이 작품은 우수 연구 센터, 제어 공학 연구소, 싱가포르 중 하나에 의해 관리되는 싱가포르 국립 연구 재단에 의해 지원되었다. MFAC는 1122703037. BKKT는 제어 공학 연구소 지원 연구를위한 과학, 기술 및 연구 기관 (싱가포르) 및 국가 기관 (프랑스) 프로젝트 번호에 따라 공동 프로그램에 의해 지원됩니다. 우리는 증명 판독 비디오 제작에 협조 원고와 양 새벽 JH 네오 씨 다니엘 HC 웡 감사합니다.

고분자 전해질 솔루션 1. 준비 <ol><li> 리아 외에서 설명하는대로, 키토산을 정제. 간략하게, 1 %를 만드는 2 % 키토산 용액 (/ V W) (v / v) 아세트산 함유하고, 진공 필터 (93)가 급 여과지. pH가 10 분 동안 1,200 XG에서 7 원심 침전 된 키토산에 안정화 될 때까지 5M 수산화 나트륨을 사용하여 여과 액을 중화. 뜨는을 가만히 따르다 및 키토산을 씻어 탈 이온수를 추가합니다. 원심 분리 및 세척 단계를 두 번 더 반복합니다. 정제 된 형태를 얻기 위해 -80 ° C 및 동결 건조 O / N에서 석출 키토산 동결. 제습 캐비닛에 정제 된 키토산을 저장합니다. </li><li> 무균 조직 배양 접시에 정제 키토산 1g을 달아. 생물학적 안전 캐비넷의 UV 램프에 최대한 가깝게 조직 배양 접시에 키토산을 넣고 15 분 동안 UV 광에 노출. 무균 핀셋을 사용하여, 유리 용기에 멸균 키토산 놓는다. 용해 키토산은 0 필터링 사용.0.5 %와 1 % 사이의 최종 농도 15M 아세트산 (W / V). </li><li> 알긴산 나트륨 0.1 g을 칭량하고 (w / v)의 용액을 1 %를 얻기 위해 탈 (DDI)의 증류수 10ml에 녹인다. 완전한 용해를 보장하기 위해 볼텍스 혼합기에서 적어도 2 시간 동안 알긴산 나트륨 염을 섞는다. 0.2 μm의 주사기 필터를 통해 알지네이트 용액 필터. 4 ° C에서 알지네이트 솔루션을 저장합니다. </li><li> 제조자가 권장하는 신경 성장 인자 (NGF), - 혈관 내피 성장 인자 (VEGF) 또는 베타 인간 재조합 성장 인자 재구성. </li></ol> IPC 섬유 2. 도면 <ol><li> 유사한 순 전하를 갖는 고분자 전해질 용액의 10 내지 20 μL의 분취 액으로 단백질, 성장 인자 또는 다른 생물 분자를 섞는다. 순 음전하 (예, 소 혈청 알부민 [BSA])와 생물학적 분자 알지네이트 용액을 혼합한다. 양전하 (예 : VEGF)와 생물 분자와 혼합되어야키토산 솔루션입니다. </li><li> 파라 필름으로 덮여 안정된 평평한 표면에 키토산 및 알긴산의 작은 분취 량 (10 ~ 20 μL)를 놓습니다. 키토산 및 알긴산 액 근접 배치 아니라 서로 접촉한다. </li><li> 가볍게 키토산과 알긴산 방울에 집게 한 쌍의 각 끝을 찍어. 함께 집게 곤란하여 고분자 전해질의 방울을 가져와. 물방울이 서로 접촉 할 때, 서서히 두 방울 (도 1a)의 인터페이스에서 IPC 섬유 그릴 수직 상방 집게를 당긴다. </li><li> 이러한주의 깊게 회전 맨드릴 상에 부착되어 플랫 고분자 담체로 집 전체에 겸자에 그려진 IPC 섬유의 단부를 배치했다 (제 3 및 4 참조). 유니폼과 IPC 섬유 beadless 형성 할 수 있도록 10mm / sec의 고정 된 속도로 맨드릴을 돌립니다. Incorporated의 버스트 방출을 일으킬 것이다 비드를 형성 할 것이다 IPC 섬유 드로잉 속도를 증가생화학 및 조기 섬유 종료. (10) </li><li> 통합의 효율성을 결정하기 위해, 남아있는 모든 액체가 1X 인산염 완충 생리 식염수 500 μL (PBS)으로 희석하여 파라 필름에 남아 수집합니다. BCA의 (BSA에 대한) 분석 (VEGF 및 NGF의 경우) 또는 ELISA 혼입 생체 분자를 검출하기 위해 적절한 분석을 통해 잔사 중의 단백질 또는 성장 인자의 함유량을 측정한다. </li></ol> 복합 하이드로 겔 풀루 란 - 덱스 트란의 비계 (PD) 다당류 및 IPC 섬유 3. 제작 <ol><li> IPC 섬유 수집을 위해 희생 풀루 란 프레임 제조 <ol><li> 풀루 란 다당류를 달아 (W / V) 수용액을 20 %를 만드는 증류 탈 (DDI) 물과 혼합. 균일 성을 보장하기 위해 풀루 란 솔루션 O / N을 섞는다. </li><li> 10cm 직경의 조직 문화 폴리스티렌 (FPC 기판) 접시에 풀루 란 용액 15g 캐스트. 37 ° C에서 풀루 란 솔루션 O / N을 건조. 7에 풀루 란 영화를 잘라MM은 7mm 사각 프레임을 X. </li></ol></li><li> 풀루 덱스 트란 폴리 사카 라이드 용액을 제조 <ol><li> 30 %를 만들기 다당류의 솔루션 풀루 란과 덱스 트란 (/ V W) : DDI 물 (3 1 비율). 폴리 사카 라이드 용액의 균질성을 확인하기 위해 O / N 섞는다. 천천히 20 %의 최종 농도를 달성하기 위해 다당류 용액에 중탄산 나트륨으로 추가 (W / V). 용액의 균일 성을 보장하기 위해 O / N을 섞는다. 4 ° C에서 다당류 솔루션을 저장합니다. </li></ol></li><li> 풀루 란 프레임 IPC 섬유를 수집 <ol><li> 악어 클립을 사용하여 희생 풀루 란 프레임 (3.1)를 붙입니다. 플라스틱 코팅 된 접착 테이프를 사용하여 회전 맨드릴의 끝에 악어 클립 및 풀루 란 프레임을 스틱. 10mm / sec의 일정한 속도로 부착 된 프레임 맨드릴을 돌립니다. 풀루 프레임은 원하는 방향으로 회전 맨드릴 상에 부착 될 수있다. </li></ol></li><li> 집게 쌍 (1 절)와 ATTAC을 사용하여 IPC 섬유 그리기H 회전 풀루 란 프레임 상에 IPC 섬유의 그린 끝. 