Abstract

   [34]graphic file with name mt0c00555_0006.jpg

   Brushite cements are promising bone regeneration materials with limited
   biological and mechanical properties. Here, we engineer a mechanically
   improved brushite–collagen type I cement with enhanced biological
   properties by use of chiral chemistry; d- and l-tartaric acid were used
   to limit crystal growth and increase the mechanical properties of
   brushite–collagen cements. The impact of the chiral molecules on the
   cements was examined with Fourier-transform infrared spectroscopy
   (FTIR), X-ray diffraction (XRD), and scanning electron microscopy
   (SEM). A 3-point bend test was utilized to study the fracture
   toughness, and cell attachment and morphology studies were carried out
   to demonstrate biocompatibility. XRD and SEM analyses showed that l-,
   but not d-tartaric acid, significantly restrained brushite crystal
   growth by binding to the {010} plane of the mineral and increased
   brushite crystal packing and the collagen interaction area. l-Tartaric
   acid significantly improved fracture toughness compared to traditional
   brushite by 30%. Collagen significantly enhanced cell morphology and
   focal adhesion expression on l-tartaric acid-treated brushite cements.

   Keywords: collagen, brushite, chiral molecules, bone regeneration, bone
   cements, tartaric acid

Introduction

   The need for bone tissue replacements has grown significantly in recent
   years with supply not meeting demand. Autografts are limited, and
   allografts or transplants face issues such as disease transmission or
   immune rejection.^[35]1−[36]4 Synthetic biomaterials such as
   bioceramics can overcome these issues.^[37]1,[38]2,[39]4 This has
   particular relevance for high-load-bearing applications, e.g., in
   maxillofacial reconstruction, as they are similar in composition to the
   inorganic-mineral component of bone^[40]5 and have shown good
   mechanical properties as well as biocompatibility in vitro and in
   vivo.^[41]6−[42]10 Ideally the implanted biomaterial should degrade
   gradually with time, thus providing space for new tissue
   ingrowth,^[43]11 yet keep its load-bearing properties until new bone
   takes over its natural function.

   Hydroxyapatite (HA)-based materials have become gold standard
   bioceramics despite being nondegradable^[44]12,[45]13 in vivo. There
   are other promising materials like α- and β-tricalcium phosphate (TCP),
   but their resorption is too fast and nonuniform,
   respectively.^[46]9,[47]14,[48]15 Dicalcium phosphate dihydrate (DCPD),
   also known as brushite, is a promising alternative with both good
   resorbability in vivo and good biocompatibility.^[49]7,[50]16 However,
   brushite materials, similar to other bioceramics, are brittle and prone
   to in vivo mechanical failure.^[51]17 This can lead to particle release
   causing inflammation and deep vein thrombosis in extreme
   cases.^[52]18,[53]19

   In native bone, collagen (mainly type I), a ubiquitous mineralizable
   protein, compensates for the brittleness of its inorganic, mineral
   phase^[54]20,[55]21 and imparts strength and toughness to the native
   tissue.^[56]22 Studies suggest that damage to the collagen triple helix
   due to either radiation or a genetic disorder such as osteogenesis
   imperfecta (a gene mutation leading to incorrect formation of α
   collagen chains) has detrimental effects on the mechanical properties
   of bone.^[57]23 Collagen also promotes cell attachment and
   proliferation^[58]24−[59]27 through integrin binding (α1β1, α2β1,
   α10β1, and α11β1) to the GFOGER sequences of collagen.^[60]28
   Integrin–collagen interactions are vital as they allow cells to
   interact with their surroundings, which induces intracellular signaling
   directing cell behavior (e.g., differentiation, contractility, and
   motility).^[61]24,[62]28 Collagen–bioceramic composites have been
   primarily produced in the form of spongelike scaffolds, which, despite
   their impressive biological properties and high ductility, are not
   strong and stiff enough for high-load-bearing
   applications.^[63]24−[64]26,[65]29 To date, only one study has
   attempted to produce high-strength collagen–bioceramic cements.^[66]17
   However, despite showing increased cell attachment compared to plain
   brushite, the mechanical properties did not improve in wet conditions
   as would be found in the body.^[67]17 Therefore, there is an increased
   demand to develop resorbable bioceramic-based materials, which are more
   resistant to mechanical failure but can interact with cells to direct
   their behavior and initiate implant to bone remodeling.

