The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

모든 실험은 스탠포드 대학 인간 피험자의 지침에 따라 수행 기관 심사위원회 프로토콜을 승인했다. 1. 관절 연골 세포의 분리 <ol><li> 슬관절 전 치환술시 폐기 조직에서 연골을 얻기 이전 8 설명한 바와 같이 해부. </li><li> 시각적으로 매끄러운 표면에 대한 연골 샘플들의 내측 또는 외측 대퇴골을 선택 및 연골 하골에 영향을주지 않고 4~5mm 연골 생검을 제거하기 위해 메스 통해 연골의 표면에 절개를. </li><li> 정제 콜라게나 제와 O / N 처리와 조직의 효소 분해하여 세포 외 기질에서 연골 세포를 분리합니다. 37 ° C에서 연골 세포 성장 미디어 콜라게나 II (125 U / ㎖) 및 콜라게나 IV (160 U / ㎖)의 1의 비율 : 1을 사용합니다. </li><li> 다음 날, 70를 통해 해리 세포를 포함하는 조직 분해를 필터링mm 크기의 나일론 메쉬 기공. 5 분 동안 600 XG에 DMEM / F12와 원심 분리기 20 ㎖로 2 회 세척 할 것. </li><li> 10 % 소 태아 혈청 (FBS)이 보충 된 DMEM / F12 재 현탁액 25 μg의 / ㎖ 아스코르브 산, 2 mM L- 글루타민, 항생제 다음 1 × 106 세포 / 60mm 배양 접시의 밀도로 격리 연골 판형 (100 U / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신, 0.25 μg의 / ㎖ fungizone). 이전에 생체 모방 히드로 겔에 캡슐에 37 ° C에서 접시를 유지하고 고밀도 단층로 기본 연골 세포 배양을 유지한다. </li></ol> 2. 생체 모방 하이드로 겔의 제조 참고 :이 방법은 허용 된 폴리 (에틸렌 글리콜 디 아크릴 레이트) (PEGDA, MW 5,000g / 몰), DPBS에 콘드로이틴 설페이트 - 메타 크릴 레이트 (CS-MA)를 함유하는 하이드로 겔의 생체 모방 합성. 광개시제의 첨가는 광 활성화 가교 결합을 가능하게한다. 최종적인 하이드로 겔 조성물이 7 %가 체중이 포함T / 볼륨 (W / V) PEGDA의 3 % CS-MA의 V / W 및 광개시제의 V / W 0.05 %. <ol><li> 메타 크릴 레이트 그룹과 CS 폴리머를 기능화하여 CS-MA를 합성. <ol><li> MES의 1.952 g 및 탈 이온수 200 ㎖의 염화나트륨 5.84 G (DiH 2 O)을 용해하여 50 mM의 2 morpholinoethanesulfonic 산 (MES) 및 0.5 M 염화 나트륨 (NaCl을)의 완충 용액을 준비합니다. 완충 용액에 콘드로이틴 설페이트 나트륨 염 5 g의 용해. </li><li> (: EDC = 1 : 2의 몰비 NHS)로 0.532 g (4.62 mmol)의 N- 히드 록시 숙신이 미드 (NHS) 및 1.771 g (9.24 밀리몰), 1- 에틸 -3- (3- 디메틸 아미노 프로필) 카르 보디이 미드 히드로 클로라이드 (EDC)를 추가 5 분을위한 솔루션 및 교반. </li><li> 용액을 24 시간 동안 실온에서 반응을 유지한다 (2 : 1 : EDC : AEMA NHS = 1의 몰비) 0.765 g (4.62 밀리몰)의 2- 아미노 에틸 메타 크릴 레이트 (AEMA)를 추가한다. </li><li> 투석 튜브 (12 ~ 14 kDa의 MWCO)를 사용하여 4 일 동안 탈 이온수에 대한 투석 (diH 2 O)에 의한 혼합물을 정화. </li><li> P 필터0.22 μm의 필터를 통해 urified 솔루션 및 건조기, 진공 O / N에서 솔루션을 포함하는 개방형 튜브를 놓습니다. 모든 튜브는 빛으로부터 보호되어 있는지 확인합니다. 