이 연구는 LSH 2Treat 프로그램 (436001003)과 네덜란드 신장 재단 (14a2d507)의 일환으로 ZonMw에 의해 재정적으로 지원됩니다. N.A.K.는 유럽 연구 위원회 (851960)의 지원을 인정합니다. 네덜란드 과학연구기구(024.003.013)가 후원한 중력프로그램 '재료 중심재생'을 감사하게 인정합니다.

저자는 공개 할 것이 없습니다.

본 명세서에 기재된 생물반응기는 관내 반사성 스캐폴드에서 염증 및 조직 재생에 대한 전단 스트레스 및 순환 스트레칭의 개별 및 결합된 효과의 기여도를 체계적으로 평가할 수 있다. 또한 이 방법을 사용하면 대표적인 결과 섹션에서 예시된 바와 같이 혈관 구조에서 다양한 분석을 수행할 수 있습니다. 이러한 결과는 TEVG 구조의 성장과 리모델링 모두에 다양한 혈역학 적재 체제(즉, 전단및 스?...

심혈관 조직 공학(TE)은 현재 사용되는 영구 심혈관 보철물(예를 들어, 혈관 이식, 심장 판막 교체)에 대한 대체 치료 옵션으로 추구되고 있으며, 이는 환자의 큰 코호트에 대해 최적이 아니다1,2,3,4. 많은 수요가 응용 프로그램은 조직 엔지니어링 혈관 이식편 (TEVGs)5,6 및 심장 판막 (TEHV)7,8을포함한다. 대부분의 경우, 심장 혈관 TE 방법론은 새로운 조직이 형성될 수 있는 유익한 발판역할을 하는 개역 가능한 생체 재료(천연 또는 합성)를 사용합니다. 새로운 조직의 형성은 체외에서 완전히 설계될 수 있으며, 세포와 함께 스캐폴드를 시드하고 이식 전에 생체 반응기에서 배양하여(체외TE)9,10,11,또는 직접 앉아서 합성 스캐폴드가 체내에서 직접 새로운 조직의 형성을 유도하기 위해 사전 배양없이 이식된다( 12) 1,12. 시험관 내 및 시투 심혈관 TE 접근법의 경우 성공적인 기능성 재생은 이식된 구조에 대한 숙주 면역 반응과 적절한 생체 기계적 적재모두에 크게 의존합니다.
심혈관 TE용 생체역학적 하중의 중요성은15. 심혈관 임플란트의 경우, 스캐폴드를 채우는 세포는 혈역학적 환경의 결과로 발생하는 순환 스트레칭및 전단 응력에 노출됩니다. 수많은 연구는 콜라겐16,17,18,19,글리코소미노글리산 (GAGs)20,및 엘라스틴(21,22)및 다양한 세포 유형에 의해 매트릭스 성분의 형성에 (순환) 스트레칭의 자극 효과를보고했다. 예를 들어, 황 외는 혈관생물반응기(23)를이용하여 체외 TEVG에서 콜라겐 및 엘라스틴의 증착 및 조직을 상승시키는 양축 스트레칭을 입증했다. 강조는 일반적으로 지배적 인 부하로 스트레칭에 놓여 있지만, 이러한 연구는 종종 샘플이 전단 흐름에 노출되는 흐름 중심의 생물 반응기를 사용합니다. 3D의 조직 형성과 염증에 대한 전단 응력의 고립 된 영향에 대해서는 상대적으로 거의 알려져 없지만 일부 데이터를 사용할 수 있습니다. 예를 들어, Hinderer 외. 및 Eoh 외. 전단 흐름, 3D 스캐폴드 미세 구조 이외에, 체외 모델시스템(24,25)에서인체 혈관 평활근 세포에 의한 성숙한 엘라스틴형성에 중요했다. 전부, 이 사실 인정은 심장 혈관 TE를 위한 순환 스트레칭 및 전단 긴장 둘 다의 관련성을 보여줍니다.