일정 속도로 IPC 섬유를 그린다. IPC 섬유의 말단부에 도달 한 후, RT에서 N / 섬유 - 온 - 프레임 구조의 O를 건조. </li><li> PD 하이드로 겔 지지체에 IPC 섬유의 포함 </li><li> . (/ V W) 나트륨 수용액 및 100 μL의 10M 수산화 나트륨을 트리 메타 11 %, 100 ㎕를 추가 풀루 덱스 트란 용액의 각 그램 가교 7 1 ~ 2 분 동안 stirplate를 이용하여 60 rpm으로 용액을 혼합하기. 트리 메타 포스페이트 나트륨과 수산화 나트륨을 첨가 한 후, 폴리 사카 라이드 용액을 거의 즉시 가교 것이다. IPC 섬유를 완전히를 포함하는 섬유 - 온 - 프레임 구조에 점성 다당류 용액을 붓고. 화학적으로 가교 결합 된 복합체 지지체 (도 1b)를 형성하기 위해 30 분 동안 60 ° C에 결합 된 덱스 트란, 풀루 란 - IPC 섬유 (PD-IPC)를 부화. </li><li> 주의 : 흄 후드 단계 3.3.2을 수행하고 적절한 보호 동등를 사용아세트산 등의 pment 부식성 및 인화성이다. </li><li> PD-IPC 지지체의 기공 형성을 유도하기 위하여, 20 분에 20 % (w / v)의 아세트산 전체 골격 잠수함. </li><li> 100 rpm으로 흔들어 섞으면 서 5 분간 1X PBS에 세척 PD-IPC 골격에 의해 미 반응 시약을 제거합니다. 이 단계를 2 번 반복합니다. </li><li> 초과 PBS를 제거하고 즉시 -80 ° CO / N의 PD-IPC 발판을 동결. 비계 어떤 제어 방출 또는 생체 활성 분석에 사용하기 전에 적어도 24 시간을 동결 건조. </li></ol> PCL 및 IPC 섬유의 복합 비계 4. 제작 주의 : 디클로로 메탄은 위험한 물질이다. 디클로로 메탄을 처리 할 때 흄 후드 및 개인 보호 장비를 사용합니다. <ol><li> 깨끗한과 패턴 PDMS 기판 만들기 <ol><li> 소프트 리소그래피 공정을 이용하여 원하는 치수의 FPC 기판의 일부를 사용하여 깨끗한 폴리 디메틸 실록산 (PDMS) 탄성 기판 만든다. patte 만들기원하는 템플릿 지형 폴리 (메틸 메타 크릴 레이트)에 표준 소프트 리소그래피 방법을 사용하여 rned PDMS 기판. (12) </li></ol></li><li> IPC 섬유 수집을 위해 희생 PCL 프레임 제조 <ol><li> PCL을 달아 (W / V) 솔루션을 0.9 %를 만들 디클로로 메탄에 용해. PDMS 기판의 모든 1cm 2 지역의 경우, 0.9 % PCL 용액 500 μl를 놓습니다. 완전히 흄 후드에서 증발 디클로로 메탄 용매를 모두 허용합니다. 원하는 두께로 필름을 두껍게 0.9 % PCL 주조의 과정을 반복합니다. PDMS 기판에서 건조 PCL 필름을 제거합니다. 적절한 크기의 구멍을 뚫는을 사용하여 PCL 프레임에 구멍을 만듭니다. (2) </li></ol></li><li> PCL 프레임 IPC 섬유를 수집 <ol><li> 악어 클립 (4.2.1)에서 구멍 희생 PCL 프레임을 부착합니다. 플라스틱 코팅 된 접착 테이프를 사용하여 회전 맨드릴에 악어 클립 스틱. PCL의 FRAM에 IPC 섬유의 그린 끝을 연결합니다10mm / 초 (제 2)의 일정 속도로 회전을 시작하기 전에 전자. IPC 섬유 드로잉 종료 후, 섬유에 프레임 구조의 O / N에서 4 ℃에서 건조. </li></ol></li><li> 섬유에 프레임이 패턴 PCL 기판으로 구성 퍼가기 <ol><li> 필요에 따라, 깨끗한 또는 패턴 PCL 기반을 만들기 위해 PDMS 기판에 0.9 % PCL 용액 500 μl를 삭제합니다. 원하는 두께와 발판을 얻었다 0.9 % PCL 용액 다층 캐스트. 완전히 흄 후드에서 증발 디클로로 메탄 용매를 모두 허용합니다. </li><li> PCL베이스의 상단에있는 섬유에 프레임 구조 (섹션 4.3.1)을 놓습니다. 원하는 두께를 얻기 위해 섬유의 프레임 구성을 여러 번에 0.9 % PCL 솔루션을 추가하고 완전히 PCL-IPC 복합 지지체 (그림 1C을) 제조, IPC 섬유를 포함. </li></ol></li></ol> 복합 IPC 공사장 공중 발판에서 생물학 작용제의 릴리스 5. 측정 <ol><li> 배치 합성 PD IPC-또는PCL-IPC 골격 및 독립 실행 형 IPC 섬유를 별도로 24 웰 플레이트에. </li><li> 발판을 담그고 독립형 IPC 섬유를 1X PBS 500 μL와 함께. 37 ° C에서 샘플을 품어. 다양한 시점 (릴리스 미디어)에서 PBS를 수집하고 1X PBS 500 μL로 교체합니다. </li><li> 통합 생체 분자 누적 방출 프로파일을 계산하는 BCA 분석 (BSA), ELISA (VEGF 및 NGF) 또는 다른 적절한 분석을 이용하여 분리 배지에서 단백질 또는 증식 인자의 양을 측정한다. </li></ol> 합성 IPC 공사장 공중 발판에서 세포의 6. 시드 출시 생물 제의 생체 활성 테스트하는 <ol><li> 적어도 20 분 동안 생물학적 안전 캐비넷에 UV 광을 사용하여 동결 건조 PD-IPC 또는 PCL-IPC 복합 지지체 멸균. </li><li> 200 ㎕의 성장 배지에 2 × 105 세포의 세포 현탁액을 수득 표준 세포 배양 기술을 사용한다. 복합체 지지체 상 농축 세포 현탁액을 시드. AfteR 20 분, 완전히 비계 잠수함하는 성장 미디어의 최고 최대 볼륨을 설정합니다. </li><li> 이러한하는, Alamar 블루 대사 활동 분석, PC12의 신경 돌기 가지 분석 또는 면역와 같은 표준 기술을 통해 세포 활성을 측정합니다. </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+pullulan-dextran+polysaccharide+scaffold+with+interfacial+polyelectrolyte+complexation+fibers:+A+platform+with+enhanced+cell+interaction+and+spatial+distribution.">Composite pullulan-dextran polysaccharide scaffold with interfacial polyelectrolyte complexation fibers: A platform with enhanced cell interaction and spatial distribution.