   The mechanical properties of bone can be largely attributed to
   nanosized mineral particles and collagen fibers forming a staggered
   structure at the sub-nanometer level, which are the building blocks of
   the hierarchically structured bone tissue.^[68]30−[69]32 Noncollagenous
   proteins (NCPs), particularly those belonging to the small
   integrin-binding ligand, N-linked glycoprotein (SIBLING) family, e.g.,
   osteopontin and bone sialoprotein 2, are known to control bone mineral
   size and regulate intra- and interfibrillar
   mineralization.^[70]33−[71]36 Recent studies suggest that matrix
   proteins composed of chiral l-amino acids play an important mineral
   modulatory role and control mineral crystal nucleation, growth, and
   morphology of bioceramics through chiral recognition.^[72]37,[73]38 It
   is suggested that α-hydroxy carboxyl acid groups recognize the [101]
   dynamic step of brushite crystal growth,^[74]18,[75]38 allowing
   compounds such as l-(+)-tartaric acid (l-(+)-Tar), already used in the
   pharmaceutical industry, to have a similar mineralization modulatory
   role,^[76]18,[77]37,[78]38 which enhances the mechanical properties of
   bioceramics.^[79]18

   Brushite mineralization involves three dynamic steps along the [101,
   001], and [201] directions, which grow evenly. The even growth of
   crystals is retained when d-(−)-tartaric acid (d-(−)-Tar) is added.
   However, l-(+)-Tar interacts strongly with the dynamic step [101]
   ({020} crystal plane) and limits brushite crystal size.^[80]18
   According to computer simulations, tartaric acid has three interaction
   sides with brushite: a carboxyl group of the tartaric acid interacts
   with calcium (hydrogen bond) and HPO[4] (hydrogen bond). A hydroxyl
   group of the tartaric acid forms another hydrogen bond with
   HPO[4.]^[81]18 The main difference between l-(+)- and d-(−)-Tar is that
   the hydroxyl group that interacts with brushite is different. In the
   former, the hydroxyl group is adjacent to the carboxyl group. In the
   latter, the hydroxyl group is located further back along the C–C chain,
   resulting in a lengthier and weaker bond. Thus, d-(−)-Tar is unable to
   pin the [101] dynamic step and stop it from propagating. In contrast,
   l-(+)-Tar does and inhibits mineral crystal growth.^[82]18,[83]39 The
   theory behind this process is described in [84]Figure [85]1

Figure 1.

   [86]Figure 1
   [87]Open in a new tab

   Theory of the selective effect of chiral Tar on crystallization of
   brushite crystals based on previous findings:^[88]18 (A) Dynamic growth
   steps of brushite [001, 101, and 201] and its {010} plane represented
   on a cubic unit cell. (B) Representation of the atomic arrangement
   within the mineral. (C) Binding of d-(−) and l-(+)-Tar to the [101]
   growth step of brushite. The carboxyl group of tartaric acid interacts
   with calcium (hydrogen bond) and HPO[4] (hydrogen bond). The hydroxyl
   group of tartaric acid forms another hydrogen bond with HPO[4]. The
   hydroxyl group to the carboxyl group determines the strength of the
   Tar–brushite interaction, the closer the better, due to bond resonance.
   Thus, l-(+)-Tar, unlike d-(−)-Tar, binds stronger and inhibits [101]
   dynamic strep growth.

   In the present study, we engineered collagen–brushite cements with much
   improved mechanical and biological properties by taking advantage of
   chiral Tar’s ability to affect crystal subunit size. We demonstrate
   that even in the presence of collagen, l-(+)-Tar decreases mineral
   crystal size, while d-(−)-Tar fails to do so. This increases the
   fracture toughness of l-(+)-Tar- and not d-(−)-Tar-treated
   collagen–brushite cements. Thus, l-(+)-Tar allowed, for the first time,
   the production of collagen–brushite cements, which show enhanced
   biological and also mechanical properties compared to currently used
   brushite bioceramics.