TE 임플란트의 성공 또는 실패에 대한 또 다른 중요한 결정요인은 이식된접목(26)에대한 호스트의 면역 반응이다. 이는 실제로 세포 유입 및 내인성 조직 형성 및 리모델링27의후속 과정을 킥스타트하기 위해 스캐폴드에 대한 급성 염증 반응에 의존하는 시투 TE 전략에서 물질 중심의 물질 중심의 물질 중심을 위해 특히 중요하다. 대식세포는 기능성 조직 재생의 중요한 시노레이터이며, 이는 다중 연구28,29,30에의해 나타났다. 상처 치유와 유사하게, 조직의 재생은 섬유아세포와 근섬유세포 와 같은 대식세포와 조직 생성 세포 사이의 파라크리노 신호에 의해 지배된다31,32,33. 새로운 조직 증착을 조정하는 것 외에도, 대식세포는이물질(34,35)의활성 재흡수에 관여한다. 이와 같이, 생체물질에 대한 체외 대식세포반응은임플란트(36,37,38)의생체 내 성공을 위한 예측 파라미터로서 확인되었다.
이식된 스캐폴드에 대한 대식대식 반응은 재료 조성 및미세구조(35,39,40)와같은 스캐폴드 설계 기능에 의존한다. 스캐폴드 특성 외에도 비계에 대한 대식대식 반응과 근섬유아세포와의 교차토크도 혈역학적 하중의 영향을 받았습니다. 예를 들어, 순환 스트레치는 대식세포 표현형41,42,43,44 및 사이토카인의 분비(43,44,45,46in 3D 일렉트로스펀 스캐폴드)의 중요한 변조기로 나타났다. 대식세포와 혈관 평활근 세포의 공동 배양 시스템을 사용하여, Battiston 등. 대식세포의 존재는 엘라스틴과 GAGs의 증가 수준으로 이어졌고 순환 스트레치 (1.07-1.10)의 적당한 수준이 콜라겐 I와 엘라스틴47의증착을 자극했다는 것을 입증했습니다. 전작에서는 전단 응력이 3D 전두엽 비계48,49로단백세포 모집을 위한 중요한 결정제이며, 전단 응력과 순환 스트레치 모두 인간 단세포와 중간엽 기질세포(50)사이의 파라크리온 신호에 영향을 미친다는 것을 입증하였다. Fahy 등은 전단 흐름이 인간단핵구(51)에의한 염증성 사이토카인의 분비를 증가시키는 것을 입증했다.
종합하면, 위의 증거는 혈역학 적재에 대한 적절한 이해와 제어가 심혈관 TE에 매우 중요하며 이를 달성하기 위해 염증 반응을 고려하는 것이 중요하다는 것을 보여줍니다. 수많은 생물 반응기는 이전에 시험관 내52,53,54,55, 56,57,57,58 또는 전 생체59,60,61 심혈관 조직의 문화를 위해 이전에 기술되었다. 그러나 이러한 모든 시스템은 가능한 한 생리적 혈역학 적재 조건을 모방하도록 설계되었습니다. 이것은 시험관 내에서 심장 혈관 조직을 만들거나 전 생체 문화를 유지하기위한 목적으로 매우 가치가 있지만, 이러한 시스템은 개별 단서의 개별 효과에 체계적인 연구를 허용하지 않습니다. 이는 이러한 생물 반응기에서 순환 스트레칭과 전단 응력의 적용이 본질적으로 연결되는 동일한 가압 흐름에 의해 구동되므로. 정확한 멀티큐 기계식 조작을 허용하는 마이크로시스템은 2D 기판62 또는 3D 하이드로겔 설정63,64에대해 설명되어 왔지만, 이러한 설정은 탄성액 3D 생물재료 스캐폴드의 통합을 허용하지 않는다.
