The authors would like to thank Prof. Steven Nutt and Mr. Yuzheng Zhang for assistant with the Focus Ion Beam, and the Center for Electron Microscopy and Microanalysis (CEMMA) at USC for electron microscopy. Research was supported by NIH-NIDCR grants; DE-13414 and DE-020099 to J.M.O.

인간의 어금니는 남부 캘리포니아 대학의 치과의 스트로우 학교에서 추출을위한 표준 절차에 따라 추출 및 기관 검토위원회의 승인을 처리했다. 1. 산 - 에칭 치아의 준비 조각 <ol><li> 복원 된 모든 충치가없는 사람의 세 번째 어금니를 선택합니다. </li><li> 어금니의 뿌리 부분을 제거하고, 길이 방향으로 두께 수냉식 저속 다이아몬드 톱을 사용하여 조각 2mm에 몰의 왕관을 잘라. 초음파로 2 분 동안이 조각을 청소 한 다음 탈 이온수로 3 회를 씻어. </li><li> , 미란 성 병변을 시뮬레이트 30 초 동안 30 % 인산으로 치아 슬라이스 에칭 후 즉시 탈 이온수로 헹구어. </li><li> 초음파로 2 분 동안 에칭 조각을 청소 한 후 탈 이온수로 3 회를 씻어. </li></ol> 성별 감정 - 키토산 하이드로 겔의 2. 준비 <ol><li> 촬영 준비키토산 원액 N <ol><li> 1 % 녹여 80 ° CO / N.에서 교반하여 1 % (v / v) 아세트산 용액 (w / v) 키토산 (중간 분자량 75-85% 탈 아세틸) </li><li> RT로 용액을 냉각 한 후, 0.45 μm의 필터를 사용하여 키토산 용액을 고를. 1 M NaOH 용액을 첨가하여 5.0으로 pH 값을 조정한다. </li></ol></li><li> 칼슘과 인산 원액의 제조 (0.1 M) <ol><li> 염화칼슘 (염화칼슘)를 밖으로 달아 0.1 M 용액을 제조 한 후 솔루션가 선명해질 때까지 소용돌이를 사용하여 섞는다. 1 M NaOH 용액을 첨가하여 11 pH 값을 조정합니다. </li><li> 인산 수소 나트륨 인산염 (Na 2 HPO 4)을 칭량하고 0.1 M 용액을 제조하고 용액을 클리어 할 때까지 소용돌이를 사용하여 혼합한다. </li></ol></li><li> 표현과 재조합 유전자 성별 감식 17-18의 정화 여기에 사용되는 재조합 전체 길이 돼지 유전자 성별 감식 (rP172)는 172 개의 아미노산을 가지고 있으며, 전체의 아날로그길이 네이티브 돼지를 P173 만 N-말단 메티오닌 등 Ser16 18 인산기 결여. <ol><li> , 탈 이온수 500 ㎖에 Lysogeny 국물 (LB) 한천 가루 20g을 추가 20 분 동안 121 ° C (액체 설정)에서 솔루션을 압력솥. ~ 55 ° C가, 한천 용액에 암피실린 (100 ㎍ / ㎖) 추가 한천 멋진하자. </li><li> 중후 때까지 각 판은 식지, 페트리 접시에 한천 솔루션을 전송 한 후 한천에 응축을 방지하기 위해 플립. 4 ° C에서 비닐 봉투에 보관 판 </li><li> 글리세롤 재고 재조합 세포 (대장균-BC21)와 행진 LB 한천 플레이트 (-80 ° C). 37 ° C에서 접시 O / N을 품어 </li><li> NZCYM 미디어 50 ㎖를 준비하고 30 분 동안 121 ° C에서 미디어를 압력솥. 미디어가 식을 때까지 허용, 50 ㎖ 미디어에 100 ㎍ / ㎖의 암피실린 50 μl를 추가합니다. </li><li> ampicill 보충 50 ㎖ NZCYM 미디어에 LB 한천 플레이트에서 단일 콜로니를 접종높은 37 ℃에서 진탕 배양기에서 세포를 O / N을 품어 </li><li> NZCYM 미디어 1 L를 준비하고 30 분 동안 121 ° C에서 미디어를 압력솥. 1,000 ml의 미디어에 100 ㎎ / ㎖ 암피실린 1 ML을 추가합니다. UV-VIS 분광 광도계를 사용하여 빈으로 광학 밀도 (OD) 측정 값이 표시됩니다. 