{"paper_id":"199cad04-c31a-45f6-b63c-373bfdc41d8f","body_text":"Wisnugroho et al. 2025   1 \n \nPolyanionic Non -Collagenous Proteins and Their \nAnalogues Promote Artificial Mineralization of  \nEmbryonic Mouse Bone \n \nMuhammad Wisnugrohoa,†, Fraser H. J. Laidlawb, Andrei V. Gromova, Colin Farquharsonc, and  \nFabio Nudelmana,*\naSchool of Chemistry, The University of Edinburgh, Joseph Black Building, Edinburgh, EH9 3FJ, UK  \nbSchool of Physics and Astronomy, The University of Edinburgh, James Clerk Maxwell Building, Edinburgh, EH9 3FD, UK \ncThe Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian, \nEH25 9RG, UK \n†Present address: Department of Physics, The University of Indonesia, Biophysics Laboratory, Depok, 16424, Indonesia   \n*Correspondence should be addressed to Fabio Nudelman. E-mail: fabio.nudelman@ed.ac.uk \n \nAbstract \n \nNon-collagenous proteins (NCPs) are specialized biomacromolecules within the extracellular matrix (ECM) that regulate the mineraliza-\ntion of calcified tissues, such as bone and dentin. Numerous in vitro studies have demonstrated that natural polyanionic NCPs and their \nanalogues can mediate intrafibrillar mineralization, characterized by the infiltration of apatite minerals into collagen fibr ils. However, \nthese studies primarily utilize self-assembled collagen fibrils or demineralized mature tissues, leaving it unclear whether pristine embry-\nonic bone ECM at a developmental stage permissive to mineral deposition can regulate intrafibrillar mineralization independen tly or \nrequires polyanionic NCP substitutes to promote the process artificially. To address this, we employed an ex vivo model of endochondral \nossification using metatarsals isolated from 15-day-old embryonic mice (E15). In addition to a supersaturated calcium (Ca) and inorganic \nphosphate (Pi) medium, we introduced fetuin -A, a native polyanionic NCP or poly-DL-aspartic acid (pAsp), commonly used as an NCP \nsubstitute. The incorporation of either additive was essential for the effective mineralization of embryonic metatarsals. Both fetuin-A and \npAsp played a direct role in facilitating the infiltration of Ca-Pi precursors into the avascular cartilaginous matrix. Raman spectroscopy \nand electron microscopy confirmed the formation of hydroxyapatite (HAp) exhibiting diverse levels of crystallinity, with fetu in-A sup-\nplementation resulting in the greatest HAp ac cumulation within the rudiments. HAp was localized in the perichondrium, a region con-\nducive to initial mineralization and enriched with a fibrillar network of collagen types I and II. Three -dimensional reconstructions im-\nplementing Dijkstra’s algorithm revealed the association between HAp and collagen fibrils either organized in an intrafibrillar, extrafi-\nbrillar, or combined arrangement. \n \nKeywords: Bone, Embryonic, Organ Explant, Mineralization, Non-Collagenous Proteins \n \nIntroduction \nBone is a calcified connective tissue that stores minerals, cre-\nates the skeletal system, and actively contributes to metabolic \nfunctions.1 In mammals, all long bones are formed via endo-\nchondral ossification, which is a replacement of hyaline carti-\nlage anlagen with bone tissue. 2 Collagen fibrils —the primary \nstructural units of cartilage and bone —are internally strength-\nened by minerals at the nano-structural level via a process called \nmineralization to improve their structural integrity and re-\nsistance to internal and external load s.3 Bone minerals consist \nof platelet-shaped hydroxyapatite (HAp) crystals with a length, \nwidth, and thickness of 12–100 nm, 10–40 nm, and 0.61–5 nm, \nrespectively.4–7 However, biogenic HAp typically exhibits a \nnon-stoichiometric phase, characterized by a high carbonate \n(CO32–) ion content, ranging from approximately 5.8% to 7.4% \nby weight, along with minor substitutions of other anions and \ncations, such as Na +, Mg 2+, K+, Cl−, and F −.8,9 This imbalance \nmakes biological HAp highly reactive and effective at exchang-\ning calcium (Ca), inorganic phosphate (Pi), and other ions, as \nbones are intricately connected with blood vessels and con-\nstantly exposed to blood flow.10  \nThe complex “intrafibrillar mineralization” mechanism by \nwhich nano-sized HAp crystals nucleate and grow within colla-\ngen fibrils during bone tissue formation has been studied for \ndecades.3,11–13 Recent in vitro  experiments have successfully \nachieved intrafibrillar mineralization by treating reconstituted \ncollagen fibrils with supersaturated Ca and Pi solution with \npoly-DL-aspartic acid (pAsp) as a negatively charged “polyan-\nionic” non -collagenous proteins (NCPs) substitute.14,15 This \npolyanionic macromolecule was demonstrated in vitro to inter-\nact with Ca and Pi ions in solution and prevent spontaneous nu-\ncleation of HAp crystals by stabilizing amorphous calcium \nphosphate (ACP) precursor phase via a polymer-induced liquid \nprecursor (PILP) process.14,16,17 This negatively charged precur-\nsor interacts with a positively charged region at the C -terminus \nend of the gap zones of collagen fibrils, which facilitates the \ninfiltration of ACP within the fibril and subsequent ACP to \nHAp transformation. 18 The PILP process was further demon-\nstrated to be used as a biomimetic system to study different pa-\nrameters controlling the mineralization process, such as the role \nof the polymer molecular weight 19 and extended beyond single \nfibril to promote the mineralization of dense collagen scaffolds \nand the remineralization of demineralized bone. 20,21 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   2 \nAdvanced remineralization studies using decalcified and chem-\nically fixed mature dentin tissue have shown that the native ex-\ntracellular matrix (ECM), which includes collagen fibrils and \nother associated macromolecules contains the information \nneeded to guide mineralization when exposed to metastable Ca-\nPi solution, resulting in the formation of ACP within the tis-\nsue.22 These experiments highlight the need to determine \nwhether the ECM of developing bone possesses inherent func-\ntional properties that regulate mineralization or whether the \npresence of polyanionic NCPs is necessary to trigger mineral \ndeposition within collagen fibrils. \nFetuin-A is an embryonic NCP with polyanionic characteristics, \nclassified as a phosphorylated glycoprotein (42 –68 kDa) and a \nmember of the cystatin superfamily.23–25 It consists of two struc-\nturally linked cystatin D1 and D2 domains, along with a third \ndomain rich in proline and glycine.26 At the onset of endochon-\ndral ossification, hypertrophic chondrocytes and cartilage ECM \nexhibit high expression of fetuin-A, suggesting its potential in-\nvolvement in tissue mineralization. 25,27 Additionally, this poly-\nanionic NCP normally circulates in the blood and extracellular \nfluid with a half-life of several days, to regulate the Ca -Pi pre-\ncursors and prevent spontaneous vascular calcification.28,29  \nPrevious comparative in vitro studies demonstrated that fetuin-\nA promotes intrafibrillar formation of HAp crystals in both the \nreconstituted collagen fibrils and decalcified bone tissue via \nmineralization by an inhibitor size exclusion (ISE) mecha-\nnism.12 During the ISE process, fetuin -A binds selectively to \nCa-Pi precursors30 and inhibits the apatite precipitation outside \nthe collagen fibrils. 