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.
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.
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.
Som rapporteret i denne protokol, 3D hydrogeler er i stand til at opretholde chondrocyt fænotype i kultur, så man undgår processen med celle dedifferentiation i fiberbrusk celler normalt støder på med monolagskulturer 15. Desuden langsigtede kulturer i chondrocytes- hydrogel konstruktion afslørede et gunstigt miljø, der fastholder de iboende celle træk forbundet med alder og sygdom.
Anvendelsen af en 3D biomimetiske hydrogel har flere fordele. Først optagelse af chondroitinsulfat (CS), en hovedkomponent findes i ledbrusk, aktivere celler til at nedbryde hydrogelmatriksen ved at udskille chondroitinase og fastsætte nyligt syntetiserede brusk extracellulære matrix 5, 16. Desuden har CS påvist at have anti-inflammatoriske egenskaber i arthritiske led. Den biomimetiske hydrogel kan også anvendes som et stillads materiale til celle levering i reparation af brusk, og kan være kemisk modificeretat fremme en bedre væv-biomateriale integration 17,18.
Anvendelsen af PEG-hydrogeler CS tillader langvarige kulturer af chondrocytter og evalueringen af biokemiske og mekaniske egenskaber. Her viser vi, hvordan denne platform kan være nyttige for de komparative analyser af forskellige kilder af differentierede chondrocytter med henblik på at fastlægge den optimale celletype til brusk teknik. Interessant chondrocytter indkapslet i hydrogeler forblive levedygtig og formere henhold til deres iboende evner. Hydrogelsammensætningen understøtninger, i virkeligheden, væksten af sunde unge og voksne chondrocytter som vist i figur 2. Sammensætningen og strukturen af de beskrevne hydrogeler fremmer også bruskvæv dannelse som angivet ved aflejring af en funktionel ekstracellulær matrix vurderet af glycosaminoglycan (GAG ) kvantificering.
En yderligere fordel er, at chondrocyt-hydrogel konstruktionerkan vurderes for de mekaniske egenskaber af det nydannede bruskvæv. Bemærk, at den unconfined kompression testen skal udføres på acellulær hydrogel til sammenligning. Hydrogelerne, faktisk har en iboende stivhed på grund af stivheden af CS dele. Unconfined komprimering stamme af 5-20% (ved en belastning på 1% / s) kan anvendes til mekanisk afprøvning af bruskvæv 11,12 siden fysiologisk belastning opleves af bruskvæv under belastningstilstand er blevet rapporteret at være 10-20 % 13,14. Svaret fra både celle-lastet og acellulære hydrogeler til mekanisk prøvning blev evalueret ved kulturen endepunkt. I det beskrevne eksempel ovenfor har vi observeret en sammenlignelig stivhed af konstruktioner indeholdende voksen og unge chondrocytter i modsætning til den nedre stivhed af konstruktioner indeholdende OA chondrocytter. Sådanne mekaniske egenskaber af cellen-hydrogel konstruktion mulighed for at vurdere de funktionelle egenskaber afdannede væv giver en dybtgående analyse af cellemodning evne.
Som konklusion kan i vid udstrækning anvendes brugen af 3D biomimetiske hydrogeler at undersøge mulighederne for forskellige chondrocytpopulation at generere bruskvæv. Udover de in vitro-undersøgelser, der beskrives her, kan in vivo transplantation af cellen lastet konstruktioner kan forestille sig at studere celle modning og regenerativ potentiale i fysiologiske kontekst. Yderligere modifikationer af hydrogel platform med yderligere biomimetiske faktorer kan også forestille sig at optimere chondrocyt proliferation og modning.
The authors have nothing to disclose.
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.
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 |