빛과 습기로부터 보호 -20 ° C에서 용해 된 폴리머를 저장합니다. </li></ol></li></ol> 3. 세포 캡슐화 <ol><li> (셀 - 라덴 3D 하이드로 겔을 펀칭에 대한) PCR 필름과 원통형 막대를 압력솥과 자외선 아래에서 조직 문화 후드에서 0.2 μm의 필터링 된 70 % 에탄올에 침수에 의해 주문 제작 원통형 겔 금형을 소독. </li><li> 기포 또는 누수를 방지하기위한 간극없이 멸균 PCR 막을 겔 몰드의 하부를 밀봉. 멸균 150mm 판에 보관하십시오. </li><li> 종래의 하이드로 겔의 생체 모방 전지 밀봉에 날, 콜라게나 제 II에서 O / N 처리 및 콜라게나 제 IV 용액 고밀도 연골 세포 단층을 해리. 콜라게나 VI 각 연골 세포 성장 미디어 콜라게나 II 125 U / ㎖와 160 U는 / ㎖를 사용하여37 ° C에서. </li><li> 50 ㎖ 튜브를 가지고 추가 5 % 중량 / 부피 (w / v)의 폴리 (에틸렌 글리콜 디 아크릴 레이트) (PEGDA, MW = 5,000g / 몰), w 3 % / V 콘드로이틴 설페이트 - 메타 크릴 레이트 (CS-MA)를, 및 DPBS에 V 광개시제 / W 0.05 %. 솔루션을 혼합하여 따로 보관 유지하는 튜브를 소용돌이. 빛으로부터 튜브를 보호합니다. </li><li> , 50 mL의 팔콘 튜브에 세포를 수집 해리 5 분 동안 460 XG에 카운트 원심. 흡 인물은 모든 셀 관으로부터 미디어 및 15 × 106 세포 / ml의 밀도로 세포를 다시 일시 중단 상기 혼합 겔 물질을 추가한다. 공기를 피하는 형성 거품 동안 30 회 철저하게 섞는다. </li><li> 피펫 (72) 사용자 정의 만든 원통형 겔 금형에 셀 하이드로 겔 현탁액 또는 단독으로 하이드로 겔의 μL 및 5 분 동안 3 mW의 / 평방 미터에 자외선 (365 nm 파장)에 대한 노출을 통해 겔화를 유도한다. 더 셀 내용 일부 하이드로 겔 솔루션을 준비합니다. 미래의 생화학 적 및 기계적 시험에 대한 음성 대조군 이러한 구조를 사용합니다. MeasuUV 강도 측정기 UV 광의 세기 재. 강도를 조정하기 위해, UV 광원 겔화시 하이드로 겔 지지체 사이의 거리를 변경. 참고 : 하이드로 겔 (CS)를 포함하기 때문에, 무 세포 기여 만 휴대 개그를 표현하기 위해 세포 함유 하이드로 겔의 생화학 개그 내용에서 제외됩니다. </li><li> 오프 쉽게 껍질의 필름을 잘라 메스를 사용하여 조심스럽게 제거합니다. 원통형 막대의 도움으로, 비중 합 젤 느슨한 세포를 씻어 살균 DPBS 5 ㎖로 6 웰 플레이트에 젤을 밀어. </li><li> 문화 아니라 각으로 연골 세포의 성장 매체의 1.5 ml에 포함 된 24 웰 플레이트의 세포 함유 하이드로 겔. 우물에 세척 하이드로 겔을 전송하는 일회용 주걱을 사용합니다. </li><li> 생존 / 세포 독성 키트를 사용하여 라이브 죽은 염색 24 시간 후 캡슐에 의해 세포 생존 능력을 평가합니다. 문화 신선한 연골 세포 촉진제로 변경 3~6주의 세포 함유 하이드로 겔과 빈 하이드로 겔2 일마다 분석을 위해 수확 전에 미디어 WTH. </li></ol> 4. RNA 추출 및 유전자 발현 분석 <ol><li> 조심스럽게 미디어를 기음과 세포 - 라덴뿐만 아니라 빈 제어 하이드로 겔에 멸균 PBS 1X했습니다. </li><li> 멸균 주걱의 도움으로, 깨끗한 1.5 ㎖의 에펜 도르프 튜브에 각각의 하이드로 겔을 전송하고 각 튜브에 트라이 시약 250 μL를 추가합니다. RNA를 분리 제조업체의 지침을 따르십시오. </li><li> RNA 분리 및 정량 사용 각 샘플에서 그것의 1 μg의 이후 대용량 cDNA를 역 전사 키트를 사용하는 cDNA로 반전 전사를 수행합니다. </li><li> 정량 PCR에 관심의 유전자의 발현의 시험에의 TaqMan 유전자 발현의 배열을 사용합니다. 