저자는이 프로젝트의 이전 반복에 기술적 공헌 가브리엘 AROM 마이클 Yeranosian에게 감사의 말씀을 전합니다. 이 프로젝트는 OREF 임상 과학자 교육 그랜트 (NL)에 의해 투자되었다, HH 리 외과 연구비 (NL), 재향 군인의 관리 BLR &amp; D 탁월성 평가 1 I01 BX00012601 (DM) 및 근골격계 이식 재단 젊은 연구자 상 (FP).

참고 : 모든 동물의 수술이 실험을 시작하기 전에 지역 수의학 직원과 동물 사용위원회에 의해 승인되었다. 전기 방사 된 폴리 카프로 락톤 비계의 1. 준비 <ol><li> w 10 %를 생성하는 1,1,1,3,3,3- 헥사 플루오로 -2- 프로판올의 과립 형태로 의료용 폴리 에스테르 말단 (ε 카프로 락톤)를 계량하고 용해 PCL 중합체 용액 w /. 균일 한 솔루션을 보장하기 위해 적어도 3 시간 동안 교반 판을 사용하여 솔루션 저어 보자. </li><li> PCL 솔루션을 Electrospin하는 발판 제조에 매우 정렬 된 PCL 섬유의 팔목을 만들 수 있습니다. <ol><li> 항상에 팬 화학 흄 후드에서 전기 방사 설치를 준비합니다. 이는 전압원, 모터 구동 수집 맨드릴, 및 진공 포트에 의해 구동되는 소스 PCL 용액에 대한 엔트리 포인트를 전기 방사에 대한 격리 된 진공 매체로서 역할을하고 갖는 것 큰 아크릴 상자로 구성되어있다. acryli를 청소C 상자 철저히 에탄올과 전기 방사 제품의 품질을 손상시킬 수있는 임의의 불순물을 제거하고 파라 필름 시트와 모든 표면을 커버. </li><li> 무딘와 10 ML의 주사기에 상기 용액의 부하 약 3 ㎖를 18 G, 1 ½ 인치 바늘을했다. 주사기를 위로 눌러 기포를 제거합니다. 프로그램 주사기 펌프에 잠금 솔루션을 제공합니다. 전압원 와이어 부착 아크릴 상자 외부에 관하여 ½ 인치의 바늘을 유지하면서 아크릴 상자에 작은 구멍을 통해 바늘을 삽입한다. </li><li> 매우 정렬 된 PCL 섬유의 집으로 30mm 회전 선반 맨드릴을 사용; 알루미늄 포일의 얇은 스트립 단단히 맨드릴을 커버. 약 15cm 거리에서 주사기 바늘 상자의 반대측에 상기 모터에 맨드릴 잠금. </li><li> 진공 포트에 플라스틱 호스를 삽입하고 흄 후드 진공 소스에 연결합니다. 진공 원을 켜고 뚜껑 아크릴 상자를 커버한다. </li><li> 에스외 2.5 ㎖ / HR 상기 프로그래머블 주사기 펌프의 주입 속도. 3,450 rpm에서 맨드릴을 운영하고 악어 클립을 사용하여 상자 외부의 바늘 끝으로 전압 소스의 양극 도선을 연결하는 모터의 전원을 켭니다. </li><li> PCL 용액의 주입이 시작되면, 전압 소스를 켜고 20 kV의 동작 전압을 설정합니다. </li><li> PCL 용액 0.5ml를으로부터 균질 커프를 만드는 12 분 동안 용액을 주입한다. 참고 : 평균적으로, 각 커프 3 ~ 4 층 비계의 총을 만드는 데 사용할 수있는 2 ~ 6 스트립 시트를 생성하기에 충분한 전기 방사 물질을 가지고있다. </li></ol></li><li> 레이저는 저 진공 설정, 10.0 keV의 착륙 전압, 6.4 mm 작동 거리, 및 3.0의 프로브 직경에서 작동 VersaLaser 커터 2.3에 여러 개의 작은 시트를 형성하기 위해 PCL 커프를 잘라. 주 :이 예에서, 컴퓨터 지원 설계는 X 35mm × 150 μm의 비계 1.5 mm 여러 장의 수득 커터를 지시하는 데 사용 된균등하게 15 % 세공 영역에 직경 150㎛의 구멍을 분산. </li><li> 플라즈마 이온 가속 PCL 표면의 친수성을 유도하기 위해 플라즈마 세정기를 이용하여 PCL 발판을 에칭. 450 mTorr로하고 치료를 높은 전력 (29.6 W)에서 30 초 동안 비계에 진공을 설정합니다. </li><li> 무균 환경에서 70 % 에탄올의 발판 목욕. </li><li> 코트 콜라겐 개별 지지체는 생체 내에서 세포 부착과 증식을 촉진한다. <ol><li> 4 ° C에서 1X PBS에서 9 : 1 : Purecol 콜라겐 3 ㎎ / ㎖ 표준 용액, 10 배 PBS 0.1 N NaOH를 하나의 2.5 무​​균 용액 (8)을 희석하여 콜라겐 코팅 용액을 만듭니다. 