ABSTRACT Heterotopic ossification (HO), the pathological formation of bone in soft tissues, is a debilitating condition, as well as one of the few instances of de novo bone formation in

adults. Chemical mapping of HO tissue showed distinct islands of calcium phosphate within phosphate-deficient, calcium-rich regions, suggesting a transition to apatitic bone mineral from a

non-phosphatic precursor. The transition of amorphous calcium carbonate (ACC), a generally suggested bone-mineral precursor, in physiological conditions was thus investigated. Here, we show

that adenosine triphosphate (ATP), present in high amounts in forming bone, stabilised ACC for weeks in physiological conditions and that enzymatic degradation of ATP triggered rapid

MINERAL LAYERS COVERING APATITE CRYSTALLITES Article Open access 24 September 2020 THE STRUCTURAL PATHOLOGY FOR HYPOPHOSPHATASIA CAUSED BY MALFUNCTIONAL TISSUE NON-SPECIFIC ALKALINE

PHOSPHATASE Article Open access 08 July 2023 SKELETAL AND EXTRASKELETAL DISORDERS OF BIOMINERALIZATION Article 16 May 2022 INTRODUCTION Heterotopic ossification (HO) is the pathological

deposition of bone in non-skeletal sites, which can occur following high-energy trauma, nervous system injury, burns, skeletal surgery, and hereditary conditions1. While appearing disordered

and growing polyaxially at the macro-scale, the microstructure of HO is nearly identical to that of skeletal bone2. Therefore, in addition to researchers aiming to prevent and treat HO,

this condition is of great interest to those aiming to stimulate bone formation, a key aim in regenerative medicine3,4,5,6. Further, HO can be a useful model to study bone development, as it

represents de novo bone formation in humans, excised and available at multiple time points, that can then be characterised and studied in detail more easily than skeletal bone7. Despite

decades of dedicated research on the subject, there is still much debate in the bone community on the exact progression from ionic calcium and phosphate to highly structured

calcium-deficient hydroxyapatite (HA) in vivo8. Popular explanations include the direct formation of HA, perhaps structured by the organic scaffold that forms prior to mineralisation, or HA

formation within the specific environment inside osseous cells or extracellular vesicles, which is then deposited onto the scaffolds9. The other prevalent theory is that HA is not formed

initially, but rather a precursive salt is precipitated, which later transforms into HA, with amorphous calcium carbonate (ACC)10, amorphous calcium phosphate (ACP)11, and polymer-induced

liquid precursors12 being the most cited. A specific quandary faced by the HO community is the sheer quantity of material required for the rapid development of dense bone. For example, the

phosphorous concentration in dense HO is estimated to be around 5 mmol cm−3 13, slightly higher than in physiological bone (4.2 mmol cm−3 14); however, the phosphorous concentration in serum

is only 0.001 mmol cm−3 15. This has led researchers to seek a specific source of phosphate, for example, dense granules in platelets, which have a high phosphate concentration of around

0.13 mmol cm−3 16. Such granules are known to aggregate around bone defects17 and, in addition to inorganic polyphosphate18, contain high levels of adenosine tri- and di-phosphate (ATP and

ADP)19. ATP has been shown to be important in HO formation, where remote hydrolysis of this molecule has been shown to mitigate ectopic bone deposition through inhibition of the SMAD 1/5/8

pathway20. The large amount of energy released through phosphate cleavage may also be important as, in addition to the materials of formation, bone development also requires a large amount

of energy21. In this study, the chemical architecture of human HO was mapped, where the presence of microregions of calcium and no phosphate suggests formation through a precursor species,

most likely amorphous calcium carbonate. The transformation of ACC in physiological conditions was then studied, both in the presence and absence of ATP, in order to present a possible

mechanism for the chemical development of HO. RESULTS CHEMICAL COMPOSITION OF HO The chemical composition of human HO was mapped through XRF scanning. Much of the excised section was

unmineralised soft tissue, evidenced by the high sulphur signal (blue), indicating the presence of cysteine-containing proteins such as collagen, and low signals for calcium (red) and

phosphorus (green) (Fig. 1A–C). Regions of colocalised calcium and phosphate (yellow) indicated the presence of a calcium phosphate salt, which may be amorphous or crystalline. However,

there were also regions with a high calcium but low phosphorous signal, seen at the mineralisation front in Fig. 1A (arrows), at the peripheries in Fig. 1B (arrows), and in a large area in

