이 작품은 UWS 연구 기금에 의해 지원되었다.

1. 키토산 접착제 준비 <ol><li> 키토산 분말은 아세트산 용액에 용해이다; 아세트산 (2 % V / V)의 주식 솔루션의 준비를 위해, 490 ML 탈 이온수 (DI-H 2 O)로 10 ML 빙하 아세트산을 추가합니다. </li><li> 아세트산의 장미 벵골 (RB) (0.01 % w / V)의 주식 솔루션의 준비를 들어, photobleaching 피하기 위해 알루미늄 호일로 싼 병에 RB의 5 밀리그램 무게. 아세트산 주식 솔루션 (섹션 1.1에 설명)의 49.5 ML을 추가 한 후 분말을 용해하는 DI-H 2 O의 0.5 ML를 추가합니다. </li><li> 키토산 용액 (1.7 % w / V)의 준비를 위해, 키토산 분말 (중간 MW, 아세틸의 85 % 정도)의 0.85 g을 무게 RB 아세트산의 산성 솔루션의 50 ML에 추가 할 수 있습니다. 컨트롤은 RB없이 아세트산 용액에 키토산을 용해하여 준비되었다. </li><li> 키토산 혼​​합물에 자석 교반기를 추가하고 실온에서 젓는다. 구성 요소의 완전한 해산을 보장하기 위해, 키토산을 흔들어2 주 5 일 아세트산의 키토산 (제어)에 대한 아세트산의 + RB. 키토산을 용해되면 솔루션 pH는 2.6 ~ 3.9에서 ~까지 증가합니다. RB는 산성 pH의에서 제대로 용해하고 그 용액의 pH 및 용해 할 수있는 확장 저어 시간의 증가가 필요합니다. </li><li> 1 시간에 3,270 XG에서 솔루션을 원심 분리기와 예를 들어, 멸균 10 ML의 주사기 사용하여 표면에 뜨는를 수집합니다. 불순물 펠렛을 방해하지 않도록하십시오. 이 단계 동안, 솔루션은 매크로 불순물과 불용성 물질에서 청소 서비스가 제공됩니다. </li><li> 멸균 주사기를 사용하여 평면 perpex 판에 (centrifuged) 키토산 용액을 주사. 건조 필름의 두께는 솔루션의 투여 량과 솔루션 확산되는 동안 표면 영역 사이의 비율에 따라 달라집니다. 솔루션은 기포의 무료이며 고르게 슬래브에 퍼져 있는지 확인합니다. ~ 13 μm의 두께로 박막을 구하려면 12cm이 표면에 ~ 1 ML 솔루션을 주입영역 (크기가 ~ 3x4 cm, 그림 1A). </li><li> 솔루션의 photobleaching 피하기 위해 예를 들어 알루미늄 호일로 만든 화면 Perspex 판을, 다룹니다. 공기가 충분히 환기가 화면 아래에있는 솔루션을 건조 보장되어 있는지 확인하십시오. </li><li> 그 결과 영화는 물에 불용성이며, macroscopically 팽창하지 않습니다 때까지 실내 온도 (25 ° C)와 압력 (1 ATM)에서 건조 할 수있는 솔루션을 둡니다. 이것은 일반적으로 2 주에 달성된다. </li><li> 조심스럽게 얇고 평평한 주걱을 사용하여 필름을 분리 :이 필름은 쉽게 손상시키지 않고 Perspex 슬래브에서 벗겨 수 있습니다. </li><li> 마이크로 미터를 사용하여 필름의 두께를 측정합니다. </li><li> 깨끗한 날카로운 가위로 사각형 스트립에 접착제를 잘라과 평면 모양을 유지하기 위해 멸균 유리 슬라이드 사이에 넣어. 빛 차폐 용 알루미늄 호일에있는 슬라이드를 정리하고 실온에서 보관하십시오. 또는 &quot;키토산 adhes&quot;접착제 장미 &quot;로 RB와 함께 또는없이 접착제 레이블을필자 &quot;각각. </li><li> 이 접착제 필름은 감마 방사선 (0.1 KGy / 24 시간 용 H) 4,5이나 (90 %) 에탄올을 사용하여 소독 할 수 있습니다 올랐다. </li></ol> 2. 접착제 광 감쇠 <ol><li> 석영 큐벳 안에 장미 접착제를 해결했습니다. </li><li> 이중 빔 분광 광도계 4를 사용 800 nm의 - 400의 범위에서 해당 감쇠 스펙트럼을 기록합니다. </li><li> 다음과 같이 맥주의 율법의 유효성 가정, 접착제의 감쇠 길이를 계산 : I = I 0 전자 도끼 어디 0 입사 빔 강도, 1 / A는 감쇠 길이이며, 여기서 x는 필름 두께입니다. </li><li> 키토산 접착제에 대한 2.1-2.3가 컨트롤 역할을하는 단계를 반복합니다. </li></ol> 3 체외 응용 프로그램에서 :. 소장에 레이저 조직 본딩 <ol><li> 수확 종아리 소장은 즉시 후 -80 동물 안락사 및 매장 ° C. </li><li> 사용하기 전에, 녹이과 수화물 트레져 아일랜드10 분 동안 DI-H 2 O에 immersing하여 ssue. </li><li> 운영 현미경 (X10)에서 # 10 블레이드를 사용하여 섹션 (2 × 1 ㎝)로 조직을 이등분. DI-H 2 O를 사용하여 섹션 습기를 유지 </li><li> (끝과 끝) 가까운 곳에 절개를 stumps 가져와. 면 팁을 사용하여 표면에서 초과 물을 닦아주십시오. </li><li> 장 (그림 2)와 전체 접촉을 보장 microforceps로 serosa 층에있는 절개를 통해 장미 접착제 스트립 (10 × 6 ㎜) 놓습니다. </li><li> 장미 접착제의 조직 유착은 멀티 광섬유 (코어 직경 200 μm) 4에 커플 링 다이오드 펌핑 고체 레이저에 의해 활성화됩니다. </li><li> 180 MW에 레이저의 전력 수준을 설정하고 표 1에 나와있는 매개 변수를 사용 ~ 5 mm의 빔 스폿 크기 ~ 6 분, 대한 접착제를 비추다. 스팟 - 비추다 접착제는 각 지점이 인접 지점으로 빔을 이동하기 전에 ~ 5 초 동안 방사선 것을 보장합니다. 티그 과정은 레이저 빔은 접착제 여러 번 전체 면적을 검색하는 보장합니다. </li><li> 조직 접합 강도를 평가하기 위해, 기계 그립을 사용하여 보정 한 열 tensiometer (Instron, MA, USA)에 샘플을 클램프. 네 별도의 두 조직 stumps까지 22mm / 분에서 그립을 이동합니다. </li><li> 단계 3.5에 설명 된대로 조직의 장미 접착제를 놓습니다. 