저자는 텍사스 A &amp; M 대학 공학을 감사하고이 연구의 금융 지원을 위해 역 (티) 실험. 린지 네일은 감사 소수 참가 텍사스 A &amp; M 대학 루이스 스톡스 얼라이언스 (LSAMP)와 국립 과학 재단 (NSF) 대학원 연구 활동 프로그램 (GRFP)의 지원을 인정합니다. 웨이 장은 감사 텍사스 A &amp; M 대학 논문 친목.

1. 합성 PCL-DA 로머 <ol><li> 아크릴화 반응을 실행합니다. <ol><li> 테프론 피복 자기 교반 바가 장착 된 250 mL의 둥근 바닥 플라스크에 PCL 디올 (M에서 N = ~ 10,000g / 몰) 20g을 단다. </li><li> DCM에서 PCL 디올을 녹인다. <ol><li> 플라스크에 120 mLlof DCM 추가 (농도 = 0.17 ㎍ / ㎖). </li><li> 또한 DCM의 증발을 방지하면서 압력 빌드 업을 피하기 위해, 플라스크의 목에 느슨하게 고무 격막을 배치. </li><li> ~ 250 rpm에서 30 분 ~ 동안 교반 솔루션은 완전히 폴리머를 용해한다. </li></ol></li><li> 용액에 4- 디메틸 아미노 피리딘 (DMAP)의 ~ 6.6 ㎎을 추가하고, 교반하면서 용해. </li><li> 플라스크의 목에 단단히 고무 격막을 놓습니다. 이 솔루션은 교반을 계속하도록 허용합니다. </li><li> 고무 격막을 통해 조심스럽게 긍정적 인 N 2 압력 바늘 입구와 오픈 바늘을 사용하여 2 ~ 3 분 동안 N 2 플라스크를 제거출구로. </li><li> N 2 입구와 출구를 분리합니다. </li><li> 고무 셉텀을 통해 삽입 된 바늘이 장착 된 유리 주사기를 통해 트라이 에틸 아민 0.56 ㎖ (4.0 밀리몰) (N 잇 3) 적가. </li><li> 고무 셉텀을 통해 삽입 된 바늘이 장착 된 유리 주사기를 통해 적가 아크릴로 일 클로라이드 0.65 ㎖ (8.0 밀리몰)을 추가한다. </li><li> 플라스크에 N 2 입구를 돌아 내용이 ~에 대해 긍정적 인 N 2 압력을 받고 30 분을 자극 할 수 있습니다. </li><li> 55 ºC로 오일 욕을 예열. </li><li> 할당 ~ 30 분 후, N 2 입구를 제거하고 응축기 격벽을 대체. </li><li> 예열 오일 배스에​​ 플라스크를 담근다. </li><li> 플라스크의 내용물을 20 시간 동안 교반한다. </li><li> 할당 된 20 시간 후, 오일 조로부터 플라스크를 제거하고 내용물을 실온으로 냉각 할 수있다. </li><li> 회전 증발기를 사용하여, 플라스크에 DCM에서 용매를 제거한다. </li></ol></li><li> 피원유 PCL-DA 제품을 urify. <ol><li> 플라스크에, 아세트산 에틸 ~ 135 mL를 추가하고, 조 PCL-DA를 녹인다. </li><li> 중력은 깨끗한 250 mL의 둥근 바닥 플라스크에 여과지 용액을 필터. (참고 : 솔루션을 쉽게 통과하지, 필터 종이에 두껍게 할 수 있으므로,주의 깊게 가열 총으로 가벼운 열을 적용하면..) </li><li> 회전 증발기를 사용하여, 용매를 플라스크로부터 에틸 아세테이트를 제거한다. </li><li> 플라스크에, DCM ~ 140 mL로 추가 조질 PCL-DA를 녹인다. </li><li> 500 mL의 분액 깔때기에 내용을 전송합니다. </li><li> 깔때기에, 2 M 탄산 칼륨 (K 2 CO 3)의 13.5를 가하여. </li><li> 깔때기를 모자. 조심스럽게 마개를 통해 압력을 해제 돌보는, 깔때기를 반전 부드럽게 한 번 또는 두 번 소용돌이에 의해 두 개의 층을 섞는다. 3 번 반복합니다. </li><li> 파라 필름의 층으로 뚜껑을 교체하고, 혼합물 O / N (~ 12 시간)을 분리 할 수​​ 있습니다. </li><li> 저부를 수집하거나250 mL의 삼각 플라스크에 ganic 층. </li><li> 플라스크에 무수 황산 마그네슘 5g ~ (황산)를 첨가하고, 가볍게 소용돌이. </li><li> 중력은 둥근 바닥 플라스크 정성 여과지 및 깨끗한 250 ㎖ 중에 혼합물을 필터. </li><li> 회전 증발기를 사용하여, 플라스크에 DCM에서 용매를 제거한다. </li><li> 높은 진공 하에서 건조 잔류 DCM을 제거합니다. (참고 : PCL-DA는 빛으로부터 멀리 보관해야합니다.) </li><li> 1 H NMR (22, 23)와 아크릴화를 확인합니다. </li></ol></li></ol> SMP 비계를 준비​​ (2) (그림 1) <ol><li> 용융 소금 템플릿을 준비합니다. <ol><li> 직경이 70 ~ 460 μm의 ± 염화나트륨 입자를 얻기 위해 425 ㎛의 체를 사용한다. (주 : 평균 입경은 ImageJ에 소프트웨어를 주사 전자 현미경 [SEM] 이미지에서 확인 될 수있다.) 14 </li><li> 3 ㎖의 유리 바이알 (ID = 12.9 mm)에, 이전에 체질 염화나트륨 1.8 g을 추가한다. </li><li> 천천히 유리 병에 네 부분, 7.5 중량 % (소금의 중량을 기준으로) 탈 이온수 (0.146 g)에 추가 할 수 있습니다. 