Fig. 1C. Within this large region of phosphate deficiency, there were clear islands of colocalisation (Fig. 1C, insert). A line scan across this region showed a near-constant sulphur signal,

a phosphorous signal that was much higher in regions of colocalisation, and a calcium signal that, while always high, was higher in regions of colocalisation (Fig. 1D). The ratio of calcium

to phosphorous was also much lower in regions of colocalisation (Fig. 1E). In contrast, XRF maps of mature physiological bone appeared to show colocalisation of calcium and phosphorous

throughout, demonstrating significantly more homogeneity than the pathological tissue (Fig. 1F, G). Given that calcium metal is highly unstable in vivo, there must be a counter-ion that

cannot be detected through XRF in these areas, the most likely choice being a carbon species. Further, as amorphous species are more easily directly substituted22, and it has been implicated

previously in bone mineralisation22,23, the phosphate-deficient phase was assumed most likely to be an amorphous calcium carbonate (ACC). ACC is highly unstable in water, rapidly

crystallising into vaterite and/or calcite. However, it can be stabilised by many species found in vivo, such as Mg2+ ions and ATP. The transition between stabilised ACC and HA was thus

studied as a possible chemical mechanism of HO, and thus also physiological bone formation and an explanation for the chemical heterogeneity seen in forming HO. TRANSFORMATION OF MG

STABILISED ACC ACC was first synthesised and stabilised with Mg2+ ions24,25. When submerged in a solution containing a physiological level of phosphate, Mg stabilised ACC fully crystallised

into calcite after 8 days, though crystallinity was evident through XRD after only 1 h (Fig. 2A). The FTIR spectra of the starting material initially showed a broad carbonate double peak,

and a peak at 862 cm−1, indicative of ACC (Fig. 2B). Post-immersion, the carbonate peak remained, though it had sharpened and the doublet was less clear, and the peak at 862 cm−1 had shifted

to a sharp peak at 871 cm−1 which, along with the new peak at 713 cm−1, indicated calcite formation. Additionally, phosphate peaks could be seen in the post-immersion FTIR spectra; however,

no crystalline phosphate salts were detected through XRD. This may indicate that the phosphate salt was amorphous or present in low quantities on the surface of the calcite mineral, giving

it an amplified signal in FTIR, a primarily surface technique. SEM images of the material pre- and post-immersion were also taken, showing the transition from associated nanoparticles (Fig. 

2C) to what appears to be several phases (Fig. 2D). TRANSFORMATION OF ATP STABILISED ACC ACC can also be stabilised with ATP. At the concentrations studied, ATP proved a more effective

stabiliser than magnesium, with full crystallisation occurring between a week and a month of submersion, compared to an hour (Fig. 3A). The transformation here also appeared more complex,

with small amounts of vaterite appearing to form from the amorphous powder within 24 h, which had disappeared by a week of incubation, to be replaced by mainly HA and a small proportion of

calcite within a month. Further, in the ATP stabilised system, the broad carbonate double peak disappeared from the FTIR spectra within a week, contrary to the Mg stabilised system, and the

phosphate peaks, which remained rounded in the Mg stabilised ACC, became notably sharp at 1 month (Fig. 3B). The emergence of PO43− absorption bands at 1036, 554 cm−1 and the weakened

vaterite peaks at 1470, 1402, 876 cm−1 after immersion for 7 days, indicated the conversion of ACP from ACC, while the sharper band at 1036 cm−1, the split bands of 603 and 564 cm−1 and the

decreased CO32− bands at 1470 and 1402 cm−1 after submersion for 1 month indicated a highly ordered HA crystal structure was eventually formed from ACC26,27. The disappearance of the

symmetric PO32− and asymmetric PO23− peaks seen in the starting material at 997 and 1242 cm−1, respectively, suggested hydrolysis of the phosphate moieties from ATP16. This process was also

visualised through SEM imaging, where a similar association of nanoparticles as in the Mg stabilised system can be seen in the ATP stabilised ACC (Fig. 3C). After 1 h, larger, distinct

spheres, more crystalline in appearance form (Fig. 3D), which then become smaller and more connected, similar to the amorphous starting material, after 1 day (Fig. 3E), and become entirely

indistinct after a week, forming an almost fluid appearance (Fig. 3F). The final form after 1 month appears to be an aggregate of particles (Fig. 3G). The composition of the initial material

was assessed through TGA and DSC, which showed three distinct weight-loss phases: weight loss before 200 °C, with a highly exothermic heat flow peak at around 100 °C, suggested loss of free

water; the weight lost between 200 and 450 °C, associated with a slightly endothermic heat flow, was assigned to the breakdown of ATP, and the large decrease in weight, with a large

exothermic heat flow peak, after 550 °C was likely the decomposition of calcium carbonate to calcium oxide and carbon dioxide28 (Fig. 3H). In order to study the conversion from CaCO3 to HA

in a shorter time, the system was investigated at an elevated temperature (60 °C) to both accelerate the crystallisation of ACC and the conversion of ACC to calcium phosphate (CaP),

mimicking the trend of conversion at 37 °C. By heating to 60 °C, ATP stabilised ACC was converted to HA in only 2 days, compared to >7 days at 37 °C, following the same phase transition

albeit with a small amount of vaterite remaining at the reaction’s conclusion (Supplementary Fig. S1A, B). This system was also used to study ATP hydrolysis, and it was found that around 18%

of the ATP was entirely released immediately, followed by a linear release up to 60% at 15 h, which tailed off until 90% of the loaded adenosine was released at 48 h (Supplementary Fig.