</a> Acta Biomater. 10 (10), 4410-4418 (2014).</li><li>Yow, S. Z., Quek, C. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-based+fibrous+scaffold+for+spatial+organization+of+encapsulated+and+seeded+human+mesenchymal+stem+cells.">Collagen-based fibrous scaffold for spatial organization of encapsulated and seeded human mesenchymal stem cells.</a> Biomaterials. 30 (6), 1133-1142 (2009).</li><li>Yim, E. K. F., Wan, A. C. A., Le Visage, C., Liao, I. C., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+human+mesenchymal+stem+cell+encapsulated+in+polyelectrolyte+complexation+fibrous+scaffold.">Proliferation and differentiation of human mesenchymal stem cell encapsulated in polyelectrolyte complexation fibrous scaffold.</a> Biomaterials. 27 (36), 6111-6122 (2006).</li><li>Liao, I. C., Wan, A. C., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+from+fibers+of+polyelectrolyte+complexes.">Controlled release from fibers of polyelectrolyte complexes.</a> J. Control. Release. 104 (2), 347-358 (2005).</li><li>Wan, A. C. A., Liao, I. C., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+fiber+formation+by+interfacial+polyelectrolyte+complexation.">Mechanism of fiber formation by interfacial polyelectrolyte complexation.</a> Macromolecules. 37 (18), 7019-7025 (2004).</li><li>Yim, E. K. F., Reano, R., Pang, S., Yee, A., Chen, C., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26 (26), 5405-5413 (2005).</li><li>Sun, H., Xu, F., Guo, D., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+NGF-microsphere+conduits+for+regeneration+of+defective+nerves.">Preparation and evaluation of NGF-microsphere conduits for regeneration of defective nerves.</a> Neurol. Res. 34 (5), 491-497 (2012).</li><li>Sim&#243;n-Yarza, T., Formiga, F. R., Tamayo, E., Pelacho, B., Prosper, F., Blanco-Prieto, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated-PLGA+microparticles+containing+VEGF+for+long+term+drug+delivery.">PEGylated-PLGA microparticles containing VEGF for long term drug delivery.</a> Int. J. Pharm. 440 (1), 13-18 (2013).</li><li>Patel, Z. S., Ueda, H., Yamamoto, M., Tabata, Y., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+release+of+vascular+endothelial+growth+factor+from+gelatin+microparticles+and+biodegradable+composite+scaffolds.">In vitro and in vivo release of vascular endothelial growth factor from gelatin microparticles and biodegradable composite scaffolds.</a> Pharm. Res. 25 (10), 2370-2378 (2008).</li><li>Sun, H., Xu, F., Guo, D., Liu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+effects+of+various+additives+and+polymers+on+nerve+growth+factor+microspheres.">In vitro evaluation of the effects of various additives and polymers on nerve growth factor microspheres.