이 작품은 NIH (보조금 1R01AR072027-01, 1R03AR069383-01), NSF 경력 상 (1653702) 및 코네티컷 대학에 의해 재정적으로 지원됩니다.

1. JBNT의 합성
참고 : JBNT 단조량은 이전에 출판 된 대로 준비되었다11.
<ol><li>화합물 A1의 합성<ol>
<li>톨루엔 25mL및 N-디메틸포르미드의 2.5mL에서 8.50g의 시아노아세이스틱산과 9.80 g의 에틸카르바메이트를 함유한 용액을 준비한다. 4.90mL의 인산화를 드롭와이즈로 넣습니다. 그런 다음 혼합물을 70 °C로 가열하고 1.5 시간 동안 계속 저어줍니다.</li>
		<li>반응 혼합물을 실온으로 식히고 얼음 물 100g을 붓습니다. 에틸 아세테이트(3 x 250mL)로 수성 층을 추출하고 100mL의 소금물로 세척합니다. 무수성 나트륨 황산나트륨 위에 유기층을 건조시키고, 진공 상태에서 휘발성을 걸러내고 증발하여 옅은 노란색 고체를 얻습니다.</li>
		<li>노란 고체내지 실리카 젤 플래시 컬럼 크로마토그래피(3% 메탄올/디클로로메탄)를 주체하여 백색 분말로서 화합물 A1의 15.61g을 산출한다.</li>
	</ol></li>
	<li>화합물 A2의 합성<ol>
<li>화합물 A1 3.12 g와 탄산칼륨 2.75 g를 N, N-디메틸포르마미드의 50mL에 섞는다. 실온에서 2시간 동안 저어줍니다.</li>
		<li>반응 현탁액에 2.6mL의 탄소 이황화를 추가합니다. 4 시간 동안 교반 유지합니다.</li>
		<li>0°C에서 반응 혼합물에 절대 에탄올을 넣습니다. 노란 침전을 걸러내고 디틸 에테르로 씻습니다. 밤새 진공 상태에서 건조하여 6.18g의 화합물 A2를 옅은 노란색 고체로 산출합니다.</li>
	</ol></li>
	<li>화합물 A3의 합성<ol>
<li>아세토닐릴의 15mL에 메틸 요오드 2.7mL를 추가합니다. 또한, 물/아세토나이트(7:3비율)의 A2~80mL 6.18g을 첨가하여 화합물 A2용액을 별도로 준비한다. 용액을 모두 섞고 실온에서 30분 간 저어줍니다.</li>
		<li>반응 혼합물을 95°C로 가열하고 3시간 동안 계속 저어줍니다.</li>
		<li>반응 혼합물을 실온으로 냉각한 다음 진공 상태에서 휘발성을 증발시보세요.</li>
		<li>에틸 아세테이트 100mL로 잔류물 3배 추출하고 소금물로 씻어내다. 무수성 나트륨 황산나트륨 위에 유기층을 건조시키고, 진공 하에서 휘발성을 피하여 노란색 고체를 얻습니다.</li>
		<li>노란색 고체내지 실리카 젤 플래시 컬럼 크로마토그래피(30% 에틸 아세테이트/헥산)를 주체하여 A3의 4.52 g를 연황고체로서 산출한다.</li>
	</ol></li>
	<li>화합물 A4의 합성<ol>
<li>에탄올 20mL에 925 μL의 아를라민을 추가하여 아일라민 용액을 준비하십시오. 이 용액을 절대 에탄올 75mL에 3.14 g의 A3를 추가하여 30분 이상 제조한 화합물 A3용액에 드롭와이즈를 추가합니다. 그런 다음 반응 혼합물을 16 시간 동안 역류로 가열합니다.</li>
		<li>반응 혼합물을 실온으로 냉각시키고 휘발성을 증발하여 노란색 고체를 줍니다.</li>
		<li>노란색 고체내지 실리카 젤 플래시 컬럼 크로마토그래피(50% 에틸 아세테이트/헥산~메탄올/디클로로메탄)를 주체하여 백색 결정으로서 1.80g의 화합물 A4를 산출한다.</li>
	</ol></li>
	<li>화합물 A5의 합성<ol>
<li>2.05 g의 과니디늄 염산염과 7.8 mL의 나트륨 에탄올 (에탄올21 wt%)을 절대 에탄올 14mL에 넣습니다. 혼합물을 45°C로 15분 동안 가열합니다.</li>
		<li>불용성 물질을 걸러내고 40mL 절대 에탄올에 A4 2.70 g을 첨가하여 제조한 화합물 A4의 용액에 직접 여과물을 첨가한다. 반응 혼합물을 16시간 동안 역류에 가열합니다.</li>
		<li>오프 화이트 고체로서 화합물 A5의 2.65 g를 얻기 위해 밖으로 침전물을 필터링합니다.</li>
	</ol></li>