12 This allows the precursors to exclusively \ninfiltrate the fibril and subsequently nucleate to form HAp crys-\ntals within the intrafibrillar compartments.12 The ISE route con-\ntrasts with the PILP process in which a liquid ACP precursor \nphase forms extrafibrillarly and enters the collagen fibrils via \ncapillary forces. 14 Despite the differences, both mechanisms \nhighlight the intricate interplay between collagen fibrils, poly-\nanionic NCP substitutes, and Ca -Pi minerals to control and fa-\ncilitate mineralization.12,14  \nNevertheless, all those studies were conducted either using self-\nassembly collagen fibrils 13–15 or decalcified mature tissue. 12,22 \nHence, it remains unclear whether the pristine and unmineral-\nized ECM of embryonic bone contains sufficient capability to \nregulate the mineralization directly or whether native polyan-\nionic NCPs and similarly charged macromolecule analogues are \nnecessary to promote artificial mineralization within embryonic \nbone. Accordingly, our present study aimed to investigate these \nissues using an ex vivo model of endochondral ossification in \nembryonic mouse metatarsals, an organ explant culture pio-\nneered by Burger and colleagues 31 with a novel upgrade on the \nincorporation of natural polyanionic NCP or its polymer ana-\nlogue. We hypothesized that the polyanionic -type macromole-\ncules: fetuin-A and pAsp are crucial to initiate mineral precur-\nsors infiltration and promote artificial minera lization within \nembryonic bone tissue. It is important to understand the role of \nthese negatively charged macromolecules and identify the best \npromoter of collagen mineralization during embryonic bone \nformation to aid the development of novel bio -inspired materi-\nals that are greatly beneficial in promoting fracture repair and \ntreating bone-related diseases. \n \nResults \nDevelopment of Embryonic Bone Mineralization Model \nThe 15th gestational days (E15) metatarsals were isolated as pre-\nviously described. 32 At this developmental stage, no matrix \nmineralization was observed at mid -diaphysis ( Figure 1 A). \nMoreover, the rudiments are still avascular and translucent, \nwith proliferating/hypertrophic chondrocytes, collagen type -II \nfibrils, and aggrecan-type proteoglycans as their primary com-\nposition.33,34 While metatarsals at E14 consist almost entirely of \nsmall-sized chondrocytes, the widening of lacunae or the begin-\nning of cartilage hypertrophy is seen in the mid -diaphysis of \nE15, which is the preliminary sign that the tissue is ready to \naccept mineral deposition.31 The cartilage matrix is pristine and \nunmineralized, and the bone collar is still absent compared to \nlater development stages.31,32 Small amounts of soft tissue, such \nas tendons and muscles remain attached to the outside of the \nrudiments, which are very difficult to remove during dissection. \nHowever, these soft tissues remove themselves after a few days \nin culture.  \nFrom this point onward, artificial mineralization of E15 meta-\ntarsals was conducted using four different incubation mediums \nat two observation times: 7 and 9 days -of-culture. The culture \nmediums were classified as cell culture medium in the absence \nof CaPi minerals (Control), medium with CaPi minerals only \n(CaPi), and medium with CaPi minerals plus polyanionic addi-\ntives (CaPi + pAsp and CaPi + fetuin-A). \n \nPhysical Appearance Change and Mineralization Features \nAfter 7 and 9 days in culture, the E15 metatarsals treated with \nCaPi + pAsp and CaPi  + fetuin-A became stiff and appeared \nwhite under the microscope compared to the Control and CaPi \nsamples ( Figure 1 B). Since all samples were washed thor-\noughly with water before observation, this physical transfor-\nmation may indicate mineral deposition within the metatarsals. \nIt should be noted that the metatarsals cultured in the CaPi me-\ndium (i.e., supersaturated Ca and Pi solution with respect to \nHAp stoichiometry 35,36) did not appear white under dark field \n(DF) optical imaging, which reflect the absence of mineral \nwithin the tissue. Comparatively, there were fewer solid precip-\nitates observed in the bottom of the culture well of both samples \nsupplemented with pAsp and f etuin-A than in the CaPi -only \nsample. Altogether, this white color transformation, an increase \nin rudiment stiffness, and less mineral sedimentation in the cul-\nture well are the first indication of a mineralization within E15 \nmetatarsals treated with NCP substitutes. \n \nOrganic and Inorganic Phases Identification \nTo investigate both the organic and inorganic phases of E15 \nmetatarsals, Raman observation on a selected region of the mid-\ndiaphysis (Figure 1B) was conducted after 7 and 9 days-of-cul-\nture. The resulting 100 spectra from each sample mapping area \nwere averaged and normalized with respect to amide III band \n(~1250 cm –1) in agreement with previous studies. 37 The com-\nbined Raman spectra in the range of 250–3000 cm−1 region was \ndisplayed for both culture days ( Figure 1 C). The vibrational \nband assignments were identified and described (Supporting In-\nformation: Table S1). \nThe peaks corresponding to the collagen, namely amides, along \nwith the ring of proline, hydroxyproline, and phenylalanine \nwere present in all samples both at 7 and 9 treatment days (Fig-\nure 1C and Supporting Information:  Table S1). Amide I and \nIII bands are indicative of the collagen phase signatures and \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   3 \nwere observed across all treatment conditions. In contrast, am-\nide II band, which is associated with other proteins and lipids \nwas exclusively detected in the Control sample on both incuba-\ntion days. The presence of glycosaminoglycans (GAGs) could \nalso be identified by the peaks corresponding to the pyranose \nring at 1045 cm−1 and the symmetry O–SO3− bond at 1062 cm−1. \nImportantly, the peaks correspond to the v1, v2, v3, and v4 modes \nof phosphate (PO 43−) were present only in the samples incu-\nbated in CaPi + fetuin-A with little change from 7 to 9 days-of-\nculture. The appearance of these peaks indicates the presence of \nHAp crystals within the metatarsals. The spectrum of samples \nincubated in CaPi + pAsp after 7 days only displayed a low -\nintensity peak at 961 cm −1, corresponding to the v1 mode of \nPO43−, whose intensity increased slightly after 9 days. In addi-\ntion, the v2 and v4 peaks were absent at 7 days and at low inten-\nsity at 9 days of incubation in CaPi + pAsp, indicating that min-\neralization was slower than with CaPi + fetuin-A, with less HAp \nformed. The absence of the v1 and v4 modes of PO43− in the Con-\ntrol and CaPi samples showed that without additives no miner-\nalization of the embryonic bone tissue takes place. \nTaken together, these results imply that fetuin -A and pAsp can \nboth promote artificial mineralization of E15 metatarsals, with \nfetuin-A being more effective. It should be highlighted that Ra-\nman spectroscopy is a surface technique, with a beam \npenetration depth of approximately up to 12 µm. Therefore, \nelectron microscopy (EM) examination is necessary to evaluate \nmineral localization deep inside the metatarsals (discussed be-\nlow). \n \nCoils Arrangement and Intrafibrillar Mineralization \nA more ordered coils structural