</a> Drug Dev. Int. Pharm. 40 (4), 452-457 (2014).</li><li>Lee, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+drug+release+from+hydrogel+matrices.">Kinetics of drug release from hydrogel matrices.</a> J. Control. Release. 2, 277-288 (1985).</li><li>Cutiongco, M. F. A., Choo, R. K. T., Shen, N. J. X., Chua, B. M. X., Sju, E., Choo, A. W. L., Le Visage, C., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+scaffold+of+poly(vinyl+alcohol)+and+interfacial+polyelectrolyte+complexation+fibers+for+controlled+biomolecule+delivery.">Composite scaffold of poly(vinyl alcohol) and interfacial polyelectrolyte complexation fibers for controlled biomolecule delivery.</a> Front. Bioeng. Biotechnol. 3 (3), 1-12 (2015).</li><li>Yow, S. Z., Lim, T. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Electroactive+Polypyrrole-Collagen+Fibrous+Scaffold+for+Tissue+Engineering.">A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering.</a> Polymers. 3 (1), 527-544 (2011).</li><li>Leong, M. F., Toh, J. K. C., 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=Patterned+prevascularised+tissue+constructs+by+assembly+of+polyelectrolyte+hydrogel+fibres.">Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres.</a> Nat. Commun. 4, 2353 (2013).</li><li>Di Benedetto, F., Biasco, A., Pisignano, D., Cingolani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+polyacrylamide+hydrogels+by+soft+lithography.">Patterning polyacrylamide hydrogels by soft lithography.</a> Nanotechnology. 16 (5), S165 (2005).</li><li>Revzin, A., Russell, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+poly(ethylene+glycol)+hydrogel+microstructures+using+photolithography.">Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography.</a> Langmuir. 17 (18), 5440-5447 (2001).</li><li>Yim, E. K. F., Liao, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+compatibility+of+interfacial+polyelectrolyte+complexation+fibrous+scaffold:+evaluation+of+blood+compatibility+and+biocompatibility.">Tissue compatibility of interfacial polyelectrolyte complexation fibrous scaffold: evaluation of blood compatibility and biocompatibility.</a> Tissue Eng. Part A. 13 (2), 423-433 (2007).</li><li>Chaouat, M., Le Visage, C., Autissier, A., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+a+small-diameter+polysaccharide-based+arterial+graft+in+rats.">The evaluation of a small-diameter polysaccharide-based arterial graft in rats.</a> Biomaterials. 27, 5546-5553 (2006).</li><li>Eshraghi, S., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+microstructural+properties+of+polycaprolactone+scaffolds+with+one-dimensional,+two+dimensional,+and+three-dimensional+orthogonally+oriented+porous+architectures+produced+by+selective+laser+sintering.">Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering.</a> Acta Biomater. 6, 2467-2476 (2010).</li></ol>