	<li>화합물 A6의 합성<ol>
<li>24.9mL의 트리에틸라민을 1분 이상 테트라하이드로푸란 120mL에 화합물 A5 2.11g과 4-디메틸아미노피리딘 1.10g을 첨가하여 얻은 슬러리 혼합물에 천천히 첨가한다. 실온에서 2분 간 저어줍니다.</li>
		<li>디테르트부틸 디카보네이트 20.7mL을 반응 혼합물에 드롭와이즈로 넣고 40시간 동안 실온에서 계속 저어줍니다.</li>
		<li>20mL의 물을 추가하여 반응을 담금질합니다. 진공 상태에서 휘발성을 증발시다.</li>
		<li>에틸 아세테이트 500mL로 적점 잔류물을 추출합니다. 이어서 250mL의 물, 구연산 75mL, 200mL의 물, 포화나트륨 200mL, 소금물 200mL로 세척한다. 무수성 나트륨 황산나트륨 위로 유기층을 건조시키고, 진공 상태에서 휘발성을 걸러내고 증발하여 빨간색/주황색 고체를 줍니다.</li>
		<li>고체내지 실리카 젤 플래시 컬럼 크로마토그래피(0-20% 에틸 아세테이트/헥산)를 주체하여 백색 폼으로서 화합물 A6 3.87g을 산출한다.</li>
	</ol></li>
	<li>화합물 A7의 합성<ol>
<li>N-메틸모포린 N-Oxide(물 50w.%, 1.64mL)를 아세톤/물(8:1, 58.5mL)에 화합물 A6(2.93g)의 용액에 넣고 실온에서 5분 간 저어줍니다.</li>
		<li>3분 동안 혼합물에 오스뮴 테트라옥사이드(4% 수성 용액)를 드롭방향으로 넣고 실온에서 24시간 동안 계속 저어줍니다.</li>
		<li>용액이 무색으로 변할 때까지 1.0M 수성 나트륨 황산염으로 반응을 담금질합니다. 진공 상태에서 휘발성을 제거하여 물에 백색 슬러리를 생성합니다. 350mL의 디클로로메탄으로 잔류물을 추출한 다음 150mL의 물로 씻어내고 150mL의 소금물로 세척합니다.</li>
		<li>무수성 나트륨 황산나트륨위에 유기층을 건조시키고, 진공 하에서 휘발성을 증발시키고, 백색 고체로서 2.45g의 화합물 A7을 산출한다.</li>
	</ol></li>
	<li>화합물 A8의 합성<ol>
<li>디클로로메탄/물(6:1, 35mL)의 35mL에 1.36 g의 화합물 A7을 혼합하고 42시간 동안 실온에서 저어줍니다.</li>
		<li>불용성 물질을 걸러내고 진공 상태에서 여과물을 농축합니다. 디클로로메탄 250mL로 생성된 잔류물을 추출한 다음 100mL의 물과 100mL의 소금물로 세척합니다. 무수성 나트륨 황산나트륨 위에 유기층을 건조시키고, 진공 상태에서 휘발성을 걸러내고 증발하여 원유 제품을 제공합니다.</li>
		<li>실리카 젤 플래시 컬럼 크로마토그래피(5-40% 에틸 아세테이트/헥산, 5% 메탄올/디클로로메탄)를 통해 1.08g의 화합물 A8을 산출한다.</li>
	</ol></li>
	<li>화합물 A9의 합성<ol>
<li>1,2-디클로로에탄의 15mL에서 830 mg의 화합물 A8용액을 준비한다. 6-벤질옥시카보라미노-2-L-아미노 헥사노산 트리메틸실릴 에틸 에스테르(649.5 mg)와 N,N-이소프로틸레틸레틸라민(591 μL)을 1,2-디클로로탄 24mL로 첨가하고 15분 이상 15분 동안 15분 이상 저어줍니다.</li>
		<li>360.3 mg의 트리아세톡시보라이드 나트륨을 용액에 넣고 실온에서 24시간 동안 계속 저어줍니다.</li>
		<li>반응 혼합물을 6mL의 물로 담금질합니다. 디클로로메탄 200mL로 반응 혼합물 3x를 추출하고 물 속 100mL의 구연산 200mL, 200mL의 물, 포화 나트륨 중탄산염 200mL, 염수 200mL로 세척한다. 무수성 나트륨 황산나트륨 위에 유기층을 건조시키고, 진공 상태에서 휘발성을 걸러내고 증발하여 원유 제품을 얻습니다.</li>
		<li>실리카 젤 플래시 컬럼 크로마토그래피(5-30% 에틸 아세테이트/헥산)에 원유 제품을 적용하여 885 mg 화합물 A9를 산출합니다.</li>
	</ol></li>
	<li>JBNT 단조량합성<ol>
