# Lack of embryonic skeletal muscle in mice leads to abnormal mineral deposition and growth

Isabella Silva Barreto, Marianne Liebi, Sophie Le Cann, Saima Ahmed, Leonard C. Nielsen, Tilman A. Grünewald, Hector Dejea, Viviane Lutz-Bueno, Niamh C. Nowlan, Hanna Isaksson

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## ► To cite this version:

Isabella Silva Barreto, Marianne Liebi, Sophie Le Cann, Saima Ahmed, Leonard C. Nielsen, et al. Lack of embryonic skeletal muscle in mice leads to abnormal mineral deposition and growth. Journal of Structural Biology, 2025, 217 (1), pp.108178. 10.1016/j.jsb.2025.108178. hal-05122116

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Submitted on 26 Jun 2025

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Research Article

# Lack of embryonic skeletal muscle in mice leads to abnormal mineral deposition and growth

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Isabella Silva Barreto<sup>a,1,*</sup>, Marianne Liebi<sup>b,c,d</sup>, Sophie Le Cann<sup>a,e</sup>, Saima Ahmed<sup>f</sup>, Leonard C. Nielsen<sup>d</sup>, Tilman A. Grünewald<sup>g,1</sup>, Hector Dejea<sup>a,h</sup>, Viviane Lutz-Bueno<sup>b,d</sup>, Niamh C. Nowlan<sup>f,i</sup>, Hanna Isaksson<sup>a</sup>

ARTICLE INFO

Keywords:

Mechanobiology

Hydroxyapatite

Zinc

Synchrotron imaging

X-ray scattering

X-ray fluorescence

ABSTRACT

Developing bones can be severely impaired by a range of disorders where muscular loading is abnormal. Recent work has indicated that the effects of absent skeletal muscle on bones are more severe early in development, with rudiment length and mineralization lengths being almost normal in muscle-less limbs just prior to birth. However, the impact of abnormal mechanical loading on the nanoscale structure and composition during prenatal mineralization remains unknown. In this exploratory study, we characterized the mineralization process of humeri from muscle-less limb embryonic mice using a multiscale approach by combining X-ray scattering and fluorescence with infrared and light microscopy to identify potential key aspects of interest for future in-depth investigations. Muscle-less humeri were characterized by initially less mineralized tissue to later catch up with controls, and exhibited continuous growth of mineral particles, which ultimately led to seemingly larger mineral particles than their controls at the end of development. Muscle-less limbs exhibited an abnormal pattern of mineralization, reflected by a more widespread distribution of zinc and homogenous distribution of hydroxyapatite compared to controls, which instead showed trabecular-like structures and zinc localized only to regions of ongoing mineralization. The decrease in collagen content in the hypertrophic zone due to resorption of the cartilage collagen matrix was less distinct in muscle-less limbs compared to controls. Surprisingly, the nanoscale orientation of the mineral particles was unaffected by the lack of skeletal muscle. The identified accelerated progression of ossification in muscle-less limbs at later prenatal stages provides a possible anatomical mechanism underlying their recovery in skeletal development.

# 1. Introduction

Bone is a dynamic tissue known to adapt to its local mechanical environment (Buckwalter et al., 1996; Frost, 2001). Its mechanical properties are dependent on the structure and properties on all hierarchical length scales (Reznikov et al., 2018). It has been shown that fetal immobilization can have dramatic effects on the developing skeleton

(Nowlan et al., 2007; Sharir et al., 2011), with the mechanical environment affecting the mechanical competence of the mineralized tissues. For instance, multiple neuromuscular diseases which cause reduced or complete lack of fetal movement in utero lead to bones more prone to fracture in infants and children (Rodriguez et al., 1988; Ward et al., 2006).

The effects of reduced movement during embryonic bone

<footnote><sup>a</sup> Department of Biomedical Engineering, Lund University, 223 62 Lund, Sweden</footnote>

<footnote><sup>b</sup> Photon Science Division, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland</footnote>

<footnote><sup>c</sup> Institute of Materials, Ecole Polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland</footnote>

<footnote><sup>d</sup> Department of Physics, Chalmers University of Technology, Gothenburg, Sweden</footnote>

<footnote><sup>e</sup> CNRS, Univ Paris Est Creteil, Univ Gustave Eiffel, UMR 8208, MSME, F-94010 Créteil, France</footnote>

<footnote><sup>f</sup> Department of Bioengineering, Imperial College London, London, UK</footnote>

<footnote><sup>g</sup> European Synchrotron Radiation Facility, Grenoble, France</footnote>

<footnote><sup>h</sup> MAX IV Laboratory, Lund University, 224 84 Lund, Sweden</footnote>

<footnote><sup>i</sup> School of Mechanical and Materials Engineering, University College Dublin, Dublin, Ireland</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: isabella.silva_barreto@bme.lth.se (I. Silva Barreto).</footnote>

<footnote n="1">¹ Present address: Aix Marseille Univ, CNRS, Centrale Med, Institut Fresnel, 13013 Marseille, France.</footnote>
development have been widely investigated in chick, mouse and
zebrafish models (Nowlan et al., 2010). A common mouse model with
muscle-less limbs is the Pax3<sup>Spd/Spd</sup> mutant, in which the lack of the
transcription factor Pax3 prevents migration of muscle progenitor cells
into the limb buds and consequently prevents the formation of skeletal
muscles (Franz et al., 1993). The resulting morphology of the developing
bones in this and other similar mice models is altered, where initially
smaller mineralized regions, more circular cross-sectional areas and
absence of or modified deltoid tuberosities are assumed to mainly be a
consequence of reduced mechanical stimulation (Sharir et al., 2011;
Nowlan et al., 2010; Gomez et al., 2007). Our group recently used
synchrotron-based phase contrast microtomography to quantify the ef-
fects of absent skeletal muscle on the growth plate which governs the
longitudinal bone growth, and bone mineralization patterns at the
microstructural level (Pierantoni et al., 2021). When skeletal muscles
were absent, a decreased volume of the mineralized region and an
altered bone geometry were observed during the early stages of bone
formation (Pierantoni et al., 2021; Sotiriou et al., 2022). Towards the
end of embryonic development these alterations became less pro-
nounced, indicating that the delayed mineralisation was partly recov-
ered (Pierantoni et al., 2021; Sotiriou et al., 2022). During these early
stages of bone formation, the density and size of the chondrocytes at the
growth plate were also altered as a result of absent skeletal muscle. To
improve our understanding of both healthy and pathological bone for-
mation, elucidating the mechanobiological characteristics on all hier-
archical length scales is crucial. However, our current knowledge on the
effects of muscular loading on the mineralization process down towards
the nanoscale is limited.