참고 : 첫 번째 독서 후이를 폐기하지 마십시오. </li><li> 단계 2.3.6에서 1,000 ml로 미디어에 2.3.5 단계에서 세포 배양의 10 ㎖를 접종한다. 즉시 접종 후 595 nm에서 광학 밀도를 참조하십시오. 성장을 추적하기 위해 주기적으로 광학 밀도의 측정 값이 표시됩니다. 참고 : OD이 촬영 될 때마다 빈을 읽어보십시오. </li><li> 박테리아의 성장은 595 nm에서 약 0.75 ~ 0.8 OD에 도달 할 때 이소 프로필 β-D-1-티오 갈 락토 피 라노 시드 (IPTG, 1 M) 700 μL로 유도한다. </li><li> OD가 1.1에 도달하면 세포를 수확. 6 패트병로 플라스크의 장입 물을 붓는다. 균형과 원심 분리기를 사용하기 전에 병을 무게. 4 ° C에서 8,554 XG에 20 분 동안 원심 분리-20 ° CO / N.에 세포 펠렛을 유지 </li><li> 세포 알약을 꺼내 50 ㎖ 튜브에 그들을 결합. 발효의 L 당 4M 구아니딘 염산의 3.5 ML를 추가합니다. 1 분 간격으로 얼음, 30 초에 초음파 처리 2 ~ 3 시간마다. </li><li> 0.5 % 포름산과 세포 부피의 6 배 희석을 확인하고 2 시간 동안 추운 방에 저어 보자. 4 ℃에서 30 분, 8,554 XG에 원심 분리기 뜨는을 유지합니다. 추운 방에서 포화 원액과 파문을 사용하여 20 % 포화 황산 암모늄을 추가, O / N. </li><li> 4 ° C에서 30 분, 8,554 XG에 원심 분리기와 펠렛을 유지합니다. C4 칼럼 (10 × 250mm, 5 μM)에 0.1 % 트리 플루오로 아세트산 (TFA), 부하의 펠릿을 재구성하고,이면 TFA에서 A = 0.1 % TFA 및 B = 60 % 아세토 니트릴의 선형 구배를 사용하여 분별 1.5 ML · 분 -1의 유량. </li><li> 드라이 아이스에 단백질 용액을 동결 및 냉동 샘플 O / N을 냉동 건조 </li></ol></li><li> 유전자 성별 감식 - 키토산의 제조 (CS-AMEL) hydrog엘 (그림 1A) <ol><li> 200 ㎍의 rp172을 1 % 키토산 용액 960 μl를 함유하는 튜브에 가입 한 후 용액이 클리어 될 때까지 소용돌이를 사용하여 혼합한다. </li><li> 유전자 성별 감식 함유 키토산 용액에 0.1 M 나 2 HPO 4 용액을 15 μL 다음 0.1 M 염화칼슘 용액 25 μl를 추가하고 5 분 동안 소용돌이를 사용하여 섞는다. </li><li> 정중 1 M NaOH 용액을 첨가하여 6.5 CS-AMEL 용액의 pH를 조정한다. pH 값이 6.5에 도달 할 때 CS-AMEL 하이드로 겔이 형성됩니다. </li></ol></li></ol> 성별 감정 - 키토산 하이드로 겔 3. 에나멜 재성장 <ol><li> 인공 타액 용액의 조제 <ol><li> 특정 염화 마그네슘의 양 (MgCl2를, 0.2 ㎜), 칼슘 염화물 (염화칼슘, 1 ㎜), 인산이 수소 칼륨 (KH 2 PO 4, 4 ㎜), 염화칼륨 (KCl을 16 ㎜) 및 염화 암모늄 (디졸브 20 mM의 HEPES에있는 NH 4 망할 CIA, 4.5 ㎜)(4 - (2 - 히드 록시 에틸) 피페 라진 -1 - 에탄 술폰산) (12)를 버퍼링. </li><li> 1 M NaOH로 7.0으로 산도를 조정하고 4 ℃에서 인공 타액 솔루션을 저장 </li><li> 용액을 사용하기 전에, 불화 나트륨 (NaF로, 300 PPM)를 추가한다. </li></ol></li><li> CS-AMEL 하이드로 겔의 응용 프로그램 <ol><li> 주사기 (그림 1B)를 사용하여 에칭 치아 슬라이스 약 20 μL CS-AMEL 하이드로 겔을 적용합니다. </li><li> 2 시간 동안 실온에서 데시 케이 터에서 하이드로 겔 덮인 치아 조각을 건조. </li><li> 인공 타액 용액 30 ㎖를 함유하는 비이커에 치아 슬라이스 전송. 알루미늄 호일로 비커를 덮고, 그 후에을 7 일 동안 37 ° C의 오븐에서 비커를 유지합니다. </li><li> 치아 조각을 제거하고 실온에서 건조기에 건조. </li></ol></li></ol> 새로 성장 층과 에나멜 사이의 인터페이스 4. 