<li>화합물 A9(0.54g)을 94% 트리플루오로아세트산/티오아니솔 용액의 10mL에 0.54g을 녹이고 실온에서 72시간 동안 저어줍니다.</li>
		<li>다이틸 에테르(80mL)를 반응 혼합물에 넣고 침전물원심을 줄입니다. 초월제를 함유한 맑은 트리플루오로아세트산을 붓고 168.6 mg의 원유 JBNT 단조량을 산출하기 위해 다이틸 에테르와 메탄올로 백색 침전물을 씻어낸다(보충파일 1).</li>
		<li>역상 컬럼을 사용하여 고성능 액체 크로마토그래피(HPLC)로 원유 JBNT 단조량을 정화합니다. 격리 프로그램은 다음과 같습니다: 용매 A: 100% 물; 용매 B: 100% 아세토닐트릴; 용매 C: pH=1 HCl 용액; 유량: 3mL/min(표 1참조).</li>
		<li>약 7.2 분에 가장 큰 피크를 수집합니다.</li>
	</ol></li>
</ol>2. JBNT/FN용 제작
<ol><li>1 mg/mL JBNT 수성 용액 의 80 μL을 1 mg/mL FN 수성 용액 및 파이펫의 40 μL에 여러 번 추가합니다.</li>
	<li>약 10s에 대한 파이펫팅 후 흰색 고체 서스펜션의 존재를 확인합니다. 참고: NM(보충파일 2)의자체 조립 프로세스를 캡처하기 위해 비디오가 촬영되었습니다.</li>
</ol>3. lyophilized 표본의 관찰
참고: 이 단계는 JBNT및 FN으로 만든 NM의 비계 구조를 보여주기 위해 수행됩니다.
<ol><li>Lyophilized 물질 관찰을 위한 FN, JBNT 및 FN/JBNT NM 솔루션을 준비합니다. FN의 100 μg/mL의 10 μL을 40 μL의 증류수로 희석하여 FN의 20 μg/mL 용액을 만듭니다.</li>
	<li>DI 수의 45 μL에 1 mg/mL JBNT의 5 μL을 희석하여 JBNT의 100 mg/mL 용액을 준비하십시오.</li>
	<li>100 μg/mL FN의 10 μL과 5 μL의 1mg/mL을 DI 수의 35 μL에 JBNT의 1 mg/mL의 5μL을 혼합하여 NM 용액을 형성합니다.</li>
	<li>FN, JBNT 솔루션 및 NM 솔루션으로 96웰의 투명한 둥근 하단 마이크로 플레이트의 3개의 웰을 각각 코팅합니다. 각 웰은 용액의 50 μL을 받습니다.</li>
	<li>아래에 설명된 바와 같이, NM의 비계 구조를 시각화하기 위해, lyophilization을 수행한다.<ol>
<li>하룻밤 -80 °C에서 접시를 동결.</li>
		<li>lyophilized 기기와 진공 펌프를 켭니다.</li>
		<li>냉각 버튼을 눌러 시스템의 온도를 -50 °C로 줄입니다.</li>
		<li>냉동 판을 lyophilized 악기에 넣고 악기의 모든 구멍을 닫습니다.</li>
		<li>시작 버튼을 클릭하여 시스템 압력을 0.9 mbar로 줄입니다.</li>
		<li>4시간 후, 대격자 버튼을 누르고 밸브를 열어 기압에 대한 시스템 압력을 증가시다.</li>
		<li>lyophilized 악기에서 접시를 꺼내.</li>
		<li>현미경을 사용하여 FN, JBNT 및 생성된 자체 조립 NM을 관찰합니다. 재료에 대한 카메라와 함께 여러 사진을 촬영합니다.</li>
	</ol>
</li>
</ol>4. 흡수 스펙트럼 측정
참고: FN 및 JBNT에 대한 스펙트럼 변경을 사용하여 자체 조립된 JBNT/FN NM12를제시합니다.
<ol><li>100 μg/mL FN의 30 μL을 증류수 15μL과 5 μL의 1 mg/mL JBNT와 혼합하여 JBNT/FN NM 솔루션을 준비합니다. 백색 고체 서스펜션은 용액을 여러 번 피펫팅한 후 볼 수 있습니다. 참고: 용액의 JBNT와 FN의 최종 농도는 각각 100 μg/mL 및 60 μg/mL입니다. NM 솔루션에 사용된 것과 동일한 농도의 JBNT 및 FN 솔루션도 제어 솔루션으로 제조되었다.</li>
	<li>JBNT 제어 솔루션을 준비하기 위해 증류수 45μL로 JBNT 1 mg/mL의 5 μL을 희석합니다.</li>
	<li>FN 제어 솔루션을 준비하기 위해 증류수 20μL로 100 μg/mL FN의 30 μL을 희석시하십시오.</li>