S1D). The initial fast release may be attributed to the free ATP in the sample. Mg stabilised ACC dispersed in 0.1X PBS and 0.15X PBS at 60 °C for 1 and 2 days both exhibited crystalline

CaCO3 as shown in Supplementary Fig. S2A, B. The 0.15X PBS applied here as a dispersant aims to maintain the same P concentration with ATP-ACC in 0.1X PBS, considering the hydrolysis of ATP.

Vaterite and calcite microparticles were formed, as shown in the SEM image (Supplementary Fig. S2C), and EDX mapping and EDX spectrum in Supplementary Fig. S2D, E demonstrate the formation

of CaP nanoparticles and crystalline CaCO3 on the microscale. The EDX mapping indicates that CaP formed on the surface of crystalline CaCO3 via a dissolution-precipitation reaction. To

examine how this process might be accelerated in vivo, the ATP-stabilised ACC system was submerged in 0.1X PBS for 1 or 7 days, as previously, before the addition of the enzyme apyrase and

then incubated for another day to accelerate the decomposition of ATP. The addition of apyrase appeared to hasten the crystallisation to HA to 7 days (Fig. 4A), compared to 1 month in the

absence of apyrase (Fig. 3A). This can be seen more clearly in the FTIR spectra, where 1 day in the presence of apyrase appeared to entirely remove both the broad carbonate peak and the peak

corresponding to crystalline vaterite, and sharpen the phosphate peak (Fig. 4B). The FTIR spectrum for the sample soaked for 7 days shows the formation of highly ordered HA crystals26,27.

TRANSFORMATION OF ACC IN HIGH PHOSPHATE CONCENTRATION (0.1 M) Finally, to understand the role of environmental phosphate concentration, both the ATP and Mg stabilised ACC sample powders were

submerged in 0.1 M (NH4)2HPO4 solution, which has 100 times the phosphate concentration of 0.1X PBS. The ATP stabilised system formed HA much more rapidly than when submerged in 0.1X PBS,

with HA peaks visible after only 5 min and clear crystallinity within a day (Fig. 5A). The Mg stabilised system, contrary to when immersed in 0.1X PBS, also formed HA in 0.1 M (NH4)2HPO4,

with additional peaks visible due to the formation of MgNH4PO4 (Fig. 5B). The FTIR spectra in Fig. 5C indicate the formation of HA crystals26,27. DISCUSSION Chemical mapping of human HO

tissue using XRF revealed numerous regions which were calcium-rich but contained no detectable phosphate (Fig. 1). This is in contrast with the mapping of mature physiological bone, where

the calcium and phosphate were localised throughout the majority of the sample. Indeed, although there were some small areas that were calcium-rich-phosphate-poor in normal bone, the mineral

composition was generally much more homogeneous, suggesting that the mineral phase in the tissue was predominantly calcium phosphate, as would be expected for human bone. The areas with no

phosphate may be undergoing remodelling and mineralisation, or this may be an artefact due to surface roughness. While any element lighter than neon cannot be categorically ruled out through

XRF, the most likely light counter-ion to calcium in vivo is a carbon species. Again, while this is possible to be any number of carbon species, such as citrate, lactate or oxalate,

carbonate is perhaps the most likely, as many forms of calcium carbonate are conserved throughout the animal kingdom29, and ACC has been implicated as a bone mineralisation precursor for

decades22,23. The conversion of ACC in physiological solutions was therefore studied. ACC is highly unstable in water30 and must therefore be stabilised by some species to exist in vivo.

Many additives, including Mg2+ ions31,32, phosphorus-containing compounds28, biomolecules33 and polymers34 have been shown to form stable ACC during its formation; two such species found in

vivo are Mg2+ ions and polyphosphates such as ATP16,24. Mg2+ was studied first, and at the concentration studied, ACC appeared to be stable for up to 1 h in 0.1X PBS, which contains the same

phosphate concentration as serum, at 37 °C (Fig. 2). However, on crystallisation, Mg stabilised ACC primarily transformed into a calcium carbonate mineral, calcite, rather than the HA that

would be expected in bone. A phosphate phase did appear to form alongside the calcite, though crucially, it was not incorporated into the carbonate phase and formed separately. As no

crystalline phosphate phase was detected through XRD, it is likely that this phase was amorphous and/or formed only in small amounts on the calcite surface. This hypothesis is supported by

the EDX imaging in Supplementary Fig. S2D, showing the phosphate phase forming a layer on top of the calcite mineral. This amorphous phase may crystallise over long time periods. Regardless,

the short-term stabilisation and final formation of mainly calcite, a mineral not found in the human body, suggest that this is, at least principally, not the process that occurs in human