샘플을 비추다과 단계 3.8에 설명 된대로 조직 접합 강도를 측정하지 마십시오. 이 예제는 제어 역할을합니다. </li></ol> 4 생체 응용 프로그램에서 :. 정강 신경 문합 <ol><li> O / isoflurane이 혼합물 (이후 유도 동안 4퍼센트, 2 %) 5 품위 Wistar 쥐. 피하 간 수술 진통제 활동을 제공하고 가능한 통증이나 불편 함을 완화하기 위해 Buprenex를 (0.03 밀리그램 / kg) 주입. </li><li> 다음 수술 절차는 운영 현미경 (X10)에서 수행해야합니다. 경사 피부 만들기오른쪽 허벅지의 dorso - 측면 부분의 길이 둔부 근육이를 통해 근육 분할 방식으로 정강 신경을 노출 3cm에 대한 절개. </li><li> 부분적으로 해부하고 바로 마이크로 가위를 사용하여 정강 신경의 adventitia을 다듬하고 멸균 거즈 나면 팁을 초과하는 물을 흡수합니다. </li><li> 마이크로 가위로 정강 신경을 잘라 요. </li><li> 마이크로 포셉을 사용하여 정강 신경 아래에 살균 키토산 스트립 (크기 5 X 4mm)을 배치합니다. </li><li> 대략적인 신경 stumps 장미 접착제 스트립 이상의 마이크로 포셉과 엔드 - 투 - 엔드. </li><li> 스트립 완전히 스트립의 레이저 조사 동안 신경의 회전을 도와드립니다 칼라를 형성 신경을 준수합니다. </li><li> ) 단계 3.6에 설명 된대로이 레이저 접착제를 장미 활성화) 및 3.7. </li><li> 작동 신경의 칼라에서 중복 장미 접착제를 잘라. </li><li> 다섯 3-0 봉합 5 근육과 피부를 닫습니다. </lI&gt; <li> 필요한 경우 표준 수술 복구 절차는 dehiscence 또는 고름 형성과 진통제 관리에 대한 일일 점검 등의 준수해야합니다. </li></ol> 5. 대표 결과 얻을 수있는 영화는 밝고은 색 장미 얇고 매끄러운 표면 (그림 1)가 있습니다. 또한 유연하고 원인이 눈물 나 다른 명백한 손상 (그림 1B)없이 작은 직경의 튜브에 와서 할 수 있습니다. 장미 접착제는 530과 562 나노 미터에 두 흡수 피크가, 녹색 레이저 ~ 12 μm (그림 3)입니다 따라서 강력하게 532 나노 미터의 접착제 및 해당 감쇠의 길이에 의해 흡수됩니다. 대조적으로, RB없이 키토산 필름 가능성이 높습니다, 약하게 산란에 의한 레이저 (1 / A ~ 162 μm)를 attenuates. 그것은 RB 대한 중요한 집계은 영화에서 발생하지 않는 스펙트럼의 줄거리에서 나타납니다. 은 단단히 접착 채권 장미레이저 조사시 장에 (그림 2) 0.9의 실패의 전형적인 최대 하중 ± 0.4 N (N = 30)을 달성. 접착제 강도는 접착 면적, 즉 15으로 나눈 최대 부하 ± 6 kPa (N = 30)로 추정되었다. 비 방사능 장미 접착제가 조직에 훨씬 적은 보세 (2 ± 2 kPa, N = 30, unpaired t-테스트 P &lt;10 6). 정강 신경의 인 생체 문합이 성공적으로 녹색 레이저와 함께 장미 접착제의 응용 프로그램과 결합에 따라 달성했다. 일주일 postoperatively, 운영 신경이 연속성에 있던 및 수리 강도는 17 살 ± 9 kPa (N = 10). <img alt="그림 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> 그림 1. A) RB-키토산 솔루션은 평면 Perspex 판 (슬래브 두께 ~ 5mm)을에 주조되어 있습니다. 그래프 용지는 건조 캐스트 단계를 촉진하기 슬래브 아래에 배치됩니다. 물 불용성 FILM은 2 주 주조 후 얻어진다. B) 장미 접착제는 유연하고 작은 실린더에 압연 할 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/4158/4158fig2.jpg" /> 그림 2. 장미 접착제는 레이저 조사 후 종아리 소장에 접착되어 있습니다. 통일 조사는 조직 - 본딩 동안 RB 접착제에 적용되지만 선택 관광은 접착제에 레이저의 사진 표백 효과를 설명하기 위해이 그림에 표시됩니다. <img alt="그림 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> 그림 3. 장미 접착제의 흡수 스펙트럼은 (임의의 단위) 530과 562 nm의 두 봉우리를 보여줍니다. 녹색 레이저 (λ = 532 nm 정도)은 따라서 강력 레이저에 노출시 접착제에 의해 흡수됩니다. RB없는 키토산 접착제이 저조한 레이저 빔을 흡수. <table border="1"><tbody><tr><td></td><td> N </td><td><s&gt; 면적 (mm 2) trong </td><td> 전원 (W) </td><td> 시간 (s) </td><td> Fluence (J / cm 2) </td><td> I (W / cm 2) </td><td> 최대로드 / 지역 (kPa) </td></tr><tr><td> 접착제 + 레이저 </td><td> 30 </td><td> 60 ± 10 </td><td> 0.18 ± 0.03 </td><td> 365 ± 5 </td><td> ~ 110 </td><td> ~ 0.9 </td><td> 15 ± 1 </td></tr><tr><td> 점착성의 </td><td> 30 </td><td> 60 ± 10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 이 ± 2 </td></tr></tbody></table> 표 1. 전설. N, 샘플 번호, 지역, 장미 접착제 (± 최대 오류를 의미)의 면적, 전력, 레이저 전원 (± 최대 오류를 의미), 시간, 조사 시간 (± 최대 오류를 의미), Fluence, 평균 레이저 fluence, I, 예상 irradiance, 최대로드 / 지역, 접착제 면적으로 나눈 값 수리 조직의 실패에서 최대로드 (± SE를 의미).