	<li>NM의 조립을 특성화하기 위해 분광계(재료 표 참조)를 사용하여 FN, JBNT 및 JBNT/FN NM 솔루션의UV-vis 흡수 분광 분광을 측정합니다.<ol>
<li>UV-Vis 버튼을 클릭합니다. 분광계의 시험 표면을 청소하고 증류수 2 μL을 떨어 뜨립니다. 190-850 nm에서 빈 것으로 측정합니다.</li>
		<li>분광계의 테스트 표면을 청소하고 FN 용액의 2 μL을 떨어 뜨립니다. FN 용액의 UV-Vis 흡수 스펙트럼을 190nm에서 850nm로 측정합니다.</li>
		<li>분광계의 테스트 표면을 청소하고 JBNT 용액의 2 μL을 떨어 뜨립니다. JBNT 용액의 흡수 스펙트럼을 190nm에서 850nm로 측정합니다.</li>
		<li>분광계의 테스트 표면을 청소하고 JBNT /FN NM 용액의 2 μL을 떨어 뜨립니다. 190nm에서 850nm까지 JBNT/FN NM 용액의 흡수 스펙트럼을 측정합니다.</li>
	</ol>
</li>
</ol>5. 전송 전자 현미경 검사법 (TEM)에 대한 JBNT / FN NM의 준비
<ol><li>기본 플라즈마 클리너로 음수 염색하기 전에 여러 그리드를 청소하십시오(재료 표참조).</li>
	<li>두 개의 서로 다른 프로세스에 따라 시편에 대한 음의 염색을 수행합니다.<ol>
<li>JBNT의 1 mg/mL의 3 μL과 분리된 그리드에 JBNT/FN NM 용액 3μL을 떨어뜨리고 2분 동안 그대로 둡니다. 그런 다음 0.5 % uranyl 아세테이트 (UA) 용액의 100 μL로 각 그리드를 헹구는 다. 필터 용지로 과도한 용액을 배출하고 그리드를 건조시다.</li>
		<li>JBNT/FN NM 용액 9μL을 2.0% UA 용액3μL과 여러 번 섞는다. 그리드의 혼합 솔루션을 파이펫하고 2 분 동안 그리드에 보관하십시오. 필터 용지를 사용하여 그리드에서 혼합 된 솔루션을 제거하고 실온에서 그리드를 건조시다.</li>
	</ol>
</li>
	<li>마지막으로, JBNT, JBNT/FN NM에 대한 표본을 5.2.1단계로 염색하고, JBNT/FN NM에 대한 표본을 TEM으로 5.2.2단계로 염색했다.</li>
</ol>6. 체외 생물학적 기능 분석
<ol><li>각각 8웰 챔버 슬라이드의 한 웰에 음수 컨트롤 (NC, PBS), JBNT, FN 및 JBNT / FN NM 솔루션의 200 μL을 추가합니다.</li>
	<li>8웰 챔버 커버글래스를 구균 밑의 소재를 코팅하기 위해 리오필화 된 악기로 얼립니다. 동결 건조 프로토콜은 3.5 단계와 동일합니다.</li>
	<li>그런 다음 10,000 개의 세포 / 우물 hMSC (Passage 3)를 우물에 넣고 5 % CO2로37 °C에서 24 시간 동안 배양하십시오.</li>
	<li>인큐베이션 후, 우물에서 세포 배양 배지를 피펫하고 1x PBS 용액으로 배양을 헹구는다.</li>
	<li>고정 용액의 100 μL을 셀과 잘 추가하고 5 분 동안 둡니다. 1x PBS로 부드럽게 헹구십시오.</li>
	<li>1% 트리톤-X 100 ~ 0.1%를 희석합니다. 세포와 잘 각 웰에 0.1 % 트리톤 X 100의 100 μL을 추가하고 10 분 동안 둡니다. 1x PBS로 부드럽게 헹구십시오.</li>
	<li>로다민 팔로이드를 1.65 μM으로 희석. 세포와 잘 각 잘 에 로다민 Phalloidin의 100 μL을 추가하고 30 분 동안 실온에서 빛으로부터 유지 챔버 커버 글래스를 배양. 1x PBS로 부드럽게 헹구십시오.</li>
	<li>형광 현미경으로 세포 이미지를 캡처합니다.</li>
	<li>각 그룹에 대한 형광 이미지의 세포 수를 계산합니다. 각 웰에 있는 세 개의 임의 영역을 평균화하여 셀 접착 밀도를 계산합니다.</li>
	<li>모든 데이터를 평균 ± 표준 편차(SD)로 표시합니다. 한 꼬리 t-테스트를 사용하여 통계 분석을 수행하고, 그 다음으로 통계적으로 유의한 것으로 간주되는 p&05를 가진 분산(ANOVA)의 분석이 뒤따릅니다.</li>
</ol>

