were observed after 36 hours (Fig. 1B and
movies S1 and S2).
The progression of mineralization was mon-
itored by in-operando Raman scanning micros-
copy of regions close to the mineralizing front
(fig. S2). The spatiotemporal variation of the
line intensity from the symmetric stretching
mode v1 of carbonate groups (1080 cm−^1 )( 18 )
is shown (fig. S2D). Signal intensity started to
increase at 5 hours, and 20 min later a strongincrease of the signal was observed. This indi-
cated that the mineralization front crossed the
observation window at this moment. Moreover,
the gradual appearance of a crystalline phase
was indicated by an increasing intensity of
the characteristic band of SrCO 3 at 1080 cm−^1
( 19 ) (Fig. 1D).
When a tendon slice was placed between
force gauges, no stress developed in an aqueous
solution or a salt solution (Fig. 1C). When trans-
ferred to the mineralizing solution, the develop-
ment of a contractile stress appeared, gradually
increasing to a maximum of 7.8 MPa within
~96 hours. Figure 2A shows the measured
contractile stress as the result of intrafibrillar
mineralization and the influence of pH value
on the stress rate. The stress generation is cor-
related with the formation of (nanometer-sized)
crystals inside the collagen fibrils.
In the presence of PAA, the SrCO 3 precursor
infiltrated the collagen fibrils, as evidenced by
a comparison of Fig. 2, B and C. In Fig. 2C,
where mineral did not nucleate within fibrils,
dehydration flattened them, whereas in Fig.
2B, the fibrils stayed nicely cylindrical even
after dehydration because they were filled
with SrCO 3 mineral (fig. S3F). The surface of
collagen fibrils was also covered by SrCO 3 nano-
particles (Fig. 2B and fig. S3F), corresponding
to an extrafibrillar mineral coating.
We found that the content of SrCO 3 crystals
in mineralized tendon amounts to about 90 wt %
after mineralization at final conditions (fig. S4).
Microcomputed tomography (mCT) demon-
strated that minerals were deposited through-
out the whole body of the tendons (fig. S5A).
Wide-angle x-ray scattering (WAXS) on min-
eralized tendons and the integration of the
resulting two-dimensional (2D) patterns re-
vealed a strong scattering peak at 17.9 nm−^1 ,
corresponding to the (111) lattice planes of
SrCO 3 crystals (fig. S5B). The orientation of
minerals in tendons was evaluated by ana-
lyzing azimuthal profiles of the integrated
small-angle x-ray scattering (SAXS) patterns
(fig. S5C) ( 20 ) to determine therparameter,
which generally is used to characterize the
degree of alignment of platelet-like minerals in
mineralized tissues. The averagerparameter
of mineralized tendon is 0.45 ± 0.03 (n= 30),
which is comparable to that of lamellar bone
( 21 ), indicating a relatively high degree of or-
ganization of SrCO 3 mineral particles along the
long axis of collagen fibrils.
We describe three aspects that have a strong
influence on the mineralization process. First,
when using a precursor solution without PAA,
no stress generation was observed. In this case
no mineral phase was formed inside the fibrils
(Fig. 2C and fig. S3A), which indicated that
molecular interactions between collagen and
mineral inside fibrils were a prerequisite for
contraction. Small SrCO 3 particles were nu-
cleated only at the surface of the tendon, as190 8 APRIL 2022¥VOL 376 ISSUE 6589 science.orgSCIENCE
Fig. 3. In-operando synchrotron SAXS during tendon mineralization of SrCO 3 (force constant mode,
zero stress).(A) Schematic of in-operando setup for synchrotron SAXS. (B) Two-dimensional SAXS patterns
of mineralized tendon at different reaction times. First- and third-order reflections from collagen axial
staggering are marked in the upper left pattern. Two peak positions of the third order (marked by yellow
boxes) are acquired by Gauss fitting of scattering peaks after radial integration. (C) Integrated SAXS intensity
of two regions of the tendon, one that was mineralized and another that remained unmineralized after
8 hours, as a function of time. Intensities were obtained by integrating within the sector area marked by a
white line in (B). (D) Correlation between tissue and fibril strain in the mineralized and unmineralized
spots (negative strains indicate contraction). For easier comparison with (C), time is also indicated on the
upper horizontal axis. (E) Optical snapshot of a tendon after 4 hours of mineralization. (F) Schematic of the
evolution of strains during tendon mineralization. In the high-magnification image and the schematic,
the mineralized regions are marked by red dashed circles. In mineralized regions, a stress (yellow arrows)
toward the central areas is generated by the contraction of the collagen matrix (decreasingDspacing). Owing
to the inhomogeneity of mineralization, strains in the tendon are also inhomogeneous.
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