내부적 GAPDH 유전자 발현 수준을 정상화. </li><li> PCR 조건 : 10이어서 우라실 DNA 글리코와 이전 증폭 물을 불 활성화하여 50 ℃에서 2 분의 인큐베이션을 포함60; 95 ° C에서 배양 분당의 Taq 중합 효소를 활성화합니다. 이어서 95 ° C에서 15 초로 구성된 PCR 사이클의 마흔을 수행하고, 60 ℃에서 1 분. </li></ol> 5. 생화학 적 분석 <ol><li> 각 셀 하이드로 겔 구조를 들어 DNA와 다음과 같은 황산 글리코 사 미노 글리 칸 (sGAG) 생산을 정량화. </li><li> 조심스럽게 미디어를 기음과 세포 - 라덴뿐만 아니라 빈 제어 하이드로 겔에 멸균 PBS 1X했습니다. 빈 튜브를 달아 티슈 페이퍼에 그것을 블로 팅 후 내부의 하이드로 겔을 넣고 하이드로 겔의 중량 젖은 무게를 기록한다. </li><li> 효소 소화에 대한 별도의 1.5 ㎖의 에펜 도르프 튜브에 -20 ° C에서 각각의 하이드로 겔을 동결 이상 (GAG에 대한) (DNA에 대한) Picogreen 분석 및 디메틸 메틸렌 블루 분석을 실행하는 소화 제품의 나누어지는을 사용합니다. </li><li> 하이드로 겔의 효소 소화 papainase 솔루션을 준비합니다. <ol><li> 제 phosphat 나트륨 7.1 g을 칭량하여 PBE 버퍼를 준비전자 이염 (나 2 HPO 4) 및 에틸렌 디아민 테트라 아세트산이 나트륨 염 (EDTA-나 2) 1.6 g을. , DH 2 O 500ml에 녹이고 6.5로 pH를 조정하고 0.22 mm 필터를 통해 정화 된 용액을 필터. </li><li> PBE 완충액 20ml에 -L- 시스테인 0.035 g을 녹인다. 솔루션 및 멸균 파파인 효소의 100 μl를 필터링합니다. </li></ol></li><li> 준비 분석을 수행 할 때, -20 ° C에서 튜브를 타고 얼어 붙은 하이드로 겔을 준비 파파인 솔루션의 300 μl를 추가합니다. 박격포와 유 봉 젤 크러시와 모터 유봉과 잘 섞는다. </li><li> 추가 파파인 용액 500 μL에 볼륨을 가져와. 제어 겔에 대해 동일한 절차를 수행한다. 16 시간 3,9 60 ° C에서 품어. 소화 하이드로 겔을 포함하는 용액 따라서 DNA와 개그 정량에 사용 할 수 있습니다. 제조자의 PR 다음 표준 람다 파아지 DNA와 Picogreen 분석법을 이용 DNA 함량을 측정otocol. </li><li> 1,9- 디메틸 메틸렌 블루 (DMMB) 표준 10 상어 콘드로이틴 설페이트와 염료 결합 분석법을 이용하여 황산 GAG 함량을 정량화. 해당 젖은 무게 GAG의 양을 분할함으로써 GAG 함량을 결정한다. </li></ol> 6. 기계적 시험 <ol><li> 10 N 하중 셀이 장착 5944 인스 트론 시험 장치를 이용하여 일축 압축 시험을 수행한다. <ol><li> 체외 배양의 바람직한 일 후, 세포 - 하이드로 겔 지지체와 무 세포 제어 하이드로 겔에 압축 시험을 수행한다. </li><li> RT에서 PBS 욕에서 시험편을 담그고 하중 조건 하에서 연골 조직에 의해 경험 생리 변형 때문에 15 % (11, 12)의 최대 변형률 1 % 스트레인 / sec의 속도로 압축하는 것은 10 내지 20 %의 것으로보고되었다 (13, 14). </li></ol></li><li> 3 차 다항식을 사용하여 변형 곡선과 곡선 맞춤 대 스트레스를 만듭니다. COMPR 결정15 %의 변형률 값의 곡선에 맞게 방정식에서 (메모리) 접선 계수. </li></ol>