물의 각 부의 첨가 후 금속 주걱으로 혼합한다. </li><li> , 유리 병 캡 조직 포장 및 원심 분리 관에 수직으로 배치합니다. 3,220 X g에서 15 분 동안 원심 분리기. </li><li> 뚜껑을 제거하고 자연 건조 O / N (~ 12 시간)을 할 수 있습니다. </li></ol></li><li> 새로운 유리 바이알에서, DCM 1 ㎖ 당 PCL-DA 0.15 g을 조합함으로써 &quot;로머 ​​용액&quot;을 준비한다. (주 :. 한 지지체를 들어, ~ 용액 1 ㎖를 제조한다)과 캡은 ~ 1 분 동안 와동 믹서 고속으로 용액을 혼합한다. </li><li> 새로운 3 mL 유리 바이알에, 10 중량 %, 2,2- 디메 톡시 -2- 페닐 아세토 페논 1- 비닐 -2- 피 롤리 디논 (DMP) (NVP)에 기초하여 &quot;광개시제 용액&quot;을 준비한다. NVP의 1 ml의 DMP의 0.115 g을 결합합니다. (빛을 차단하기 위해) 알루미늄 호일에 포장, 모자 및 와류 믹서에 빠른 속도로 솔루션을 혼합 : (180 μL가 필요합니다 ~, 하나의 발판은. 참고)~ 1 분. (참고 : 고급에서 제조하는 경우, 용액을 냉각하고 빛으로부터 보호되어야한다.) </li><li> 알루미늄 호일 (캡 제외) 거대 단량체 용액을 함유하는 바이알을 랩 (빛을 차단하는) 피펫을 통해 15 부피 %를 추가 개시제 용액 (거대 단량체 용액의 총 부피를 기준으로). 캡 ~ 1 분 동안 와동 믹서 고속으로 용액을 혼합한다. </li><li> 알루미늄 호일 (캡 제외) 용융 염 템플릿을 함유 바이알을 랩 (빛을 차단하기 위해) 및 피펫을 통해 미리 제조 로머 / 광개시제 용액을 추가 (~ 0.6 ㎖에 또는 템플릿까지 완전히 피복). </li><li> , 유리 병 캡 조직 포장 및 원심 분리 관에 수직으로 배치합니다. 템플릿 전반 로머 용액을 분배 1,260 XG에서 10 분 동안 원심 분리기. </li><li> 알루미늄 호일을 제거 병을 뚜껑을 벗기다, 3 분 동안 자외선 (365 nm의 25 승)에 노출. 공기 건조 O / N. </li><li> &quot;소금 함유 scaffol 제거득점과 유리 병의 상단을 골절 후 핀셋으로 유리 병에서 D &quot;. </li><li> (1 부피 : 부피 1) 400 ㎖의 비이커에, ~ 용매를 물 / 에탄올 200 ㎖를 준비한다. </li><li> 매일 용매 변경 4 일 동안 용매 물 / 에탄올에 빠져들 골격을 유지한다. </li><li> 용매 및 자연 건조 O / N에서 비계를 제거합니다. </li></ol> 3. SMP 비계에 Polydopamine 코팅을 적용 (그림 1) <ol><li> 테프론 피복 교반 바가 장착 된 400 ml의 비이커에, 도파민 염산염 용액 ~ 200 ㎖ (10 mM 트리스 완충액에 2 ㎎ / ㎖, pH는 8.5, 25 ºC)를 준비한다. ~ 150 rpm으로 저어. </li><li> 거리의 절반 발판을 발판으로, ~, 일회용 바늘 (게이지 = 20 길이 = 40 mm)를 놓습니다. 바늘 허브 주위에 와이어를 감 쌉니다. </li><li> 비이커의 림에 와이어를 고정함으로써 도파민 교반 용액에 (솔루션면 위에 바늘 허브)와 발판을 담근다. </li><li> 바늘 허브에 주사기를두고 지지체의 공기를 끌어을 사용하여 발판을 탈기. (주 : 더 많은 공기가 제거 될 수 없으며 용액이 완전히 지지체에 침투되면 탈기 완료). </li><li> 16 시간 동안 교반 도파민 솔루션에 빠져들 골격을 유지한다. </li><li> 솔루션에서 비계를 제거하고 바늘을 제거합니다. 24 시간 동안 실온에서 진공 오븐에서 탈 이온수로 세정하고 건조. </li><li> 1 시간 동안 85 ºC 오븐에서 비계를 놓습니다. </li><li> 발판은 실온으로 냉각 할 수 있습니다. 