This study presented the effects of OPN on regulating sex-specific calcification in valve leaflet tissues and hydrogel based tissue mimics. The authors evaluated both diseased valve leaflets from female and male patients and also implemented porcine VIC laden hydrogels as tissue model to study the effects of OPN and related regulator. The finding is very interesting and clinical relevant and has great potential impact in the treatment. However, there are also some unclear description and missing experiments.

Major comments
1. Since the authors only used three female and two male tissue samples, the authors should have a section to discuss the limitation of the sample numbers and related results.

1. Fig. 3A, 4A, 6A. Please do not use "fibroblast". This is confusing.

2. Fig. 3C and E. It seems that only OPN gene expression changed, but not protein level. Please comment whether this is the case for the tissues. Please add related discussion.

3. Fig. 4B Female in CM and Fig. 6B Female Control. Based on the reviewer's understanding, these two figures should be comparable/same. But it seems that Fig. 6B had more calcification. Please clarify and choose typical figures.

4. The authors showed the alteration of OPN and correlated calcification. A simple experiment is to add exogenous OPN and evaluate the anti-calcification effect, which is missing.