균질 용액을 완전히 혼합되도록. </li><li> 코트 개별 1.5 mm X 35mm X 상기 콜라겐 용액의 박층 막으로 150 μm의 발판. 무균 환경에서 24 시간 동안 건조시킵니다. </li></ol></li><li> 5-0 Vicryl 봉합사를 사용하여 스택과 네 개의 개별 1.5 mm X 35mm X 150 μm의 발판을 부착Krackow가 주입을위한 준비가 최종 0.6 mm 두께, 다층, 콜라겐 코팅 발판을 만들 스티치 사용. </li></ol> 2. 쥐 수술 프로토콜 <ol><li> 흡입 챔버에서 래트를 배치하고 2 L / 분으로 산소 2 % 이소 플루 란을 전달하여 마취를 유도한다. 쥐를 확인하는 것은 적절 발을 뒷다​​리에 압력을 가하지하고 응답을 평가하여 마취. </li><li> 비 멸균 다시 테이블에서 피하 25 ㎎ / ㎏ 암피실린 및 0.03 ㎎ / ㎏ 부 프레 노르 핀을 주입. </li><li> 눈 안과 연고를 적용합니다. 수술 뒷다리에서 모피를​​ 클립과 세 클로르헥시딘의 교류 스크럽 70 % 에탄올로 수술 부위를 예비 학교. </li><li> 저체온증을 방지하기 위해 가열 패드, 수술대에 쥐를 전송합니다. 보안 코 콘, 그리고, 2 L / min의 산소에 2 % 이소 플루 란과 절차를 통해 전달 코 콘 마취를 유지한다. 노출 수술 사지를 떠나, 균 방식으로 드레이프. </li><li&gt; 수술을 시작하기 전에, 먼저 발 압력을 뒷다리에 대한 응답의 부족으로 적절한 마취를 확인합니다. </li><li> 슬개골의 수준에 중심을 무릎에 2cm 긴 수직 절개 내측을 확인합니다. 절개 무릎 위에 중심 될 때까지 옆으로 피부를 집어 넣으십시오. </li><li> 바로 슬개골 내측에 절단 및 대퇴사 두근과 원위 경골 결핵의 슬개 건 삽입의 수준으로 musculotendinous 접합의 수준에 근접하게 확장하여 내측 슬개골 arthrotomy를 만들기 위해 메스를 사용합니다. 슬개골 또는 대퇴사 두근 힘줄을 잘라 않도록주의하십시오. </li><li> 슬개 건에 불과 측면 무릎 캡슐을 통해 1cm 세로 절개를함으로써 측면 출시 슬개골. </li><li> 무릎이 확장되어 있는지 확인합니다. 미세 가위를 가지고 내측하는 측면에서 슬개골 아래를 통과. 신근기구 양쪽로 번역 될 수 있도록 위를 몇 번 확산. </li><li> t을 휘어 동안그 무릎, 무릎 관절의 내부를 노출시키기 위해 측방 슬개골 번역. 과간 노치와 대퇴골의 명확한 시각화를 확인합니다. 메스를 사용하여 노치에서 ACL과 PCL을 가로로 쪼개다. </li><li> 1.6 mm의 K-와이어로드 파워 드릴. 과간 노치에서 ACL 기원에 K 와이어 팁을 놓습니다. superolaterally 드릴과 메스에 필요한 모든 연부 조직을 제거, 대퇴골의 측면 측면에서 출구 지점을 시각화. 이식에 대한 명확한 통행을 보장하기에 몇 시간에서 K-와이어를 전달합니다. </li><li> 경골 고원에 ACL 공간에 K-와이어를 놓습니다. anterolaterally 드릴과 전 외측 경골 근위부에 종료 지점을 시각화. K-와이어 경골 나오는 지점이 완전히 가시화 될 수 있도록 필요에 따라 부드러운 조직을 취소 메스를 사용합니다. </li><li> 대퇴골 터널을 통해 (이상적으로 더 이상 2보다 인치) 단축 키스 바늘을 전달합니다. K의 눈을 통해 그라프의 한쪽 끝에서 두 봉합 단부 스레드eith 바늘. 대퇴 터널을 통해 이식의 한쪽 끝을 당겨 바늘을 사용합니다. </li><li> 경골 터널을 통해 이식의 다른 쪽 끝을 전달하는 이전 단계를 반복합니다. </li><li> 그림 8 자 스티치와 골막 또는 다른 부드러운 주변 조직에 이식의 대퇴골 끝을 부착하기 위해 4-0 Vicryl 봉합사를 사용합니다. 수동 장력 확장의 무릎에 이식. 그림 8 자 스티치와 골막 또는 다른 부드러운 주변 조직에 이식 경골 끝을 붙입니다. </li><li> 그림 8 자 스티치 과거의 각 끝에 1-2 mm로두고, 양쪽 끝 초과 이식을 잘라 가위를 사용합니다. </li><li> 무릎을 확장 및 슬개골을 줄일 수 있습니다. 4-0 vicryl을 사용하여, 슬개골의 외측 아 탈구를 방지, 내측 관절낭을 닫습니다 단일 그림 8 자 스티치를 배치합니다. </li><li> 기본 근육을 봉합하지 않도록주의, 실행 표피 하 5-0 Monocryl 또는 Vicryl 봉합사로 피부를 닫거나 피부가 폐쇄되면 눈에 보이는 봉합있다. </li><li> 인제피하 부 프레 노르 핀과 CT 쥐 수술 후 삼일의 총 매 12 시간. 