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BACKGROUND:
Type I collagen gel is used as a biomimetic matrix for 3D chondrocyte culture, but does not form a shape quickly enough for use in bioprinting. Alginate is traditionally used for 3D chondrocyte culture but does not have as favorable cell survivability or glycosaminoglycan (GAG) production (an element of the extracellular matrix) as collagen gel alone. However, alginate has the ability to become crosslinked in the presence of calcium, conferring a robust shape. Combining the two materials could potentially provide a bioink for use in cartilage bioprinting by providing the proper material properties as well as supporting chondrocyte survival and GAG production.
METHODS:
Bovine chondrocytes were harvested and cultured under standard conditions until passage 3. Cells were trysinized, counted, and resuspended at a concentration of 10 x 106 cells per mL in 3% (w:v) medium viscosity alginate (Sigma) or a combined alginate-collagen gel made of type I collagen (PureCol, Advanced Biomatrix) and 3% alginate in a 1:1 ratio. Cell suspensions were extruded through a 20-gauge needle dropwise into sterile 100mM CaCl2, forming beads. Beads were transferred to standard culture media and cultured for 7 days. 10 beads per group were harvested on day 1 and 7 for analysis. Beads were weighed and placed in a papain digest solution at 60oC for 24 hours. The resulting digest material was analyzed for DNA using Hoechst 33258 and GAG using a modified dimethylmethylene blue (DMB) assay.
RESULTS:
DNA: In the alginate-only gel beads, day 7 DNA was 61.1% of day 1 (p=0.0008) while in the combined gel beads it was 75.9% (p=0.0004). Initial day 1 DNA was 2.4 fold higher in the combined gel beads (p<0.0001).
GAG: In alginate-only beads, GAG concentration increased 89.7%, from 126.9 μg/mL to 240.8 μg/mL (p<0.0001). The combined gel beads saw a 45.1% increase from 210.9 μg/mL to 305.9 μg/mL (p=0.0016).
CONCLUSIONS:
Bovine chondrocytes cultured in combined alginate-collagen beads for 7 days had superior cell survivability and higher GAG concentrations when compared to cells cultured in beads containing only alginate. This hybrid material holds promise as a bioink in 3D bioprinting due to the crosslinking capabilities of alginate combined with the biomimetic effects of collagen on cell survival and GAG production.