마지막 원통형 비계 ~ 6mm 직경 X ~ 5mm 높이됩니다. </li></ol> 4. 평가 &quot;셀프 피팅&quot;행동 <ol><li> 두께 ~ 5mm 인 경질 플라스틱 시트를 이용한 &quot;CMF 요철 결함 모델&quot;을 준비한다. 그림 2A에서 입증 된 바와 같이, ~ 6mm보다 약간 적은 평균 직경 플라스틱 시트 내의 빈 공간을 만들기 위해 드릴을 사용합니다. </li><li> AB에서eaker ~ 60 ºC의 온도 (식염수 임상 사용을 나타내는) 열 DI 물. </li><li> 의 비커 ~ 60 ºC 물에 비계를 놓습니다. 물에 모든 영역을 노출, 수면 아래의 발판을 밀어 핀셋을 사용합니다. 1 ~ 2 분 동안 또는 발판이 눈에 띄게 (그림 2B)을 부드럽게 될 때까지 계속합니다. </li><li> 비커에서 비계를 제거하고 즉시 모델의 결함으로 (손으로) 키를 누릅니다. </li><li> RT (~ 5 ~ 10 분) (그림 2C)을 식 힙니다. </li><li> 새로운 고정 임시 모양과 상대적으로 단단한 상태 (그림 2D)의 반환을 관찰하기 위해 결함에서 제거합니다. </li></ol> 5. 시험 형상 기억 동작 <ol><li> 동적 기계 분석기를 사용하여 (DMA 본원에서 사용 된 바와 같이, 예를 들면 TA 인스트루먼트 Q800)를, 2 사이클 (N) 형상으로 고정 인 (R의 F)를 결정하기 위해 이상 지지체에 변형 제어형 환상 열적 기계적 압축 시험을 실행할복구 (R의 R) (그림 3) 모양입니다. <ol><li> 5 분 60 ° C (T 높은) 평형. </li><li> 50 % / 분에서 최대 변형 (ε 분 = 50 %)에 압축. </li><li> ε 분 (5 분)에서 잡으십시오. </li><li> 25 ° C (T 낮음)으로 냉각시키고, 10 분 임시 모양을 수정하는 유지한다. </li><li> 부하를 제거합니다. </li><li> (U ε) 스트레스없는 상태에서 궁극의 변형을 측정합니다. </li><li> 60 ° C (T 높음)로 재가열하고 10 분 영구 형상을 회복하기 위해 유지한다. </li><li> 복구 된 변형 (ε의 p)를 측정한다. </li><li> 아직 (T 높음) 60 ℃에서, 2 차 사이클을 시작 (N = 2) 1 차 사이클 후에 회수 높이 (N = 1)의 50 %로 압축하여 발판을한다. </li><li> 5.1 반복.3-5.1.8 N = 2. </li><li> 다음 식을 이용하여 N = 1, R 2의 F 및 R의 R을 계산한다 : R의 F (N) = [U ε (N) / ε 분] R의 R (N) = ε 분 - ε의 P (N)] / [ε M - ε의 P (N-1)] </li></ol></li></ol> 6. 떠올리 기공 크기와 기공 상호 접속 <ol><li> 주사 전자 현미경을 사용하여 (SEM, 예를 들면 본원에서 사용 FEI 콴타 SEM)을, 공극 크기를 관찰하고 상호 기공. <ol><li> 핀셋을 사용하면, SMP의 발판을 개최 1 분 동안 액체 N 2에 잠수함합니다. </li><li> 깨끗한 면도날과 발판의 중앙을 따라 액체 N 2, 골절에서 제거합니다. </li><li> 카본 테이프를 사용하여, 시료에 SMP 골격 반쪽들 중 하나를 부착골절면이 위를 향하게하여 무대. </li><li> 금-PT (~ 4 ㎚)와 코트를 스퍼터. </li><li> 10 ~ 15 kV의 (그림 4A)의 권장 가속 전압의 SEM 이미지를 캡처합니다. </li></ol></li></ol> 체외에서 생체 활성 7. 테스트 <ol><li> 50 ㎖ 원심 분리 튜브로, 24 ~ 1X SBF 30ml의 추가. </li><li> 원래, 원통형 성형 영구 모양의 발판을 얻습니다. 깨끗한 블레이드를 사용하여 (원형 모서리에서) 반 비계를 잘라. </li><li> 준비된 원심 분리기 튜브와 캡으로 개별 발판 절반을 놓습니다. </li><li> SBF 변경없이 정적 조건 하에서 수조에서 37 ℃에서 튜브를 유지한다. </li><li> 십사일 후, 24 시간 동안 SBF 공기 건조에서 비계를 제거합니다. </li><li> 탄소 테이프를 사용하여, 골절면이 위를 향하도록하여 샘플 무대에 발판을 부착합니다. </li><li> 금-PT (~ 4 ㎚)와 코트를 스퍼터. </li><li> 재에서 SEM 이미지를 캡처10 ~ 15 kV의 (그림 4B)의 가속 전압을 칭찬. </li></ol>