arrangement of collagen fibril \nis correlated to intrafibrillar mineralization, or it can also be in-\nterpreted as fibril preparation to receive minerals deposit.38 The \nquantification of coil arrangement level (CAL) values ( Equa-\ntion 1) on Raman spectra resulted in the highest overall value \non CaPi + fetuin -A treatment on both 7 and 9 days of c ulture. \nThe CAL mean values (Supporting Information:  Table S2 ) \nwere increased as follows: Control (0.511 ± 0.08) < CaPi + \npAsp (0.667 ± 0.07) < CaPi (0.669 ± 0.13) < CaPi + fetuin -A \n(1.078 ± 0.06) for day 7, and Control (0.534 ± 0.09) <  CaPi \n(0.641 ± 0.13) < CaPi + pAsp (0.705 ± 0.13) < CaPi + fetuin-A \n(1.411 ± 0.16) for day 9. With prolonged incubation time, the  \nCAL variability of the additive -supplemented samples in-\ncreased, signifying that the fibrils became more ordered with \ntime in culture as a result of mineral deposition within the fi-\nbrillar compartment. \nStatistical analysis revealed that samples with CaPi only and \nCaPi plus additives have a significant increase in CAL ( p < \n \nFigure 1. Optical images of E15 metatarsals on (A) day 0 in which the mineralized core was absent at the mid-diaphysis (black asterisk) and (B) \nafter 7 and 9 days-of-culture. The red boxes indicate the Raman mapping area in each sample. (C) Raman spectra of E15 metatarsals cultured for \n7 and 9 days with each line representing a different treatment: Control (black), CaPi (red), CaPi + pAsp (blue), and CaPi + fetuin-A (green) (Inset: \namide bands of collagen in 1200 –1700 cm−1 range). Amide II band (purple asterisk) was only visible in the Control samples at both incubation \ndays. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   4 \n0.001) compared to the Control sample (Figure 2A), and a sig-\nnificant increase ( p < 0.001) in the value for CaPi  + fetuin-A \nfrom day 7 to day 9 of culture ( Figure 2 B). The absence of \nPO43− peaks in the CaPi sample suggests that, even though no \nmineral was formed, enough Ca and Pi infiltrated into the tissue \nto induce coil arrangement changes in the collagen fibrils. \nThese observations also demonstrate the direct role of fetuin-A \nand pAsp in facilitating the infiltration of Ca-Pi precursors into \nthe avascular cartilage rudiments to affect coil arrangements \nand promote mineralization. \n \nMineral Development in Relation to Tissue Mineralization \nTo investigate the correlation between the formation of HAp \ncrystals and the collagen matrix, Raman quantitative analyses \non several aspects (Figure 2C–H) were determined and com-\npared. Tissue mineralization was evaluated by both the mineral \nto GAGs ratio (MGR) and the mineral to matrix ratio (MMR) \nvalues. The MGR distribution within the mineralized matrix of \nthe GAGs-rich ECM (Equation 2) depicted that while all sam-\nples with NCP substitutes have mineral deposition in their \nECM, there were differences in the mineralization level be-\ntween them; with fetuin-A supplemented samples producing the \nhighest overall MGR value on both culture days. The  MGR \nmean values (Supporting Information:  Table S2 ) were in-\ncreased as follows: Control = CaPi (null) < CaPi + pAsp (0.171 \n± 0.05) < CaPi + fetuin-A (2.785 ± 0.18) for day 7, and Control \n= CaPi (null) < CaPi + pAsp (1.091 ± 0.13) < CaPi + fetuin -A \n(3.912 ± 0.29) for day 9. All additive-treated samples were sig-\nnificantly different (p < 0.001) from Control and CaPi samples \n(Figure 2 C). Moreover, the MGR of the pAsp and fetuin -A \nsupplemented samples increased with time in culture ( p < \n0.001) suggesting a greater mineral deposition into the tissue \nwith prolonged culture time (Figure 2D). \nFurthermore, when the overall quantity of minerals was com-\npared to the total collagen matrix (MMR, Equation 3), the CaPi \n+ fetuin-A sample on both culture days also yielded the highest \noverall value when compared with the other treatment condi-\ntions. The MMR mean values (Supporting Information:  Table \nS2) were increased as follows: Control = CaPi (null) < CaPi + \npAsp (0.827 ± 0.24) < CaPi + fetuin -A (3.071 ± 0.36) for day \n7, and Control = CaPi (null) < CaPi + pAsp (1.104 ± 0.28) < \nCaPi + fetuin -A (4.151 ± 0.64) for day 9. The MMR for the \nCaPi + fetuin-A sample was higher (p < 0.001) when compared \nto the CaPi + pAsp sample ( Figure 2E). This also implies that \nthe rate of mineralization is faster in the presence of fetuin -A \nthan pAsp. Accordingly, when the culture time was prolonged \nto 9 days, the ratio increased (p < 0.001) further for both fetuin-\nA and pAsp treated samples ( Figure 2 F), indicating the in-\ncrease of tissue mineralization level during this period. \nMature minerals have higher crystallinity values due to the \ncrystal apatite having a more perfect lattice organization with \nfewer ionic substitutions or stoichiometric arrangement. 39 The \ncrystallinity index (CI ) ( Equation 4 ) of the CaPi + fetuin -A \nsamples was greater than that of the other samples at both cul-\nture time points. The CI mean values (Supporting Information:  \nTable S2) were increased as follows: Control = CaPi (null) < \nCaPi + pAsp (0.271 ± 0.04) < CaPi + fetuin -A (0.341 ± 0.01) \nfor day 7, and Control = CaPi (null) < CaPi + pAsp (0.313 ± \n0.01) < CaPi + fetuin-A (0.352 ± 0.01) for day 9. The lower CI \nin the pAsp compared to the fetuin-A supplemented samples (p \n< 0.001) is reflective of a well-defined and organized HAp crys-\ntal structures with fetuin -A addition (Figure 2 G). The \nrespective CI of both NCP supplemented samples increased ( p \n< 0.001) from day 7 to day 9 of the culture suggesting that crys-\ntal growth and maturation was a time dependent process (Fig-\nure 2H). \nIn general, tissue mineralization (MGR and MMR) has a paral-\nlel correlation to mineral crystallinity (CI). All quantitative \nanalyses yielded the same results, in which the CaPi + fetuin-A \nsample at both culture days resulted in the highest overall value \nin contrast to the other treatments. Concisely, artificial HAp \ncrystals formation inside E15 metatarsals was successfully ini-\ntiated and promoted by adding natural polyanionic NCP, such \nas fetuin-A (1 mg/mL) or its polymer substitute, such as pAsp \n(25 µg/mL) in a supersaturated Ca (2.5 mM) and Pi (1 mM) \nsolution. Collagen-associated mineralization occurred in fetuin-\n \nFigure 2. Statistical analysis of E15 metatarsals culture with (A –B) \nCAL, (C–D) MGR, (E–F) MMR, and (G–H) CI as compared to each \nspecific medium (left side) and culture time (right side). Zero (null) \nvalues for MGR, MMR, and CI in the Control and CaPi samples due \nto the absence of v1PO43− peaks. *p < 0.05; **p < 0.01; ***p < 0.001. \n. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   5 \nA supplemented cartilage as indicated by the highest coil struc-\ntural change and tissue mineralization is correlated with mineral \ncrystallinity, which reveals the interplay between col lagen fi-\nbrils, polyanionic NCP substitutes, and Ca-Pi minerals. \n \nMineral Localization in Cartilaginous Tissue \nTransmission electron microscopy (TEM), scanning -transmis-\nsion electron microscopy (STEM), and energy dispersive X-ray \nspectroscopy (EDX) observations were used to investigate min-\neral localization in the tissue sections. In accordance with the \nRaman measurement, mineral presence was absent in the Con-\ntrol and CaPi samples after 7 and 9 days of culture (Figure 3–\nTEM). Mineral was sparsely detected in the metatarsals in CaPi \n+ pAsp sample at 7 and 9 days -of-culture. In contrast, CaPi + \nfetuin-A revealed the presence of minerals throughout the inner \nregion (i.e., near cartilage core) of the metatarsals. These TEM \nfindings have also confirmed that mineralization takes place in-\nside the metatarsals rather than just on their surface. Minerals \nin the CaPi + pAsp and CaPi + fetuin -A samples have similar \nplate-shaped crystals with a width/thickness of about 5 –20 nm \nand a length of 30–100 nm. The minerals of the CaPi + fetuin -\nA samples were more neatly distributed along the collagen fi-\nbrillar network, a pattern not so obvious in the CaPi + pAsp \nsamples. In agreement with the previous data, this indicates that \nfetuin-A is a robust promoter of crystal apatite deposition. \nElemental mapping after 9 days -of-culture of the samples with \nNCP additives showed the presence of both Ca and P, confirm-\ning that the tissue is deposited with calcium -phosphate mineral \n(Figure 3–EDX). Combined with bright field (BF) and high an-\ngle annular dark field (HAADF) imaging, the analysis indicated \nthat in the CaPi + fetuin -A samples, the mineral was arranged \nadjacent to collagen fibrils ( Figure 3 –STEM). Overall, the \nmineral within the CaPi + fetuin-A sample was shown to local-\nize to the perichondrium, forming an electron-dense layer about \n30–40 µm thick (Figure 4A). As the perichondrium in E15 met-\natarsals contains both collagen types -I and -II fibrils, the min-\neralized collagen type -I fibrils within this layer represents the \ninitiation of the bone collar, which fully forms at later stages of \nbone development. 31,34,40,41 Within CaPi + fetuin -A supple-\nmented metatarsals, some regions of the collagen fibril ( d~20 \nnm) were mineralized, and a cloud -like mineral complex was \nattached to the fibril surface (Figure 4B). These findings are in \nagreement with the role of fetuin-A in promoting collagen min-\neralization.12,18 \nInterestingly, not only the collagen matrix but also the cells \nwithin the cartilage core of fetuin -A treated samples were \n \nFigure 3. Comprehensive TEM (day 7 and day 9) and STEM -EDX (day 9) observations on the inner region (i.e., near cartilage core) of E15 \nmetatarsals cultures. Irregular-shaped cells (blue asterisks) were adjacent to the unmineralized collagen fibrillar (green arrowhea ds) network in \nControl and CaPi samples. The presence of minerals (orange arrowheads) not associated with collagen fibrils was detected in t he CaPi + pAsp \nsamples. A distinguishable pattern between unmineralized (green arrowheads) and mineralized (red arrowheads) collagen fibrils was observed in \nCaPi + fetuin-A supplemented samples, which matched with their Ca and P distribution. BF: bright field; HAADF: high angle annular dark field; \nSTEM: scanning-transmission electron microscopy; EDX: energy dispersive x-ray. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   6 \nheavily calcified. The cells were relatively small ( d~4–8 µm) \nwith irregularly shaped morphologies ( Figure 4 C–D). These \ncells possibly undergo shrinkage, necrosis, and apoptosis be-\ncause the culture conditions were insufficient to maintain cell \nsurvival, which was followed by rapid mineral deposition and \nHAp crystallization within their apoptotic cytoplasm.  \n \nThree-Dimensional (3D) Structure of Mineralized Embry-\nonic Metatarsal \nTo reconstruct and visualize the calcified collagen matrix, fo-\ncused-ion beam scanning electron microscopy (FIB -SEM) to-\nmography was conducted on CaPi + fetuin -A treated samples \nsince these were the only ones that contained overt areas of min-\neralization. Large area mapping of a cross section through the \nmetatarsal using the backscattered electron (BSE) revealed the \ndistribution of HAp (Figure 5A), and two areas of the perichon-\ndrium at the mid-diaphysis were selected as the region of inter-\nests (ROIs) (Figure 5A inset) for tomography. In contrast to the \ninitial E15 metatarsal ( Figure 1 A) before culture ( L~0.85–1 \nmm), the cartilage rudiments become relatively longer ( L~1.2–\n1.5 mm) after being cultured with CaPi + fetuin-A (Figure 5A), \nindicating tissue growth. However, it is important to note that \nthis growth is minimal (Δ L~0.2–0.5 mm) in comparison to the \ntypical elongation of cultured embryonic cartilage rudiments \nduring endochondral ossification (ΔL~1.5–2 mm), likely due to \nculture conditions that were insufficient to support prolonged \ncell survival.31,32 \nA 3D reconstruction of the transverse volumetric stacks showed \nthat the perichondrium layer was heavily mineralized in the lat-\neral direction towards the cartilage core at the mid -diaphysis \n(Figure 5B). A cytoplasm portion of the irregular -shaped cell \nadjacent to the perichondrium layer was fully embedded in min-\neralized ECM, which is portrayed as a 3D mineralized peri-\nchondrium model. Parallel to the perichondrium layer a fila-\nmentous structure was observed from longitudinal image stacks \nand displayed as a 3D filamentous model (Figure 5C). This fil-\namentous structure was likely constructed from numerous col-\nlagen fibrils that aggregated into a collagen fiber together with \nHAp crystal deposited intra - or extrafibrillarly. It was difficult \nto differentiate between collagen fibrils/fibers and HAp crystals \nas they were closely packed together with similar image conflu-\nency and contrast. However, implementing a Dijkstra’s—short-\nest path method—algorithm42 (Supporting Information: Figure \nS2) to follow the trace or common path of the collagen fibrils \nand combining it with the previous model, it was revealed that \nnumerous smaller filamentous-like structures and rough apatite \nmorphology were responsible for the 3D collagen + HAp model \nat the perichondrium (Figure 5C and Supporting Information: \n \nFigure 4. Selected STEM (HAADF) images of E15 metatarsals culture with CaPi + fetuin-A addition. (A) The highly mineralized perichondrium \nlayer extends to the inner region of the cartilage tissue, where numerous calcified cells (yellow asterisks) reside. (B) The partially mineralized \ncollagen fibril (red arrows) with the mineral-fetuin-A complex adhered to the surface in a cloud-like structure (yellow arrowheads). At the cartilage \ncore, minerals were also heavily localized in (C) the cells and (D) some regions of the collagen matrix either still unmineralized (green arrowheads) \nor mineralized (red arrowheads). \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   7 \nVideo S1). Since collagen fibrils often interweave, branch, and \noverlap, this algorithm purposely computes optimal paths \nwithin two or more adjacent voxels that minimize intensity dif-\nferences or follow geometric constraints through noisy BSE \ndata. When the structural paths are discontinuous and the volu-\nmetric morphology is spherical at specific site, the algorithm is \nunable to reconstruct fibrils, resulting in HAp classification. \nTherefore, each filament structure was identified as a collagen \nfibril that had a length of approximately 1–6 µm. All these col-\nlagen fibrils were interconnected and embedded within dense \nHAp crystals. \nAccording to this 3D model, these HAp crystals were portrayed \nto be associated with collagen fibrils either organized in an in-\ntrafibrillar, extrafibrillar, or combined arrangement. This model \nprojection also reinforces the TEM and STEM results (Figure \n3 and Figure 4), indicating that HAp crystals in CaPi + fetuin -\nA samples were organized in a regular pattern along collagen \nfibrillar network. It should be highlighted that extrafibrillar \ncrystal arrangement is predominant within the perichondrium at \nthis time studied. Nevertheless, further evaluations are neces-\nsary to determine the exact location of HAp crystal deposition. \n \nDiscussion \nOur ex vivo embryonic metatarsal culture provides a highly suit-\nable and physiological ECM for investigating early bone min-\neralization. This model more accurately replicates the initial \nbone tissue mineralization process compared to earlier in vitro \napproaches, such as the mineralization of self-assembled colla-\ngen fibrils or the remineralization of demineralized mature bone \ntissue. Uncalcified embryonic metatarsal tissue at E15 provides \nan authentic and functional cartilaginous ECM template for \nmineralization.31,32 Moreover, the absence of blood vessels in \nthe tissue at this gestational age infers that the mineral infiltra-\ntion solely depends on a simple diffusion or convection mecha-\nnisms.33,34,40,43 These characteristics create an excellent model \nfor investigating the functional role of embryonic bone ECM \nand polyanionic NCP substitutes in regulating Ca-Pi precursors \nto promote tissue mineralization during endochondral ossifica-\ntion. To the best of ou r knowledge, this is the first ex vivo at-\ntempt to utilize natural polyanionic NCP or its analogue to pro-\nmote artificial mineralization of the embryonic long bone. \nDuring embryonic bone development, the blood carries the Ca-\nPi ions within the vascular network in its supersaturated and \n \nFigure 5. (A) Overlay area of E15 metatarsals cultured with CaPi + fetuin -A. Within the perichondrium of the mid -diaphysis (inset: close up \narea), the ROIs were selected for serial surface imaging in (B) transverse (blue dashed box) and (C) longitudinal (green dashed box) orientations \n(x, y, and z axes match between the direction in the inset and each 3D model). The representative 3D models from volumetric stacks were displayed \nas (B) a mineralized perichondrium model composed of the irregular cell (red) embedded within mineralized ECM (green) and (C) a filamentous \nmodel composed of calcified collagen fibrils orientated parallel to the perichondrium. The shortest path method or Dijkstra’s  algorithm imple-\nmentation on the filamentous model exposes a structure of collagen fibrils (yellow) embedded with in minerals (green semi-transparent volume). \nIntrafibrillar HAp crystals deposited within the projected collagen fibrillar network are indicated by blue arrows. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   8 \nbalanced state with respect to HAp stoichiometry, [Ca] = 2.05–\n2.58 mM and [Pi] = 0.78–1.49 mM.35,36 The avascular cartilage \nanlagen takes up the mineral precursors from the surrounding \nextravascular fluid after they are released from adjacent capil-\nlaries via paracellular pathways 44,45 and larger negatively \ncharged macromolecules (42–68 kDa), such as albumin and fe-\ntuin-A enter the interstitial fluid through the opening of the tran-\nsendothelial channel to continuously maintain the Ca -Pi ionic \nequilibrium and prevent spontaneous apatite nu cleation.46–48 \nAccordingly, we chose two polyanionic macromolecules \nknown to regulate the mineral precursors in vitro : pAsp (25 \nµg/mL with approximate size of 11 kDa or d~2.9 nm) and fe-\ntuin-A (1 mg/mL with approximate size of 64 kDa or d~5.3 nm) \nin addition to the serum-equivalent Ca and Pi concentrations of \n2.5 mM and 1 mM, respectively.12,14,35 \nThe first intriguing point of discussion is the inability of E15 \ncartilage rudiments to mineralize spontaneously upon exposure \nto a supersaturated CaPi solution, despite the rudiments pos-\nsessing a functional ECM and being at a developmental stage \nconducive to mineral deposition. It must be noted that these \nconditions are different from those of normal culture conditions \n(e.g. tissue grown in 95% air and 5% CO 2 supply31,32) where \nmatrix mineralization continues for several days. In this latter \ncase, the cells play a critical role in controlling matrix homeo-\nstasis and mineral deposition. In the case of our system, miner-\nalization only occurred within the tissue in the presenc e of ei-\nther fetuin-A or pAsp along a supersaturated CaPi medium. The \nhydrated state of cartilage maintains its structural integrity by \nintermolecular electrostatic -steric forces predominantly be-\ntween negatively charged chondroitin sulfate networks within \nthe GAGs chain.49 Together with small cartilage pores (d~6–14 \nnm), these properties may provide high resistance to fluid flow \nand water redistribution on the tissue because of low permea-\nbility.50–52 Moreover, in unloaded cultures, the mineral infiltra-\ntion rate into the tissue is also impaired in compliance with pre-\nvious studies.53 Therefore, it is unlikely that supersaturated Ca-\nPi medium alone can promote spontaneous cartilage minerali-\nzation as observed in our work. Without a mineral regulator, \nlike fetuin -A or pAsp, Ca -Pi precursors nucleate faster and \nspontaneously precipitate within the solution rather than diffuse \ninto the cartilage anlagen.  \nThere is a possibility that when the Ca and Pi concentrations \nfall below those selected in our study (i.e., [Ca] < 2.5 mM and \n[Pi] < 1 mM), the precursors may not undergo spontaneous nu-\ncleation in solution, allowing them to infiltrate the tissue with-\nout the necessity of either fetuin-A or pAsp as a mineralization \nregulator. However, a cartilage ECM has been shown to tolerate \nsubstantially higher Ca concentrations in the presence of normal \nPi levels without inducing apatite precipitation, a phenomenon \nnot observed in aqueous solutions. 54,55 Under these conditions, \nprecursors would merely diffuse in and out of the tissue, making \nmineralization unfeasible. \nNative polyanionic NCPs and their analogues, such as fetuin-A \nand pAsp inhibit crystal nucleation and precipitation in solu-\ntion, subsequently promoting intrafibrillar mineralization of \ncollagen in vitro.12,14  Our results show that the incorporation of \nfetuin-A or pAsp within Ca and Pi-rich fluid is necessary to in-\nitiate mineral precursor infiltration and promote embryonic \nbone mineralization. One possible explanation is that these pol-\nyanionic macromolecules m ay form a semi -liquid or amor-\nphous complex with the Ca-Pi precursors in solution as widely \npostulated.12,14 This amorphous \"ACP -NCPs\" complex may \nadhere to the cartilage rudiment surface and progressively dif-\nfuse through the pores of a dense perichondrium layer to pro-\nmote mineralization.  \nOur Raman measurements at the mid -diaphysis region dis-\nclosed that the embryonic cartilage supplemented with addi-\ntives beside CaPi solution, especially with fetuin -A treatment \nresulted in similar spectra (Figure 1C) to the bone, even when \ncompared to calcified cartilage. 