## 2 The Role(s) of Non-Collagenous Bone Matrix Proteins in Bone
### Discrepant Results in the Literature and the Path Forward  
Closer scrutiny of the findings reported in Table 2 reveals that there are discrepancies and sometimes diverging results (i.e., studies claiming an increase, decrease, or non-change in the same parameter) between different reports. This has also been recently pointed out by [PERSON] and [PERSON] in a comment piece, noting the lack of reproducibility in the results of bone properties in OC\({}^{-/-}\) mice [116]. Several factors may contribute to the observed differences: briefly, intrinsic factors such as the nature and state of the specimen and the anatomical site of bone, as well as extrinsic factors such as the techniques employed to characterize the sample and the associated sample preparation. The inbred strain, age, and gender of the knockout mice models used can greatly impact the resulting attributes of bone, as demonstrated by Bgn\({}^{-/-}\) mice [96, 117]. In addition, the sample preparation can introduce artifacts into the sample to be imaged/analyzed. One aspect that eludes most studies is the hydration state of bone. Bone is an inherently hydrated material that will dehydrate in the high vacuum environment of the electron microscope, and the degree of hydration of bone influences its structure and mechanical behavior, especially tissue viscoelasticity. Recently, the presence of bound water and its significance in determining the bone mechanics of WT and SLRP-knockout mice have been demonstrated, highlighting the differential roles of bound water versus simple hydration in bone toughness [98]. Lastly, the length scale at which characterization has been carried out is crucial for correctly interpreting the results. Some results are not in actuality debatable; rather, they are inferred results tested at different levels of bone's structural hierarchy using whole-bone vs. sectioned/machined samples [118]. It can be concluded that, following the principle that \"smaller-is-stronger\", the mechanisms of bone failure differ at different length scales,showing higher strength and ductility in smaller specimens when only tissue was inspected. This small-scale characterization can only be attained with the use of electron microscopy, which provides resolutions lower than the limit diffraction of light (<200 nm). The semi-conflicting results in the literature and their lack of reproducibility highlight the need for correlative experiments where imaging, chemical analysis, and mechanical testing are performed simultaneously within the same electron microscope sample holder. Here, we suggest some specific strategies. To address the problem of sample dehydration, a one-hour to overnight immersion of specimens in phosphate-rich water solution is the standard procedure followed prior to mechanical testing [119], although the possible effect of this procedure on demineralization has not been quantified. Constant dripping or complete submersion of the hydrating media during macroscale testing is also widely accepted as a technique to rehydrate the bone [120; 121]. These procedures appear to be effective in keeping the specimens wet at the macroscale but when the length scale of the test is reduced to a few um, adapting these same procedures to retain water becomes a real challenge. For correlative imaging-mechanical testing, improvements and advances in electron microscopy have enabled us to test the mechanical properties of hydrated bone specimens in these microscopes. One emerging technique is liquid-phase electron microscopy--including environmental SEM (ESEM) and liquid-cell electron microscopy--for multimodal and correlative analysis of undecalcified and hydrated bone samples [122; 123]. These emerging electron microscopy modalities show great promise for the imaging of biomaterials in their near-native states without following the harsh sample preparation protocol as in conventional EMs, which operate under high vacuum and without significant compromise of the spatial and temporal resolutions [123].