bone formation. ATP is found ubiquitously in vivo and has also been shown to stabilise ACC in a concentration-dependent manner; up to 12 days in PBS, at higher ATP concentrations than those

used in this study28,33. The concentration used here, 1 mM, was higher than normally found in serum; however, local concentrations of ATP can reach 3–5 mM with the accumulation and

activation of platelets following trauma35. Interestingly, following submersion, some vaterite crystallisation occurred within 1 h; however, this appeared to resolubilise with the formation

of an amorphous phase, with the final apatite mineral forming between 7 days and 1 month (Fig. 3). The significant delay in crystallisation, and the final product being HA, make ATP a more

likely stabiliser of ACC in vivo that Mg. Of particular interest was the formation of the X-ray amorphous phase at 7 days (Fig. 3A), which suggested that the pathway of HA formation

underwent a metastable precursor rather than direct ionic substitution from vaterite. While direct substitution is possible in vaterite, but notably not the thermodynamically stable calcite

formed in the Mg stabilised system, this process is far more favourable in ACC22. Further, the lack of carbonate peaks suggested that this amorphous phase was not simply the reprecipitation

of ACC, but a different phase, most probably ACP. Indeed, a study synthesising HA from ACC in non-physiological conditions found that this reaction progressed _via_ ACP36. This is an

exciting finding, as ACP is also commonly implicated as a precursor in human bone mineralisation37. As HO is commonly defined as bone formation in soft tissues, and thus HO and bone are

identical, at least at the microscale2, it was assumed that HA would be the final mineral produced in both cases. However, interestingly, a calcium non-phosphate mineral appeared in the

samples tested up to 3 years post-injury. This is long after HO is considered metabolically ‘mature’, usually between 6 months and 1 year, though this can depend on multiple factors38.

Indeed, it has been shown that HO can continue to remodel after 3 years39. It may be that the bone in this particular patient was still indeed maturing, or else that the non-phosphatic

calcium species is stable and persists indefinitely. Due to differing formation pathways, it may be that HO and skeletal bone are chemically distinct. Indeed, studies that examine HO tend to

focus on clinical and morphological aspects, relying on radiographic, microscopic and histological examination, techniques incapable of probing the mineral chemistry7,39. Further studies

examining the chemistry of HO, including further XRF studies, XRD analyses and Raman spectroscopy, are required to confirm or refute this hypothesis. The conversion of ACC to calcite or HA

could be thermodynamically driven, although it is still unclear whether the conversion to HA undergoes an internal structural bulk rearrangement of the metastable precursor or a

dissolution-reprecipitation process40. The reappearance of an amorphous phase at day 7 may indicate the latter in this case (Fig. 3A). The fate of ACC during crystallisation may be

determined by the priorities between its conversion to calcite and CaP; this process is likely governed by the more kosmotropic phosphate ions and polyphosphate molecules, relative to the

lighter and more chaotropic Ca2+ ions acting as diffusive fillers in the phosphate network41. In the ATP-stabilised ACC system, the incorporation of phosphate and gradual formation of

phosphate framework within the ACC precursor happened before the crystallisation of ACC into stable calcite. A similar phenomenon has also been reported for peptides rich in aspartic acid

and glutamic acid, which accelerate the conversion of ACC into HA at phosphate concentrations (50 and 500 mM) much higher than physiological conditions22. The crystallisation of ACC into

calcite may take the dominant role in the Mg system due to the poor stability of Mg-stabilised ACC, leading to crystallisation before phosphate incorporation. In vivo, of course, there are

many enzymes capable of breaking down ATP, and it is far more likely that it is broken down in this way than by thermal hydrolysis. These include enzymes, such as apyrase and tissue

non-specific alkaline phosphatases, that cleave one phosphate unit sequentially from ATP, ADP and AMP, resulting in adenosine and three orthophosphate units per ATP molecule42,43,44. Enzymes

also exist in vivo, such as ectonucleotide pyrophosphatase/phosphodiesterase 1, that cleave two phosphate units, producing pyrophosphate and AMP. Pyrophosphate is a known inhibitor of

mineralisation in the body and has also been shown to stabilise ACC45. However, pyrophosphate can also be degraded enzymatically into two orthophosphate units, simultaneously removing a

mineralisation inhibitor and producing material for apatite formation21. Other phosphatic compounds found in vivo, such as inorganic polyphosphates, which have also been shown to stabilise

ACC, may act similarly46. To examine the effect of phosphatase enzymes, apyrase was added to the system, and indeed far more rapid crystallisation into HA was observed (Fig. 4). However, the

important implication of this is the possibility of localised transition through enzymatic activity: the initial ACC laydown is stabilised by ATP then when locally broken down by enzymatic

activity of cells, the dual action of removal of ATP stabilisation and release of phosphate triggers local transition from ACC to HA. This might be a reasonable mechanistic explanation for

the nodular colocalisation of calcium and phosphate within a phosphate-depleted region of HO (Fig. 1C). Clearly localisation of this effect is important, as a high concentration of phosphate

triggers rapid phase transformation of ACC to apatite regardless of stabilisation (Fig. 5). The key factors affect the transformations of ACC occurred under different conditions in the