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D., Shirazi-Adl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-rate+dependent+stiffness+of+articular+cartilage+in+unconfined+compression.">Strain-rate dependent stiffness of articular cartilage in unconfined compression.</a> Journal of biomechanical engineering. 125, 161-168 (2003).</li><li>Armstrong, C. G., Bahrani, A. S., Gardner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+measurement+of+articular+cartilage+deformations+in+the+intact+human+hip+joint+under+load.">In vitro measurement of articular cartilage deformations in the intact human hip joint under load.</a> The Journal of bone and joint surgery. American. 61, 744-755 (1979).</li><li>Macirowski, T., Tepic, S., Mann, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+stresses+in+the+human+hip+joint.">Cartilage stresses in the human hip joint.</a> Journal of biomechanical engineering. 116, 10-18 (1994).</li><li>Benya, P. D., Shaffer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+chondrocytes+reexpress+the+differentiated+collagen+phenotype+when+cultured+in+agarose+gels.">Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.</a> Cell. 30, 215-224 (1982).</li><li>Buckwalter, J. A., Mankin, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage:+tissue+design+and+chondrocyte-matrix+interactions.">Articular cartilage: tissue design and chondrocyte-matrix interactions.</a> Instructional course lectures. 47, 477-486 (1998).</li><li>Wang, D. A., 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=Multifunctional+chondroitin+sulphate+for+cartilage+tissue-biomaterial+integration.">Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration.</a> Nat Mater. 6, 385-392 (2007).</li><li>Simson, J. A., Strehin, I. A., Allen, B. W., Elisseeff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+and+fusion+of+meniscus+fibrocartilage+using+a+novel+chondroitin+sulfate+bone+marrow+tissue+adhesive.">Bonding and fusion of meniscus fibrocartilage using a novel chondroitin sulfate bone marrow tissue adhesive.</a> Tissue engineering. Part A. 19, 1843-1851 (2013).</li></ol>