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The authors have nothing to disclose.

이 프로토콜은, 자기 피팅 동작 polydopamine 코팅, PCL 계 지지체의 제조뿐만 아니라, 생체 활성 및 osteoinductivity 설명 불규칙한 CMF 뼈 결함의 치료에서 관심을 만든다. 프로토콜의 측면은 다양한 비계 기능을 변경하는 변경 될 수있다. 이 프로토콜은 UV 경화를 허용하는 PCL 디올의 아크릴화로 시작합니다. 보고 된 예에서, PCL 디올 M n을 ~ 10,000g / 몰이다. 그러나, 적절 PCL-DA, ...

현재 부교감 악안면 (CMF) 골 결손 치료의 황금 표준으로 간주, 수확자가 이식의 이식은 복잡한 이식 절차, 공여부의 이환율과 제한된 가용성 (1)에 의해 방해된다. 특별한 어려움은 성형 및 골 유착을 얻고 그래프트 재 흡수를 방지하기 위해, 결함에 단단히 강성 자가골 고정된다. 조직 공학은 autografting하는 대안 전략 및 합성 골 대체물 (예를 들면 뼈 시멘트) 2,3로 조사되었다. 조직 공학 방법의 성공에 중요한 속성의 특정 세트 발판이다. 우선 골융합을 달성하기 위해, 지지체는 인접한 뼈 조직 (4)에 밀착 형성한다. 발판은 세포 이동, 영양 확산과 neotissue 증착 4,5 허용, osteoconductive해야한다. 이 동작은 일반적으로 생분해 SCA 달성된다ffolds는 고도로 상호 연결된 기공 형태를 나타내는. 뼈 조직 (5) 주변과의 통합 및 결합을 촉진하기 위해 마지막으로, 지지체는 생체 활성해야합니다. 여기, 우리는 이러한 특성을 가진 조직 공학 발판을 준비하는 프로토콜을 제시한다. 중요한 것은,이 지지체는 형상 기억 행동 6 불규칙한 CMF 결함에 &quot;자기 끼움&quot;할 수있는 능력을 나타낸다. Thermoresponsive 형상 기억 폴리머 장치 (SMPS)는 7,8 열에 노출시에 형상 변화를 겪는 것으로 알려져있다. SMP의 영구 형상 및 임시 형태를 유지하고, 영구 형상 복구 &quot;스위칭 세그먼트&quot;결정 &quot;netpoints&quot;(즉, 화학적 또는 물리적 가교)로 구성된다. 스위칭 세그먼트 중 유리 전이 (T의 g)에 대응하는 열 전이 온도 (T 트란스)를 나타낼 또는 중합체의 천이 (T의 m)을 용융. 으로결과는 SMP의 순차적 T &lt;T 트랜스에서 임시 형태로 고정 T&gt; T 트랜스에서 임시 모양으로 변형하고, T&gt; T 트랜스의 영구적 인 형태로 회수 할 수있다. (6)을 다음과 같이 따라서, SMP의 발판은 CMF 결함에서 &quot;셀프 피팅&quot;달성 할 수있다. 노출 후 식염수 (T&gt; T 트랜스), SMP의 발판이 모양 복구 결함 경계에 발판의 확장을 촉진하여, 손으로 누르면 불규칙한 결함에로 일반적으로 제조 된 원통형의 발판을 허용, 가단 될 것 따뜻하게. (T &lt;T 트랜스)를 냉각시, 비계 모양의 정착이 결함 내 새 임시 형태를 유지하면서, 상대적으로 더 단단한 상태로 반환합니다. 이 프로토콜에서, SMP의 발판은 생분해 성 고분자는 조직 재생 및 기타 생물 의학 응용 프로그램에 9-11에 대한 광범위하게 연구, 폴리 카프로 락톤 (PCL)에서 제조된다. 형상 기억하십시오, 일PCL의 전자의 m T는 T 트랜스 역할 및 PCL (12)의 분자량에 따라, 43과 60 사이에서 변화 ºC. 이 프로토콜에서, 지지체의 T 트랜스 (즉 T의 m)은 56.6 ± 0.3 ºC 6입니다. osteoconductivity을 달성하기 위해, 프로토콜은 용매 캐스팅 미립자 침출 (SCPL) 6,13,14 방법에 기초하여 고도로 상호 연결된 기공 PCL 기반 SMP 골격을 위해 개발되었다. 디 아크릴 레이트, 폴리 카프로 락톤 (PCL-DA) (M = N ~ 10,000g / mol)을 신속하게, 광 화학적 가교 결합을 허용하기 위해 이용하고, 염 템플리트 위에 용매 캐스팅을 허용하는 디클로로 메탄 (DCM)에 용해시켰다. 광화학 요법 및 용매 증발에 이어, 염 템플릿을 물에 침출하여 제거 하였다. 염 평균 크기는 골격의 기공 크기를 조절한다. 중요한 것은, 염 템플릿 전에 기공 interconnectivi을 달성 캐스팅 용매 물과 융합시켰다타이. 생체 활성은 기공 벽 (6)에 polydopamine 코팅의 현장 형성에 의한 SMP 지지체에 부여했다. 생체 활성은 종종 유리 또는 유리 - 세라믹 충전제 (15)의 포함으로 골격에 도입된다. 