여기에서, 우리는 유일하게 전단 응력과 순환 스트레칭의 분리를 가능하게하고 기계적으로 그들의 개별및 결합된 효력을 조사하는 것을 돕는 관 생물 반응기 시스템의 응용을 제시합니다. 이 시스템은 조직 설계 혈관 이식의 광범위한 테스트를 허용 (예를 들어, 합성 또는 자연 기원, 다른 마이크로 아키텍처, 다양한 다공성). 전단 응력과 스트레치의 적용을 효과적으로 분리하기 위해, 생체 반응기에서 사용하는 주요 개념은 (1) 뚜렷한 펌프 시스템을 사용하여 전단 응력 및 스트레치 제어의 분리와 (2) 스캐폴드의 자극을 계산 구동 치수로 '내부 아웃'방식으로 분리하는 것이다. 흐름은 유동 펌프의 사용을 통해 관 비계의 외부 표면에 적용되는 반면, 스캐폴드의 일주 스트레칭은 별도의 스트레인 펌프를 사용하여 스캐폴드가 장착되는 실리콘 튜브를 확장하여 유도된다. 구성을 포함하는 실리콘 튜브및 유리 튜브의 치수는 스캐폴드(흐름에 의한) 및 외주 스트레치(튜브 팽창로 인한)의 전단 응력이 서로 크게 영향을 미치지 않도록 하기 위해 전산 유체 역학 시뮬레이션을 사용하여 신중하게 선택및 검증된다. 이 내부 아웃 디자인은 몇 가지 실용적인 근거가 있습니다. 발광 유체 압력(생리적 적재와 유사)에 의해 스트레치가 적용되는 경우, 본질적으로 샘플 설계가 누출되지 않습니다. 또한, 시료를 스트레칭하는 데 필요한 압력은 시료 강성에 의해 완전히 결정되며, 이는 시간이 지남에 따라 샘플과 샘플 내에서 다를 수 있어 스트레치를 제어하기가 어려워집니다. 이 생물 반응기는 실리콘 튜브 주위에 조직 설계 이식편을 장착하고 접목의 외벽에 벽 전단 응력 (WSS) 응용 프로그램을 허용하고 내부에서 접목을 가압합니다. 이러한 방식으로, 시간이 지남에 따라 샘플과 샘플 내의 동일한 하중 조건을 보장할 수 있으며, 또한, 다공성 혈관비계(19)에공통적으로 도공되는 바와 같이 시료가 새는 것을 허용한다. 이 내부 아웃 생물 반응기는 특히 전통적인 혈관 생물 반응기 설치가 더 적합한 시험관 내의 토착 혈관의 엔지니어링보다는 전단 및 / 또는 스트레치의 효과에 대한 체계적인 연구를 위한 것입니다. 생물 반응기 설계 도면에대한 그림 1A-B를 참조하고, 생체 반응기의 주요 구성 요소 뒤에 기능적 설명 및 근거에 대한 해당 표 1을 참조하십시오.
생물 반응기의 사용은 우리가 시상 심혈 관 조직19,43,44에대한 반사 전기 스프폴드에서 염증과 조직 형성에 전단 스트레스와 순환 스트레칭의 개인 및 결합 된 영향을 조사하는 우리 그룹에 의해 최근 연구의 시리즈에 기초하여 입증된다. 이를 위해, 우리는 인간 대식세포와 근섬유아세포를 모노 또는 공동 배양에서 사용하여 시투 재생 폭포의 다양한 단계를 시뮬레이션했습니다. 우리는 인간 대식세포에 의한 사이토카인 분비액이 이러한 비계에서 인간 근섬유세포에 의해 매트릭스 증착 및 조직에 영향을 미치는 순환 스트레치 및 전단 응력 모두에 의해 뚜렷하게 영향을 받는것을 입증했으며, 파라크리노 신호 및 직접 접촉19,43,44를통해. 특히, 이러한 연구는 전단 스트레스와 스트레칭의 결합 된 응용 프로그램의 경우, 조직 형성과 염증에 미치는 영향은 두 부하 중 하나에 의해 지배된다는 것을 밝혔다, 또는 두 부하의 시너지 효과가있다. 이러한 연구 결과는 TE 공정에서 기계적 환경의 기여를 더 잘 이해하기 위해 두 부하를 분리하는 관련성을 보여줍니다. 이러한 이해는 관련 혈역학 적재 기조에서 스캐폴드 설계 파라미터를 체계적으로 최적화하기 위해 적용될 수 있습니다. 또한, 이러한 잘 조절된 환경에서 의 기계형 데이터는 최근 TEVGs65 또는 TEHV66에대해 보고된 바와 같이, 시투 조직 리모델링 과정을 예측하기 위해 개발되고 있는 수치 모델에 대한 입력으로 작용할 수 있으며, 예측 능력을 더욱 향상시킨다.