The authors have nothing to disclose.

이 프로토콜에보고 된 바와 같이, 3D 하이드로 겔은 일반적으로 단층 배양 물 (15)가 발생 섬유 연골 세포로 세포의 탈분화 과정을 회피 배양 연골 세포 표현형을 유지할 수있다. 또한, chondrocytes- 하이드로 겔 구조의 장기 문화는 연령과 질병에 관련된 고유 세포 기능을 유지하는 유리한 환경을 밝혔다. 3D 생체 모방 하이드로 겔의 사용은 몇 가지 장점을 갖는다. 우선, 콘드로이틴 설페이트의 포함 (CS), 관절 연골에서 발견되는 주요 성분은, chondroitinase 분비하여 하이드로 겔 매트릭스를 분해 및 세포 외 매트릭스 5, 16 새로 합성 된 연골 누워 세포를 가능하게한다. 또한, CS는 도시 된 관절염 관절의 항 염증 특성을 가지고있다. 하이드로 겔은 또한 생체 모방 연골 복구 세포 전달을위한 발판 재료로서 사용될 수 있고, 화학적으로 변형 될 수있다좋은 생체 조직 (17, 18)를 통합 촉진한다. PEG-CS 하이드로 겔의 사용은 장기간의 연골 세포의 배양 및 생화학 적 및 기계적 성질의 평가를 허용한다. 여기에서 우리는이 플랫폼은 연골 엔지니어링을위한 최적의 세포 유형을 정의하기 위해 차별화 된 연골 세포의 다양한 소스의 비교 분석에 유용 할 수 방법을 보여줍니다. 흥미롭게도, 하이드로 겔에 캡슐화 된 연골 세포는 자신의 고유 능력에 따라 가능한 유지 및 증식. 하이드로 겔 조성물 지지체는 사실상 건강한 소아 및 성인 연골 세포의 성장은도 2에 도시 된 바와 같이. 기술 된 하이드로 겔의 조성과 구조는 연골 조직의 형성을 촉진하는 글리코 사 미노 글리 칸에 의해 평가 기능 외 기질의 증착 (GAG로 나타낸 바와 ) 정량. 추가적인 장점은 연골 - 하이드로 겔 구조이다새로 형성된 연골 조직의 기계적 특성에 대해 평가 될 수있다. 일축 압축 시험 비교를 위해 무 세포 하이드로 겔에서 수행되어야합니다. 하이드로 겔은 사실 때문에 CS 부분의 강성 극한 강도를 갖는다. (변형 속도 / s 1 %에서) 5-20%의 일축 압축 변형은 하중 조건 하에서 연골 조직에 의해 경험 생리 균주 보낸 연골 조직 (11, 12)의 기계적 시험에 적용될 수는 10-20 인 것으로보고되었다 % (13, 14). 기계적 시험에 세포 - 라덴과 무 세포 모두 하이드로 겔의 응답은 문화 엔드 포인트에서 평가 하였다. 설명 된 예에서는 OA 연골 세포를 포함하는 구조체의 하부의 강성 대조적 성인 및 청소년 연골 함유 구조체의 강성을 비교 관찰 위에. 셀 하이드로 겔 구조의 기계적 특성의 기능적 특성의 평가를 허용세포 성숙 능력에 대한 심층적 인 분석을 제공 형성 조직. 결론적으로, 연골 조직을 생성하기 위해 다른 연골 세포 모집단의 잠재력을 연구하는 3D 생체 모방 하이드로 겔의 사용은 광범위하게 적용될 수있다. 여기에 기술 된 시험 관내 연구 이외에도, 세포 함유 구조체의 생체 내 이식 생리 컨텍스트에서 세포 성숙 및 재생 가능성을 연구하기 위해 구상 될 수있다. 추가적인 생체 모방 인자 하이드로 플랫폼의 추가 수정은 연골 세포의 증식 및 성숙을 최적화하기 위해 구상 될 수있다.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

