Enhanced mineralization in joints with OA lesions has been widely noticed. The features of the mineralized crystals in OA joints is less described due to the technique limitation. The present paper described the mineral crystals in OA joint using state-of-the-art micro-focus small-angle X-ray scattering with a 5-um spatial resolution. It is interesting. However, major revision is required.
The big concern is the data interpreting. First, how did the the upper-most mineralization and the rest of the calcified cartilage were defined, for example, within the top two tidemarks. Further, please clarify the location of tested subchondral bone. If it is the plate bone, please use subchondral plate bone other than subchondral bone, to make a easier follow-up. __ adding to the M&M; section.
Could the rest of the calcified cartilage be replaced by the deeper calcified cartilage while the upper-most one be defined as the superficial calcified cartilage? That may be easier to be followed when reading the data. With that definition about the loaction, this review suggest a brief summary of the present obtaining in the first paragraph in discussion section. For example, the superficial vs. deep zone of the calcified cartilage; cartilage vs. subchondral plate bone; medial vs. lateral compartment of the cartilage; patients vs. healthy donors.
The authors wrote that a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32nm thicker crystals vs. rest calcified cartilage. There are 0.2nm thicker mineral crystals in less OA sites, the lateral compartment vs. healthy knees. Opposite was found in subchondral bone. The crystal thickness in subchondral bone was lower in those with increasing histopathological OA severity (OARSI Score). As far as the understanding by this reviewer, the present data indicated that the less degenerated cartilage, lateral compartment, has thicker crystals, while the more degenerated cartilage, the medial compartment, has thinner crystals; the subchondral plate bone in patients has further thinner crystals, and the healthy donor has the thinnest crystals in subchondtal bone. If so, the data have to be carefully interpreted following a consistent philosophy, and the conclusions have to be re-considered because there are seem quite a few contradiction points in the discussion. For example, it is strange to read that unloading relates to growth of mineral crystals or thicker crystals. During the process of endochondral ossification, the cartilage is replaced by the subchondral bone. Chondrocyte-to-osteoblast lineage continuum has been evidenced in vitro and in vivo (Ono et al. 2014; Yang et al. 2014; Zhou et al. 2014). Could the calcified cartilage frontier to be a kind of new formed bone? The authors had suggested in previously publication (19) that the thickening of bone structures is a synchronous process with cartilage degeneration during OA progression. On page 14 line 38, the authors wrote that mineralization is an independent process compared to cartilage degeneration. Is any contradiction here? In publications, cartilage degeneration is linked with cartilage mineralization (Zhang M1, Wang H2, Zhang J1, Zhang H1, Yang H1, Wan X1, Jing L1, Lu L1, Liu X1, Yu S1, Chang W3, Wang M4. Unilateral anterior crossbite induces aberrant mineral deposition in degenerative temporomandibular cartilage in rats. Osteoarthritis Cartilage. 2016 May;24(5):921-31. Zhang J1, Liao L1,2, Zhu J3, Wan X1, Xie M1, Zhang H1, Zhang M1, Lu L1, Yang H1, Jing D4, Liu X1, Yu S1, Lu XL5, Chen C6, Shan Z3, Wang M1. Osteochondral Interface Stiffening in Mandibular Condylar Osteoarthritis. J Dent Res. 2018 May;97(5):563-570.).
The authors wrote that calcified cartilage has been reported to have a higher degree of mineralization than subchondral bone (15), the hardness and stiffness of the tissue are between those of subchondral bone and articular cartilage (16, 17). Please try to explain this phenomenon in discuss for the functional meaning.
It is possible that the calcified cartilage undergoes structural modification during OA. However, the changes in tidemark multification (20, 21) can not be taken as the evidence (page 5, line 20) because the newly formed tidemark is derived from newly calcified cartilage (21-23), say the calcification front, other than modification of the already calcified cartilage represented by the old tidemark.
Page 5 line 50, Mineral crystal thickness change with tissue maturation (30). What tissue? What kind of change, increasing or decreasing?
Page 10 line 51, in all other groups, what other groups? please provide the name of the groups and make the reading easier.
In the statistical section, please explain how the adjustment of age and BMI were performed.
The authors wrote that (page 12 line 15) the thickness of the mineral crystal is regulated by the structure of the organic matrix (43). please explain that possible differences of the organic matrix between calcified cartilage and subchondral plate bone. The authors provided non-organic constitution explain, that is fewer non-stoichiometric substitutions of carbonate for phosphate, that is plausible. May authors in reference #45 had provided some information on the organic matrix differences?

The work is much improved. However, a few minor points have to be revised.
The lateral compartment is generally less loaded than in medial compartment of knee. The authors explained the present difference between the thickness of the mineral crystals, in both tissues of the lateral compartment in OA compared with healthy knees, as loading-related differences. The authors discussed the similar difference between cartilage and bone with the tissue structure involving matrix arrangement. Bio-mechanical factor is the key initiator of OA. The crystal formation is one of the responses in cartilage to loading. Is it possible that a thicker crystal might be an earlier stage response to less harmful loading, while a thinner one a later stage response to more harmful loading in the process of remodeling?

Page 4 line 20-22: These properties allow cartilage to dissipate and distribute loading, protecting the underlying subchondral bone(4,5). Please reconsider the word “protecting”, even though here is a citation of the references #4,5. Cartilage and bone here function more likely as a unit.
Page 13, line 20-22: The geometry, as well as orientation and architecture of the mineral crystals in
osteochondral mineralized tissues factor in the strength of the tissues(43). The sentence seems not complete.

This paper brings about a new insight for tidemarks formation and the growth of mineral crystals during cartilage calcification as a response to loading under physiological and pathological conditions. The comparison between lateral and medial compartment seems less depth, thus leaves a space for further investigations.