Long bones form through a combination of intramembranous (pri-
mary) and endochondral (secondary) ossification, where the former
corresponds to direct apposition of disorganized woven bone by osteo-
blasts and the latter by a gradual replacement of a cartilaginous template
(Olsen et al., 2000; Shapiro, 2008). In long bones, the primary ossifi-
cation centre is formed in the midshaft of the bone through intra-
membranous ossification, followed by radial growth through
intramembranous ossification of the bone collar and simultaneous lon-
gitudinal growth through endochondral ossification at the growth plate.
As development progresses, the disorganized woven bone is remodelled
into more well-aligned lamellar bone. Some aspects of the mineraliza-
tion pathway are still being debated, such as the origin, delivery system
and crystallization events of hydroxyapatite (HA, Ca₅(PO₄)₃(OH)) at the
sites of mineralization. Once at the mineralization site, HA has been
found to form small, spherulitic nanoscale mineral foci which then
elongate their c-axis along the direction of the collagen fibril long axes
and ultimately form cross-fibrillar mineral at the microscale (Buss et al.,
2020). Recent evidence suggests that the formation of the nanoscale
mineral spherulites starts in the interfibrillar compartment, to then
spread across and into the fibrils (Macías-Sánchez et al., 2022). The
stenciling principle introduced by Reznikov et al. (2020) describes a
controlled and spatially precise mineralization process on the sub-
micron scale of the skeletal regions to mechanically optimized loca-
tions and geometries, while at the same time keeping the neighbouring
connective tissue soft.

To study the structure and composition of bone at the nanoscale,
several high-resolution techniques can be used (Grünewald et al., 2024).
Two-dimensional scattering techniques such as small- and wide-angle
X-ray scattering (SAXS, WAXS) can estimate the size and orientation
of the nanoscale HA mineral particles (Peyrin, 2009). SAXS and WAXS
have been combined with conventional tomography (Wittig and Birke-
dal, 2022; Seidel et al., 2012), and more recently also with tensor to-
mography (SASTT, WASTT) (Liebi et al., 2015; 2018; Grünewald et al.,
2020) to study bone around implants and biomaterials (Liebi et al.,
2021; Casanova et al., 2023), mineral orientation and crystal structure
(Mürer et al., 2021; Grünewald et al., 2023). Using scattering and
electron microscopy techniques, mineral has been observed to reside in
both intra- and extrafibrillar spaces, with up to 80 % of the mineral

fraction being extrafibrillar (Reznikov et al., 2018; McNally et al., 2012; Schwarcz et al., 2014; Micheletti et al., 2023; Schwarcz et al., 2020), and a localized presence of misaligned extrafibrillar mineral particles with different lattice parameters has been found (Grünewald et al., 2020). The orientation of collagen fibres can be studied using polarized light, due to their intrinsic birefringence (Wolman and Kasten, 1986), and different types of polarized light microscopies (PLM) have been used on collagenous tissues such as bone (Bromage et al., 2003). To quantify the molecular and elemental composition of bone, spectroscopy techniques such as Fourier-transform infrared (FTIR) (Boskey and Camacho, 2007) and X-ray fluorescence (XRF) (Peyrin, 2009) are often used. The primary element of interest in mineralized tissues is Ca, which is the main component of HA. However, trace elements such as zinc (Zn) and iron (Fe) are also of interest (Percival, 1999). Zn has been shown to play a particularly important role during bone growth and bone mineralization (Ortega et al., 2004; Litchfield et al., 1998; Kim et al., 2009; Yamaguchi, 1998), presumably through its role as a co-factor for matrix metalloproteases (MMPs) and alkaline phosphatase (ALP). A recent *in vitro* study on bone mesenchymal stem cells also found that the biomineralization of the surrounding extracellular matrix (ECM) starts with Zn-HA nucleation within the cell (Procopio et al., 2019). While the techniques presented above have been used to study bone in its mature state or in *in vitro* systems, there are very few to no studies using these techniques on embryonic bone development and none have investigated the mechanobiological effects of reduced loading during this process.

To improve our limited understanding of how muscular loading af-
fects bone formation at the nanoscale, this exploratory study aims to
identify key factors of early embryonic long bone mineralization which
could be impacted by the absence of skeletal muscle and thus should be
the focus of future in-depth studies. This was achieved using a multi-
modal approach quantifying the effects of muscle loading over extensive
areas, including the growth plate and bone collar, at multiple embryonic
development stages and across several length scales. Specifically, we use
a range of high-resolution scattering- and spectroscopy-based 2D and 3D
imaging techniques (SASTT, SAXS, WAXS, PLM, FTIR and XRF) to study
the maturation of the nanoscale structural organization in terms of both
HA and collagen, as well as the distribution of trace elements, in
Pax3<sup>Spd/Spd</sup> embryonic mice from start of mineralization to shortly
before birth.

## 2. Materials and methods

2.1. *Samples*

This study used mice (n = 45, C57BL/6 strain) with and without
skeletal muscles (Pax3<sup>Spd/Spd</sup>). Pax3<sup>Spd/Spd</sup> (Splotch-delayed) muscle-
less mice were interbred by spontaneous mating, where ~ 25 % of the
embryos become muscle-less (Tajbakhsh et al., 1997). The absence of
the transcription factor Pax3 prevents muscle progenitor cell migration
into the limb buds, which in turn prevents the development of limb
skeletal muscles (Franz and Kothary, 1993).

