Minimizing Engineered Auricular Cartilage Contracture By Maximizing Construct/Cage Contact: The Importance of Injection Molding

Purpose:

As human auricular chondrocytes (HAuCs) remodel their environment and secrete extracellular matrix they exert intrinsic contractile forces. Previously we demonstrated that an external cage scaffold protected demolded chondrocyte-seeded type I collagen constructs from extrinsic compressive forces from the skin and soft tissue. We hypothesize that by allowing the HAuC-seeded collagen hydrogels to polymerize within cages via injection molding, the resulting construct will have increased surface area contact with the external scaffold, equating to a greater number of microscopic attachments between collagen polymers and the cage.

 

Methods:

Disc-shaped cages were designed using SolidWorks, then 3D-printed with polylactic acid on a MakerBot printer. HAuCs were harvested from discarded otoplasty remnants, then expanded to passage 3. HAuCs were mixed with10mg/mL type I collagen at 25million cells/mL. In group 1 a 2mm-high HAuC-seeded collagen sheet gel was allowed to polymerize for 30min under standard cell culture conditions, then an 8mm biopsy punch was used to create 8mmX2mm discs that were then placed in well plates (“naked” (N) discs). In group 2, the same method was followed, but following punch biopsy, the discs were placed within individual cages before being moved to a well plate (“caged” (C)). In group 3, cages were placed within polydimethylsiloxane molds that contoured to the exterior of the cage, then HAuC-seeded collagen was injected directly into the cages and allowed to polymerize for 30 minutes under standard cell culture conditions before being moved to a well plate (“injection molded” (IM)). All groups were maintained with DMEM/F12 with 10% FBS and 1% Pen-Strep for 28 days. Groups were photographed on day 0 and day 28. Constructs underwent microCT for volume calculation on day 28. Images were analyzed in ImageJ.

 

Results:

On day 0, the average base areas of the 3 groups were compared (N: avg=49.6+/-2.5mm²) (C: avg=49.8+/-2.0mm²) (IM: avg=53.9+/-2.4mm²). Unpaired t-tests between groups showed significant area difference between N and IM (p=0.0233) and between C and IM (p=0.0192). On day 28, measurements were repeated (N: avg=36.6+/-2.3mm²) (C: avg=39.9+/-7.5mm²) (IM: avg=53.8+/-1.2mm²). Unpaired t-tests between groups showed significant area difference between N and IM (p<0.0001) and C and IM (p=0.0035). Constructs were imaged via microCT and volume was calculated on day 28 (N: avg=46.5+/-4.9mm³) (C: avg=51.4+/-1.6mm³) (IM: avg=123.7+/-22.9mm³). Unpaired t-tests between groups showed significant volume difference between N and IM (p<0.0001) and between C and IM (p=0.0001). Early evidence of auricular cartilage formation was seen histologically.

 

Conclusions:

External scaffolding of “maturing” auricular cartilage results in significantly decreased loss of volume. Although PLA cages have been thought to confer protection against volume loss by shielding constructs from overlying compressive external forces, these in vitro studies demonstrate that it is the interaction between the hydrogel and the cage that is responsible for the minimal loss of volume observed in the injection molded group. Injection molding of HAuC-seeded collagen directly within our cages allows the hydrogel to polymerize and maximize attachment to the scaffold. This technique can be applied to in vivo constructs to develop mature elastic cartilage that maintains anatomically complex shapes.