ATP-ACC conversion system are summarised in Fig. 6. CONCLUSIONS ACC was stabilised by ATP in a physiological solution, and the incorporation of different contents of ATP significantly

affected its conversion pathway. In this study, the transformation followed an ACC → ACP → HA pathway after submersion in a physiological phosphate solution for 1 month. The transformation

process can be triggered by the breakdown of ATP, resulting in HA formation, suggesting that a carbonate-based precursor could reasonably exist in HO and, by extension, form physiological

bone. The breakdown of ATP can be triggered by enzymes, both removing its stabilising effect and increasing the local phosphate concentration. Local enzymatic degradation of ATP through

cellular activity could lead to the apatitic nodules seen in the otherwise phosphate-deficient regions of HO. METHODS MATERIALS Calcium chloride dihydrate was purchased from G-Biosciences

(St Louis, MO, USA). Sodium carbonate decahydrate was bought from Alfa Aesar (Lancashire, UK). Magnesium chloride hexahydrate, adenosine 5’-triphosphate (ATP) disodium salt hydrate, Apyrase

from potatoes, Dulbecco’s phosphate buffered saline (PBS) with calcium chloride and magnesium chloride was ordered from Sigma-Aldrich Company Ltd. Ammonium phosphate dibasic was obtained

from Fluka (Germany). Optimal cutting temperature (OCT) compound was supplied by VWR Chemicals (Leuven, Belgium). All of the chemicals were used without further purification. HO TISSUE Human

HO tissue was acquired from the Human Biomaterials Resource Centre at the University of Birmingham, UK, the details of which are listed in Supplementary Table S1. The HO samples were

defrosted from −80 °C and embedded in optimal cutting temperature (OCT) compound under vacuum conditions (20 kPa). The embedded samples were then sectioned into 100-μm thickness slices in a

cryostat using a Leica microtome with a tungsten carbide blade. The resulting slices were air-dried on glass slides. The mature physiological bone block (thickness: <1 cm) was obtained

from a human femur, which was cut to create a macroscopically flat surface to image. The bone block was not embedded in resin in order to minimise the background effects of the chemical

preparation. Tissue transfer and handling were conducted under the approval of the National Research Ethics Service (15/NW/0079) and in accordance with the Human Tissue Act 2004. AMORPHOUS

CALCIUM CARBONATE (ACC) SYNTHESIS ACC was fabricated by combining equal volumes of equal molarity of CaCl2 and Na2CO3 solutions25. To prepare Mg-stabilised ACC, CaCl2 (0.1 M, 200 mL), Na2CO3

(0.1 M, 200 mL) and MgCl2 (0.5 M, 40 mL) solutions were combined through vigorous magnetic stirring for 15 s, with no pH adjustment. Following precipitation, the suspension was centrifuged

(1 min at 4000 rpm), and the supernatant fluid was removed to recover the particles. These were washed thrice with absolute ethanol and vacuum dried for 12 h to yield a dry powder. The same

procedure was followed to produce the ATP-stabilised ACC, but the initial CaCl2 and Na2CO3 solutions were at a concentration of 25 mM, and no MgCl2 solution was added; instead, 1 mM of ATP

was added to the CaCl2 solution prior to combining with the Na2CO3 solution. TRANSFORMATION OF ACC IN PHOSPHATE SOLUTIONS ACC powders were dispersed in 40 mL of solution (0.1X PBS, 0.15X PBS

or 0.1 M (NH4)2HPO4) at a concentration of 0.5 mg mL−1, under magnetic stirring at 250 rpm at 37 or 60 °C. Aliquots were taken at each time point and recovered by centrifugation and washing

with ethanol. To study the effects of enzymatic degradation, apyrase (70 units) was added for 24 h, after either 1 day or 7 days, to ACC powders (20 mg) immersed in 0.1X PBS (40 mL).

PHYSICOCHEMICAL CHARACTERISATION OF POWDERS The crystallinity and phase composition of the materials were determined by powder X-ray diffraction (XRD) using a D8 Advance (Bruker) X-ray

diffractometer with Cu-Ka radiation (l = 1.5406 Å). Fourier-transform infrared (FR-IR) spectra were obtained using a Tensor 27 (Bruker) spectrometer, measured from 4000 to 400 cm−1. Peak

assignments were obtained from26,27,47,48,49. Scanning electron microscopy (SEM) images were attained using a TM3030 (Hitachi), in secondary electron mode, with an accelerating voltage of 15

 kV. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements were carried out on a STA 449 F3 system (Netzsch) with a heating rate of 10 K min−1 from room

temperature to 800 °C in a nitrogen flow. X-RAY FLUORESCENCE The chemical composition of the HO sections was mapped using a Tornado M4 micro X-ray fluorescence (XRF) system (Bruker Nano