22 embryos with muscle-less forelimbs and 23 littermates with
normal skeletal muscle (controls) were harvested at different develop-
mental stages (Theiler stages TS) based on the Theiler morphological
criteria (Theiler, 1989). Stages were included from the first sign of
mineralization (TS23, approximately embryonic day 14.5) to shortly
before birth (TS27, approximately embryonic day 18.5). The experi-
mental protocol adhered to the ARRIVE guidelines and was conducted in
accordance with European legislation (Directive 2010/63/EU), under
project license number P39D18B9C. The humeri were carefully
dissected, fixed and stored in 70 % ethanol until embedding. The humeri
were divided into subgroups to be assessed with SASTT, SAXS, WAXS,
XRF, FTIR and PLM (Table 1) each with field of view (FOV) and spatial
resolution presented in Table 2. Some humeri were investigated with
multiple techniques.
<caption>**Table 1**
The number of humeri evaluated per technique and development stage.</caption>

The techniques used in this study to assess the structure and composition of humeri from muscle-less (M) forelimbs and their littermate controls (C) were: Fourier transform infra-red (FTIR) microspectroscopy and polarized light microscopy (PLM); small-angle X-ray scattering tensor tomography (SASTT); combined micrometre resolved small- and wide-angle x-ray scattering with x-ray fluorescence (µSAXS/WAXS/XRF); nanometre resolved x-ray fluorescence (nXRF).

<table><caption>**Table 2**</caption><thead><tr><th rowspan="2">Developmental stage</th><th colspan="2">TS23</th><th colspan="2">TS24</th><th colspan="2">TS26</th><th colspan="2">TS27</th></tr><tr><th>C</th><th>M</th><th>C</th><th>M</th><th>C</th><th>M</th><th>C</th><th>M</th></tr></thead><tbody><tr><td>FTIR/PLM</td><td>N<br>= 4</td><td>N<br>= 4</td><td>N<br>= 4</td><td>N<br>= 4</td><td>N<br>= 5</td><td>N<br>= 4</td><td>N<br>= 4</td><td>N<br>= 4</td></tr><tr><td>SASTT</td><td>N<br>= 1</td><td>N<br>= 1</td><td>N<br>= 1</td><td>N<br>= 1</td><td>N<br>= 1</td><td>N<br>= 1</td><td>N<br>= 1</td><td>N<br>= 1</td></tr><tr><td>µSAXS/WAXS/<br>XRF</td><td>—</td><td>—</td><td>N<br>= 1</td><td>N<br>= 1</td><td>—</td><td>—</td><td>N<br>= 1</td><td>N<br>= 1</td></tr><tr><td>nXRF</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>N<br>= 1</td><td>N<br>= 1</td></tr></tbody></table>

Approximate field of view (FOV) and spatial resolution obtained per technique. Given the time constraints and limitations of each technique, the FOVs were adapted or multiple FOVs were acquired when necessary to fit as much of the regions of interest as possible. The regions of interest were the growth plate / mineralization front, which spans a few hundred µm in length (Pierantoni et al., 2021), and the bone collar, which spans a few tens of µm in thickness (Sharir et al., 2011).

<table><thead><tr><th></th><th>Pixel size (µm)</th><th>FOV (µm)</th></tr></thead><tbody><tr><td>SASTT</td><td>25</td><td>500–1500 x 500–1500</td></tr><tr><td>FTIR</td><td>4.6</td><td>300 x 800</td></tr><tr><td>µSAXS/WAXS/XRF</td><td>2</td><td>500 x 500</td></tr><tr><td>PLM</td><td>0.5</td><td>1000 x 1000</td></tr><tr><td>nXRF</td><td>0.1</td><td>40 x 80–200</td></tr></tbody></table>

## 2.2. Synchrotron small-angle X-ray scattering tensor tomography (SASTT)

SASTT was performed on humeri from 8 forelimbs (Table 1) at the cSAXS beamline, Paul Scherrer Institute (PSI), Switzerland.

Measurements were conducted over a FOV including at least one mineralization front of a growth plate and the bone collar to evaluate both new bone formation as well as a few days older tissue. Scans were acquired using a beam energy of 11.2 keV, step size of 25x25 µm², exposure time of 30 ms and a sample detector distance of 2.165 m. The samples were scanned with isotropic angular sampling in both rotation and tilt directions, acquiring 2D projections of scattering patterns at equally spaced rotations between 0° and 180° at 0° tilt and between 0° and 360° at 6 tilt angles, while ensuring equal angular sampling (Liebi et al., 2015; Liebi et al., 2018). No substantial effect of radiation was identified on the mineral structure, as the SAXS signal remained unchanged between the start and end of each measurement (Supporting Information, Methods S1 and Fig. S1).

All scattering patterns were integrated in two q-regions related to the scattering from the mineral particles (Fig. 2.A). To evaluate mineral particle orientation, the reciprocal space map in each voxel was reconstructed in the q-range 0.5–0.6 nm⁻¹ (Fig. 2.A, green shaded area) using spherical integral geometric tensor tomography (SIGTT) (Nielsen et al., 2023) with the software package MUMOTT version 0.2 (Nielsen et al., 2023). To evaluate mineral particle thickness T, reconstruction using the zonal spherical harmonics tensor tomography (ZHTT) method (Liebi et al., 2015; 2018; 2021) was performed in 200 logarithmically spaced q-bins in the q-range 0.1–4 nm⁻¹ (Fig. 2.A, red line) and fitted assuming a platelet shape (Turunen et al., 2014; Bünger et al., 2010), as previously described (Silva Barreto et al., 2020). For more information on the SASTT measurements and data analysis, see Supporting Information, Method S1 and Table S1.