특성 새로 성장 층과 멜 계면의 미세 구조는 obser이었다전자 현미경 (SEM) 및 높은 회전 투과 전자 현미경 (HR-TEM)을 주사함으로써 VED. TEM 샘플은 집속 이온 빔 (FIB) 기술은 다음과 같이되어있는 절차를 사용하여 제조 하였다. <ol><li> FIB-SEM 악기로 샘플을로드하고 운영 지침에 따라 모든 정렬을 마무리합니다. 참고 : 미세 Z 조정이 최소 2 번 수행해야합니다. </li><li> 기본 구조 (그림 2A)를 보호하기 위해 샘플에 탄소 층 (15 × 3 μm의) 보증금. </li><li> 공장은 탄소 층의, 상하과 오른쪽에서 조심스럽게 샘플은 샘플의 얇은 조각을 준비하고, 다음이 얇은 조각의 바닥면을 잘라. 참고 : 각 분쇄 공정 (그림 2B) 전에 이온 빔 정렬을 확인합니다. </li><li> 용접 백금 얇은 조각에 팁과 샘플 조각 (그림 3C)를 분리하기 위해 왼쪽을 잘라. 참고 : 용접 단계 이전에 이온 빔 정렬을 확인합니다. </li><li> 티셔츠를 들어 올려그는 천천히 샘플 팁 및 리프트 아웃 TEM 그리드에 층상 샘플을 탑재 PT. </li><li> 두께가 100nm 이하 (그림 3D)가 될 때까지 샘플과 팁, 얇은 샘플을 분리합니다. TEM 관찰을 수행하는 기기에서 샘플을 제거합니다. 참고 : 각 숱이 프로세스가 완료되기 전에 이온 빔 정렬을 확인합니다. </li></ol> 새로 성장 층의 바인딩 및 기계적 성질 5. 평가 <ol><li> 결합력은 초음파 처리에 의해 평가된다. 10 분 동안 초음파 세척기 (42 kHz에서, 100 W)에 수리 치아 슬라이스 유지하고 후방 산란 전자-SEM 분석을 수리 층과 천연 에나멜 사이의 계면을 관찰한다. </li><li> 각 수리 에나멜 표면에 20 테스트 포인트 (N = 3) 베르 코 비치 팁과 나노 압입에 의해에서 경도와 탄성 계수를 측정한다. </li></ol> 6. 면역 형광 염색법 <ol><li> 트리스 buffe와 치아 조각을 세척15 분 동안 R 식염수 (TBS). </li><li> 15 분 동안 TBS에서 1 % 소 혈청 알부민 (BSA)으로 차단. </li><li> 액체를 제거하고 TBS (0.1 % BSA, 0.3 % 트리톤 X-100)에 희석 1 ° AB (1:500 치킨 안티 성별 감정)를 이용하여 시료의 O / N을 배양한다. </li><li> 30 분 TBS와 함께 샘플을 세척하고 TBS에 희석 2 ° AB (1:100 안티 닭 FITC)와 O / N을 품어. </li><li> 30 분 TBS와 함께 샘플을 세척하고 또 다른 30 ​​분 동안 건조 둡니다. </li><li> 형광 현미경으로 관찰한다. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+incorporation+inside+of+organic+single+crystals+grown+in+agarose+hydrogels.">Gel incorporation inside of organic single crystals grown in agarose hydrogels.</a> Crystengcomm. 13, 1060-1062 (2011).</li><li>Ruan, Q., Zhang, Y., Yang, X., Nutt, S., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+amelogenin-chitosan+matrix+promotes+assembly+of+an+enamel-like+layer+with+a+dense+interface.">An amelogenin-chitosan matrix promotes assembly of an enamel-like layer with a dense interface.</a> Acta Biomater. 9, 7289-7297 (2013).</li><li>Hu, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+cDNA+sequence,+and+alternative+splicing+of+porcine+amelogenin+mRNAs.">Cloning, cDNA sequence, and alternative splicing of porcine amelogenin mRNAs.