Gmbh, Berlin, Germany) equipped with a rhodium X-ray tube operating at 50 kV and 400 μA, a 25-μm spot size, 20 μm spot distance, 60 ms exposure time and operated at a reduced pressure of 50

mbar. Note that XRF can only detect elements heavier than neon. RELEASE OF ADENOSINE To measure the formation and release of adenosines, including ATP and its dephosphorylated forms, ATP-ACC

(50 mg) was placed into dialysis tubing and transferred into 0.1X PBS (100 mL) at 60 °C under mild agitation (250 rpm). The released sample aliquots (2 mL) were collected at time points

between 5 min and 48 h, with the replacement of fresh 0.1X PBS (2 mL). The concentration of adenosines in each aliquot was found by measuring the absorbance at a wavelength of 258 nm (Cecil

2021 UV spectrophotometer) and then finding the concentration from a standard curve of known adenosine concentration. The original ATP content in the sample was found by immersing the sample

(10 mg) in 0.1 M HCl (5 mL) overnight to completely dissolve the ACC and measuring its UV absorbance. The payload of ATP in ATP-ACC was calculated to be 16.73% based on the below equation.

$${{{{{\rm{Payload}}}}}}=\frac{{{{{{\rm{Mass}}}}}}\; {{{{{\rm{of}}}}}}\; {{{{{\rm{ATP}}}}}}\; {{{{{\rm{recovered}}}}}}\; {{{{{\rm{from}}}}}}\; {{{{{\rm{the}}}}}}\; {{{{{\rm{obtained}}}}}}\;

{{{{{\rm{ATP}}}}}}\mbox{-}{{{{{\rm{ACC}}}}}}\; {{{{{\rm{powder}}}}}}}{{{{{{\rm{Mass}}}}}}\; {{{{{\rm{of}}}}}}\; {{{{{\rm{ATP}}}}}}\mbox{-}{{{{{\rm{ACC}}}}}}\; {{{{{\rm{powder}}}}}}}$$ All of

the experiments in the paper were repeated thrice. DATA AVAILABILITY The authors declare that the data supporting the findings of this study are available within the paper and its

supplementary information files. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request. REFERENCES * Shore, E. M.

& Kaplan, F. S. Fibrodysplasia ossificans progressiva and progressive osseous heteroplasia. _Clin. Rev. Bone Miner. Metab._ 3, 257–259 (2005). Article  Google Scholar  * Robinson, T. E.,

Cox, S. C. & Grover, L. M. Heterotopic ossification following traumatic blast injury. _Cardiovascular Calcification and Bone Mineralization_, 297–315.

https://doi.org/10.1007/978-3-030-46725-8_14 (Humana, 2020). * Matsumoto, Y. et al. Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show

increased mineralization and cartilage formation. _Orphanet J. Rare Dis._ 8, 1–14 (2013). Article  Google Scholar  * Hrkac, S. et al. Heterotopic ossification vs. fracture healing:

extracellular vesicle cargo proteins shed new light on bone formation. _Bone Rep._ 16, 101177 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Barradas, A. M. C., Yuan, H.,

van Blitterswijk, C. A. & Habibovic, P. Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms. _Eur. Cells Mater._ 21, 407–429

(2011). * Krishnan, L. et al. Delivery vehicle effects on bone regeneration and heterotopic ossification induced by high dose BMP-2. _Acta Biomater._ 49, 101–112 (2017). Article  CAS  PubMed

  Google Scholar  * Eisenstein, N. M., Cox, S. C., Williams, R. L., Stapley, S. A. & Grover, L. M. Bedside, benchtop, and bioengineering: physicochemical imaging techniques in

biomineralization. _Adv. Healthc. Mater._ 5, 507–528 (2016). Article  CAS  PubMed  Google Scholar  * Olszta, M. J. et al. Bone structure and formation: a new perspective. _Mater. Sci. Eng. R

Rep._ 58, 77–116 (2007). Article  Google Scholar  * Michigami, T. Skeletal mineralization: mechanisms and diseases. _Ann. Pediatr. Endocrinol. Metab._ 24, 213–219 (2019). * Weiner, S.,

Mahamid, J., Politi, Y., Ma, Y. & Addadi, L. Overview of the amorphous precursor phase strategy in biomineralization. _Front. Mater. Sci. China_ 3, 104–108 (2009). Article  Google

Scholar  * Lotsari, A., Rajasekharan, A. K., Halvarsson, M. & Andersson, M. Transformation of amorphous calcium phosphate to bone-like apatite. _Nat. Commun._ 9, 1–11 (2018). Article 

CAS  Google Scholar  * Jee, S. S., Culver, L., Li, Y., Douglas, E. P. & Gower, L. B. Biomimetic mineralization of collagen via an enzyme-aided PILP process. _J. Cryst. Growth_ 312,

1249–1256 (2010). Article  CAS  Google Scholar  * Jaovisidha, S. et al. Influence of heterotopic ossification of the hip on bone densitometry: a study in spinal cord injured patients.