## 2.3. Synchrotron scanning small-/wide-angle X-ray scattering (SAXS, WAXS) and X-ray fluorescence (XRF)

Simultaneous micrometre resolved SAXS, WAXS and XRF was performed on humeri from 4 forelimbs (Table 1) at the ID13 beamline, European Synchrotron Radiation Facility (ESRF), France. Measurements were conducted over a FOV including at least one mineralization front of a growth plate and the bone collar, using a beam energy of 13 keV, 50 ms exposure time, spot and step size of 2x2 µm². Diffraction patterns were collected using an Eiger 4 M X detector at a sample-detector distance of 93 mm and XRF spectra were collected using a single element Vortex EM detector (Hitachi High-Technologies Corp, US) at a sample-detector distance of 20 mm and at an angle of approximately 70° with respect

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Morphological representation of controls and muscle-less limb humeri. Representative microscope images of humeri from control and muscle-less limbs at TS23 (A) and TS27 (B). Sections from muscle-less humeri at TS23 exhibited sectioning artefacts such as folding, which did not occur in any of the control samples. The hypertrophic zones are indicated with black arrowheads (►◄) and the proliferative zone by an asterisk (*).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** 3D nanostructural orientation of mineralized tissue, based on SASTT. A) Radially integrated scattering intensity curve, showing the q-region used for orientational analysis with SIGTT (green shaded area), an example of a reconstructed reciprocal space map and the corresponding estimated nanoscale mineral particle orientation, as well as a fit of the I(q) curve for mineral particle thickness T (red line) after q-resolved ZHTT reconstruction. B) Average relative anisotropy and symmetric intensity of the mineral particles across all sample-related voxels. Error bars indicate the standard deviation within each sample. C) Glyph render at longitudinal (purple) and transversal (red) views of the mineralized humeri, reconstructed using SIGTT (band-limit $ℓ \le 6$), showing the main orientation of the nanoscale mineral particles (glyph direction), mean scattering intensity (glyph size) and relative anisotropy (color scale), respectively indicating the amount of crystalline mineral particles present and their degree of alignment. The deltoid tuberosity is visible in TSS26 control (asterisk). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

to the incident x-ray beam. The scattering patterns were analyzed using in-house codes in MATLAB (MathWorks Inc. US), following our previously described protocols (Silva Barreto et al., 2020) for mineral particle orientation, thickness T as well as the length L and width W of coherently scattering domains in the 002- and 310-direction (Fig. 3.A),

respectively. The µXRF spectra were analyzed using the XRF analysis program PyMCA (Solé et al., 2007) for elemental intensities at each scanned point, following previously described protocols (Silva Barreto et al., 2020). Based on our previous studies (Dejea et al., 2023; Silva Barreto et al., 2020; Sharma et al., 2024), Ca and Zn were chosen for
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** HA mineral particle dimensions acquired by 2D SAXS and WAXS. A) Radially integrated scattering intensity curve, showing the q-regions of SAXS and WAXS, as well as the fit related to thickness (T, red line) and the peaks related to length (L) and width (W) of coherently scattering domains in the 002- and 310-direction, respectively. B) Spatial distribution maps of mineral thickness in the control and muscle-less humeri at TS27, showing the regions of interest which were further analyzed; mineralization front (MF) and bone collar (BC). C) Average mineral particle dimensions of all sample-related points (per sample) at the first 50 µm of the mineralization front, in a region 100–200 µm further into mineralization as well as two areas of approximately 20x20 µm² each within the bone collar. Error bars indicate the standard deviation within each area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

more detailed micrometre resolved analysis (Fig. 5.A).

To gain more detailed information on the most active regions of mineralization, nanometre resolved XRF was performed at NanoMAX beamline, MAX IV, Sweden, on the two TS27 embryonic forelimbs imaged at ESRF. Measurements were conducted over smaller FOVs of the mineralization front at the growth plate, approximately 200 µm into mineralization and the bone collar, using a beam energy of 12 keV, 100 nm step size (~80 nm spot size) and 80 ms exposure time. The XRF spectra were collected using a single-element silicon drift detector (RaySpec, UK) coupled to an Xpress 3 pulse processor (Quantum Detectors, UK) at a sample-detector distance of approximately 25 mm and at an angle of 90° with respect to the incident x-ray beam. The sample was rotated 15° towards the XRF detector. The elemental concentrations at each scanned point were estimated in PyMCA (Solé et al., 2007) using a thin film standard (RF-C00, AXO Dresden GmbH, Germany), as previously described (Silva Barreto et al., 2020). Based on previous studies (Dejea et al., 2023; Silva Barreto et al., 2020; Sharma et al., 2024), Ca, Zn and Fe were chosen for more detailed analysis.

For detailed information on the SAXS, WAXS and XRF measurements

and data analysis, see Supporting Information, Method S2.

## 2.4. Polarized light microscopy (PLM)

To evaluate collagen fiber orientation, PLM measurements were conducted on humeri from 33 forelimbs (Table 1) using a birefringence imaging microscope (Exicor Birefringence Microimager, Hinds Instruments, Inc., OR, USA). Images were captured with a FOV including the whole humerus using a CCD camera and a 10X objective, resulting in a spatial resolution of 0.5 µm. A stroboscopic LED of wavelength 475 nm was used to calculate the Mueller matrix components in order to quantify and retrieve maps of the collagen fibers angle of their optical fast axis and retardance (phase shift between transmitted waves, related to alignment) (Han and Chao, 2006; Nichols et al., 2014). For detailed information on the PLM measurements, see Supporting Information, Method S3.
## 2.5. Fourier transform infra-red microspectroscopy (FTIR)

To evaluate the molecular composition, FTIR measurements were conducted on the same sections of the 33 humeri as measured with PLM. Measurements were conducted in transmission over FOVs of the growth plate (including the mineralization front) and the bone collar using a Bruker Hyperion 3000 IR microscope (Bruker Corp, USA) with a 64 x 64 focal plane array detector. Measurements were acquired with a spatial resolution of 2.3 µm, spectral resolution of 4 cm⁻¹ and 64 repeated scans. Mineral, collagen (matrix) and mineral to matrix ratio (M:M) were estimated from the spectra (Fig. 4.A), following previously described protocols (Silva Barreto et al., 2020). For detailed information on the FTIR measurements and data analysis, see Supporting Information, Method S4.

## 2.6. Statistics

For all data acquired in this study, mean and standard deviation were calculated. For the FTIR and PLM data, Wilcoxon rank sum test was performed to test for statistically significant differences between muscle-less and control humeri at each development stage. For the SASTT, SAXS, WAXS and XRF data, the low sample numbers make statistical testing unfeasible. Instead, we focus on descriptive findings that were similar across several developmental stages.