<table><tbody><tr><td>advanced Dulbecco&amp;rsquo;s modified EagleMedium (aDMEM)</td><td>Gibco</td><td>12491-015</td><td>cell culture medium for fibroblasts</td></tr><tr><td>Aqua Stabil</td><td>Julabo</td><td>8940012</td><td>prevent microorganism growth in bioreactor-hydraulic reservoir</td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T4648</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td>to spin down cells and conditioned medium</td></tr><tr><td>Clamp scissor - &quot;kelly forceps&quot;</td><td>Almedic</td><td>P-422</td><td>clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td></tr><tr><td>CO2 cell culture incubators</td><td>Sanyo</td><td>MCO-170AIC-PE</td><td>for cell culturing</td></tr><tr><td>Compressed air reservoir</td><td>Festo</td><td>CRVZS-5</td><td>smoothing air pressure fluctuations and create time delays for pressure build-up</td></tr><tr><td>Custom Matlab script to calculate the maximum stretches</td><td>Matlab</td><td>R2017. The Mathworks, Natick, MA</td><td>calculate the minimum and maximum outer diameter of the electrospun scaffold</td></tr><tr><td>Data acquisition board</td><td>National Instruments</td><td>BNC-2090</td><td>data processing in between amplifier system and computer</td></tr><tr><td>Ethanol</td><td>VWR</td><td>VWRK4096-9005</td><td>to keep sterile working conditions</td></tr><tr><td>Fetal bovine calf serum (FBS)</td><td>Greiner</td><td>758087</td><td>cell culture medium supplement; serum-supplement</td></tr><tr><td>Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>flow culture chamber compartments (autoclave at step 1, step 7)</td></tr><tr><td>Glass Pasteur pipet</td><td>Assistant</td><td>HE40567002</td><td>apply vacuum on electrospun scaffold (autoclave at step 1)</td></tr><tr><td>Glass tubes of the flow culture chamber</td><td>Custon made, Equipment &amp;amp; Prototype Center, Eindhoven University of Technology</td><td>n.a.</td><td>part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td></tr><tr><td>GlutaMax</td><td>Gibco</td><td>35050061</td><td>cell culture medium amino acid supplement, minimizes ammonia build-up</td></tr><tr><td>High speed camera</td><td>MotionScope</td><td>M-5</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td><td>Nikon</td><td>JAA616AB</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>Hose clip</td><td>ibidi GmbH</td><td>10821</td><td>block medium flow (autoclave at step 1, step 7)</td></tr><tr><td>Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td></tr><tr><td>Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td><td>ibidi GmbH</td><td>10902</td><td>set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td></tr><tr><td>Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td><td>Swann Morton</td><td>0301; 0933</td><td>to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td></tr><tr><td>LabVIEW Software</td><td>National Instruments</td><td>version 2018</td><td>to control the stretch applied to the scaffolds</td></tr><tr><td>Laminar flow biosafety cabinet with UV light</td><td>Labconco</td><td>302310001</td><td>to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td></tr><tr><td>Large and small petri dishes</td><td>Greiner</td><td>664-160</td><td>for sterile working conditions</td></tr><tr><td>L-ascorbic acid 2-phosphate (vitamin C)</td><td>Sigma</td><td>A8960</td><td>cell culture medium supplement, important for collagen production</td></tr><tr><td>LED light cold source KL2500</td><td>Zeiss</td><td>Schott AG</td><td>to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td></tr><tr><td>Luer (female and male) locks and connectors, white luer caps</td><td>ibidi GmbH</td><td>various, see (https://ibidi.com/26-flow-accessories)</td><td>to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td></tr><tr><td>Measuring amplifier (PICAS)</td><td>PEEKEL instruments B.V.</td><td>n.a.</td><td>to amplify the signal from the pressure sensor and feedback to LabView</td></tr><tr><td>Medium reservoir (large syringes 60 mL) and reservoir holders</td><td>ibidi GmbH</td><td>10974</td><td>medium reservoir (autoclave at step 1, step 7)</td></tr><tr><td>Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td><td>Rubber BV</td><td>1805</td><td>to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td></tr><tr><td>Motion Studio Software</td><td>Idtvision</td><td>2.15.00</td><td>to make the high speed time lapse images for stretch monitoring</td></tr><tr><td>Needle (19G)</td><td>BD Microlance</td><td>301700</td><td>together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td></tr><tr><td>Needle driver</td><td>Adson</td><td>2429218</td><td>to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td></tr><tr><td>Paper tissues</td><td>Kleenex</td><td>38044001</td><td>for cleaning of the equipment with 70% ethanol</td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td>quick fix if leakage occurs</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Lonza</td><td>DE17-602E</td><td>cell culture medium supplement; prevent bacterial contamination</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P4417-100TAB</td><td>for storage and washing steps (autoclave at step 1)</td></tr><tr><td>Plastic containers (60 mL) with red screw caps</td><td>Greiner</td><td>206202</td><td>to prepare the fibrinogen solution</td></tr><tr><td>Pneumatic cylinder</td><td>Festo</td><td>AEVC-20-10-I-P</td><td>to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &amp;micro;m wall thickness, 20 mm length)</td><td>SyMO-Chem, Eindhoven, The Netherlands</td><td>n.a.</td><td>produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td></tr><tr><td>Pressure conduit without holes (for static control)</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td></tr><tr><td>Pressure sensor and transducer</td><td>BD</td><td>TC-XX and P 10 EZ</td><td>the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td></tr><tr><td>Proportional air pressure control valve and pressure sensor</td><td>Festo</td><td>MPPES-3-1/8-2-010, 159596</td><td>provides compressed air to the pneumatic actuated pump</td></tr><tr><td>Roswell Park Memorial Institute 1640 (RPMI-1640)</td><td>Gibco</td><td>A1049101</td><td>cell culture medium for monocyte/macrophage</td></tr><tr><td>Safe lock Eppendorf tubes (1.5 mL)</td><td>Eppendorf</td><td>30120086</td><td>multiple applications (autoclave at step 1)</td></tr><tr><td>Sodium dodecyl sulfate solution 20%</td><td>Sigma</td><td>5030</td><td>Used to clean materials, at a concentration of 0.1%.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Silicone O-rings</td><td>Technirub</td><td>1250S</td><td>to prevent leakage (autoclave at step 1, step 7)</td></tr><tr><td>Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td><td>Rubber BV</td><td>1805</td><td>to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td></tr><tr><td>Sterile tube (15 mL)</td><td>Falcon</td><td>352095</td><td>multiple applications</td></tr><tr><td>Suture, 5-0 prolene with pre-attached taper point needle</td><td>Ethicon, Johnson&amp;amp;Johnson</td><td>EH7404H</td><td>Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td></tr><tr><td>Syringe (24 mL)</td><td>B. Braun Melsungen AG</td><td>2057932</td><td>to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td></tr><tr><td>Syringe filter (0.2 &amp;micro;m)</td><td>Satorius</td><td>17597-K</td><td>to filter the fibrinogen solution</td></tr><tr><td>T150 cell culture flask with filter cap</td><td>Nunc</td><td>178983</td><td>to degas culture medium</td></tr><tr><td>T75 Cell culture flask with filter cap</td><td>Nunc</td><td>156499</td><td>to culture static control samples</td></tr><tr><td>Teflon bellow</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Tray (stainless steel)</td><td>PolarWare</td><td>15-248</td><td>for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td></tr><tr><td>Tweezers</td><td>Wironit</td><td>4910</td><td>sterile handling of individual parts (autoclave at step 1 and 7)</td></tr><tr><td>Ultrapure water</td><td>Stakpure</td><td>Omniapure UV 18200002</td><td>to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td></tr><tr><td>UV light</td><td>Philips</td><td>TUV 30W/G30 T8</td><td>for decontamination of grafts and bioreactor parts before seeding</td></tr></tbody></table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

