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>

<ol>
	<li>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=Protein-+mediated+enamel+mineralization.">Protein- mediated enamel mineralization.</a> Frontiers in Bioscience. 17, 1996-2023 (2012).</li><li>Palmer, L. C., Newcomb, C. J., Kaltz, S. R., Spoerke, E. D., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+Systems+for+Hydroxyapatite+Mineralization+Inspired+By+Bone+and+Enamel.">Biomimetic Systems for Hydroxyapatite Mineralization Inspired By Bone and Enamel.</a> Chem. Rev. 108, 4754-4783 (2008).</li><li>Onuma, K., Yamagishi, K., Oyane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleation+and+growth+of+hydroxyapatite+nanocrystals+for+nondestructive+repair+of+early+caries+lesions.">Nucleation and growth of hydroxyapatite nanocrystals for nondestructive repair of early caries lesions.</a> J. Cryst. Growth. 282, 199-207 (2005).</li><li>Xie, R. Q., Feng, Z. D., Li, S. W., Xu, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDTA-Assisted+Self-Assembly+of+Fluoride-Substituted+Hydroxyapatite+Coating+on+Enamel+Substrate.">EDTA-Assisted Self-Assembly of Fluoride-Substituted Hydroxyapatite Coating on Enamel Substrate.</a> Cryst. Growth Des. 11, 5206-5214 (2011).</li><li>Li, L., 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=Bio-Inspired+Enamel+Repair+via+Glu-Directed+Assembly+of+Apatite+Nanoparticles:+an+Approach+to+Biomaterials+with+Optimal+Characteristics.">Bio-Inspired Enamel Repair via Glu-Directed Assembly of Apatite Nanoparticles: an Approach to Biomaterials with Optimal Characteristics.</a> Advanced Materials. 23, (2011).</li><li>Yin, Y. J., Yun, S., Fang, J. S., Chen, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+regeneration+of+human+tooth+enamel+under+near-physiological+conditions.">Chemical regeneration of human tooth enamel under near-physiological conditions.</a> Chem. Commun. , 5892-5894 (2009).</li><li>Fan, Y., Sun, Z., 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=Controlled+remineralization+of+enamel+in+the+presence+of+amelogenin+and+fluoride.">Controlled remineralization of enamel in the presence of amelogenin and fluoride.</a> Biomaterials. 30, 478-483 (2009).</li><li>Li, L., 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=Repair+of+enamel+by+using+hydroxyapatite+nanoparticles+as+the+building+blocks.">Repair of enamel by using hydroxyapatite nanoparticles as the building blocks.</a> J. Mater. Chem. 18, 4079-4084 (2008).</li><li>Chen, 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=Acellular+synthesis+of+a+human+enamel-like+microstructure.">Acellular synthesis of a human enamel-like microstructure.</a> Advanced Materials. 18, (2006).</li><li>Yamagishi, K., 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=A+synthetic+enamel+for+rapid+tooth+repair.">A synthetic enamel for rapid tooth repair.</a> Nature. 433, 819-819 (2005).</li><li>Bleek, K., Taubert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+polymer-controlled,+bioinspired+calcium+phosphate+mineralization+from+aqueous+solution.">New developments in polymer-controlled, bioinspired calcium phosphate mineralization from aqueous solution.</a> Acta Biomaterialia. 9, 6283-6321 (2013).</li><li>Fletcher, J., Walsh, D., Fowler, C. E., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+mats+of+PVP/ACP+nanofibres+for+remineralization+of+enamel+tooth+surfaces.">Electrospun mats of PVP/ACP nanofibres for remineralization of enamel tooth surfaces.</a> Crystengcomm. 13, 3692-3697 (2011).</li><li>Fang, P. -. A., Conway, J. F., Margolis, H. C., Simmer, J. P., Beniash, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+self-assembly+of+amelogenin+and+the+regulation+of+biomineralization+at+the+nanoscale.">Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 14097-14102 (2011).</li><li>Yang, X., 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=How+Amelogenin+Orchestrates+the+Organization+of+Hierarchical+Elongated+Microstructures+of+Apatite.">How Amelogenin Orchestrates the Organization of Hierarchical Elongated Microstructures of Apatite.</a> Journal of Physical Chemistry B. 114, 2293-2300 (2010).</li><li>Li, H., Fujiki, Y., Sada, K., Estroff, L. 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.