56,57 Additionally, significant \nchanges to the coil structural arrangement (Figure 2A–B) indi-\ncated that the collagen fibrils become more ordered due to the \ndeposition of crystals within the fibrils thus aligning with pre-\nvious studies. 38 It is noteworthy that CaPi -only treatment also \nyielded a change in the coil structure without mineralization and \nthe value is comparable to that observed in the CaPi + pAsp \ntreated rudiments. In contrast, the CaPi + fetuin-A gave a higher \nvalue that may a ccount for the association of the mineral with \ncollagen fibrils, implying this association was also absent in the \nCaPi + pAsp samples. Although electron microscopy in combi-\nnation with 3D reconstruction showed the mineral following a \nfilamentous structure, indicative of its association with collagen \nfibrils (Figure 5C), it could not allow for a clear distinction of \nwhether it is deposited within the gap zones or on the fibril sur-\nface. Thus, we concur that the HAp crystals either organized in \nan intrafibrillar, extrafibrillar, or combined arrangement. \nIt is possible that in the presence of CaPi only, small quantities \nof mineral precursors can enter the cartilage rudiments and alter \nthe coil arrangement in the ECM. However, native NCPs, such \nas chondrocalcin, osteopontin, osteocalcin, and bone sialopro-\ntein are possibly absent in the cartilage tissue at this stage of \ndevelopment (i.e., primary ossification) and only synthesized at \na later ossification stage in accordance to previous in vivo stud-\nies.58–61 Therefore, it is likely that the unexpected mineral pre-\ncursors that bypass the tissue hydraulic permeability barrier and \ninfiltrate the cartilage anlagen without the support of polyan-\nionic additives cannot crystallize properly within collagen fi-\nbrils or they are only sufficient to alter the coil structural order \nmoderately. \nWe reveal that the perichondrium of the developing \"cartilage \nto bone\" anlagen is the specific region that is heavily mineral-\nized (Figure 4A). Our finding is similar to in vivo observations \nthat endochondral ossification of long bone begins with the for-\nmation of a mineralized \"bone collar\" layer around this site, \nwhich contains overlapping collagen type-I and type-II compo-\nsition at this gestational period.31,34,40 This finding is not surpris-\ning because it is established that type -II collagen plays an im-\nportant role during endochondral ossification of bone tissues. 62 \nRecent in vivo observations on the endochondral ossification of \nmice auditory ossicles and the bony labyrinth tissues demon-\nstrated that mineralization with high mineral density take place \nwithin the conjoint type-I and -II collagen network, suggesting \na potential link b etween the presence of collagen type -II and \nhypermineralization of bone during endochondral ossifica-\ntion.63 Proteoglycans associated with collagen type -II exhibit a \nhigher molecular mass compared to those bound to collagen \ntype-I fibrils. 64 Given the propensity of Ca ions to localize at \nproteoglycan sites, 65,66 the presence of collagen type -II within \nthe osteoid matrix—predominantly consisting of collagen type-\nI fibrils—may facilitate enhanced mineral deposition. This no-\ntion is supported by our observation of a highly mineralized re-\ngion within the perichondrium,  consistent with the elevated \nmineralization typically associated with type-I and -II collagen-\ncontaining tissues. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   9 \nAn important aspect to consider is which type of collagen in the \ncartilage anlagen contains the intrafibrillar deposition of crystal \napatite. Although collagen type-II has a similar 67 nm periodic \nspacing as collagen type -I in accordance to previous studie s,67 \ncollagen type -II cannot be mineralized intrafibrillarly in vitro \nby using pAsp. 68 Moreover, in vivo  cartilage mineralization \nstudies have also demonstrated that early crystal nucleation is \nlocalized within extrafibrillar sites, where proteoglycans are \nheavily distributed and not within collagen type -II fibrils. 65,69 \nTherefore, we propose that if intrafibrillar mineral deposition \noccurs inside E15 cartilage rudiments as the collagen becomes \nmore ordered following the increase of CAL values and as il-\nlustrated by the 3D model within perichondrium, it likely occurs \nexclusively within collagen type-I fibrils. \nOur work reveals that fetuin-A promotes embryonic bone min-\neralization more effectively than pAsp. Despite the similar pol-\nyanionic characteristics of pAsp to the acidic domain of NCP, \nthis macromolecule is not an NCP like fetuin -A. We assume \nthat fetuin -A o r other natural NCPs have specialized mecha-\nnisms for regulating the mineral precursors in the tissue, which \nare beyond the electrostatic attraction between mineral ions and \nthe charged surface of macromolecules.70 Furthermore, we con-\nsidered the possibility that both pAsp and fetuin -A facilitate \ncollagen mineralization of embryonic bone through the PILP \nprocess.14 However, the Raman spectra obtained are distinctly \ndifferent between each additive with the PO43− intensity of pAsp \nsignificantly lower compared to fetuin -A treatment. Smaller \npAsp molecules complexed with Ca-Pi precursors would be ex-\npected to penetrate faster and deeper into the dense-packed col-\nlagen fibrils. If the PILP mechanism is the optimal way for em-\nbryonic bone mineralization, pAsp should have a higher pro-\nmoting influence than fetuin -A and not otherwise. Hence, we \nhighlight that the ISE process, which emphasizes the regulation \nof mineralization dynamics as the most favourable route during \nembryonic bone mineralization, and this aligns with previous \nstudies.12  \nOverall, we demonstrate that artificial mineralization of embry-\nonic cartilage anlagen can be initiated and promoted with an \nexternal mineral supply and polyanionic NCP substitutes. Tis-\nsue mineralization is concentrated within the perichondrium, \nand the HAp crystals are associated with collagen fibrils either \norganized in an intrafibrillar, extrafibrillar, or combined ar-\nrangement. Nevertheless, both intra- and extrafibrillar mineral-\nization are fundamental to the development of embryonic bones \nand the determination of their final mechanical properties. \n \nConclusions \nThis study represents the first demonstration of embryonic bone \nartificial mineralization by utilizing natural polyanionic non -\ncollagenous protein or its analogue within an ex vivo model of \nendochondral ossification. While embryonic bone tissue pos-\nsesses an inherent molecular composition and functional ad-\nvantages, its extracellular matrix alone is insufficient to directly \npromote mineralization. However, the presence of a polyan-\nionic macromolecule, such as fetuin-A or poly-DL-aspartic acid \nwithin a supersaturated calcium -phosphate medium exerts a \nsignificant and direct influence on promoting embryonic bone \nmineralization, potentially by stabilizing the amorphous min-\neral phase. The mi neralized embryonic metatarsals have or-\nganic and inorganic phases that are comparable to the bone tis-\nsue with hydroxyapatite as the sole mineral phase according to \nRaman spectroscopy analysis. Electron microscopy observation \ncombined with a three-dimensional reconstruction depicted that \nhydroxyapatite crystals are heavily localized within the peri-\nchondrium in association with collagen fibrils either organized \nin an i ntrafibrillar, extrafibrillar, or combined arrangement. \nOverall, fetuin-A is an effective pr omoter of early mineraliza-\ntion during embryonic endochondral ossification. \n \nExperimental Section \nReagents and Solutions \nAll chemicals were purchased from Sigma -Aldrich (Dorset, \nUK), unless otherwise stated.  \n \nAnimal Welfare \nPregnant female C57BL/6 mice were purchased from Charles \nRivers Lab (Kent, UK) and maintained under conventional \nhousing conditions with 12 hours light/dark cycle. All animal \nexperiments were approved by The Roslin Institute’s Animal \nUsers Committee and th e animals were maintained in accord-\nance with UK Home Office guidelines for the care and use of \nlaboratory animals. \n \nIsolation and Culture of Embryonic Mouse Metatarsals \nPregnant female mice were sacrificed by cervical dislocation \nand their E15 embryos were collected following decapitation in \naccordance with home office guidelines in the UK. The middle \nthree metatarsals were isolated and dissected under a dissecting \nmicroscope in accordance with previous protocols.32 Through-\nout the dissection procedure, the metatarsals were kept under \npreparation medium, which composed of 0.8 mL alpha mini-\nmum essential medium ( α-MEM) (without ribonucleosides), \n10.45 mL sterile phosphate-buffered saline (PBS), and 22.5 mg \nbovine serum albumin (BSA) (Fraction V). \nFor the mineralization process, metatarsals were cultured in 24-\nwell plates with each well containing one metatarsal in 600 µL \nof specific culture medium with the final concentration as de-\nscribed (Supporting Information: Table S3). Each specific cul-\nture medium was buffered to normal blood pH 7.4 with 0.25 \nmM hydroxyethylpiperazine ethane sulfonic acid (HEPES) so-\nlution and incubated in closed environment under physiological \nbody temperature (37 °C) without a carbon dioxide (CO 2) sup-\nply. The culture medium was changed every 3 days throughout \nthe culture period and the rudiments were collected after 7 and \n9 days of culture. Metatarsals were individually washed several \ntimes with ultrapure water to remove excess mi nerals on the \nsurface and fixed with 2.5% glutaraldehyde in 0.1 M sodium \ncacodylate buffer solution pH 7.4 at 37 °C for 1 hour. Metatar-\nsals were then stored in 0.1 M sodium cacodylate buffer solu-\ntion pH 7.4 at 4 °C until required for processing. \n \nRaman Spectroscopy \nMetatarsals were characterized using a Thermo Scientific \nDXR3 Raman microscope equipped with 785 nm laser using \n10X objective with NA = 0.25 and 3 µm laser spot size. Each \nmetatarsal was analyzed following optimal measurement pa-\nrameters of 10 mW laser power and 12 s exposure time, with \neach spectrum as the average of 3 accumulations. Raman map-\nping of each metatarsal within an area of mid -diaphysis region \nwas carried out using 5 µm step between measurement points \nwith focus tracking under dark field (DF) imaging condition to \nproduce a consisted minimum 100 spectra. Spectra were \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   10 \nfluorescence corrected (5 th order) by the instrument OMNIC \nsoftware to exclude background variations. A total of resultant \n800 spectra (4 culture medium treatments for 2 time points) \nwere then processed by OriginPro 2024b software for subse-\nquent asymmetric least squares (ALS) backgr ound subtraction \nand 5th order fast Fourier transform (FFT) filter smoothing. \nFor quantitative analysis, integrated peak areas (ʃA) of the spec-\ntra were carefully selected (Supporting Information: Figure S1 \nand Table S4) based on the Raman assessment studies of carti-\nlage and bone tissue.37,56,57,71 The ʃA values under the normalized \ncurve within each band region were determined by OriginPro \n2024b software with interpolation to rectangle edges. Raman \nvisualization was made by averaging and normalizing all repre-\nsentative spectra relative to amide III band in tensity in accord-\nance with previous studies.37  \nCoil arrangement level  (CAL). A relative quantification to \nmeasure structural changes in collagen fibril as a consequence \nof intrafibrillar mineralization. 38 This value was calculated by \ndividing the α-helical (ordered) proteins band (Amide III) to the \nrandom-coil (disordered) proteins band (Amide I). \nCAL =\n∫𝐴Amide III\n∫𝐴Amide I (1) \nMineral to GAGs ratio (MGR). A relative estimation of the total \nmineralized matrix in comparison to ECM rich GAGs or rela-\ntive amount of bone to cartilage tissue .72,73 This value was de-\ntermined by dividing the pyranose ring-symmetric stretch of O-\nSO3− band (GAGs) to the primary phosphate band (v1PO43−). \nMGR =\n∫𝐴𝑣1PO4\n3−\n∫𝐴GAGs (2) \nSince the GAGs band highly overlaps with the tertiary phos-\nphate band ( v3PO43−) in agreement with previous studies37, all \nʃAGAGs values for each specific treatment were approximated \nfrom the subtraction with the ʃAv3PO43− value of pure HAp min-\neral spectrum ( rruff-database, https://rruff.info/hydroxylap-\natite/R050512). \nMineral to matrix ratio (MMR). A relative determination of the \noverall ratio between mineral and collagen in the tissue, which \nis correlated to the bone ash fraction. 74 This value was calcu-\nlated by dividing the primary phosphate band ( v1PO43−) to the \nα-helical (ordered) proteins band (Amide III). \nMMR =\n∫𝐴𝑣1PO4\n3−\n∫𝐴Amide III (3) \nCrystallinity index  (CI). A relative quantification of apatite \nphase, which represents the average size of crystallites, perfec-\ntion, and ordering in a crystal.75 This value was determined by \nusing the width of the primary phosphate band, which is math-\nematically described as the reciprocal of the full width at half \nmaximum (FWHM) of the v1PO43− band. \nCI = 4.9\nFWHM of 𝑣1PO4\n3− (4) \nThe value of 4.9 cm−1 corresponds to the average FWHM of the \nstandard magmatic apatite (Conodont or phosphatic microfos-\nsil) and biological apatite have CI value in range of ≤ 0.5.76  \nStatistical analysis. A two-way analysis of variance (ANOVA) \nwas performed with Bonferroni test to make a comparison of \nboth culture treatments and culture times for each specific quan-\ntitative value. Differences were considered significant at p < \n0.05, highly significant at p < 0.01, and very significant at p < \n0.001. \n \nElectron Microscopy \nFor electron microscopy, metatarsals were post-fixed in 1% os-\nmium tetroxide in 0.1 M sodium cacodylate buffer solution pH \n7.4 for 45 minutes, then washed in three (10 minutes) changes \nof 0.1 M sodium cacodylate buffer solution pH 7.4. Next, spec-\nimens were dehydrated in 50%, 70%, 90%, and 100% ethanol \nthree times for 15 minutes each, followed by two (10 minutes) \nchanges in propylene oxid e. Samples were then embedded in \nTAAB 812 epoxy resin. \nTransmission electron microscopy (TEM). The resin-embedded \nsamples were cut (1 µm thickness) on a Leica Ultracut ultrami-\ncrotome, stained with toluidine blue, and viewed in a light mi-\ncroscope to select suitable areas for investigation. The ultrathin \nsections (60 nm thickness) were cut from selected areas and \ntransferred to quantifoil TEM grids, stained in uranyl acet ate \nand lead citrate solutions, then examined with JEOL JEM-1400 \nPlus TEM. Representative images were collected on a GATAN \nOneView camera. \nScanning-transmission electron