Q2. What are the future works in this paper?
The interplay of ACPCs with 26 chondrocytes and MSCs supported neo-cartilage synthesis in layered co-cultures, indicating the possibility to use ACPCs also as a complementary cell source in cartilage constructs to produce functionally relevant differentiated tissue and to also act as a pool of stem cells for further growth and remodeling. Consequently, future studies focusing on in-depth biomechanical characterization will play an important role in the field of cartilage tissue engineering. Importantly, further research on ACPC biology, 3D culture and bioprinting will be required to fully recapitulate the zonal organization of native cartilage. Even though ACPC-laden hydrogels showed a lower production of ECM components compared to MSC-laden ones, ACPCs displayed distinctive phenotypic features, particularly a low expression of collagen type X and a high expression of PRG4, suggesting a priming toward a phenotype similar to superficial zone chondrocytes.
Q3. What is the common concern associated with MSC use for articular cartilage repair?
Chondrocyte hypertrophy and associated calcified cartilage production is a common concern associated with MSC use for articular cartilage repair.
Q4. What is the role of the bioink in the development of cartilage?
Modification of the bioink may be necessary to enhance the quality of the cartilage tissue in future experiments, as cell response can be tuned modifying the microenvironment of the hydrogel[71].
Q5. What is the way to improve the mechanical properties of cartilage?
As the compressive mechanical properties of their hydrogel-only constructs did not reach those of adult articular cartilage, combination with reinforcement strategies or bioreactor culture will be recommended to fully address the complex mechanical behavior of cartilage under compression, but also in response to tensile and shear stresses.
Q6. What is the effect of the density and stiffness of the gelMA network?
At the polymer concentration used in this study, the density and stiffness of the gelMA network would limit cell migration, while mass transfer and diffusion of bioactive molecules are largely unhindered within the same23gel matrix[58].
Q7. What are the advantages of using ACPCs in cartilage repair?
ACPCs are promising sources for cartilage regenerative medicine and biofabrication, and the encapsulation in gelMA hydrogels allowed the formation of 3D cartilage constructs in vitro.
Q8. What could be done to reduce UV-A light?
to further reduce potential concerns of UV-A light, crosslinking chemistries that require reduced UV exposure, such as thiol-ene click reactions [65], or even alternatives based on visible light [46], could be applied in the future for encapsulating ACPCs, and in general for bioprinting.
Q9. What is the role of pluronic in the biofabrication of large joints?
reinforcing strategies, such as the co-printing with stiffer materials will be required to provide biomechanical stability, especially in the biofabrication of large joint components[62].
Q10. What is the role of ACPCs in cartilage repair?
The interplay of ACPCs with26chondrocytes and MSCs supported neo-cartilage synthesis in layered co-cultures, indicating the possibility to use ACPCs also as a complementary cell source in cartilage constructs to produce functionally relevant differentiated tissue and to also act as a pool of stem cells for further growth and remodeling.
Q11. What is the role of the cell-laden hydrogel in the development of cartilage?
In the quest for therapies that enhance cartilage healing, hydrogel-based constructs are particularly appealing for regenerative medicine, as they allow encapsulation of cells in a highly hydrated environment, analogous to that of native cartilage[4].
Q12. What software was used for slicing the condyle?
A computer aided manufacturing software (CAM, BioCAM, regenHu) was used for slicing the condyle model and the g-code was generate with the BioCAD software (regenHu).
Q13. What was used as a platform for cell encapsulation and 3D culture?
A photosensitive, gelatin methacryloyl (gelMA) hydrogel bioink was used as a platform for cell encapsulation and 3D culture, and the overall production of cartilage ECM by all three cell types was assessed, together with the mechanical properties of cultured constructs.
Q14. What is the role of pluronic in the biofabrication of ACPCs?
ACPCs were viable after the process of printing, pluronic removal and UV crosslinking, with cell viability values comparable to those observed for MSCs undergoing the same process, showing that the process is also non-harmful for ACPCs.
Q15. What is the role of gelMA in the development of cartilage?
Previous research already demonstrated that gelMA provides a permissive environment for neo-cartilage formation, using encapsulated chondrocytes[47,48], and MSCs [49].
Q16. Where did the cells with the intense staining be found?
for all the samples, a layer of spread, elongated cells, with intense and continuous PRG4 positive staining was found at the outer rim of the hydrogel.
Q17. What is the role of biomechanical characterization in cartilage engineering?
future studies focusing on in-depth biomechanical characterization will play an important role in the field of cartilage tissue engineering.
Q18. What is the only effective communication between the cells in adjacent layers?
in these layered co-culture models, the only effective communication between the cells in adjacent layers is through secreted factors.
Q19. What was the RNA isolation done on cells?
RNA isolation was performed on cells at passage 3 using the RNAeasy mini kit (Qiagen, Germany), following the instructions of the manufacturer.