주입시 어떤 배수 수술 부위 나 상처 열개를 확인합니다. 파행과 붓기는 수술 후 처음 몇 일 정상, 그러나 신속하게 수의학 직원들과 함께 어떤 수술 후 문제를 해결. 수술 절개 부위가 완전히 치유 될 때 동물은 수술 후 이주에서 사회 주택으로 복귀 할 수있다. </li></ol> 3. 데이터 수집 프로토콜 <ol><li> 희생 이때, 개흉술이어서 CO 2 밀폐 챔버 내에서 개별적으로 쥐 질식. </li><li> 고관절에서 분리하여 외과 적 재구성과 반대측 사지를 모두 수확. <ol><li> 복원 된 사지를 들어, 만 대퇴골, 경골 및 이식을 분리하는 미세 절개하여 후방 십자 인대 수술 중단 기본 ACL에서 잔존물을 포함한 모든 연부 조직을 제거합니다. </li> &lt;리&gt;는 반대측 사지를 들어, 기본 ACL을 제외한 모든 연부 조직뿐만 아니라 미세 절개에 의한 대퇴골과 경골을 제거합니다. </li><li> 대퇴골 그라프 경골 단지의 각 끝에서 뼈의 ¾에 1cm 남기고 모두 제거 할 때에는 드레 멜으로 회전 도구를 사용합니다. </li><li> 이 과정과 생체 역학 테스트를 통해 정기적으로 자주 거짓 결과를 변경할 수있다 수확 무릎의 탈수를 방지하기 위해 생리 식염수와 인대 영역을 스프레이. </li></ol></li><li> 조직 학적 분석을 위해 48 시간 동안 25 ℃에서 각각 4 % 파라 포름 알데히드 용액에서 각 무릎을 고정한다. 다음으로, 전체에 대한 탈회 Immunocal 시약 용액 무릎 잠수함; 이 과정은 샘플의 석회화 내용에 따라 달라집니다 5 일까지 걸릴 수 있습니다. 샘플 품질이 저하 될 수 있습니다 불완전 탈회 등의 진행 상황을 평가하기 위해 매일 각각의 샘플을 확인합니다. 원하는대로, 절편 수행, 슬라이드 장착하고, 염색이 완료되면. </li><li> Perform 생체 역학 테스트는 조직 설계 인대의 기능적 능력을 평가합니다. <ol><li> 개별적으로 각 뼈의 골단의 주위에 28 G 아연 도금 강철 와이어를 배치하여 대퇴골과 경골을 고정합니다. 이것은 뼈보다는 관심 인대에서 시료의 인장 조기 장애 부정확 생체 역학 테스트 데이터를 방지하는 것이다. </li><li> 냄비 크릴 레이트 (PMMA) 골 시멘트의 혼합물에 대퇴골. 이렇게하려면 두 개의 시멘트 성분을 혼합하고 즉시 완전히 자유롭게 돌출 골단과 연결된 인대와 시멘트 냄비에 뼈의 골간을 감싸는 금속으로 대퇴골을 확보하기 위해 점성 혼합물을 사용합니다. 자연 자유 라디칼 중합이 점차 밀가루 반죽 재료로 결국 고체 강화 된 매트릭스에 혼합 점성 구성 요소를 변환 할 수 있습니다. 참고 :이 프로세스는 몇 분 정도 걸립니다 수동으로 만든 루스의 온도를 평가하여 모니터링 할 수 있습니다나머지 시멘트; 온도가 일시적으로 발열 중합 반응 동안 증가 물질이 응고 후 RT로 가라 앉을 것이다. </li><li> 이상적인 기계적 시험 20 ° 굴곡에서 무릎 인대를 유지하면서 제외하고, 경골을 확고히하기위한 위와 같은 과정을 반복합니다. </li><li> 인장 시​​험 장치에 접합 대퇴골 - 경골 그래프트 착체를 장착하고 실패 장력의 시작으로부터 시간의 함수로서 기록 하중과 변위를 준비한다. 이 예에서는 1 KN 로드셀 인스 트론 모델 5564를 사용 하였다. </li><li> 사전 장력은 0.5 N / 분의 램프 속도에서 2 N 그래프트하고 0.5 mm / 초의 변형 속도로 실패 그래프트 테스트. 이 과정에서 인대가 중간 물질에 실패되어 있고 뼈 대퇴골과 경골이 안전한지 및 조기 부정확 시험 인대의 생 역학적 특성을 평가 할 수있는, 실패하지 않도록주의. </li><li> 생성 된 하중 - 변위를 사용하여곡선은 시험 인대 실패 부하 및 강성을 계산. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