Methods

Composite Cement Preparation

   Brushite cements were prepared using a standard protocol of a
   solid–liquid biphase reaction as described elsewhere.^[89]18
   β-tricalcium phosphate (β-TCP, 1.8 g) Ca[3](PO[4])[2] (Sigma-Aldrich)
   was mixed with 1.2 g of monocalcium phosphate monohydrate (MCPM)
   Ca(H[2]PO[4])[2].H[2]O (abcr GmbH, Germany) using a mortar and pestle
   until no clumps were visible. Gypsum CaSO[4]·H[2]O was also used in
   some cases and was added to the powder mixture at 100, 50, 34, 25, 17,
   10, 5, and 0% w/w. The cement’s liquid phase was prepared by hydrating
   (0.5, 1, 1.5, and 2% w/v) bovine tendon powder (Collagen Solutions
   Ltd., U.K.) in 0.5 M d-(−)-Tar and l-(+)-Tar overnight. Consequently,
   the mixture was homogenized using an IKA T18 Ultra-Turrax homogenizer
   (IKA-Werke GmbH, Germany) for 5 min on ice (1 min on, 20 s off). For
   some samples, a mixture of NCPs purified from bovine bone was also
   mixed into the collagen suspension at a concentration of 150, 100, or
   50 μg/mL (for the purification protocol, see section 2.2). Distilled
   water was used as the liquid phase in the control group. The cement
   reaction was induced by mixing, on a frozen glass slab using a spatula,
   the two phases at powder-to-liquid ratios (P/L) of 2.5, 3, 3.5, 4, and
   4.3 g/mL. After a workable paste is formed, it was transferred to molds
   for further processing and left at room temperature for 30 min. The set
   cements were removed from the molds and incubated in distilled water
   for 24 h at 37 °C.

NCP Purification

   Cortical bone was obtained from veal shank cross sections. The bone was
   cleaned from muscle and bone marrow and broken up into 1 g pieces.
   After freezing them in liquid nitrogen, they were ground into a powder
   using a biopulverizer (Biospec Products). The organic phase of bone is
   primarily collagenous with small amounts of NCPs. Thus, the collagenous
   phase was removed before the NCPs as described elsewhere.^[90]40 To
   extract collagen type I, the bone powder was incubated at 4 °C on a
   rotor in 4 M guanidine-HCl in a 50 mM Tris–HCl buffer (pH 7.4) for 48 h
   (50 mL/g of bone). The supernatant was removed daily by centrifuging at
   1000g for 15 min and stored at 4 °C for later use. NCPs were then
   extracted by incubating the remaining bone powder pellet in 0.5 M
   ethylenediaminetetraacetic acid (EDTA) in 50 mM Tris–HCl (pH 7.4) at 4
   °C for 48 h again removing and storing the supernatant daily. To
   prevent protein breakdown, extraction buffers were supplemented with a
   protease inhibitor cocktail (100 mg/mL benzamidine, 1 mg/mL leupeptin,
   and 0.05 M phenylmethylsulfonyl fluoride). The NCP extract from both
   days was combined and concentrated using a Centricon Amicon Ultra 15,
   Ultracel 10k centrifugal filter (10 kDa cutoff) (Merck Millipore,
   Ireland). Consequently, the concentrate buffer was changed to 5 mM
   ammonium bicarbonate (NH[4]HCO[3]) at pH 8, which supercharged NCPs to
   allow easier detection using mass spectrometry.^[91]41 All reagents,
   unless otherwise stated, were acquired from Sigma-Aldrich.

Proteomic Analysis: Liquid Chromatography-Mass Spectrometry (LC-MS)

   The NCP-containing extract was analyzed using a Thermo Scientific
   Ultimate 3000 HPLC and Orbital Fusion MS, quadrupole-Orbitrap-linear
   ion trap hybrid (Thermo Scientific) for 1 h. Data was visualized using
   Scaffold (Proteome Software Inc.) ([92]Supporting Information Table
   S1). Pathway enrichment analysis to determine the biological pathways
   of the identified NCPs was carried out using the Reactome plug-in of
   Cytoscape (Institute of Systems Biology) ([93]Supporting Information
   Table S2).

Fracture Toughness Testing

   Cement specimens for testing were rectangular with a V-shaped notch at
   the center (20 mm length, 4 mm width, 2 mm thickness, 1.6 mm deep
   notch). Before testing, each specimen was submerged in a water bath at
   37 °C for 24 h. Fracture toughness was determined according to a
   three-point bend test (ASTM specification E 399-90, single-edged
   V-notch beam (SEVNB) loaded in the transverse direction) carried out on
   a MACH-1 system (McGill University, Canada) using a 25 kg load cell and
   a speed of 0.3 mm/min (ASTM spec C 1421-10).