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Molecular composition at the growth plate and bone collar acquired by FTIR. A) Representative FTIR spectrum from one pixel within the bone collar, indicating the peaks related to phosphate (mineral) and amide I (collagen, matrix) content. B) Average mineral to matrix ratio (M:M) ratio in the bone collar, within two areas in each sample of approximately 40x40 µm². C) Representative collagen and mineral maps from growth plates at TS27, indicating the proliferative and hypertrophic zone, as well as the mineralization front (MF). The maps were aligned to the onset of mineralization (0) and longitudinally averaged from 200 µm into the unmineralized cartilage (−200) to 200 µm into the mineralized bone. D) Average longitudinal profiles of mineral (i) and collagen (ii) content, as well as M:M ratio (iii), across the growth plates and mineralization front. The profiles were aligned at M:M ratio of 1.5, which was found to be the best match of the onset of mineralization for all samples, stages and groups. The increased collagen content in the proliferative zone of the TS27 muscle-less humeri (arrowhead) and lack of synchronized decrease in collagen within the hypertrophic zone are indicated (dashed lines).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Spatial distributions of Ca and Zn content acquired by micrometre resolved XRF. A) Representative XRF spectrum from one pixel, indicating the peaks related to Ca and Zn (further analyzed by µXRF) content as well as Fe (further analyzed by nXRF). B) Combined spatial distribution maps of Ca (red) and Zn (green) of one control humeri and one muscle-less humeri at TS27; overlap of the two elements is yellow. White boxes represent the further analyzed regions of interest: first 50 µm of mineralization front (MF) and 100–200 µm into mineralization, as well as the bone collar (BC). C) Averaged Zn and Ca intensities within the regions of interest at TS24 and TS27, the error bars represent the standard deviation within the areas. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

### 3. Results

#### 3.1. Morphology

Microscopy images taken during the FTIR measurements showed a morphology of humeri from muscle-less limbs with less humeral features compared to controls (Fig. 1). This confirms data presented in earlier studies using histological methods of this and similar animal models (Sharir et al., 2011; Nowlan et al., 2010; Gomez et al., 2007; Pierantoni et al., 2021; Sotiriou et al., 2022; Ahmed et al., 2023). While the size of the mineralized region in muscle-less humeri were similar to their controls at TS27, alterations in the length of their hypertrophic zones were apparent (Fig. 1.B).

#### 3.2. 3D structural characterization

The 3D SASTT characterization of the nanoscale mineral particle orientation in the mineralized regions revealed that the average amount of crystalline mineral particles present (indicated by mean scattering intensity) in the muscle-less humeri and their alignment (relative anisotropy) were similar to controls during early mineralization (TS23-TS24, Fig. 2.B-C). The size of the mineralized region of the muscle-less humeri remained small until TS27 (Fig. 2.C), at which stage both the

size of the mineralized regions and the alignment of the mineral particles were similar to controls (Fig. 2.B-C). Both groups exhibited similar orientation of the nanoscale mineral particles across development (glyph orientation, Fig. 2.C). In both groups, the mineral particles exhibited a low degree of alignment with the long bone axis (Supporting Information Video S1 and Fig. S2). Instead, they were more diagonally oriented around the shaft at all development stages, as well as along structural features such as the deltoid tuberosity (TS26 control, asterisk, Fig. 2.C). At TS27 this orientation around the shaft was less clear and some regions of the bone collar showed a higher alignment with the long bone axis in both groups (Supporting Information Fig. S2, asterisks).

The 3D SASTT characterization of the nanoscale mineral particle thickness revealed that during early mineralization (TS23-TS24), the humeri from muscle-less forelimbs had thinner mineral particles than controls (Supporting Information Fig. S3, muscle-less: 1.45 ± 0.16 nm, controls: 1.60 ± 0.12 nm). However, by late mineralization (TS26-TS27) the particle thickness was similar (muscle-less: 1.66 ± 0.13 nm, controls: 1.66 ± 0.13 nm). Despite the similar particle thickness at TS26 and TS27, muscle-less humeri showed a more spatially homogenous distribution of thicker particles across the rudiment compared to controls, which instead exhibited more structural heterogeneity with thicker particles at the mineralization front as well as close to the forming bone collar (Supporting Information Fig. S3, arrowheads), with a sharp
difference between these and their lower values at the center of the bone
(Supporting Information Fig. S3, star).

3.3. 2D structural characterization

The 2D SAXS and WAXS characterization of the nanoscale mineral
particle dimensions retrieved from the fits of the mineral shoulder,
[002]- and [310] peaks (Fig. 3.A) showed that the mineral particles of
the humeri from muscle-less forelimbs exhibited a more widespread
spatial distribution of thicker particles at TS27 compared to controls
(Fig. 3.B, Supporting Information Fig. S4). As opposed to controls,
muscle-less humeri did not show a clear localization of thicker, longer
and narrower mineral particles in less mature regions such as the
mineralization front, compared to their bone collars (Fig. 3.C, Sup-
porting Information Fig. S4) (Silva Barreto et al., 2020). Additionally,
the muscle-less humeri exhibited an increasing trend between TS24 and
TS27 within the bone collar in all mineral particle dimensions (Fig. 3.C,
Supporting Information Fig. S4), with the muscle-less humerus at TS27
having on average larger particles in the bone collar compared to the
control.

In humeri from both control and muscle-less forelimbs at all stages studied by 2D SAXS and PLM, the collagen and mineral particles in the bone collar were aligned with the long bone axis (Supporting Information Fig. S5). At TS27, internal collagen and mineral were also aligned with the main directions of their internal trabecular-like structures. Initially, lack of muscle was associated with a lower degree of mineral particle orientation within the bone collar (Supporting Information Fig. S5.B) but did not appear to affect collagen alignment (Supporting Information Fig. S5.A). As development progressed, alignment increased in both groups and the muscle-less humeri caught up in mineral degree of orientation by TS27.