The authors have nothing to disclose.

IPC 섬유 개의 반대로 대전 된 고분자 전해질의 상호 작용에 의해 형성된다. 프로세스는 안정된 섬유 형성을위한 자기 조립 공정을 용이하게 고분자 전해질의 계면에서의 복잡한 추출을 이용한다. IPC 섬유 형성의 메카니즘은 유사 대전이 고분자 전해질 복합체 과정에서 혼입 될 수있는 생리 활성 물질로 첨가되도록. 10,11는 반대로, 반대로 대전 된 고분자 전해질로 생리 활성 물질의 첨가?...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pullulan&#160;</td>
			<td>Hayashibara Inc Okayama Japan</td>
			<td></td>
			<td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma Aldrich</td>
			<td>D1037</td>
			<td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Trimetaphosphate</td>
			<td>Sigma Aldrich</td>
			<td>T5508</td>
			<td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td>
		</tr>
		<tr>
			<td>Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>448877</td>
			<td>MW 190-310 kDa. Acetylation degree of 75 to 85%. Purification of chitosan is required to create stable IPC fibers.</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td></td>
			<td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&#160;</td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt from brown algae, low viscosity</td>
			<td>Sigma Aldrich</td>
			<td>A2158</td>
			<td>Dissolve in water overnight. Filter through sterile 0.2&#181;m syringe filter before use. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td></td>
			<td>Dissolve in sterile PBS and filter using 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>This kit was used to measure BSA release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Human Recombinant Vascular Endothelial Growth Factor</td>
			<td>R&#38;D systems</td>
			<td>293-VE</td>
			<td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5 mg/ml.</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt From Porcine</td>
			<td>Sigma Aldrich</td>
			<td>H3393</td>
			<td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&#160;</td>
		</tr>
		<tr>
			<td>ScanVac Coolsafe Lyophilizer</td>
			<td>Labogene</td>
			<td>7.001.200.060</td>
			<td>This is a non-programmable freeze dryer that operates at -105 to -110 &#176;C, This can be replaced by other standard lab lyophilizers.</td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL)</td>
			<td>Sigma Aldrich</td>
			<td>181609</td>
			<td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>V800151</td>
			<td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td>
			<td>Dow Corning</td>
			<td>(240)4019862</td>
			<td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td>
		</tr>
		<tr>
			<td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td>
			<td>R&#38;D systems</td>
			<td>256-GF</td>
			<td>Reconstituted in sterile DI water to a final concentration of 100 &#181;g/ml. Aliquot and store in -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells (hMSC)</td>
			<td>Cambrex</td>
			<td></td>
			<td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td>
		</tr>
		<tr>
			<td>Rat PC12 Pheochromocytoma Cells&#160;</td>
			<td>ATCC</td>
			<td></td>
			<td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td>
		</tr>
		<tr>
			<td>Grade 93 filter paper</td>
			<td>Whatman</td>
			<td>Z699675</td>
			<td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH 7.</td>
		</tr>
		<tr>
			<td>Swing bucket centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>To be used during the purification of chitosan using 1200 x g speed.&#160;</td>
		</tr>
		<tr>
			<td>Motor with mandrel rotating at constant speed</td>
			<td>Rhymebus</td>
			<td>RM5E</td>
			<td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>FirstBase</td>
			<td></td>
			<td>Sterilize through filtration (0.2 &#181;m filter) and autoclave.&#160;</td>
		</tr>
		<tr>
			<td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td>
			<td>Greiner</td>
			<td></td>
			<td>The TCPS dish is used for casting of pullulan frame.&#160;</td>
		</tr>
		<tr>
			<td>Human VEGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DVE00</td>
			<td>The ELISA kit is used for detection of VEGF in the release medium.</td>
		</tr>
		<tr>
			<td>Human NGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY256</td>
			<td>&#160;The ELISA kit is used for detection of NGF in the release medium.</td>
		</tr>
		<tr>
			<td>Plastic Coated Adhesive Tape</td>
			<td>Bel-Art</td>
			<td>9040336</td>
			<td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td>
		</tr>
	</tbody>
</table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