   The equation used to calculate the fracture toughness was the following
   graphic file with name mt0c00555_m001.jpg

   where
   graphic file with name mt0c00555_m002.jpg

   for which K[IC] = fracture toughness (MPa·m^1/2), P[Q] = maximum force
   (N), S = support span (m), B = specimen thickness (m), W = specimen
   width (m), and a = notch length (m).

   The SEVNB was preferred over other methods because it is a
   reproducible, inexpensive, and widely accepted method for testing
   porous ceramics.^[94]18 As ceramics are very brittle materials with
   very low strain to failure, only P[Q] and not strain is considered when
   calculating K[IC]. Nevertheless, the area under the force–displacement
   graphs were calculated for some specimens to examine the impact of
   collagen on cement ductility.

Compressive Strength Testing

   Cylindrical specimens (6 mm diameter, 12 mm in length) were prepared
   using a Teflon mold. To ensure they are flush on both sides, they were
   polished using 600 and then 800 grit paper. Before testing, each
   specimen was submerged in a water bath at 37 °C for 24 h. The
   compression test was carried out using a 2 kN Zwick Roell static
   material testing machine (Zwick Roell, Germany) at a compression head
   movement of 0.3 mm/min.

X-ray Diffraction (XRD)

   The cements produced were examined using an X-ray diffractometer (D8
   Discovery Bruker AXS, Karlsruhe, Germany), with a copper source at 40
   kV and 40 mA, to understand the impact of chiral Tar on the mineral
   crystals of the material. The diffraction spectra were obtained by
   measuring four frames with 300 s per frame (λ = 1.54056). EVA software
   (Bruker AXS, Karlsruhe, Germany) was used to identify the compounds
   present in the cements, and a python script was used to visualize the
   data obtained.

Fourier-Transform Infrared Spectroscopy (FTIR)

   The cements produced were analyzed using an FTIR spectrometer
   (PerkinElmer, Liantrisant, U.K.) to see if there is any interaction
   between chiral Tar and the brushite mineral. Cement pieces were ground
   up into a powder and placed onto the pedestal of the device. A
   transmittance spectrum was obtained using a wavelength range of
   400–4000 cm^–1 with a 1 cm^–1 resolution. The resulting spectra were
   analyzed using the know-it-all software (Biorad, Hercules) and plotted
   using a python script.

Scanning Electron Microscopy (SEM)

   Mineral particle size was analyzed through images taken using an FEI
   Inspect F-50 FE-SEM operated at 10 kV (FEI, Hillsboro). Sample
   conductivity was improved through sputter-coating a 4 nm layer of
   platinum using a Leica Microsystems EM ACE600 sputter coater (Leica
   Microsystems, Germany). The average crystal size was determined using a
   MATLAB (The MathWorks Inc., Natick) script.

Cell Culture

   Human bone marrow mesenchymal stem cells (hMSCs) (PromoCell GmbH,
   Germany) were expanded and maintained in cell medium (Dulbecco’s
   modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 1%
   fungizone, 1% penicillin/streptomycin, 2 mM l-glutamine, and 0.5%
   nonessential amino acids (NEEA) (100×)) at 37 °C with 5% CO[2] for 5
   days in a 75 cm^2 Corning culture flask. When 80% cells were confluent,
   they were trypsinized from the culture flask using trypsin–EDTA spun
   down at 1000 rpm for 5 mins. Afterward, they were resuspended in media
   before further processing. All reagents were acquired from
   Sigma-Aldrich, U.K.

   For cell attachment and morphology studies, cells were seeded onto the
   cements at a density of 5000 cells/cm^2 and incubated at 37 °C with 5%
   CO[2] in cell medium with no FBS for 4 h for cell attachment and
   morphology studies.