<table><tbody><tr><td>juvenile chondrocytes (Clonetics Normal Human Chondrocyte Cell System )</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>adult chondrocytes (Clonetics Normal Human Chondrocyte Cell System)</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>poly(ethylene glycol diacrylate)</td><td>Laysan Bio</td><td>ACRL-PEG-ACRL-1000-1g</td><td></td></tr><tr><td>2-morpholinoethanesulfonic acid</td><td>Sigma</td><td>M5287</td><td></td></tr><tr><td>photoinitiator</td><td>Irgacure&amp;nbsp;</td><td>2959</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>chondroitin sulfate sodium salt&amp;nbsp;</td><td>Sigma</td><td>C9819</td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma</td><td>130672</td><td></td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td><td>Sigma</td><td>E1769</td><td></td></tr><tr><td>2-aminoethyl methacrylate</td><td>Sigma</td><td>516155</td><td></td></tr><tr><td>dialysis tubing</td><td>Spectrum Laboratories&amp;nbsp;</td><td>132700</td><td></td></tr><tr><td>Collagenase 2</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004177</td><td></td></tr><tr><td>Collagenase 4</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004189</td><td></td></tr><tr><td>DMEM/F12 media</td><td>HyClone, Thermo Scientific&amp;nbsp;</td><td>SH3002301&amp;nbsp;</td><td></td></tr><tr><td>live/dead assay</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/L3224&quot;&gt;L3224&lt;/a&gt;</td><td></td></tr><tr><td>Tri reagent</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Invitrogen</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/P11496&quot;&gt;P11496&lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt&amp;nbsp;</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>C1276</td><td></td></tr><tr><td>1,9-dimethylmethylene blue&amp;nbsp;</td><td>Sigma</td><td>341088</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>UV light equipment - XX-15LW Bench Lamp, 365 nm</td><td>UVP</td><td>95-0042-07</td><td></td></tr><tr><td>Instron 5944 testing system&amp;nbsp;</td><td>Instron Corporation</td><td>E5940</td><td></td></tr></tbody></table>