신체에서 직접 재생을 유도하는 재생을 유도하는 재생 가능한 생체 재료의 사용은 번역 관점에서 매력적인 전략입니다. 이러한 물질은 이식 시 염증 반응을 유도하며, 이는 물질의 후속 흡수와 새로운 조직의 재생의 원동력이다. 이 전략, 또한 시투 조직 공학에서 로 알려진, 조직 설계 혈관 이식 등 심장 혈관 교체를 얻기 위해 추구. 염증 및 재생 공정 은 스캐폴드 (즉, 스트레치 및 전단 응력)에 대한 국소 생체 역학 적 단서에 의해 결정됩니다. 여기서는 관 비계에서 스트레치및 전단 응력을 분리할 수 있는 맞춤형 생물 반응기의 사용을 자세히 설명합니다. 이를 통해 인간 대식세포와 근섬유아세포아제를 이용한 역동적인 공동배양 실험에 기초하여 입증되는 잘 조절된 기계적 하중의 영향으로 관 비계의 염증및 재생 능력의 체계적이고 표준화된 평가를 가능하게 한다. 바이오 반응기의 구성 및 설정, 비계 및 세포 파종의 준비, 스트레치 및 전단 흐름의 적용 및 유지 보수, 분석을 위한 샘플 수확 등 이 접근법의 주요 실용적인 단계는 자세히 설명합니다.