3.4. Molecular characterization

The 2D FTIR characterization of the phosphate (mineral) and
collagen (matrix) content (Fig. 4.A) revealed that the bone collar of
muscle-less humeri at TS23 and TS24 were in the lower range of mineral
to matrix ratios (M:M) compared to controls (Fig. 4.B). At the TS26 and
TS27, however, the M:M ratio was similar between the two groups, but
the muscle-less humeri exhibited a larger variation. Due to folding ar-
tefacts in the sections of the muscle-less humeri at TS23 (Fig. 1.A), we
focused the profile analysis of the growth plate on the other stages. At
TS27, the larger hypertrophic zones of the muscle-less humeri were
accompanied by extended regions of the characteristic decrease in
collagen content related to the degradation of the cartilage template
prior to the mineralization front (Fig. 4.C, Fig. 4.D, ii dashed lines). In
general, the collagen content was slightly elevated in muscle-less humeri
at TS27, in particular within the proliferative zone (Fig. 4.C, Fig. 4.D, ii
arrowhead). However, the typical decrease in collagen content within
the hypertrophic zone was less pronounced in the muscle-less humeri
compared to controls, as well as less spatially synchronized between
samples and development stages, as indicated by their flatter average
profiles (Fig. 4.D, ii). Especially at TS27, at which stage the decrease in
collagen was most clear and synchronized in the controls, the lack of
synchronization in the muscle-less humeri were demonstrated by a
larger standard deviation within this region.

The effects of absent skeletal muscle on the growth plate and
mineralization front were most pronounced at TS24, with clear differ-
ences in profiles (Fig. 4.D). The pattern of mineral development in
muscle-less humeri was also altered at the mineralization front of the
growth plate, resulting in an unchanged M:M ratio (~2 a.u.) across
development, as opposed to controls in which it increased (Fig. 4.D).

3.5. Elemental characterization

Micrometre resolved XRF characterization of the elemental

distributions of Ca and Zn revealed that the Zn distribution was spatially
similarly at TS24 in control and muscle-less humeri (Fig. 5 and Sup-
porting Information Fig. S6). At TS27, however, the muscle-less hu-
merus showed a homogenous spatial distribution of Zn almost
everywhere in the ECM, while in the control Zn was primarily localized
to the mineralization front and outlining the bone collar (Fig. 5.B). In the
control, the highest Zn content was found within the first 50 µm of the
mineralization front, followed by a lower Zn content 100–200 µm into
mineralization (Fig. 5.C). In the muscle-less humerus, the Zn content
was also high in the first 50 µm of the mineralized front but remained
elevated further into mineralization as well as on average within the
bone collar (Fig. 5.D). Ca remained low at the mineralization front
throughout development in both groups (Fig. 5.C), with higher Ca
content in the bone collar. At TS24, microscale Ca appeared lower in the
bone collar of the muscle-less humerus, but at TS27 it had reached
similar values as the control (Fig. 5.C).

Nanometre resolved XRF characterization of the elemental distri-
butions of Ca, Zn and Fe revealed that Ca was localized into clusters in
the ECM of the TS27 control and Zn together with Fe were mainly
present at the edges of these (Fig. 6.A-B). In the muscle-less TS27,
however, both Ca and Zn had a more homogenous distribution, similarly
as observed with micrometre resolved XRF (Fig. 5.B). In muscle-les
humeri, Zn increased further into the mineralized structures and only
Fe had an increased localization at their edges compared to their core
(Fig. 6.B). A lower local nanoscale Ca and a higher Zn concentration was
observed both at the mineralization front and in the bone collar of the
muscle-less humerus compared to the control (Fig. 6.C). In the bone
collar of the control, there was almost no Zn present.

4. Discussion

In this study, we assessed how lack of muscles affects the early
mineralization process in humeri from embryonic mice using a wide
range of novel high-resolution tissue characterization techniques. This
enabled us to identify changes in the mineral nanostructure, as well as
the molecular and elemental distribution with high spatial resolution.
To our knowledge, this study is the first to investigate the effect of lack of
embryonic skeletal muscles on bone formation using synchrotron X-ray
scattering and fluorescence, as well as infrared and polarized light mi-
croscopy. This exploratory multimodal approach provides a step to-
wards a better understanding of the mechanisms involved during
embryonic mineralization, highlighting potential aspects that needs
further focus in future studies. Interestingly, the findings of this study
indicate that lack of skeletal muscles could result in an abnormal min-
eral particle deposition and growth, with the muscle-less humeri
exhibiting seemingly larger mineral particles just prior to birth. This
could possibly be due to a more widespread distribution of Zn, indi-
cating more active but undirected ongoing mineralization throughout
the entire muscle-less humeri. Keeping in mind that these findings need
to be confirmed on a larger sample set, the implication of each identified
potential key aspect is discussed below.

4.1. Lack of skeletal muscles might lead to a delayed and dysregulated
mineral deposition and growth

During early mineralization, muscle-less humeri were smaller with
lower Ca content compared to controls (Figs. 2, 5). By TS27, however,
both the size of the mineralized region and overall Ca had increased to
similar levels as controls. Additionally, while the humeri of the controls
quickly attained a high level of mineralization, muscle-less humeri
exhibited a more spread out, but steady increase throughout the inves-
tigated development stages which ultimately reached similar levels as
controls (Fig. 4.B). These results indicate that the recovery in size is a
result from an upregulated mineral deposition in muscle-less humeri
some time between TS24 and TS27, at which point they seemingly de-
posit and form mineral substantially faster than controls. Their more
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Spatial distributions of Ca, Zn and Fe content acquired by nanometre resolved XRF. Approximate position of the different regions is indicated by the red rectangles on the schematics, enlarged for visualization purposes. A) Spatial concentration maps at the mineralization front in one control and one muscle-less TS27. All maps are saturated to highlight the internal structure. B) Spatial concentration maps of mineralized structures in the remodelling zone, approximately 200 µm into mineralization. Average profiles across the edge and into a mineralized structure (across regions indicated by red boxes on the elemental maps), normalized to the individual maximum values to highlight the trends in concentration. C) Average concentrations in the bone collar, within two areas in each sample of approximately 20x20 µm². (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

homogenously widespread spatial distribution of large mineral particles and their increase in particle size differed from controls (Fig. 3). As previously discussed (Silva Barreto et al., 2020), during normal development the particles instead become thinner and shorter between TS24 and TS27, with a clear localization of less elongated, bulkier particles at the mineralization front. That the larger mineral particles of muscle-less humeri were not accompanied by an increase in Ca could indicate that 1) instead of forming more particles, the deposited mineral aids the growth of those already present. As recent evidence suggests mineral formation to start outside of the fibrils (Macías-Sánchez et al., 2022) and a difference in lattice parameters between intra- and extrafibrillar particles (Grünewald et al., 2020), it could also indicate that 2) their ratio between intra- and extrafibrillar mineral are altered and that there is a potentially higher presence of newly formed mineral. Additionally, differently shaped particles could also cause an alteration in thickness and thus the results could also indicate that 3) the mineral particles are differently organized, with different packing degrees and shape. While this study cannot confirm these hypotheses, it is highly likely that not only one of these processes occur, but rather that the observed more widespread distribution of apparently larger particles is due to a combination of them.