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유펑 첸 박사는 나노드 테라피의 공동 창립자입니다.

이 연구에서는 DNA에서 영감을 얻은 JBNT와 FN으로 형성된 자체 조립 된 생체 Mimetic NM을 개발했습니다. JBNT 용액을 준비할 때, PBS가 조립을 억제하는 JBNT의 응집을 유발하기 때문에 JBNT 리오필화 분말은 PBS 대신 물에 용해되어야 합니다. 또한, NM은 PBS의 염이 NM 섬유와 번들되기 때문에 NM의 나노 피브릴 구조를 관찰하려는 경우에도 물로 조립되어야 하며, 이는 이미지의 해상도를 크게 감소시킬 수 있?...

인간 중간엽 줄기 세포(hMSC)는 조직1의재생 및 유지에 도움이 되는 다른 중간엽 혈통을 따라 자가 재생 및 자가 분화의 잠재력을 보여주었습니다. 분화 잠재력에 기초하여, hMSC는 중간 엽 조직 상해 및 조혈 장애 치료2에대한 후보로 간주됩니다. hMSC는 조직 수리, 혈관 신생, 염증 감소3을증가시켜 상처 치유를 촉진하는 능력을 보여주었습니다. 그러나 생화학적 또는 생체 재료 의 지원 없이, hMSC가 표적 조직에 도달하고 원하는 위치에서 기능하는 효율은 낮다4. 병변을 고수하기 위해 hMSC를 유치하기 위해 다양한 엔지니어링 스캐폴드가 활용되었지만, 긴 뼈 의 중간에 있는 성장판 골절과 같은 일부 부위는 기존의 사전 제작 된 비계로 쉽게 접근 할 수 없으며 불규칙한 모양의 부상 부위에 완벽하게 맞지 않을 수 있습니다.
여기서, 우리는 그 자리에서 스스로 조립하고 표적 영역에 도달하기 어려운 주입할 수 있는 생체 모방 나노 물질을 개발했습니다. 주사용 바이오 스캐폴드 NM은 야누스 염기 나노튜브(JBNT)와 섬유네틴(FN)으로 구성된다. 또한 로제트 나노 튜브 (RNTs)로 알려진 JBNTs는 DNA 기본 쌍, 특히 티민과 아데닌, 여기에5,6,7에서파생됩니다. 도 1에서볼 수 있듯이, 나노튜브는 유래 된 DNA 염기의 6 분자가 수소 결합을 통해 자가 조립하여 비행기6을형성할 때 형성된다. 6개의 분자는 그 때 길이200-300 μm까지 할 수 있는 강한 pi-stacking 상호 작용7을통해 평면에 서로 적층됩니다. JBNT는 FN이 그들과 반응할 수 있도록 형태학적으로 콜라겐 섬유를 모방하도록 설계되었습니다.
FN은 세포외 매트릭스(ECM)9에서발견될 수 있는 고분자 성 접착제 당단백질이다. 이들은 ECM의 다른 성분, 특히 콜라겐10의다른 성분에 줄기 세포의 부착을 중재할 수 있다. 우리는 JBNT를 형태학적으로 모방하여 FN이 비동적 결합을 통해 몇 초 안에 NM을 형성할 수 있도록 설계했습니다. 따라서, NM은 기존의 제조된 스캐폴드에 의해 접근할 수 없었던 뼈 골절 부위에 주입되는 유망한 바이오 스캐폴드이다. 여기서, 주 사용 NM은 시험관 내에서 hMSC 앵커리지를 향상시키는 우수한 능력을 제시, 조직 재생을위한 발판으로 봉사 할 수있는 자신의 잠재력을 전시.