<table><tbody><tr><td>Human third molar&amp;nbsp;</td><td>Ostrow School of Dentistry of the University of Southern California&amp;nbsp;</td><td>N/A</td><td>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></tr><tr><td>Recombinant pocine amelogenin</td><td>Expression and purification&amp;nbsp; in lab</td><td>N/A</td><td>rP172, full-length&amp;nbsp;</td></tr><tr><td>Chitosan&amp;nbsp;</td><td>Sigma-Aldrich</td><td>448877</td><td>Medium molecular weight, 75-85% deacetylated</td></tr><tr><td>Phosphoric acid&amp;nbsp;</td><td>VWR</td><td>AA033266</td><td></td></tr><tr><td>Acetic acid glacial</td><td>VWR</td><td>A036289</td><td></td></tr><tr><td>Sodium hydroxide</td><td>VWR</td><td>BDH9292</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Dibasic sodium phosphate anhydrous</td><td>VWR</td><td>BDH0316</td><td></td></tr><tr><td>BL21-CodonPlus (DE3)-RP&amp;nbsp;</td><td>Agilent Technologies Inc.</td><td>230255</td><td></td></tr><tr><td>Ammonium sulfate</td><td>VWR</td><td>BDH8001</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>VWR</td><td>AAAL06374</td><td></td></tr><tr><td>Acetonitrile</td><td>VWR</td><td>BDH1103</td><td></td></tr><tr><td>Magnesium chloride&amp;nbsp;</td><td>VWR</td><td>BDH0244</td><td></td></tr><tr><td>Potassium dihydrogen phosphate&amp;nbsp;</td><td>VWR</td><td>BDH9268</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>VWR</td><td>BDH0258</td><td></td></tr><tr><td>Ammonium chloride&amp;nbsp;</td><td>VWR</td><td>AAAA15000</td><td></td></tr><tr><td>HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td><td>VWR</td><td>AAA14777</td><td></td></tr><tr><td>Sodium fluoride&amp;nbsp;</td><td>VWR</td><td>AA11561</td><td></td></tr><tr><td>Tris-buffered saline</td><td>Bio-Rad</td><td>170-6435</td><td>10&amp;times; TBS</td></tr><tr><td>Bovine serum albumin</td><td>EMD Millipore&amp;nbsp;</td><td>12659</td><td>CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>EMD Millipore&amp;nbsp;</td><td>TX1568-1</td><td></td></tr><tr><td>Chicken Anti-Amelogenin</td><td>N/A</td><td>N/A</td><td>A gift from Prof. Malcolm Snead, University of Southern California</td></tr><tr><td>Bovine Anti-Chicken IgY-FITC</td><td>Santa Cruz Biotechnology</td><td>Sc-2700</td><td></td></tr><tr><td>High Performance Liquid Chromatography System</td><td>Agilent Technologies Inc.</td><td>Varian Prostar 210</td><td></td></tr><tr><td>C4 column</td><td>Phenomenx&amp;nbsp;</td><td>Jupiter 5&amp;mu; 300A</td><td></td></tr><tr><td>Scanning Electron Microscopy&amp;nbsp;</td><td>JEOL&amp;nbsp;</td><td>JSM-7001</td><td></td></tr><tr><td>FIB-SEM&amp;nbsp;</td><td>JEOL&amp;nbsp;</td><td>JIB-4500</td><td></td></tr><tr><td>Transmission Electron Microscopy&amp;nbsp;</td><td>JEOL</td><td>JEM-2100F</td><td></td></tr><tr><td>Digital low speed diamond saw</td><td>MTI Corporation</td><td>SYJ-150</td><td></td></tr><tr><td>Fluorescence microscopy</td><td>Leica</td><td>DMI3000 B</td><td></td></tr><tr><td>Ultrasonic cleaner&amp;nbsp;</td><td>Branson&amp;nbsp;</td><td>2510</td><td>42 kHz, 100 W</td></tr><tr><td>Nano-indenter&amp;nbsp;</td><td>Agilent Technologies Inc.</td><td>MTS XP</td><td></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

Research

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

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

줄기 세포 - 로드된 키토산 Microspheres의 배달을 위해 콜라겐 히드로겔을 구축

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

연구

생체공학

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

주 사용 Hydrogels 키토 산 기반 및 3D 세포 배양에서의 응용의 준비

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

화학

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

조직 공학 알긴산 미세 입자와 Thermogelling 폴리 (N- 이소 프로필) -graft - 콘드로이틴 황산 복합 재료의 합성

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

키토산 필름에 인간 치주 인대 세포 스페로이드의 형성

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

발생학

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

기계론적 연구 및 재생 요법을 향한 강력한 도구로 인간의 치아에서 오가노이드를 확립

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

미세중력에서 치과 및 호흡기 세포와 장기의 전파

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

사기 질 기관 이용과 환경에 노출 된 쥐의 악의 니에서의 마이크로 해 부

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

의학

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

조직 공학 응용 분야를 위한 크기 조절 및 무유제 키토산-제니핀 마이크로젤 제조

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

생물학

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

생체 재료에 대한 세포 독성 및 세포 반응에 접근

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

에 대한 성별 감정 - 키토산 하이드로 겔의 개발

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