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Reconstr. Microsurg. 19, 17-20 (2003).</li><li>Menovsky, T., Beek, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser,+fibrin+glue,+or+suture+repair+of+peripheral+nerves:+a+comparative+functional,+histological,+and+morphometric+study+in+the+rat+sciatic+nerve.">Laser, fibrin glue, or suture repair of peripheral nerves: a comparative functional, histological, and morphometric study in the rat sciatic nerve.</a> J. Neurosurg. 95, 694-699 (2001).</li><li>Garc&#237;a, P., Jorge, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+the+mechanical+behavior+of+a+cyanoacrylate+and+a+bioadhesive.">Comparative study of the mechanical behavior of a cyanoacrylate and a bioadhesive.</a> J. Mater. Sci. Mater. Med. 15, 109-115 (2004).</li><li>Lauto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+extracellular+matrix+with+chitosan+adhesive+film+for+sutureless+tissue+fixation.">Integration of extracellular matrix with chitosan adhesive film for sutureless tissue fixation.</a> Lasers Surg. Med. 41, 366-371 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 산성 pH의에서 RB의 해산이 연장 키토산 솔루션의 감동을 필요로하지만 접착제 제조가 간단한 드라이 주조 공정을 기반으로했다. 그것이 물 불용성 필름 될 때까지이 솔루션가 건조하게하는 것이 중요합니다. 체중 물 콘텐츠가 건조 필름 6 ~ 10 % 일 때 발생합니다. 불용성 영화는 일반적으로 온도와 압력 (~ 25 ° C 및 ~ 1 ATM)의 표준 조건에서 이주 드라이 주조 후 얻을 수 있습니다. 이에 레...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 장미 벵골 </td><td> 시그마 - 알드리치 </td><td> 632-69-9 </td><td></td></tr><tr><td> 키토산 (중간 MW) </td><td> 시그마 - 알드리치 </td><td> 10318AJ </td><td></td></tr><tr><td> 빙하 아세트산 </td><td> 시그마 - 알드리치 </td><td> 08050051 </td><td> 2퍼센트 DI 물에 브이 / v </td></tr><tr><td> 자기 교반기 </td><td> Heidolph </td><td> MR 헤이 믹스 S </td><td></td></tr><tr><td> 원심 분리기 </td><td> Beckman 보습 바로 앞에 달린 풀 베는 날 </td><td> Allegra X-12R </td><td></td></tr><tr><td> 분광 광도계 </td><td> Varian </td><td> 캐리 4000 UV-표시 </td><td></td></tr><tr><td> 레이저 </td><td> CNI 레이저 </td><td> MGL-532 </td><td></td></tr><tr><td> 마이크로 미터 </td><td> Mitutoyo </td><td> 시리즈 227 </td><td></td></tr><tr><td> 외과 현미경 </td><td> Zeiss </td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