<table><tbody><tr><td>1,2-dichloroethane</td><td>Alfa Aesar</td><td>39121</td><td></td></tr><tr><td>2-cyanoacetic acid</td><td>Sigma-Aldrich</td><td>C88505</td><td></td></tr><tr><td>4-Dimethylaminopyridine</td><td>TCI America</td><td>D1450</td><td></td></tr><tr><td>8 wells Chambered Coverglass</td><td>Thermo Fisher</td><td>155409</td><td></td></tr><tr><td>96-well plate</td><td>Corning</td><td>353072</td><td></td></tr><tr><td>absolute ethanol</td><td>Thermo Fisher</td><td>BP2818500</td><td></td></tr><tr><td>acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>34851</td><td></td></tr><tr><td>allylamine</td><td>Sigma-Aldrich</td><td>145831</td><td></td></tr><tr><td>Basic Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC32G</td><td></td></tr><tr><td>citric acid</td><td>Sigma-Aldrich</td><td>251275</td><td></td></tr><tr><td>concentrated hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Deionized water</td><td>Thermo Fisher</td><td>15230147</td><td></td></tr><tr><td>dichloromethane</td><td>Sigma-Aldrich</td><td>270997</td><td></td></tr><tr><td>diethyl ether</td><td>Sigma-Aldrich</td><td>296082</td><td></td></tr><tr><td>Di-tert-butyl dicarbonate</td><td>Sigma-Aldrich</td><td>361941</td><td></td></tr><tr><td>ethyl acetate</td><td>Sigma-Aldrich</td><td>319902</td><td></td></tr><tr><td>ethylcarbamate</td><td>Sigma-Aldrich</td><td>U2500</td><td></td></tr><tr><td>Fibronectin</td><td>Thermo Fisher</td><td>PHE0023</td><td></td></tr><tr><td>Fixative Solution (4 % formaldehyde prepared in PBS)</td><td>Thermo Fisher</td><td>R37814</td><td></td></tr><tr><td>guanidinium hydrochloride</td><td>Alfa Aesar</td><td>A13543</td><td></td></tr><tr><td>hexanes</td><td>Sigma-Aldrich</td><td>227064</td><td></td></tr><tr><td>Human mesenchymal stem cells</td><td>Lonza</td><td>PT-2501</td><td></td></tr><tr><td>methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>methyl iodide</td><td>Sigma-Aldrich</td><td>289566</td><td></td></tr><tr><td>N,N-Diisopropylethylamine</td><td>Alfa Aesar</td><td>A17114</td><td></td></tr><tr><td>N,N-dimethylformamide</td><td>Sigma-Aldrich</td><td>227056</td><td></td></tr><tr><td>N-Methylmorpholine N-oxide</td><td>Alfa Aesar</td><td>A19802</td><td></td></tr><tr><td>Osmium tetraoxide</td><td>Alfa Aesar</td><td>45385</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15140163</td><td></td></tr><tr><td>Phosphate Buffer Solution</td><td>Thermo Fisher</td><td>20012050</td><td></td></tr><tr><td>phosphoryl chloride</td><td>Sigma-Aldrich</td><td>201170</td><td></td></tr><tr><td>potassium carbonate</td><td>Sigma-Aldrich</td><td>347825</td><td></td></tr><tr><td>reverse phase column</td><td>Thermo Fisher</td><td>25305-154630</td><td></td></tr><tr><td>Rhodamine Phalloidin</td><td>Thermo Fisher</td><td>R415</td><td></td></tr><tr><td>silica gel</td><td>TCI America</td><td>S0821</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>sodium ethoxide</td><td>Alfa Aesar</td><td>L13083</td><td></td></tr><tr><td>sodium periodide</td><td>Sigma-Aldrich</td><td>71859</td><td></td></tr><tr><td>sodium sulfate</td><td>Sigma-Aldrich</td><td>239313</td><td></td></tr><tr><td>sodium sulfite</td><td>Sigma-Aldrich</td><td>S0505</td><td></td></tr><tr><td>sodium triacetoxyborohydride</td><td>Alfa Aesar</td><td>B22060</td><td></td></tr><tr><td>spectrophotometer(NanoDrop One/One&lt;sup&gt;c&lt;/sup&gt; UV-Vis)</td><td>Thermo Fisher</td><td>ND-ONE-W</td><td></td></tr><tr><td>Stem Cell Growth Medium BulletKit</td><td>Lonza</td><td>PT-3001</td><td></td></tr><tr><td>tetrahydrofuran</td><td>Sigma-Aldrich</td><td>401757</td><td></td></tr><tr><td>thioanisole</td><td>Sigma-Aldrich</td><td>T28002</td><td></td></tr><tr><td>toluene</td><td>Sigma-Aldrich</td><td>179418</td><td></td></tr><tr><td>triethylamine</td><td>Alfa Aesar</td><td>A12646</td><td></td></tr><tr><td>trifluoroacetic acid</td><td>Alfa Aesar</td><td>A12198</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Fisher</td><td>HFH10</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Thermo Fisher</td><td>25200056</td><td></td></tr></tbody></table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

