Eklem Kondrosit Kültür ve Kıkırdak Üretimi için 3D hidrojel iskeleleri
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
Bu protokolde bildirildiği gibi, 3D hidrojeller genellikle tek tabakalı kültürler 15 ile karşılaştı fibrocartilage hücrelerine hücre dediferansiyasyon süreci kaçınarak, kültürde kondrosit fenotipi muhafaza edebiliyoruz. Ayrıca, hidrojel yapısının chondrocytes- uzun süreli kültürleri yaş ve hastalık ile ilişkili içsel hücre özelliklerini korumaktadır elverişli bir ortam ortaya koymuştur.
3B biomimetik hidrojel kullanılması birçok avantaj vardır. İlk olarak, kondroitin sülfat dahil (CS), eklem kıkırdağının içinde bulunan başlıca bir komponent, kondroitinaz salgılayarak hidrojel matrisi bozan ve ekstra hücresel matris 5, 16 yeni sentezlenmiş kıkırdak bırakmaya hücreleri sağlar. Buna ek olarak, CS gösterilmiştir artritik eklem anti-inflamatuar özelliklere sahip. Biomimetik hidrojel kıkırdak tamir hücre dağıtımı için bir iskele malzemesi olarak kullanılabilir, ve kimyasal olarak modifiye edilebilirDaha iyi doku biyomalzeme entegrasyon 17,18 kolaylaştırmak için.
PEG-CS hidrojellerin kullanımı, uzun süreli kondrositlerin kültürleri ve biyokimyasal ve mekanik özelliklerinin değerlendirilmesine olanak verir. İşte biz bu platformu kıkırdak mühendisliği için en uygun hücre tipini tanımlamak amacıyla farklılaştırılmış kondrosit çeşitli kaynaklardan karşılaştırmalı analizler için yararlı olabilir nasıl gösterir. İlginçtir, hidrojeller kapsüllü kondrositlere kendi içsel yeteneklerine göre canlı kalır ve çoğalır. Hidrojel bileşimi destekleri, aslında, sağlıklı genç ve erişkin kondrositlerin büyümesi Şekil 2'de gösterildiği gibi. Açıklanan hidrojel bileşimi ve yapısı da kıkırdak doku formasyonunu teşvik glikozaminoglikan ile değerlendirilen fonksiyonel hücre dışı matris birikimi (GAG ile gösterildiği gibi ) ölçümü.
Buluşun bir başka avantajı, kondrosit hidrojel yapıları olanYeni oluşan kıkırdak dokusunun mekanik özellikleri için değerlendirilebilir. Serbest basınç testi karşılaştırma için aselüler hidrojel yapılmalıdır unutmayın. Hidrojeller, aslında, bağlı CS parçalarının sertliği için temel bir sertliğe sahiptir. (Bir gerilme oranı / s% 1 at)% 5-20 serbest basınç gerilme yükleme koşulları altında kıkırdak dokusu yaşadığı fizyolojik zorlanma beri kıkırdak dokusunun 11,12 mekanik testler için uygulanabilir 10-20 olduğu rapor edilmiştir % 13,14. Mekanik test hücre yüklü ve hücresel olmayan iki hidrojellerin tepkisi, kültür son nokta değerlendirilmiştir. Tarif edilen örnekte, OA kıkırdak ihtiva eden konstruktların düşük katılıkta aksine, yetişkin ve juvenil kondrositlerin ihtiva eden konstruktların benzer bir sertliğe gözlenen, yukarıda. Hücre-hidrojel yapısının bu tür mekanik özellikleri fonksiyonel özelliklerinin değerlendirilmesine olanakHücre olgunlaşma yeteneği derinlemesine analizini veren oluşturulan doku.
Sonuç olarak, kıkırdak doku üretmek için farklı bir kondrosit nüfusun potansiyel çalışma 3D biomimetik hidrojellerin kullanımı yaygın olarak uygulanabilir. Burada tarif edilen in vitro çalışmaların yanısıra hücre-yüklü yapıları in vivo nakli fizyolojik bağlamda hücre olgunlaşmasını ve rejeneratif potansiyel çalışma tasavvur edilebilir. Ek biomimetik faktörlerle hidrojel platformun bundan başka değişiklikleri de, kondrosit çoğalmasını ve olgunlaşmasını optimize etmek için tasavvur edilebilir.
Disclosures
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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