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Mu et al. Microstructures 2023;3:2023030 https://dx.doi.org/10.20517/microstructures.2023.05 Page 13 of 21

phenotype and subsequent calcification [209,210] . PPi is identified as the principal inhibitor of HAp deposition,
evidenced by antagonizing the ability of Pi to crystalize with Ca to inhibit biomineralization and acquired
[32]
clinical conditions regarding pathological calcification upon its deficiency . OPN is found to be associated
with nascent mineralization foci during bone mineralization and present at interfaces where mineralization
is needed to be quenched [211,212] .

Several approaches to regulating the metabolisms of these natural inhibitors exhibited promising results in
treating pathological mineralization. For example, Vitamin K supplementation showed the effect of
reducing the progression of vascular mineralization and subsequent arterial stiffness [33,213] . Bisphosphonates,
derivatives of PPi, stabilize the Pi groups by a phosphorus-carbon-phosphorus backbone. Compared to PPi,
[32]
which can be easily hydrolyzed, bisphosphonates have extended plasma half-life and improved stability .
Bisphosphonates showed their potency to reduce fractures in postmenopausal osteoporosis . They have
[214]
also been found to reduce soft tissue calcification and interfere with osteoclast functions [33,204] . Alendronate, a
widely prescribed bisphosphonate, has shown good tolerance and improvements in several patients with
brain calcification . Other bisphosphonates, such as etidronate, pamidronate, and ibandronate, have been
[167]
demonstrated to inhibit aortic calcification both in vitro and in vivo [202,215-218] . In addition to bisphosphonates,
a few proteins associated with the metabolism of PPi have also been identified to be promising for targeted
treatment of pathological mineralization, such as ectonucleotide pyrophosphatase/phosphodiesterase-1
(ENPP1), ATP binding cassette subfamily C member 6 (ABCC6), progressive ankylosis protein (ANK), and
tissue-nonspecific alkaline phosphatase (TNAP) [219-222] . Inspired by the inhibitory effect of acidic urinary
macromolecules (e.g., OPN) on the formation of CaO in vitro, polymeric carboxylic amino acids, poly-L-
x
glutamic and aspartic acid, were found to inhibit the crystallization of CaO x [223] . A similar inhibitory effect
was also found to be achieved by acidic polyanion poly (acrylic acid) [224-226] . Additionally, sodium thiosulfate
(STS) treatment was found to inhibit calcium salt precipitation in calciphylaxis [227-230] . Nevertheless, these
results were limited either in the long-term efficacy or potential toxicity of the STS treatment .
[33]
Moreover, the presence of trace elements has been shown to play a role in the crystal formation kinetics or
external morphology of a growing crystal. It could be promising in designing therapeutic approaches again
pathological mineralization [102,231] . For example, Mg, Zn, aluminum (Al), Fe, and Cu are indicated as growth
inhibitors of CaO at very low concentrations [232-235] . Adding AlCl or FeCl to transplanted valves effectively
3
x
3
delayed the onset of valve calcification by blocking TNAP activity .
[236]
CONCLUSIONS AND FUTURE PERSPECTIVES
Our understanding of pathological/ectopic mineralization has been dramatically improved in parallel to
bone biology and advanced characterization techniques widely used in the field of material science [22,92,201] .
Significant progress has been made in elucidating the properties of pathological crystals and the underlying
mechanisms of pathological/ectopic calcification that occurred in soft tissues over the years. By elucidating
the physiological mechanisms of bone mineralization and their relationship with mineralization
phenomena, we can develop targeted therapeutic interventions to prevent, manage, or treat these disorders.
However, there are still some debatable questions regarding (a) the composition and structure of
pathological crystals mediated by cellular activities and factors in the surrounding environment; (b) the
dynamics of ion transport; (c) the involvement of cells and related cellular activities; (d) the interactions
between pathological crystals and the surrounding tissues; and (e) how these contribute to disease
progression . Even though attempts have been made to simulate the crystallization process, it is still
[36]
challenging to comprehend the in situ crystallization process due to the involvement of cellular activities.
Nevertheless, a more comprehensive understanding of how the structures are formed, progressed, and
adapted to changing needs enables us to conceive new insights into the progression of the pathological