Immunostaining

   Cells were then washed with warm 1× phosphate-buffered saline (PBS)
   twice before being fixed with 4% formaldehyde at room temperature for
   15 min followed by permeabilization with a solution of 0.1% Triton
   X-100 in PBS at room temperature for 5 min. To block nonspecific
   binding, the samples were incubated in 1% bovine serum albumin (BSA) in
   PBS for 1 h at room temperature. After blocking, an anti-vinculin mouse
   primary antibody at a dilution of 1:500 in 1% BSA in PBS was added to
   the samples and incubated for 1 h at room temperature. The samples were
   consequently washed with 0.5% Tween 20 in PBS three times for 5 min. A
   Cy3 anti-mouse secondary antibody (1:200) (Jackson ImmunoResearch
   Laboratories Inc.), Phalloidin Alexa Fluor 488 (1:100) (Thermofisher
   Scientific, U.K.) in 1% BSA in PBS, was consequently added to the
   samples and incubated for 1 h at room temperature followed by another
   three 5 min washes with 0.5% Tween 20 in PBS. The nuclei of the cells
   were stained using Vectorshield-DAPI (Vector Laboratories) before being
   imaged with a fluorescent microscope (Zeiss, GmbH, Germany). All
   reagents unless otherwise stated were sourced from Sigma-Aldrich, U.K.

Statistical Analysis

   Statistical significance (P < 0.05) between groups was shown using
   analysis of variance (ANOVA) and post-hoc paired t-tests between
   individual groups using GraphPad Prism 8 (GraphPad Software).
   Corrections for multiple comparisons were done using the Holm–Sidak
   method.

Results and Discussion

Material Characterization of Brushite–Collagen Composite Cements with
Tartaric Acid

   The FTIR spectra of collagen–brushite cement samples treated with
   chiral Tar ([95]Figure [96]2
   A) show prominent carboxyl group peaks (COO−) at 1406 and 1589
   cm^–1,^ ^[97]42 which are absent in the control condition.

Figure 2.

   [98]Figure 2
   [99]Open in a new tab

   Summary of material characterization of brushite–collagen–NCP cements.
   (A) FTIR spectra of brushite cements produced conventionally (control)
   and brushite–collagen cements produced by adding l-(+) or d-(−)-Tar.
   Peaks associated with carboxyl groups in the 1400–1600 cm^–1 range can
   be clearly seen in both tartaric acid groups but are absent in the
   control group. The peaks appear shifted compared to the carboxyl group
   peaks present in pure d and l Tar. (B) XRD spectrum of the l-(+)-Tar
   group shows a decrease in peak intensity associated with the [010]
   plane of the brushite crystal compared to the control and d-(−)-Tar
   group. (C) SEM images and particle size analysis of the cements
   indicate that the particle size in the l-(+)-Tar groups (i, ii) is
   significantly smaller compared to the d-(−)-Tar (iii) and control (iv)
   groups. Legend: graph shows mean ± standard deviation (SD), n = 4
   (images), multiple t-tests with ***p < 0.001.

   This suggests the participation of chiral Tar in the cement reaction
   where COO^– interacts with the brushite crystal to form stable bonds.
   The equivalent carboxyl group peaks in pure Tar are observed at 1550
   and 1740 cm^–1, respectively^[100]18 ([101]Figure [102]2
   ). The shift in peaks confirms the formation of new interactions
   between Tar and brushite^[103]18 for both enantiomers of the molecule.
   The interaction of chiral Tar with brushite is consistent with previous
   findings^[104]18 and occurs despite the addition of collagen and NCPs.
   Strong brushite-associated phosphate peaks can be seen at 500–580 and
   980–1130 cm^–1^[105]43 in all conditions, suggesting that the cement is
   indeed composed of brushite. To examine a possible brushite–collagen
   interaction, an in-depth analysis of the FTIR spectra was carried out.
   The FTIR spectrum of l-(+)-Tar–brushite cement (no collagen) was
   subtracted from the spectra of l-(+)-Tar-treated collagen–brushite
   cements at collagen concentrations of 0.5, 1, and 2% w/v to reduce
   background noise. The resulting spectra were compared to the FTIR
   spectra of pure collagen and the l-(+)-Tar–brushite, no collagen, group
   ([106]Figure [107]3).

Figure 3.