생체 모방 NM은 줄기 세포 앵커리지를 향상시킬 수 있는 조직 공학 생물학적 스캐폴드로 개발되었습니다. 생체 모방 NM은 수성 용액의 자체 조립을 통해 JBNT및 FN에서 형성됩니다. JBNT는 내부 소수성 중공 채널과 외부 친성 성 표면으로 길이 200-300 μm을 측정합니다. JBNT는 양수 충전되며 FN은 음수 로 충전됩니다. 따라서 중립 수성 용액에 주입하면 비일관성 결합을 통해 결합되어 NM 번들을 형성합니다. 자체 조립 공정은 화학 개시기, 열원 또는 자외선 없이 몇 초 이내에 완료됩니다. NM 솔루션의 pH가 FN(pI 5.5-6.0)의 등광점보다 낮으면, NM 번들은 양전하 FN의 존재로 인해 자체 방출됩니다.
NM은 세포외 매트릭스 (ECM)를 형태학적으로 모방하는 것으로 알려져 있으므로 hMSC 접착력을 향상시키는 우수한 플랫폼을 제공하는 주사용 스캐폴드로 사용할 수 있습니다. 세포 밀도 분석 및 형광 이미징 실험은 NM이 음의 대조군과 비교하여 hMSC의 앵커리지를 크게 증가시켰다는 것을 나타냈다.