## 4.2. Skeletal muscles might not govern nanoscale orientation during embryonic long bone development

It is well accepted that the nanoscale collagen and mineral in adult bone organizes according to the directions of the loads it is subjected to; where different skeletal muscle attachments are responsible for the specialized macroscopic shape and the predominant orientation in the shaft of long bones is parallel to their long axis (Buckwalter et al., 1996; Frost, 2001). Despite this, we did not find any effect of skeletal muscles on the nanoscale orientation during embryonic bone development at the probed spatial resolution (Fig. 2, Supporting Information Fig. S2). The nanoscale structure in both groups became increasingly more aligned with age, with both collagen and mineral following the direction of the structural features (Fig. 2, Supporting Information Fig. S5). This indicates that 1) lack of skeletal muscles does not override the genetic template for the structural organization of the mineralized fibrils, 2) there is another key player than solely skeletal muscles which influence their organization and 3) woven bone is not fully randomly organized, as the initially laid down woven bone has most likely not yet been remodelled into lamellar bone (the timespan from start of mineralization to the latest development stage prior to birth is about 4 days).
However, an increase in alignment could be seen internally in both
groups, with the structures at the mineralization front being less aligned
compared to more mature tissue such as the bone collar. This also in-
dicates that there may still be some minor loading induced on the bones
from the surrounding environment even when skeletal muscles are ab-
sent (Nowlan et al., 2012), which potentially is enough to guide the
developing bones at this stage.

Surprisingly, the SASTT characterization revealed that the nano-
structure was not oriented parallel to the long bone axis (Fig. 2) as
observed in the 2D PLM and SAXS measurements (Supporting Infor-
mation Fig. S5) and previously reported for adult bone (Buckwalter
et al., 1996; Frost, 2001), but instead around the shaft and following
structural features such as the deltoid tuberosity (Supporting Informa-
tion Video S1 and Fig. S3). While not directly comparable, a similar
difference in the alignment between new and older bone has also been
observed in 2-month old infants (Roschger et al., 2001). Additionally, a
recent study investigating bone fracture healing with SASTT (Casanova
et al., 2023) found that the nanostructure of the uninjured cortical
(lamellar) bone to be longitudinally oriented, while the newly regen-
erated (woven) bone exhibited a more twisted orientation similar to
what we found in the embryonic bones. This is also supported by recent
findings where 3D surface geometry was studied in vitro. Substrate
curvature was found to be important for tissue formation, which may
explain the developing twisted-plywood-like structures in tissues
(Schamberger et al., 2024) Most studies of postnatal and adult bone have
so far used 2D techniques to assess nanoscale orientation, but these
orientational differences between woven and lamellar bone, as well as
the observed discrepancies between 2D and 3D measurements in this
study, suggest that the current notion of the nanoscale mineral and
collagen being aligned with the long bone axis might be an over-
simplification. Additionally, it highlight the need for further 3D in-
vestigations and raises the question if the orientation of bone nanoscale
structure may be more complex than previously reported.

4.3. Refined bone modelling could be inhibited by lack of skeletal muscle

In the controls, the mineralization front exhibited highly localized clusters of Ca in the ECM, and the trabecular-like internal structures deeper into mineralization were outlined by Zn and Fe (Fig. 6). In the muscle-less humeri, however, Ca was instead distributed homogenously within the ECM. Fe was still localized to the edges of their internal structures, but also remained more elevated inside them while following the changes in Ca. Literature regarding the role of Fe in bone mineralization is sparse, but some *in vitro* studies have found that Fe deficiency affects bone fragility, architecture, turnover, cell differentiation and activity, matrix formation and mineralization (Balogh et al., 2018). In muscle-less humeri, Zn was not localized at the edges of the internal structures, but instead increased with distance into them. Zn has a stimulatory effect on bone formation (Yamaguchi, 1998) and Zn deficiency has been found to affect bone length and morphology of the growth plate (Kim et al., 2009). Zn is a co-factor to many MMPs crucial to bone mineralization (Ortega et al., 2004; Litchfield et al., 1998) as well as expressed by bone-lining cells responsible for modelling the internal tissue into trabeculae (Ortega et al., 2004). Additionally, Zn can substitute Ca in HA, which has been shown to occur during early stages of *in vitro* mineralization in bone mesenchymal stem cells (Procopio et al., 2019). While the data of this study cannot reveal in which form Zn and Fe are present, we and others have previously reported a clear localization of Zn during normal bone development to the regions where new mineral is being deposited (Silva Barreto et al., 2020; Brister et al., 2020), outlining heterotopic ossifications in healing tendons (Sharma et al., 2024), as well as in proximity to MMP13 during bone fracture healing (Dejea et al., 2023).

The specific localization of Zn in controls combined with their more
specialized microstructure (Fig. 5) and localized differences in mineral
particle size (Fig. 3) suggest that normal humeri undergo refined

microstructural modelling as their tissue matures. The more homoge-
nous distribution of Zn, Ca and larger mineral particles in muscle-less
humeri instead indicate that while they ultimately mineralize to a
similar extent, they do not undergo as controlled ossification as during
normal development. Additionally, the lack of a synchronized reduction
in collagen content within the hypertrophic zones of muscle-less humeri
(as shown by the flatter collagen profiles in Fig. 4.D ii) indicate that the
cartilage template is not broken down and mineralized in the same
organized manner as during normal development (Ortega et al., 2004).
Thus, skeletal muscles seem to promote mineralization of certain re-
gions, while inhibiting others. This finding adds to our current under-
standing of the Stenciling principle (Reznikov et al., 2020), which
describes how certain enzymes and inhibitors determine the location
and progression of mineralization by negative regulation (inhibiting the
inhibitor). In light of this principle, perhaps the presence of skeletal
muscles fine-tunes the negative regulation of bone mineralization, while
the lack of them results in an initially delayed, but ultimately unre-
stricted and unrefined mineralization.