광화학 조직 접합 (PTB)는 두 조직 가장자리 1,2 사이에 장미 벵골 (RB)의 솔루션을 적용하여 달성 조직 수리에 대한 sutureless 기술입니다. 이 그 다음 선택적으로 RB에 의해 흡수되는 레이저로 했소 있습니다. 그 결과 광화학 반응은 아마도 최소한의 열 생산 3 조직의 콜라겐 섬유를 crosslink. 이 보고서에서, RB는 레이저 활성화되어 새로운 조직 접착제를 조작하는 얇은 키토산 필름에 통합되었습니다. 키토산 및 ~ 0.1 중량 %의 RB를 포함에 따라 접착제 필름, 고체 상태 레이저 (λ = 532 nm의, Fluence ~ 110 J / cm 2, 스폿 사이즈 ~ 0.5 cm)에 의해 제조와 종아리 소장과 쥐 정강 신경에 접착 아르 . 개인용 컴퓨터와 인터페이스 단일 열 tensiometer은, 접합 강도를 테스트하는 데 사용됩니다. 15 세기 ± 6 kPa (N = 30)와 단단히 장에 RB-키토산 접착제 채권. 접착 강도는 2 &amp;까지 떨어plusmn, 2 kPa (N = 30) 레이저가 접착제에 적용되지 않을 때. 정강 신경의 문합은 봉합를 사용하지 않고 완료 할 수 있습니다. 소설 키토산 접착제는 해당 채권이 photochemically 조직에 가공되​​어 봉합이 필요하지 않습니다.

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

봉합은 일반적으로 수술 절차 동안 조직을 복구 할 필요합니다. 그들은 침략하고 조직에 손상을 줄 수 있으므로 그러나, 그 응용 프로그램은 문제가 될 수 있습니다. 소설 조직 접착제의 제조 및 응용 방법이 여기에보고됩니다. 이 접착제 필름은 레이저 활성화되고 봉합의 사용을 필요로하지 않습니다.

제조 및 레이저 조직 복구에 장미 벵골 - 키토산 영화의 응용

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

fabrication-application-rose-bengal-chitosan-films-laser-tissue

Research

JoVE Journal

Bioengineering

12.1K 조회수.  University of Western Sydney, NSW Australia. 봉합은 일반적으로 수술 절차 동안 조직을 복구 할 필요합니다. 그들은 침략하고 조직에 손상을 줄 수 있으므로 그러나, 그 응용 프로그램은 문제가 될 수 있습니다. 소설 조직 접착제의 제조 및 응용 방법이 여기에보고됩니다. 이 접착제 필름은 레이저 활성화되고 봉합의 사용을 필요로하지 않습니다.

비디오: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

심근 조직 공학을위한 생물학 유래 주사용 재료의 제조

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

연구

생체공학

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

전기 자극의 손쥐 Myoblast의 전구 세포 및 응용에서 공학 골격근 조직도

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

전기 방사를 적응하는 것은 트라이 계층에 대한 세 가지 독특한 환경을 제공하기 위해<em&gt; 체외</em&gt;기도 벽의 모델

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

혈관 유래 미세 유체 네트워크의 이미지 유도, 레이저 기반의 제조

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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 ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

혈관 시각화를 위한 이중 래스터 스캐닝 광음향 소형 동물 이미지

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...