   Figure 3
   [108]Open in a new tab

   2800–3600 and 1200–1700 cm^–1 ranges of the FTIR spectrum for pure
   (100%) collagen, l-(+)-Tar-treated plain brushite (LTa_control), and
   l-(+)-Tar-treated collagen–brushite cements (LTa_Col_0.5-2%) (from
   which the l-(+)-Tar-treated plain brushite control spectrum
   (LTa_control) was subtracted). Amide A and B associated with N–H
   stretching and asymmetrical CH[2] stretching, respectively, are the
   main characteristic collagen peaks in the 2800–3600 cm^–1 range. Pure
   brushite treated with l-(+)-Tar shows four water-associated peaks. The
   four water peaks can also be seen in the modified (subtracted) FTIR
   spectra of l-(+)-Tar-treated collagen-containing brushite cements;
   however, one (3275 cm^–1) is shifted toward the amide A peak,
   suggesting that it is a composite peak of amide A (3300 cm^–1) and −OH
   stretch (3270 cm^–1). The amide B peak at 2951 cm^–1 can also be seen
   in the l-(+)-Tar-treated collagen–brushite spectra. Within the
   1200–1700 cm^–1 range, the l-(+)-Tar-treated collagen–brushite
   composite cements do not showcase characteristic amide II and III
   collagen peaks but show a characteristic amide I peak at 1647 cm^–1
   with a shoulder at 1640 cm^–1 at higher collagen concentrations. The
   increased water-associated peaks in the composite cement samples
   despite the subtraction of control signify the existence of additional
   water in their structure likely due to collagen. (Note: Cements were
   stored in water and dried before analysis. However, some residual water
   might still have been present.) Legend: Label explanation; LTa (cement
   sample treated with l-(+)-tartaric acid), Col (cement sample contains
   collagen), and 2% (% collagen content in the liquid phase of cement
   reaction).

   In the 3600–2800 cm^–1 range, pure brushite shows −OH stretching at
   3536, 3481, 3270, and 3154 cm^–1 attributed to water in its
   structure.^[109]43 Pure collagen exhibits two major peaks in that range
   at 3300 and 2964 cm^–1 associated with amide A (N–H stretching) and
   amide B (CH[2] stretching), respectively^[110]44 ([111]Figure [112]3
   ). An amide A location at around 3300 cm^–1 signifies increased
   involvement of collagen amides in hydrogen bonds.^[113]44 The modified
   (subtracted) spectra of the brushite–collagen cements show all
   water-associated −OH stretching peaks, suggesting that more water is
   present in its structure compared to control, likely because of
   collagen-mediated water retention. Importantly, the 3270 cm^–1 peak is
   shifted to 3275 cm^–1, toward the collagen amide A peak ([114]Figure
   [115]3). A similar peak (at 3282 cm^–1) is also observed in a
   brushite–collagen composite sponge containing a high amount of collagen
   ([116]Supporting Information Figure 7), suggesting that the
   brushite–collagen cement peak at 3275 cm^–1 is likely associated with
   amide collagen groups, which form bonds with the brushite mineral. An
   additional peak at 2950 cm^–1 not observed in pure brushite but present
   in pure collagen can also be seen in the brushite–collagen cement
   groups, suggesting that collagen is indeed present.

   In the 1700–1200 cm^–1 FTIR spectrum range, brushite shows two
   water-associated peaks (−OH bending) at 1647 and 1210 cm^–1.^[117]43
   Collagen showcases peaks associated with amide I (carbonyl stretching),
   amide II (N–H bending), and amide III (C–H stretching) at 1600–1700,
   1539, and 1235 cm^–1, respectively ([118]Figure [119]3
   ).^[120]44 The FTIR spectra of the collagen–brushite cements show peaks
   at 1647 and 1207 cm^–1. This could be associated with additional water
   due to the presence of collagen, but particularly the peak at 1647
   cm^–1 could also signify N–H bending (amide I). This is further
   evidenced when magnifying the region and observing a second shoulder
   placed at 1640 cm^–1 particularly for brushite cements with higher
   collagen composition ([121]Figure [122]3). The equivalent amide I peak
   for the brushite–collagen sponge is seen at 1638 cm^–1 ([123]Supporting
   Information Figure S7). The amide II and III peaks could not be
   observed in the collagen–brushite cement groups. However, amide II and
   III peaks are smaller than amide I and often harder to identify
   particularly when the collagen content is low.