이 생물 반응기는 혈관 조직 성장에 전단 스트레스와 순환 스트레칭의 개별적이고 결합 된 효과를 연구하고 3D 생체 물질 스캐폴드에서 리모델링하기 위해 개발되었다. 생물 반응기의 설계는 다양한 적재 조건하에서 최대 8개의 혈관 구조를 배양할 수 있게한다(도 1A). 혈관 구조는 둘레 스트레치와 WSS가 독립적으로 제어될 수 있는 유량 배양챔버(도 1B)에</stron...

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

이 프로토콜의 목표는 관형 전기 스캐폴드에서 인간 대식세포와 근섬유세포의 동적 공동 배양을 실행하여 재료 중심 조직 재생을 조사하는 것이며, 전단 응력과 순환 스트레칭의 분리를 가능하게 하는 생체 반응기를 사용하는 것입니다.

유량 및 스트레치 하에서 생체 재료의 염증 및 재생 능력을 평가하는 다중 큐 생물 반응기

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Research

JoVE Journal

Engineering

5.5K 조회수.  Eindhoven University of Technology. 이 프로토콜의 목표는 관형 전기 스캐폴드에서 인간 대식세포와 근섬유세포의 동적 공동 배양을 실행하여 재료 중심 조직 재생을 조사하는 것이며, 전단 응력과 순환 스트레칭의 분리를 가능하게 하는 생체 반응기를 사용하는 것입니다.

비디오: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

타악기 생물 반응기를 사용하여 생물 공학 기반 혈관의 이식

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

연구

생체공학

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 ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

조직 공학을위한 Electrospun의 Fibres의 Postproduction 처리

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

조직 공학을위한 이축 기계 로딩 생물 반응기의 설계

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

연구 조직 이식에 세균성 부착을 병렬 플레이트 관류 시스템의 생체 외에서 모델

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

발생학

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

혈관 세포 리모델링의 조사에 생리의 체외 모델 및 응용 프로그램과 함께 병리학 혈액 흐름

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

동적 세포 배양을 위한 출구 분별과 통합된 다중 스트림 관류 생물반응기

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

조직과 세포를 유지하고 평가하기 위한 펌프리스 다중 채널 흐름 배양

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

저자 스포트라이트: 최적의 조직 유지 관리 및 실시간 평가를 위한 다중 채널 흐름 배양을 통한 안구 생리학 연구 발전 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.