ACL 부상은 현 시점에서 재구성에 대한 제한 옵션 정형 외과 스포츠 수술의 일반적인 조건입니다. 생체 내에서 재생을 허용 ACL에 대한 적절한 대체 조직 공학을 개발하기 위해, 적절한 동물 모델이 요구된다. 흉선 쥐에서 전방 십자 인대 재건술의 재현 모델을 사용하여 생체 내 주입 같이 본 연구에서는 생분해 설계 이식의 제조는 설명한다. 이 모델은 세포 이식하고 통합하는 성장 인...

전방 십자 인대 (ACL) 파열은 무릎 하나의 가장 일반적인 인대 부상 중 하나입니다. 파열 ACL의 수술 개입없이 치유 할 수없는 때문에, 일상 생활뿐만 아니라 스포츠에 참여의 활동에 제한은 매년 억 달러 (3)의 추정 비용으로, 매년 2 수술을 175,000 명 이상의 환자를 구동한다. 현재, 이식 또는 동종 이식 건 중 하나는 인대 재건술에 사용됩니다. 높은 성공률 모두 이식과 동종 이식 교체로 달성 될 수 있지만, 심각한 합병증이 복원 옵션 4와 연결되어 있습니다. 이식 조직은 특히 재 파열 또는 멀티 인대 손상의 경우에, 공여부의 이환율과 관련된 공급에 제한됩니다. 한편, 동종 이식 조직 지연된 그래프트 통합 불리한 염증 반응, 이론적 감염성 위험 및 제한 좌제와 링크LY 5. 합성 비 분해성 이식은 1970 년대와 1980 년대에 개발되었지만 조기 이식 파열, 이물 반응, 골 용해 및 활액막염 (6)에 의해 방해되었다. 이러한 심각한 문제의 결과로서, 미국에서의 임상 사용할 수있는 합성 이식은 현재 존재하지 않는다. 때문에 기존의 이식 옵션 및 생물학, 공학, 재생 의학의 최근의 발전에 이러한 제한, ACL 이식을위한 조직 설계 솔루션에 큰 관심이 있었다. 현재 조직 공학 전략 영구 합성 재료 주입 7과 관련된 제한을 회피하면서 호스트 조직 내 증식을 허용하는 생물 분해성 및 합성 물질을 사용한다. 폴리 카프로 락톤 (PCL)는 FDA가 접착 장벽을 포함한 의료 분야의 다수의 승인과 8 드레싱 감겨 생분해 성 고분자, 즉 U를했습니다혈관, 뼈, 연골, 신경, 피부, 식도 조직 공학 5,9-16 포함한 다양한 애플리케이션에 나오지. 바람직한 생체 적합성, 상대적으로 긴 생체 내 반감기, 충분한 기계적 강도, 높은 탄성 조직 공학에서의이 중합체의 인기에 기여한다. 상처 치유의 쥐 모델에서 이식 전기 방사 PCL은 비 면역 원성 및 이상 반응 (13)없이 지방 조직으로 통합하는 것으로 나타났다. 전기 방사 된 PCL의 SEM 이미지는도 1에 도시된다. 크고 작은 동물 모델에서 현재 FDA 규제 기준, 유효성과 안전성으로 미국에서 임상 시험에 이동 PCL 또는 기타 설계 ACL 이식을 위해 필요한 것입니다. 또한, 생체 내 조건은 종종 시험관 조직 설계 ACL 이식의 특성을 보강 할 수 있습니다. 굴곡 digitor와자가 전방 십자 인대 재건술의 쥐 모델음 longus 건은 이전에 기본 ACL이 절단 대퇴 및 경골 터널을 뚫고하고, 이식이 통과하고 봉합 17-22로 제 위치에 고정 된 한에서, 기술되었다. 이 논문에서는 설계 ACL 교체의 평가보다는 이식 기반 재건 (그림 2)이 모델의 변형 예를 설명한다. 많은 동물 모델은 인대 조직 공학을 위해 존재하지만, 래트는 여러 이유로 큰 모델에 비해 유리하다. 이러한 장점은 쉽게 사육 및 처리, 적은 수의 윤리적 고려 사항 및 비용 절감의 17, 23 등이 있습니다. 또한, 쥐 모델 연골, 힘줄, 뼈 조직 공학 (24)를 포함하여 정형 조직 재생을위한 모델로서 광범위하게 사용되어왔다. 특히, 흉선 누드 쥐 최종 주입 O를 허용, 세포 매개 성 면역 반응 (25)의 부족으로 인해 선택되었다이 모델 F 이종 도너 세포는 또한 미래의 설계 그래프트 향상시킬. 이 방법의 논문에서는 전방 십자 인대 재건술의 흉선 쥐 모델의 제조 및 무 세포의 수술 주입, 생분해 성 고분자 이식에 대해 설명합니다.