</a> Journal of Dental Research. 75, 1735-1741 (1996).</li><li>Bromley, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelogenin+Processing+by+MMP-20+Prevents+Protein+Occlusion+Inside+Calcite+Crystals.">Amelogenin Processing by MMP-20 Prevents Protein Occlusion Inside Calcite Crystals.</a> Cryst. Growth Des. 12, 4897-4905 (2012).</li><li>Fan, Y., Sun, Z., Abbott, C., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+enamel+inspired+nanocomposite+fabrication+through+amelogenin+supramolecular+assembly.">A enamel inspired nanocomposite fabrication through amelogenin supramolecular assembly.</a> Biomaterials. 28, 3034-3042 (2007).</li><li>Wright, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+etiologies+and+associated+phenotypes+of+amelogenesis+imperfecta.">The molecular etiologies and associated phenotypes of amelogenesis imperfecta.</a> Am. J. Med. Gent. A. 140 (23), 2547-2555 (2006).</li><li>Eimar, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+enamel+hardness+by+its+crystallographic+dimensions.">Regulation of enamel hardness by its crystallographic dimensions.</a> Acta Biomaterialia. 8, 3400-3410 (2012).</li><li>Ruan, Q., Siddiqah, N., Li, X., Nutt, S., Moradian-Oldak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelogenin-chitosan+matrix+for+human+enamel+regrowth:+effects+of+viscosity+and+supersaturation+degree.">Amelogenin-chitosan matrix for human enamel regrowth: effects of viscosity and supersaturation degree.</a> Connect Tissue Res. , (2014).</li><li>Pouget, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Initial+Stages+of+Template-Controlled+CaCO3+Formation+Revealed+by+Cryo-TEM.">The Initial Stages of Template-Controlled CaCO3 Formation Revealed by Cryo-TEM.</a> Science. 323, 1455-1458 (2009).</li><li>Gebauer, D., Volkel, A., Colfen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Prenucleation+Calcium+Carbonate+Clusters.">Stable Prenucleation Calcium Carbonate Clusters.</a> Science. 322, 1819-1822 (2008).</li></ol>