_Spinal Cord_ 36, 647–653 (1998). Article  CAS  PubMed  Google Scholar  * Obrant, K. J. & Odselius, R. The concentration of calcium and phosphorus in trabecular bone from the iliac

crest. _Calcif. Tissue Int._ 39, 8–10 (1986). Article  CAS  PubMed  Google Scholar  * Mostellar, M. E. & Tuttle, E. P. Effects of alkalosis on plasma concentration and urinary excretion

of inorganic phosphate in man. _J. Clin. Invest._ 43, 138–149 (1964). Article  CAS  PubMed  PubMed Central  Google Scholar  * Müller, W. E. G. G., Tolba, E., Schröder, H. C. & Wang, X.

Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. _Macromol. Biosci._ 15, 1182–1197 (2015). Article  PubMed  Google Scholar  *

Sharif, P. S. & Abdollahi, M. The role of platelets in bone remodeling. _Inflamm. Allergy Drug Targets_ 9, 393–399 (2010). Article  CAS  PubMed  Google Scholar  * Boonrungsiman, S. et

al. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. _Proc. Natl Acad. Sci. USA_ 109, 14170–14175 (2012). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Badran, Z., Abdallah, M. N., Torres, J. & Tamimi, F. Platelet concentrates for bone regeneration: current evidence and future challenges. _Platelets_ 29, 105–112

(2018). * Peterson, J. R. et al. Treatment of heterotopic ossification through remote ATP hydrolysis. _Sci. Transl. Med._ 6, 255ra132 (2014). Article  PubMed  PubMed Central  Google Scholar

  * Hughes, E. A. B. B., Robinson, T. E., Bassett, D. B., Cox, S. C. & Grover, L. M. Critical and diverse roles of phosphates in human bone formation. _J. Mater. Chem. B_ 7, 7460–7470

(2019). Article  CAS  PubMed  Google Scholar  * Müller, W. E. G. et al. Nonenzymatic transformation of amorphous CaCO3 into calcium phosphate mineral after exposure to sodium phosphate in

vitro: implications for in vivo hydroxyapatite bone formation. _ChemBioChem_ 16, 1323–1332 (2015). Article  PubMed  Google Scholar  * Pellegrino, E. D. & Biltz, R. M. Calcium carbonate

in medullary bone. _Calcif. Tissue Res._ 6, 168–171 (1970). Article  CAS  PubMed  Google Scholar  * Opitz, P. et al. Insights into the in vitro formation of apatite from Mg-stabilized

amorphous calcium carbonate. _Adv. Funct. Mater._ 31, 2007830 (2021). Article  CAS  Google Scholar  * Jiang, J., Gao, M. R., Qiu, Y. H. & Yu, S. H. Gram-scale, low-cost, rapid synthesis

of highly stable Mg-ACC nanoparticles and their long-term preservation. _Nanoscale_ 2, 2358–2361 (2010). Article  CAS  PubMed  Google Scholar  * Song, Q. et al. Contribution of biomimetic

collagen-ligand interaction to intrafibrillar mineralization. _Sci. Adv._ 5, 9075–9104 (2019). Article  Google Scholar  * Nakayama, M. et al. Stimuli-responsive hydroxyapatite liquid crystal

with macroscopically controllable ordering and magneto-optical functions. _Nat. Commun._ 9, 1–9 (2018). Article  Google Scholar  * Qi, C. et al. ATP-stabilized amorphous calcium carbonate

nanospheres and their application in protein adsorption. _Small_ 10, 2047–2056 (2014). Article  CAS  PubMed  Google Scholar  * Meldrum, F. C. Calcium carbonate in biomineralisation and

biomimetic chemistry. _Int. Mater. Rev._ 48, 187–224 (2003). Article  CAS  Google Scholar  * Brečević, L. & Nielsen, A. E. Solubility of amorphous calcium carbonate. _J. Cryst. Growth_

98, 504–510 (1989). Article  Google Scholar  * Seknazi, E. et al. From spinodal decomposition to alternating layered structure within single crystals of biogenic magnesium calcite. _Nat.

