resulting contractile stresses. The correspond-
ing mineralization processes of the collagen
matrix and the formation of internal stresses
were monitored by in-operando x-ray scatter-
ing in the case of SrCO 3. A custom-made me-
chanical testing setup equipped with a reaction
chamber and an optical microscopy system
was used to investigate the stress generation
during mineralization of unmineralized turkey
leg tendons with SrCO 3 (Fig. 1A and fig. S1).
Additionally, the local formation and morphol-
ogy of the mineral were imaged by in-operando
Raman spectroscopy and electron microscopy,
respectively.
As a biological source of parallel collagen
fibrils known to be able to mineralize in vivo,
we used slices of unmineralized turkey tendon
and immersed them into a SrCO 3 precursor
phase. In our experiments, we used a solution
of 200mg/ml polyacrylic acid (PAA), 10 mM
Sr2+, and CO 32 −, whereby the PAA molecules
stabilize the ions in solution through thepolymer-induced liquid precursor process ( 7 ).
These precursors first infiltrated the collagen
fibrils of the tendon and subsequently acted
as nucleation sites, and thereby led to gradual
mineral deposition. Initial mineral agglom-
erates could be observed after 4 hours (Fig.
1B) with a roughly ovoid morphology. This
might be caused by the faster diffusion of
the precursor along the longitudinal direction
of tendon fibers than along the transverse
direction. Fully impregnated tendon slicesSCIENCEscience.org 8 APRIL 2022¥VOL 376 ISSUE 6589 189
Fig. 2. Stress generation in tendons in SrCO 3
solution with different pH values.(A) Contractile
stress of tendon slices as a function of time in
mineralizing solution (pH = 9.0, red curve; pH = 8.75,
dark red curve; pH = 8.5, olive curve) and without
PAA (teal curve). The inset shows the slope
(stress rate) of three curves with different pH
values at 12 hours after the start of mineralization.
(B) Scanning electron microscopy (SEM) image
of mineralized tendon. Fibrils show some extrafibrillar
mineral but did not shrink by dehydration. (C) SEM
image of tendon treated in salt solution without
PAA. Collagen fibrils are laterally shrunk by
dehydration and look almost like ribbons, because
there is no mineral precipitated inside.Fig. 1. Stress generation in tendons during min-
eralization with SrCO 3 .(A) Schematic of the in-
operando mechanical testing setup. The two ends of
a tendon slice are fixed by clamps. One of the
clamps is connected to a load cell to monitor the
change of stress. The tendon slices are immersed
in a reaction chamber containing mineralizing
solution; the shape evolution of tendon slices is
recorded by optical microscopy from the top.
(B) Time series of optical images from the top view
of a tendon slice at different reaction times of
mineralization. The black spots are mineralized regions.
They gradually grow to realize the full mineralization
of the tendon. (C) Contractile stress curves of a
tendon slice in various media (water, salt, solution).
Small peaks in the curve are caused by the exchange
of solution. (D) In-operando Raman mapping of a
mineralized region on the surface of a tendon
sample. The plotted signal (1080 cm−^1 ) indicates
the progression of crystalline SrCO 3.RESEARCH | REPORTS