4.4. Accelerated mineral deposition could provide an underlying mechanism for late-stage skeletal recovery in muscle-less limbs

The increased presence of Zn in the muscle-less humeri at TS27 in-
dicates that the mineralization process has been upregulated, leading to
enhanced mineralization across the rudiment. Well in line with the
findings of the current study, we previously showed that the difference
in mineralized size between muscle-less and control humeri is most
pronounced at TS24, and that the muscle-less humeri catch up by TS27
(Pierantoni et al., 2021; Sotiriou et al., 2022). Thus, a crucial process
occurs between TS24 and TS27, i.e. within only ~ 3 days, which results
in an increase in mineralized volume. As the embryos grow and the free
space within the uterus is reduced, passive mechanical stimuli may be
exerted on the bones from movements of their mother and control lit-
termates (Nowlan et al., 2012). The introduction of these more general
forces could provide the trigger behind the accelerated mineralization
towards later development stages, and its potentially more uniform
stimuli could explain a more spatially homogenous mineral formation.

4.5. *Limitations*

The allocated time provided by synchrotrons is highly limited. Thus,
the sample numbers assessed with these techniques were few and the
findings should be considered as indications to guide further in-depth
investigations. However, this was complemented with lab-based tech-
niques on both structure and composition where it was possible to assess
larger sample sizes. In this study, most samples were sectioned and
evaluated in 2D. This provided high resolution data, enabling the pos-
sibility to observe localized patterns at the mineralization front and
internal structures. However, evaluating 2D sections of 3D objects in-
creases the impact of local variations. This limitation was partly
compensated for by analyzing the mineral particle distribution and
thickness in 3D with SASTT. This novel technique clearly highlighted
information that was lost in 2D and demonstrated the need for future
studies to also incorporate 3D characterization techniques. SASTT in-
volves repeated radiation, and while we found no indication of sub-
stantial effect of radiation on the mineral structural parameters that
were evaluated in this study, there may have been damage on the
collagen through protein disintegration as recently presented (Sauer
et al., 2022). While we were able to include many complementary tissue
characterization techniques, the sample preparation methods did not
allow us to perform high-quality histology on the same limbs. However,
the animal model is reproducible and have been extensively character-
ized with histology in previous studies (Nowlan et al., 2010; Pierantoni
et al., 2021; Sotiriou et al., 2022; Ahmed et al., 2023). Additionally, the
focus of this study was on the mineral component of the bone. However,
the mineral organization and orientation are strongly linked to the
underlying collagen fibril network. Recently, Ahmed et al. (2023) showed that absence of embryonic skeletal muscles alters the localization and structure of several collagens (primarily II and X, but also I, V, VI and XI) in the developing embryonic long bone. However, the role of loading on the collagen network of the developing skeleton is still poorly understood and in need of further investigation, e.g. into the effect on fibril orientation.

## 5. Conclusions

This study provides new insights into how the mineralization process of long bones is affected by skeletal muscle. It highlights that a lack of skeletal muscle during prenatal bone formation could lead to a delayed but ultimately upregulated and unrefined ossification. This abnormal mineralization process could be one mechanism behind how bones in muscle-less limbs catch up in size with controls towards the end of embryonic development, but also why they still exhibit less optimized morphologies and abnormal mechanical properties. These potential key alterations due to lack of skeletal muscle provide insight into which aspects of embryonic mineralization that are in need of further investigation to improve our current understanding of the mechanisms involved during bone formation.

### CRediT authorship contribution statement

**Isabella Silva Barreto:** Writing – original draft, Methodology, Investigation, Formal analysis. **Marianne Liebi:** Writing – review & editing, Validation, Supervision, Software, Resources, Methodology, Investigation, Formal analysis. **Sophie Le Cann:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Saima Ahmed:** Writing – review & editing, Resources, Methodology. **Leonard C. Nielsen:** Writing – review & editing, Software, Resources, Methodology. **Tilman A. Grünewald:** Writing – review & editing, Resources. **Hector Dejea:** Writing – review & editing, Resources. **Viviane Lutz-Bueno:** Writing – review & editing, Resources. **Niamh C. Nowlan:** Writing – review & editing, Validation, Methodology, Funding acquisition, Conceptualization. **Hanna Isaksson:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

We acknowledge the Paul Scherrer Institut, Villigen, Switzerland for providing beamtime at the cSAXS beamline for the SASTT experiment under proposal 20170791. We acknowledge the European Synchrotron Radiation Facility, (ESRF) Grenoble, France for providing beamtime at the ID13 beamline for the µSAXS/WAXS/XRF experiment under proposal IH-SC-1584 and the Partnership for Soft Condensed Matter (PSCM) for support during the preparation of this experiment. We acknowledge the MAX-IV Laboratory for beamtime on the NanoMAX beamline for the nXRF experiment under proposal 20230100. Research conducted at MAX IV, a Swedish national user facility, is supported by Vetenskapsrådet (Swedish Research Council, VR) under contract 2018-07152, Vinnova (Swedish Governmental Agency for Innovation Systems) under contract 2018-04969 and Formas under contract 2019-02496. Lund University Bioimaging Centre (LBIC), Lund University, is gratefully acknowledged for providing experimental resources. We also thank Dr. Vivien Sotiriou at Imperial College London, Dr. Michiel Op de Beeck, Dr. Susanne Strömblad and Dr. Bryan Falcones Olarte at Lund University for support during the sample preparation of this study. This project

received funding from the Knut and Wallenberg Foundation (KAW Grant agreement no. 2017.022), the Royal Society through a International Exchange Grant and the European Research Council under the European Union's Seventh Framework Programme (ERC Grant agreement no. 336306). M.L. and L.C.N. acknowledge funding from the European Research Council (ERC Grant agreement no. 949301) for the development of the software package MUMOTT.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jsb.2025.108178.

### Data availability

Data will be made available on request.

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