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">Any comparable tensile testing apparatus is suitable</td>
		</tr>
	</tbody>
</table>

athymic rat model for evaluation of engineered anterior cruciate ligament grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

하나의 외과 의사 92 쥐 수술의 우리의 경험에 의하면, 상처의 완료에 절개에서 수술 시간을 의미하는 것은 4.7 분, 표준 편차, 16.9 분이었다. 희생의 시점에서, 쥐 356 ± 23g의 무게. 모든 쥐들이 수술을 허용하고, 합병증을 경험하지. 즉시 수술 후, 쥐가 수술 말단에 무게를 견딜 언급,하지만 약간의 미약을 전시했다. 일주함으로써 수술 후 모든 쥐없이 감지 할 수있을 정도의 미약으로 ambulating했다. ?...

Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

엔지니어링 전방 십자 인대 이식의 평가 흉선 쥐 모델

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

athymic-rat-model-for-evaluation-engineered-anterior-cruciate

Research

JoVE Journal

Bioengineering

10.3K 조회수.  University of California Los Angeles. Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

비디오: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

조직 평가에 대한 자기 공명 Elastography 방법론은 구조 성장하는 기술

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

연구

생체공학

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

외과 검색, 분리 및<em&gt; 체외</em&gt; 조직 공학 응용 프로그램에 대한 인간의 전방 십자 인대에서 파생 된 세포의 확장

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

티슈 엔지니어링 혈관 이식 및 이후의 이식을위한 수술 기법<em&gt; 생체</em&gt; 모니터링

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

의 응용 프로그램<em&gt; 생체</em&gt; 조직 평가를위한 쥐 앞정 전방의 기능 테스트는 골격근 수리 엔지니어링

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

3D 인쇄 가이드 및 반지 스태킹 방법을 사용하여 엔지니어링 혈관 이식의 크기 조정

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

운동 조건의 혈청을 이용한 인대 구조의 치료 : Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

높은 처리량의 사용 모델 IgG1 조 세포에서의 생산을 위한 Microbioreactor 시스템 자동화

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...