3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

PEG 및 CS 부분 (그림 1)를 포함하는 생리 활성 하이드로 겔은 인간의 관절 연골 세포의 2,3,5,7의 문화와 성숙을위한 이상적인 플랫폼을 나타냅니다. 다양한 연령 및 질환 상태에서 연골 세포는 배양 한 방법 및 그 표현형, 유전자 발현 및 생성 된 연골 조직의 생화학 적 및 기계적 성질에 대해 분석 할 수있다. 세 연골 세포 샘플, juvenile- 6개월, adult- 34년 및 osteoarthritic- 74년는 3D 생체 모방 히드로 겔 문화 3 주 후에 수확했다. 유전자 발현이 정상 연골, 청소년 및 성인 둘 분석, 연골 세포 및 유전자 Col2a1 Col6a1의 발현 증가를 보였다. Col6a1 (도 2)의 발현을 유지 유리한 생체 모방 환경에서 배양 표현형 불구 연골의 손실을 표시하는 동안 반대로, 병든 연골 Col2a1의 극적인 감소를 나타내었다. CS-PEG 하이드로 겔은 또한, 연골 세포가 증식하는 동적 환경 역할 PEG 및 CS의 기존 매트릭스를 소화하고 그들의 pericellular 및 세포 외 기질, 성숙한 연골 조직의 주성분을 침착. 체외 배양 3 주 후 연골 세포의 확장, 이러한 능력을 감안할 때이 Picogreen 염료와 DNA의 정량에 의해 추정 할 수있다. 세포의 세 그룹의 비교 분석은 OA 연골 세포는 배양 1 일째에 비해 극적으로 감소 발휘하면서 청소년 및 성인 인구의 셀 밀도는 변하지 것을 보여준다. DNA 함량의 감소는 장기 배양 (도 3) 중에 가능한 세포 사멸을 시사 다른 OA 연골 위해 아니지만 관찰되었다. 분비 매트릭스는 배양 3 주 후에 종료점 단계 DMMB 염료 결합 분석하여 설페이트 화 - GAG 함량으로 정량 하였다. GAG 컨텐츠 recor 같은 하이드로 겔의 습윤 중량을 기준으로 규격화DED 효소 소화와 무 세포 기여 전에 뺀다. 도 4에 도시 된 바와 같이, 연골 세포는 하이드로 겔의 3 차원 배양 3 주 동안 GAG 유사한 상당한 양의 증착. 물리적 골격의 존재는 일축 압축 시험의 2,3를 통해 샘플의 생체 역학적 특성의 평가를 할 수 있습니다. 무 세포 하이드로 겔은 압축 계수의 감소를 겪는 동안, 세포 - 라덴 구조는 문화 3 주 (그림 4) 후 압축 계수를 유지했다. 표현형, 여기에 설명 된 생화학 및 생 역학적 분석 평가 및 엔지니어링 연골에 대해 서로 다른 연골 세포 인구의 잠재력을 이해 때문에 유용하다. <img alt="그림 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> 3D의 연골 세포의 막 다른 1. PEG-CS 생체 모방 하이드로 겔 그림스트럭처. 연골 세포는 폴리 (에틸렌 글리콜 디 아크릴 레이트) (PEGDA) 콘드로이틴 설페이트 - 메타 크릴 레이트 (CS-MA) 및 주문 제작 겔 원통형 금형에 캐스팅을 포함하는 혼합물에 재현 탁된다. 자외선 노출 후, 젤은 금형에서 수집 및 세포 생존 라이브 죽은 염색 24 시간 후 캡슐에 의해 평가된다 응고. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53085/53085fig2.jpg" /> 그림 3D 생체 모방 하이드로 겔에서 배양 된 연골 세포에서 연골 유전자의 2 식입니다. 3D 생체 모방 히드로 겔 문화 3 주 후에 청소년, 성인 및 OA 연골 세포로 연골 마커 Col2a1 및 Col6a1의 정량적 유전자 발현. 값이 1 일 때 오류 B에서 유전자 발현 수준으로 정상화된다ARS는 평균 ± SD 나타냅니다. * P는 &lt;양측 &#39;학생의 t 분포&#39;에 의해 결정되는 바와 같이, 0.05. Smeriglio 등에서 수정. (2) <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53085/53085fig3.jpg" /> 3D 생체 모방 하이드로 겔에서 배양 된 연골 세포 그림 3. DNA 콘텐츠. 3D 생체 모방 히드로 겔 문화 3 주 후에 청소년, 성인 및 골관절염 연골 세포에서 Picogreen 분석에 의한 DNA 정량. 값은 하루 1에서 DNA 수준으로 정상화되는 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;BR /&gt; 그림 4. 개그 콘텐츠 측정 및 1 일에서 3 차원 생체 모방 히드로 겔 문화 3 주 후에 인간의 연골 세포의 일축 압축 시험. 1 일에서 연골 세포와 문화 3 주 후에 DMMB 분석에 의해 (A) 개그 정량 3D 생체 모방 하이드로 겔. 값은 무게 (전주) 적시에 정규화 및 μg의 / G로 표시됩니다. 하루에 1에서와 3D 생체 모방 히드로 겔 문화 3 주 후에 무 세포 및 세포 함유 하이드로 겔의 (B) 압축 계수 (kPa의). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">보려면 여기를 클릭하십시오 이 그림의 더 큰 버전.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

관절 연골 세포 문화와 연골 생성을위한 3D 하이드로 겔 공사장 공중 발판

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

20.0K 조회수.  Stanford University. Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes. 

비디오: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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 ...

연구

화학

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

생체공학

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

계층화 된 알긴산를 구축 : 이기종 세포 인구의 공동 문화를위한 플랫폼

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

연골 재생을위한 3D 자기 줄기 세포 집계 및 생물 반응기 성숙

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

3 차원 펩 티 드 건설 기계에 포유류 세포 배양

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

활액 유체에 Culturing 인간 관절 Chondrocytes을위한 3D 시스템

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

생물학

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Chondrocyte - 아가로 오스 Hydrogels의 기계적 자극

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

관절 질환 모델링 및 약물 테스트를 위한 무릎 관절 칩 생성

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

탈세포화된 연골 세포외 기질 하이드로겔의 합성

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...