The authors have nothing to disclose.

에나멜의 미네랄 함량이 높은 그것을 인간의 몸에서 가장 단단한 광물 조직을 만드는 동안,이 바이오 세라믹은 종종 충치 또는 부식으로 발생하는 탈염 공정에 영향을 받기 쉽다. 유전자 돌연변이는 Amelogenesis 부전증 (20)라는 에나멜 기형의 상속 질병의 시리즈로 이어지는 얇은 또는 연약한 사기질을 일으킬 수 있습니다. 불소 또는 CPP-ACP를 포함하는 구강 건강 관리 제품은 몇 년 초...

Dental enamel is the hard mineralized surface of human teeth. It is composed of numerous needle-like apatite crystals, which are bundled in organized, parallel prisms to ensure the unique mechanical strength and biological protection that enamel provides1-2. Unlike other mineralized tissues, such as bone and dentin, mature enamel is acellular and cannot regenerate itself after substantial mineral loss1-2, which often occurs as dental caries or erosion. Commercially available products such as fluoride containing varnishes, tooth pastes and mouthwashes are effective in re-mineralizing enamel but none of them have the potential to promote the formation of organized apatite crystals. Clinically, the conventional treatment for enamel repair involves a filling procedure with artificial materials such as amalgam, ceramics, or composite resin 3. However, these materials usually do not interface well with the natural tissue surrounding the lesion, because their structures, components and properties are different from the natural enamel. As a result, secondary caries frequently develops overtime at the interface between the tooth and foreign materials. Therefore, in situ regrowth of enamel with a dense interface is an attractive target for the fields of materials science and stomatology. One particularly promising way to achieve this purpose is biomimetic synthesis of enamel-like material on the enamel surface. Recently, numerous in vitro attempts have been made to prepare enamel-like materials using biomimetic systems that contain nano-apatites or different organic additives 4-12. However, developing the optimal biomimetic strategy to promote remineralization crystals to achieve a perfect dense interface is still a challenge. 
During enamel mineralization, the oriented growth and elongation of apatite crystals is regulated by an amelogenin-rich matrix 1,13-14. To mimic the organic matrix in developing enamel, herein we describe a detailed protocol for fabricating an amelogenin-chitosan (CS-AMEL) hydrogel for in situ enamel regrowth on an acid-etched enamel surface used as a model for erosive lesions. As &#8220;the most versatile growth media&#8221; for crystals 15, hydrogel matrices have an advantage over a solution system since clinically they are easier to handle. Moreover, CS-AMEL is biocompatible, biodegradable, and has unique antimicrobial and adhesion properties that compare favorably with other biomimetic systems for dental applications 16. Importantly, the in situ mineralization of apatite crystals on the enamel surface provides a dense interface between the repaired layer and the natural enamel, which can potentially improve the durability of restorations and prevent the formation of new caries at the margin of the restoration.

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			<td height="21" style="height:21px;width:331px;">Name</td>
			<td style="width:209px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:516px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:331px;">Material/ Reagent</td>
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			<td style="width:164px;"></td>
			<td></td>
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			<td height="63" style="height:63px;width:331px;">Human Third Molar&#160;</td>
			<td style="width:209px;">Ostrow School of Dentistry of the University of Southern California&#160;</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">The human molars were extracted following the standard procedures for extraction at the Ostrow School of Dentistry of the University of Southern California and handled with the approval of the Institutional Review Board.</td>
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			<td height="42" style="height:42px;width:331px;">Recombinant Pocine Amelogenin</td>
			<td style="width:209px;">Expression and purification&#160; in lab</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">rP172, full-length&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chitosan&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">448877</td>
			<td style="width:516px;">medium molecular weight, 75-85% deacetylated</td>
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			<td height="21" style="height:21px;width:331px;">Phosphoric Acid&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA033266</td>
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			<td height="21" style="height:21px;width:331px;">Acetic Acid Glacial</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">A036289</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Hydroxide</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9292</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Calcium Chloride&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">223506</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dibasic Sodium Phosphate Anhydrous</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0316</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">BL21-CodonPlus (DE3)-RP&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">230255</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Sulfate</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH8001</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Trifluoroacetic Acid</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAL06374</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetonitrile</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH1103</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Magnesium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0244</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Dihydrogen Phosphate&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9268</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0258</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAA15000</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAA14777</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Fluoride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA11561</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tris-Buffered Saline</td>
			<td style="width:209px;">Bio-Rad</td>
			<td style="width:164px;">170-6435</td>
			<td style="width:516px;">10&#215; TBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Serum Albumin</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">12659</td>
			<td style="width:516px;">CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Triton X-100</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">TX1568-1</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chicken Anti-Amelogenin</td>
			<td style="width:209px;">N/A</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">A gift from Prof. Malcolm Snead, University of Southern California</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Anti-Chicken IgY-FITC</td>
			<td style="width:209px;">Santa Cruz Biotechnology</td>
			<td style="width:164px;">Sc-2700</td>
			<td style="width:516px;"></td>
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		<tr height="32">
			<td height="32" style="height:32px;width:331px;">Equipments</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:331px;">High Performance Liquid Chromatography System</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">Varian Prostar 210</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">C4 column</td>
			<td style="width:209px;">Phenomenx&#160;</td>
			<td style="width:164px;">Jupiter 5&#956; 300A</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Scanning Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JSM-7001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FIB-SEM&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JIB-4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Transmission Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL</td>
			<td style="width:164px;">JEM-2100F</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital Low Speed Diamond Saw</td>
			<td style="width:209px;">MTI Corporation</td>
			<td style="width:164px;">SYJ-150</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescence Microscopy</td>
			<td style="width:209px;">Leica</td>
			<td style="width:164px;">DMI3000 B</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultrasonic Cleaner&#160;</td>
			<td style="width:209px;">Branson&#160;</td>
			<td style="width:164px;">2510</td>
			<td style="width:516px;">42 kHz, 100 W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nano-indenter&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">MTS XP</td>
			<td style="width:516px;"></td>
		</tr>
	</tbody>
</table>