microscopy  (STEM) and en-\nergy dispersive X -ray spectroscopy  (EDX). Ultrathin sections \n(60 nm thickness) on quantifoil TEM grids were fitted on \nSTEM grid holder, then viewed using Zeiss Crossbeam 550 \nequipped with a field -emission gun operation on dual channel \nSTEM detector: bright field (BF) and high angle annular dark \nfield (HAADF), operating at 20 kV accelerating voltage with \n300 pA probes current and 3 mm working distance. Selected \nareas were further investigated for its elemental composition by \nEDX. \nFocused-ion beam scanning electron microscopy  (FIB-SEM) \nand serial surface imaging . The chosen sample in resin block \nwas attached to the carbon-covered stub, mounted to the holder, \nthen observed with Zeiss Crossbeam 550 field -emission gun \n(FEG) FIB-SEM using two detectors: secondary electron (SE) \nand backscattered electron (BSE), operatin g at 1 kV accelerat-\ning voltage with 200 pA probe current and 5 mm working dis-\ntance. Large area mapping of the sample using the BSE was \ncarried out first via ATLAS 5 softwar e to produce an overlay \narea of the metatarsal and specific region of interest (ROI) at \nthe perichondrium was selected. The stage was then tilted to 54o \nto set the sample to be perpendicular to the FIB column. A plat-\ninum layer was deposited on top of ROI to protect the sample \nsurface, then a trench was milled using FIB beam (30 kV; 15 \nnA) to create a cross -sectional of the ROI. After polishing of \nthe ROI cross-section, automated serial milling and slice imag-\ning took place to create both SE and BSE image sta cks. Each \nslice (20 nm thickness) was generated with 300 pA probe cur-\nrent with scan speed = 4, N = 40, dwell time = 300 µs, and pixel \nsize (X, Y) = 6.5 nm. \nImage analysis and three-dimensional (3D) reconstruction. The \nobtained BSE image stacks were carefully aligned and seg-\nmented using Avizo 9.0 and Dragonfly software, respectively. \nThe raw 3D model was then processed in Blender 3.6 software \nby a self -made geometry nodes (Supporting Information: Fig-\nure S2 ) based on Dijkstra’s shortest path algorithm. 42,77 This \nalgorithm was used to find the globally optimal fibril pathways \nbetween two or more selection (seed) points inside a given vol-\nume. The representative pathways were combined with the raw \n3D model to create a new volumetric 3D model to illustrate and \ndifferentiate between inorganic minerals and collagen fibrils. \nThe video of collagen fibrils pathway tracking and mineral lo-\ncalization (Supporting Information: Video S1) was generated \nand edited in Blender 3.6 software using the same image stacks \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 12, 2025. ; https://doi.org/10.1101/2025.06.10.658745doi: bioRxiv preprint \n\nPolyanionic Non-Collagenous Proteins and Their Analogues Promote Artificial Mineralization of Embryonic Mouse Bone \nWisnugroho et al. 2025   11 \nsegmentation of mineralized perichondrium layer in longitudi-\nnal direction which was transferred from Dragonfly software. \nThe video was rendered using cycles render engine and OptiX \ndenoising optimization with a high resolution of 1080p, 30 \nframes per second (fps), and very high contrast adjustment. \n \nAssociated Content \nData Availability Statement \nThe data that support the findings of this study are available \nfrom the authors upon reasonable request. \n \nSupporting Information \nThe Supporting Information is available. \nIntegrated peak areas (ʃA) selection, Dijkstra’s shortest path \ngeometry nodes, Raman spectra vibrational band assign-\nments and quantitative assessment values, and final concen-\ntration of mineralization mediums (PDF).  \nVideo S1 (MP4). \n \nAuthor Information \nCorresponding Author \n*Fabio Nudelman – School of Chemistry, The University \nof Edinburgh, Joseph Black Building, Edinburgh, UK;  \n: https://orcid.org/0000-0001-7464-4309;  \nEmail: fabio.nudelman@ed.ac.uk \nAuthors \n†Muhammad Wisnugroho – School of Chemistry, The \nUniversity of Edinburgh, Joseph Black Building, Edin-\nburgh, UK; : https://orcid.org/0009-0003-1761-6629  \nPresent Address: †Department of Physics, The University of \nIndonesia, Biophysics Laboratory, Depok, Indonesia \nFraser H. J. Laidlaw – School of Physics and Astronomy, \nThe University of Edinburgh, James Clerk Maxwell Build-\ning, Edinburgh, UK; : https://orcid.org/0000-0002-5907-\n0447  \nAndrei V. Gromov – School of Chemistry, The University \nof Edinburgh, Joseph Black Building, Edinburgh, UK; : \nhttps://orcid.org/0000-0001-9254-4192   \nColin Farquharson – The Roslin Institute and Royal \n(Dick) School of Veterinary Studies, The University of Ed-\ninburgh, Easter Bush, Midlothian, UK; : https://or-\ncid.org/0000-0002-4970-4039  \n \nAuthor Contributions \nThe manuscript was written through contributions of all au-\nthors. All authors have given approval to the final version of the \nmanuscript.  \n \nConflict of Interest \nThe authors declare no conflict of interest. \n \nAcknowledgements \nThe authors acknowledge financial support from the Biotech-\nnology and Biological Sciences Research Council (BBSRC) in \nthe form of an Institute Strategic Programme Grant \n(BBS/E/RL/230001C). The authors thank the Indonesia En-\ndowment Fund for Education (LPDP) from the Ministry of Fi-\nnance, Republic of Indonesia for granting the scholarship and \nsupporting this research. Electron microscopy data acquisitions \nwere performed at the TEM (the Wellcome Trust Multi -User \nEquipment Grant WT104915MA) and Cryo FIB-SEM (EPSRC \ngrant No. EP/P030564/1) facilities at the University of Edin-\nburgh. The authors also thank Martin Singleton and Stephen \nMitchell from Wellcome Trust Centre for Cell Biology, Univer-\nsity of Edinburgh for their assistance regarding the TEM usage. \nFor the purpose of open access, the authors have applied a CC-\nBY public copyright licence to any Author Accepted Manu-\nscript version arising from this submission. \n \nAbbreviations \nα-MEM, alpha minimum essential medium;  ΔL, length differ-\nence; ʃA, integrated peak areas;  3D, three -dimensional, ACP, \namorphous calcium phosphate; ALS, asymmetric least squares; \nANOVA, analysis of variance; BF, bright field; BSA, bovine \nserum albumin; BSE, backscattered electron; Ca, calcium; \nCAL, coil arrangement level; CI, crystallinity index;  CO2, car-\nbon dioxide; CO32–, carbonate; d, diameter; DF, dark field; E15, \nembryonic phase at 15th days; ECM, extracellular matrix; EDX, \nenergy dispersive X -ray spectroscopy;  EM, electron micros-\ncopy; FEG, field-emission gun; FIB-SEM, focused -ion beam \nscanning electron microscopy; FFT, fast Fourier transform; \nFWHM, full width at half maximum; GAGs, glycosaminogly-\ncans; HAADF, high angle annular dark field; HAp, hydroxyap-\natite; HEPES, hydroxyethylpiperazine ethane sulfonic acid; \nISE, inhibitor size exclusion; L, length; MGR, mineral to GAGs \nratio; MMR, mineral to matrix ratio; NCPs, non -collagenous \nproteins; pAsp, poly-DL-aspartic acid; PBS, phosphate -buff-\nered saline;  Pi, inorganic phosphate; PILP, polymer -induced \nliquid precursor; PO43−, phosphate; ROIs, regi on of interest; \nTEM, transmission electron microscopy; SE, secondary elec-\ntron; STEM, scanning-transmission electron microscopy;  \n \nReferences \n(1) Roohani, I.; Cheong, S.; Wang, A. 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