   Further evidence for the presence of collagen in the scaffold is
   revealed by an SEM image ([124]Supporting Information Figure S1) of the
   cement. Collagen fibers and “sheets” interact with the brushite
   crystals. Further analysis of SEM images shows that l-(+)-Tar
   effectively reduces the crystal size (1.6 ± 0.1 μm) of brushite, while
   d-(−)-Tar, despite interacting with the mineral, does not have a
   considerable effect on the crystal size (5 ± 0.3 μm) compared to the
   control group (4.5 ± 0.2 μm) ([125]Figure [126]1
   C).

   From a morphological perspective, the crystals showed the typical
   trapezoidal shape with large {010} faces in the control group
   ([127]Figure [128]2
   Ciii). d-(−)-Tar did not alter the size significantly, but the particle
   edges appeared more rounded ([129]Figure [130]2Civ). In the presence of
   l-(+)-Tar, brushite crystals exhibited a smaller, spheroid-like
   morphology without prominent {010} faces ([131]Figure [132]2Ci). The
   addition of NCPs did not further decrease the crystal size (1.8 ± 0.1
   μm) of brushite or affect its shape even at a concentration of 150
   μg/mL ([133]Figure [134]2Cii) despite the literature suggesting that
   NCPs have a significant impact on mineralization.^[135]35 It is hard to
   pinpoint the exact reason, but it is likely that the cement reaction is
   too fast for NCPs to affect mineralization at a low concentration. A
   study using osteopontin, an NCP, confirmed the protein’s ability to
   control mineralization using microliter amounts of reagent and allowing
   them to react for at least 48 h. Very high nonphysiological amounts of
   NCPs might influence the cement reaction. However, producing them at
   that scale was not feasible in this study.

   The XRD ([136]Figure [137]2
   B) spectra of the cements treated with chiral Tar confirm that the
   crystal size of the l-(+)-Tar groups, as shown in the previous
   work,^[138]18 is due to l-(+)-Tar binding to the {010} face, inhibiting
   its growth. Thus, effectively reducing the ratio between the basal and
   side faces yields a smaller particle. The normalized XRD pattern shows
   attenuated peaks, i.e., (020) and (040), related to the {010} face for
   the l-(+)-Tar groups. This attenuation is not observed in the d-(+)-Tar
   and control group. It should be noted that the brushite cement reaction
   is dependent on both the dissolution rate of β-TCP as well as brushite
   crystal growth. Previous studies showed^[139]18 that the chiral
   selection described is not associated with the dissociation step of
   β-TCP during the cement reaction, but only to the brushite dynamic
   growth step. The dissociation step is the same for both enantiomers of
   Tar.^[140]18 The addition of collagen did not influence the brushite
   cement reaction, and all characteristic brushite peaks were present
   without additional ones. It has been suggested that collagen can starve
   the cement reaction of water due to its hydrophilicity, leading to
   remnant β-TCP and MCPM peaks, which signal an incomplete
   reaction.^[141]17 However, collagen was fully hydrated overnight and
   homogenized, alleviating the issue.

Improvement of Mechanical Properties

   According to the Hall–Petch law, mechanical properties of hard ceramics
   are greatly increased if the crystal size is reduced.^[142]45
   Controlling the crystal size of brushite cements with l-(+)-Tar should
   allow for the production of brushite–collagen cements with mechanical
   properties that do not compromise them in wet conditions and are
   superior to current brushite materials. Indeed, the fracture toughness
   of collagen-containing cements made with l-(+)-Tar was not compromised
   in wet conditions compared to pure brushite at a P/L of 3. In contrast,
   the material prepared with d-(+)-Tar showed significantly lower
   fracture toughness ([143]Figure [144]4
   A) compared to the other two groups. Cements were also prepared using
   collagen suspended in HCl instead of Tar. However, these could not be
   tested as they were too brittle to be handled, suggesting that collagen
   has likely detrimental effects on cement integrity and strength.

Figure 4.