development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

여기에 설명 된 프로토콜의 효과는 전자 현미경 (SEM), 선택 영역 전자 회절 (SAED) 및 X 선 회절 (XRD) 분석을 스캔에 의해 입증된다. 칠일위한 유전자 성별 감식-키토산 (CS-AMEL) 하이드로 겔에 의한 수리 후, 15 ㎛의 두께를 에나멜 조의 층이 에칭 된 에나멜 표면에 형성 하였다. 새로 성장 된 층 (도 3a의 화살표) 표면에 수직 인 C 축을 따라 우선적으로 성장하고, 50 ~ nm의 직경이 결정의 고도?...

Watch this Scientific Journal Video about Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface at JoVE.com

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

이 문서에서는, 우리는 표면의 에나멜 재건을위한 유전자 성별 감식 - 키토산 하이드로 겔을 제조하는 프로토콜을 설명합니다. 하이드로 겔의 인회석 결정의 현장 성장 조직은 수복물의 효율성과 내구성을 향상하는 조밀 한 에나멜 복원 인터페이스를 형성했다.

에 대한 성별 감정 - 키토산 하이드로 겔의 개발<em&gt; 체외</em고밀도 인터페이스&gt; 에나멜 재성장

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

development-amelogenin-chitosan-hydrogel-for-vitro-enamel-regrowth

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JoVE Journal

Bioengineering

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

14.9K 조회수.  University of Southern California.  이 문서에서는, 우리는 표면의 에나멜 재건을위한 유전자 성별 감식 - 키토산 하이드로 겔을 제조하는 프로토콜을 설명합니다. 하이드로 겔의 인회석 결정의 현장 성장 조직은 수복물의 효율성과 내구성을 향상하는 조밀 한 에나멜 복원 인터페이스를 형성했다. 

비디오: Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

심장 조직 공학을위한 섬유소 히드로겔에 Cardiomyocytes의 캡슐

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

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생체공학

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 차원 자기 조립 펩티드 히드로겔에서 인간 신경 전구 세포의 배양

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Biofabrication와 바이오 전자 인터페이스 브리지

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

테인 매개 인공 단백질 히드로 겔의 합성

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

낮은 풍부 단백질을 검출하기위한 플라즈마 또는 소변의 하이드로 겔 나노 입자 수확

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

혈관 세포 리모델링의 조사에 생리의 체외 모델 및 응용 프로그램과 함께 병리학 혈액 흐름

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

개발<em&gt; 체외</em&gt; 안구 플랫폼은 콘택트 렌즈를 테스트하는

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

생체 외에서 Neuroinflammation의 모델링에 대 한 기본 Glial 세포의 3 차원 하이드로 겔 문화 개선

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

셀 회전 타원 체 형성을 유발 하는 모델 시스템에 3D 생체 외에서 Bioprintable Alginate/젤라틴 하이드로 겔

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...