그러나, 이들 원치 않는 취성 기계적 특성을 야기 할 수있다. 도파민은 기판의 다양한 16-19에 부착, polydopamine 얇은 층을 형성하는 것으로 나타났다. 이 프로토콜에서, SMP 지지체 모든 기공 벽 표면에 6 polydopamine nanothick의 피막을 형성하는 도파민 약간 염기성 용액 (PH = 8.5)를 실시 하였다. 개선 된 세포 접착을위한 표면의 친수성을 향상시키고 확산 외에 polydopamine는 모의 체액 (SBF) 18,20,21 노출시 하이드 록시 아파타이트의 형성 (HAP)의 관점에서 생물 것으로 밝혀졌다. 마지막 단계에서, 코팅 된 지지체를 85 ºC (T&gt; T 트랜스) WH 열처리를 노출무형 문화 유산은 치밀화를 폴딩 할 이어집니다. 열처리는 이전 PCL 결정질 도메인 가까이 근접 14 재구성 아마도 인해, 골격 형상 기억 행동 필수적인 것으로 관찰되었다. 우리는 또한, 불규칙한 모델 결함 내에서 자체 맞는 행동을 특징 짓는 용어로 변형 제어 순환 열 기계 압축 테스트 메모리 동작을 형성 (즉, 형상 회복과 정착을 형성)하는 방법을 설명하는 형태 기공, 체외 생체 활성에. 발판의 특성을 조정하는 전략도 제공됩니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone-diol (Mn ~ 10,000 g/mol)</td>
			<td>Sigma-Aldrich</td>
			<td>440752</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>D65100</td>
			<td>Dried over 4A molecular sieves</td>
		</tr>
		<tr>
			<td>4-dimethylaminopyridine (DMAP)</td>
			<td>Sigma-Aldrich</td>
			<td>D5640</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine (Et3N)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A24109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Carbonate (K2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous magnesium sulfate (MgSO4)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-dimethoxy-2-phenyl acetophenone (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>196118</td>
			<td></td>
		</tr>
		<tr>
			<td>1-vinyl-2-pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer (2mol/L)</td>
			<td>Fisher</td>
			<td>BP1759</td>
			<td>Used at 10 mM concentration, pH = 8.5</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>VWR</td>
			<td>47729-972</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Transilluminator (365 nm, 25 W)</td>
			<td>UVP</td>
			<td>95-0426-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Mechanical Analyzer (DMA)</td>
			<td>TA Instruments</td>
			<td>Q800</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Sputter Coater</td>
			<td>Cressington</td>
			<td>208HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>FEI</td>
			<td>Quanta 600</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a bioactive, pcl-based "self-fitting" shape memory polymer scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

