관절 연골 세포 문화와 연골 생성을위한 3D 하이드로 겔 공사장 공중 발판
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| juvenile chondrocytes (Clonetics™ Normal Human Chondrocyte Cell System ) | Lonza | CC-2550 | |
| adult chondrocytes (Clonetics™ Normal Human Chondrocyte Cell System) | Lonza | CC-2550 | |
| poly(ethylene glycol diacrylate) | Laysan Bio | ACRL-PEG-ACRL-1000-1g | |
| 2-morpholinoethanesulfonic acid | Sigma | M5287 | |
| photoinitiator | Irgacure | 2959 | |
| sodium chloride | Sigma | S9888 | |
| chondroitin sulfate sodium salt | Sigma | C9819 | |
| N-hydroxysuccinimide | Sigma | 130672 | |
| 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride | Sigma | E1769 | |
| 2-aminoethyl methacrylate | Sigma | 516155 | |
| dialysis tubing | Spectrum Laboratories | 132700 | |
| Collagenase 2 | Worthington Biochemical | LS004177 | |
| Collagenase 4 | Worthington Biochemical | LS004189 | |
| DMEM/F12 media | HyClone, Thermo Scientific | SH3002301 | |
| live/dead assay | Life Technologies | L3224 | |
| Tri reagent | Life Technologies | AM9738 | |
| Quant-iT™ PicoGreen® dsDNA Assay Kit | Invitrogen | P11496 | |
| Sodium phosphate dibasic | Sigma | S3264 | |
| Ethylenediaminetetraacetic acid disodium salt | Sigma | E5134 | |
| L-Cysteine | Sigma | C1276 | |
| 1,9-dimethylmethylene blue | Sigma | 341088 | |
| Instruments | |||
| UV light equipment – XX-15LW Bench Lamp, 365nm | UVP | 95-0042-07 | |
| Instron 5944 testing system | Instron Corporation | E5940 |
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