우리의 연구는 JBNT와 FN의 NM의 형성이 빠르다는 것을 것을을 발견했습니다, 이는 10 초에 일어났습니다. 도 2에도시된 바와 같이, JBNT 용액이 FN 용액과 혼합되고 여러 번 배관되었을 때 백색 플록을 얻었다. NM의 형성 과정은 완전히 생물모방이다. 외부 자극이 필요하지 않습니다. 제조 과정은13을교차연결하기 위한 자외선 또는 화학 개시자를 기반으로 하는 ?...

Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

이 프로토콜의 목표는 야누스 염기 나노튜브(JBNT)와 섬유네틴(FN)을 가진 생물미메틱 나노매트릭스(NM)의 조립을 보여주는 것이다. 인간 중간엽 줄기 세포(hMSC)와 공동 배양할 때, NM은 hMSCs 접착을 장려하는 우수한 생체 활성을 나타낸다.

야누스 베이스 나노튜브와 줄기 세포 접착을 위한 섬유네틴을 갖춘 바이오미메틱 나노 매트릭스의 제조

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

fabrication-biomimetic-nano-matrix-with-janus-base-nanotubes

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

4.0K 조회수.  University of Connecticut. 이 프로토콜의 목표는 야누스 염기 나노튜브(JBNT)와 섬유네틴(FN)을 가진 생물미메틱 나노매트릭스(NM)의 조립을 보여주는 것이다. 인간 중간엽 줄기 세포(hMSC)와 공동 배양할 때, NM은 hMSCs 접착을 장려하는 우수한 생체 활성을 나타낸다.

비디오: Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

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 ...

연구

생체공학

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

심장 조직 공학을위한 모세관 력 리소그래피

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

ECM 단백질 나노 섬유 및 나노 구조 엔지니어링 사용하여 표면 시작 회의

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

섬유 조직의 그라데이션으로 전기 방사 나노 섬유 공사장 공중 발판

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

셀 동작의 Nanotopographical 변조를위한 나노 패턴 기판 사용 스티치 기법을 확장

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

로컬 컨트롤 표면 점착을 고르지 Nanopatterns Dendrimer 기반: Chondrogenic 차별화 하는 방법

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

셀 시드를 위한 사용자 지정 Agarose 우물 및 자기 조립 금형 3D 인쇄를 사용 하 여 조직 반지 제작

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

열 Nanoimprinting 기술과 인간의 내 피 식민지 형성 세포의 응답의 심사에 의해 그라데이션 Nanopattern의 제조

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

연골 재생을 촉진하기 위한 층별 야누스 베이스 나노매트릭스의 제작 및 특성 분석

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...