그 결과 PCL 기반 SMP 골격 자체 피팅 모델 CMF 결함으로 (그림 2) 할 수있다. 짧은 노광 후 염수 (~ 60 ° C)을 가온, 원통형 지지체는 지지체가 수동으로 가압 모델 결함 내에 확장 될 수 있도록 연화. RT로 냉각시킨 후, 지지체는 결함으로부터 제거시에 유지되어 새로운 임시 형상으로 고정된다. SMP 지지체의 형상 기억 거동 형상 고정 인 (R의 F)와 형상 복구 (R의...

Watch this Scientific Journal Video about Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold at JoVE.com

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

생리 활성, PCL 기반 &quot;셀프 피팅&quot;형상 기억 폴리머 비계의 제작

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

fabrication-bioactive-pcl-based-self-fitting-shape-memory-polymer

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JoVE Journal

Bioengineering

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

12.5K 조회수.  Texas A&M University. Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

비디오: Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

활성 세포 배양을위한 형상 기억 폴리머

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

연구

생체공학

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

키토산 기반, 레이저 활성화 박막 외과 접착제, &#39;SurgiLux&#39;: 준비 및 데모

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

에 대한 기계적 적응 고분자 나노 복합 재료의 환경 제어 Microtensile 테스트<em&gt; 생체</em&gt; 특성

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

미리 설계된 크기와 모양의 중합체 및 바이오 하이브리드 섬유의 미세 유체 제조

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

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 ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

제조 및 Macrostructure를 포함 하는 광학 조직 팬텀의 특성화

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

서 주 신장 조직 세그먼트와 소설 비 계 없는 건설 기술 관

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Stretchable, 듀얼 채널, 미세 기관 칩의 확장 가능한 제작

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...