Commun._ 10, 1–9 (2019). Article  CAS  Google Scholar  * Liu, Z. et al. Shape-preserving amorphous-to-crystalline transformation of CaCO3 revealed by in situ TEM. _Proc. Natl Acad. Sci. USA_

117, 3397–3404 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Qi, C., Musetti, S., Fu, L. H., Zhu, Y. J. & Huang, L. Biomolecule-assisted green synthesis of

nanostructured calcium phosphates and their biomedical applications. _Chem. Soc. Rev._ 48, 2698–2737 (2019). Article  CAS  PubMed  Google Scholar  * Dong, L. et al. Highly hydrated

paramagnetic amorphous calcium carbonate nanoclusters as an MRI contrast agent. _Nat. Commun._ 13, 1–13 (2022). Article  CAS  Google Scholar  * Beigi, R., Kobatake, E., Aizawa, M. &

Dubyak, G. R. Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. _Am. J. Physiol._ 276, C267–C278 (1999). * Sawada, M., Sridhar, K.,

Kanda, Y. & Yamanaka, S. Pure hydroxyapatite synthesis originating from amorphous calcium carbonate. _Sci. Rep._ 11, 1–9 (2021). Article  Google Scholar  * Nitiputri, K. et al.

Nanoanalytical electron microscopy reveals a sequential mineralization process involving carbonate-containing amorphous precursors. _ACS Nano_ 10, 6826–6835 (2016). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Shehab, D., Elgazzar, A. H. & Collier, B. D. Heterotopic ossification. _J. Nucl. Med._ 43, 346–353 (2002). PubMed  Google Scholar  * Isaacson, B. M.,

Brown, A. A., Brunker, L. B., Higgins, T. F. & Bloebaum, R. D. Clarifying the structure and bone mineral content of heterotopic ossification. _J. Surg. Res._ 167, e163–e170 (2011).

Article  PubMed  Google Scholar  * Uskoković, V. & Uskoković, D. P. Nanosized hydroxyapatite and other calcium phosphates: chemistry of formation and application as drug and gene

delivery agents. _J. Biomed. Mater. Res. B Appl. Biomater._ 96, 152–191 (2011). Article  PubMed  Google Scholar  * Uskoković, V. Disordering the disorder as the route to a higher order:

incoherent crystallization of calcium phosphate through amorphous precursors. _Cryst. Growth Des._ 19, 4340–4357 (2019). Article  Google Scholar  * Orriss, I. R. Extracellular pyrophosphate:

the body’s “water softener”. _Bone_ 134, 115243 (2020). Article  CAS  PubMed  Google Scholar  * Hamczyk, M. R. & Villa-Bellosta, R. Pyrophosphate metabolism and calcification. _Aging_

10, 3652–3653 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Buchet, R. et al. Hydrolysis of extracellular ATP by vascular smooth muscle cells transdifferentiated into

chondrocytes generates PI but not PPI. _Int. J. Mol. Sci._ 22, 1–16 (2021). Article  Google Scholar  * Merle, M. et al. Pyrophosphate-stabilised amorphous calcium carbonate for bone

substitution: toward a doping-dependent cluster-based model. _CrystEngComm_ 24, 8011 (2022). Article  CAS  Google Scholar  * Ben, Y., Blum, Y. D., Moshe, H. & Ashkenazi, B. Amorphous

calcium carbonate stabilized with polyphosphates or bisphosphonates. US patent US11052107B2 (2021). * Xu, Y. et al. Microscopic structure of the polymer-induced liquid precursor for calcium

carbonate. _Nat. Commun._ 9, 1–12 (2018). Google Scholar  * Sun, S., Mao, L. B., Lei, Z., Yu, S. H. & Cölfen, H. Hydrogels from amorphous calcium carbonate and polyacrylic acid:

bio-inspired materials for “mineral plastics”. _Angew. Chem. Int. Ed. Engl._ 55, 11765–11769 (2016). Article  CAS  PubMed  Google Scholar  * Sui, C. et al. Synthesis of mesoporous calcium

phosphate microspheres by chemical transformation process: their stability and encapsulation of carboxymethyl chitosan. _Cryst. Growth Des._ 13, 3201–3207 (2013). Article  CAS  Google

Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Healthcare Technologies Institute, School of Chemical Engineering, University of Birmingham, Birmingham, B15 2TT,

UK Cong Sui, Thomas E. Robinson, Richard L. Williams, Neil M. Eisenstein & Liam M. Grover Authors * Cong Sui View author publications You can also search for this author inPubMed Google

Scholar * Thomas E. Robinson View author publications You can also search for this author inPubMed Google Scholar * Richard L. Williams View author publications You can also search for this

author inPubMed Google Scholar * Neil M. Eisenstein View author publications You can also search for this author inPubMed Google Scholar * Liam M. Grover View author publications You can

also search for this author inPubMed Google Scholar CONTRIBUTIONS C.S. conceived of, performed, and analysed experiments, prepared figures and wrote the original manuscript, T.E.R. edited

the figures and wrote the revised manuscript, R.L.W. and N.E. acquired and analysed the human bone tissue, L.M.G. supervised the work and edited the manuscript. CORRESPONDING AUTHOR

Correspondence to Liam M. Grover. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Communications Chemistry_ thanks

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metabolism of adenosine triphosphate as an explanation for the chemical heterogeneity of heterotopic ossification. _Commun Chem_ 6, 227 (2023). https://doi.org/10.1038/s42004-023-01015-z

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