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.
Zoals gemeld in dit protocol, 3D hydrogels kunnen chondrocyte fenotype te behouden in de cultuur, het vermijden van het proces van cel dedifferentiation in fibrocartilage cellen meestal ondervonden met monolaagkweken 15. Bovendien langdurige kweken van de chondrocytes- hydrogel construct bleek een gunstige omgeving die de intrinsieke cel functies die samenhangen met de leeftijd en ziekte handhaaft.
Het gebruik van een 3D biomimetische hydrogel heeft verschillende voordelen. Ten eerste, het opnemen van chondroïtinesulfaat (CS), een belangrijke component in gewrichtskraakbeen staat cellen aan de hydrogel matrix afbreken secreteren chondroitinase en bepaalt nieuw gesynthetiseerde kraakbeen extracellulaire matrix 5, 16. Bovendien is CS aangetoond anti-inflammatoire eigenschappen in het gewrichtsontsteking hebben. De biomimetische hydrogel kan ook worden gebruikt als een stellage materiaal voor cellulaire levering kraakbeenherstel, en kunnen chemisch gemodificeerdbeter weefsel-biomateriaal integratie 17,18 vergemakkelijken.
Het gebruik van PEG-hydrogelen CS maakt langdurige kweken van chondrocyten en de evaluatie van de biochemische en mechanische eigenschappen. Hier laten we zien hoe dit platform nuttig kan zijn voor de vergelijkende analyse van de verschillende bronnen van gedifferentieerde chondrocyten om de optimale celtype kraakbeen techniek definiëren. Interessant chondrocyten ingekapseld in hydrogels levensvatbaar blijft en prolifereren op basis van hun intrinsieke capaciteiten. De hydrogelsamenstelling dragers, namelijk de groei van gezonde jeugdige en volwassen chondrocyten zoals getoond in figuur 2. De samenstelling en de structuur van de beschreven hydrogelen bevordert ook de vorming van kraakbeenweefsel zoals aangegeven door de afzetting van een functionele extracellulaire matrix bepaald door glycosaminoglycaan (GAG ) kwantificering.
Bijkomend voordeel is dat de chondrocyten-hydrogel constructenkan worden beoordeeld op de mechanische eigenschappen van het nieuw gevormde kraakbeenweefsel. Merk op dat het onbeperkte compressietest worden uitgevoerd op de acellulaire hydrogel ter vergelijking. De hydrogels, in feite, een intrinsieke stijfheid als gevolg van de stijfheid van het CS resten. Onbeperkte compressie stam van 5-20% (bij een vervormingssnelheid 1% / s) kunnen worden toegepast voor het mechanisch testen van kraakbeenweefsel 11,12 omdat de lichamelijke belasting ervaren door kraakbeenweefsel onder belastingstoestand werd gemeld dat 10-20 13,14%. De reactie van zowel cel-beladen en acellulair hydrogels mechanische testen werd geëvalueerd aan de cultuur eindpunt. In het beschreven voorbeeld hierboven zagen we een vergelijkbare stijfheid van de constructen bevattende volwassen en jonge chondrocyten in tegenstelling tot de lagere stijfheid van de constructen die OA chondrocyten. Dergelijke mechanische eigenschappen van de cel-hydrogel construct kan de beoordeling van de functionele eigenschappen van devormden weefsel geven van een grondige analyse van de cel rijping vermogen.
Concluderend kan het gebruik van de 3D biomimetische hydrogels het potentieel van verschillende chondrocyte populatie studeren om kraakbeenweefsel te genereren schaal worden toegepast. Naast de in vitro studies beschreven, kunnen in vivo transplantatie van de cellen beladen constructen worden beoogd om celrijping en regeneratieve mogelijkheden bestuderen van de fysiologische context. Verdere modificaties van de hydrogel platform bijkomende biomimetische kunnen eveneens worden beoogd om chondrocyte proliferatie en maturatie optimaliseren.
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 |