이 글에서, 우리는 다양한 생체 분자의 서방에 대한 IPC 섬유와 복합 발판을 만드는 노력했다. 본 연구에서 사용 된 생체 분자의 특성을 표 1에서 발견된다. IPC 섬유는 제 PD-IPC 복합체 지지체 (도 1B)를 만들기 위해 PD 친수성 하이드로 겔에 포함 하였다. 모델 분자 BSA 먼저 제어 생체 릴리스 복합체 지지체를 사용하는 타당성을 결정하기 위하여 시험 하였다. BSA는 45 ± 0.97 %의...

Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

생체 분자 시간적으로 방출 제어용 고분자 전해질 계면 섬유의 복합 공사장 공중 발판

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

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Bioengineering

8.3K 조회수.  National University of Singapore. Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

비디오: Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

탄성 PGS는 아티 조직 공학 공사장 공중 발판

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

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Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D 섬유소의 준비는 줄기 세포 배양 용도 공사장 공중 발판

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

라만 분광학을 사용하여 세포와 세포외 기질의 비 접촉, 레이블이없는 모니터링

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

바인더로 박테리아 셀룰로오스를 활용하여 강력한 천연 섬유 프리폼 제조

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

결합 표면의 접목 및 Postmodification 과정에 의한 빛의 응답 멤브레인의 제조

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

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

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

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

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

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

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

제어 약물 릴리스에 대한 자기장 응답 하이브리드 젤라틴 마이크로 겔을 교류

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Decellularized 폐 공사장 공중 발판을 사용하여 ESC 파생 마우스기도 상피 세포의 생성

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

헤파린 결합 도메인 골 형성 성장 인자 전달을위한 고분자 전해질 복합

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...