 분석

철 염화물에 의한 쥐 혈전증 모델

흐름하에있는 혈소판 탈식 및 분비의 라이브 셀 이미징

기계적으로 분리된 Stromal Vascular Fraction과 Fibrin Hydrogel의 조합: 처리 프로토콜

기계적으로 분리된 Stromal Vascular Fraction과 Fibrin Hydrogel의 조합: 처리 프로토콜

백혈구 - 혈소판 풍부한 섬유소, 소설 생체 재료의 특성

요약

더 많은 비디오 탐색

이 비디오의 챕터

ASC의 분화를 제어하는​​ Bilayered 히드로겔 꾸민

준비를 한 다음 감염을 방지하기 위해 새로운 접근 방식으로 PRP

미세 유체 흐름 챔버 지혈 및 혈소판 수혈을 모델링하기 위해 재구성 된 혈액을 사용하여

향상된 세포 접착 폴리 테트라 플루오로 에틸렌에 젖은 화학 및 펩티드 고정화

혈소판 수혈 및 지혈을위한 미세 유체 유량 용기 모델은 실시간으로 혈소판 증착 및 섬유소 형성을 측정합니다

교양된 혈관 세포에 부착 혈소판 백혈구 신규 모집 조사 하 층 플로우 기반 분석

철 염화물에 의한 쥐 혈전증 모델

흐름하에있는 혈소판 탈식 및 분비의 라이브 셀 이미징

기계적으로 분리된 Stromal Vascular Fraction과 Fibrin Hydrogel의 조합: 처리 프로토콜

기계적으로 분리된 Stromal Vascular Fraction과 Fibrin Hydrogel의 조합: 처리 프로토콜

ASC의 분화를 제어하는 Bilayered 히드로겔 꾸민