   [145]Figure 4
   [146]Open in a new tab

   Graphs showing the mechanical optimization of
   brushite–collagen–noncollagenous protein (NCP) cements. (A) Comparison
   of the fracture toughness between conventionally produced plain
   brushite cements made with water (H[2]O-3-ctrl) and brushite–collagen
   cements produced with d-(-) and l-(+)-Tar. Optimization shows that
   brushite–collagen (bru–col) cements produced with l-(+)-Tar have
   similar mechanical properties to control and significantly superior
   properties to the material produced with d-(−)-Tar at a P/L of 3.
   Legend: explanation of the x axis sample labeling notation. (B)
   Comparison of the fracture toughness of the l-(+)-Tar-treated
   collagen–brushite cement for different powder-to-liquid ratios.
   Optimization shows that a 3.5 powder/liquid ratio produces the toughest
   material (tougher than plain brushite cements). (C) Comparison of the
   fracture toughness of cements produced with different l-(+)-Tar
   concentrations. Optimization shows no significant difference between
   groups. (D) Comparison of fracture toughness for different % w/v
   collagen in the liquid part of the l-(+)-Tar-treated brushite–collagen
   cements. Optimization shows no significant difference between groups.
   The 0.5 and 1.5% w/v collagen groups were chosen to explore the
   biological response of the brushite cements compared to control. The 2%
   w/v group was avoided due to manufacturing difficulties. (E) Comparison
   of the fracture toughness after incorporation of NCPs (μg/mL) in the
   liquid part of the cement reagents. Optimization shows no significant
   difference between groups. NCPs (50 μg/mL) were chosen for further
   experiments to examine potential biological benefits. Legend: graph
   shows mean ± standard error of the mean (SEM), n = 5, multiple t-tests
   with *p < 0.05.

   Collagen type I is highly hydrophilic.^[147]17,[148]46 Wettability is
   essential for cell spreading and attachment^[149]47 but often leads to
   weaker mechanical properties of collagen-based
   biomaterials.^[150]17,[151]25 Interestingly, water is thought to
   increase fracture toughness in native bone^[152]48 by forming thin
   layers between adjacent mineral slabs as well as collagen fibers
   holding them together via capillary action while allowing some degree
   of movement.^[153]49,[154]50 Indeed, capillary forces increase as the
   particle size decreases.^[155]51 Shape also affects capillary action
   with symmetric shapes, e.g., spheres, experiencing larger forces from
   water/air compared to tapered/nonsymmetrical shapes.^[156]51

   In composite bioceramics, mineral particles are usually nonsymmetrical
   and randomly oriented. Thus, introducing water will destabilize the
   system particularly when a second phase such as hydrophilic collagen is
   introduced. This is the case for the d-(−)-Tar-treated condition
   ([157]Figure [158]4
   C). In contrast, l-(+)-Tar creates small, spherelike particles,
   effectively increasing the interaction area and capillary forces
   between the phases and exhibiting adequate fracture toughness despite
   the introduction of collagen ([159]Figure [160]4C).

   Cement reactions with powder-to-liquid ratios (P/L) exceeding 3 g/mL,
   that were not possible for the plain brushite control group, were
   feasible for the l-(+)-Tar group due to the ability of l-(+)-Tar to
   effectively reduce the mineral particle size of brushite–collagen
   cements, thus further improving the fracture toughness of the material.
   We show that as P/L increases, so does the fracture toughness of the
   material up to a P/L of 3.5. At 3.5 g/mL, the fracture toughness of the
   materials was 0.27 MPa.m^1/2, which to our knowledge is the toughest
   collagen–brushite cement to date ([161]Figure [162]4
   B). Higher P/L cements were possible, but their fracture toughness
   decreased as the cement mixture was increasingly hard to work with and
   to homogenize properly.

   Tar molarity and collagen and NCP concentrations did not affect the
   fracture toughness of the cements ([163]Figure [164]4
   C–E). Collagen concentrations higher than 2% w/v were not chosen as the
   liquid phase of the cement reaction was too viscous to work with.
   Taking the findings into consideration, l-(+)-Tar-treated cements with
   a P/L of 3.5, NCP concentration of 50 μg/mL, and collagen
   concentrations of 0.5 and 1.5% w/v were picked for further biological
   characterization. The fracture toughness and compressive strength of
   the 0.5 and 1.5% w/v collagen l-(+)-Tar-treated group were (0.27 ± 0.02
   MPa·m^1/2 and 13 ± 2 MPa) and (0.23 ± 0.02 MPa·m^1/2 and 10 ± 1 MPa) in
   wet conditions, respectively. Comparatively, the pure brushite control
   group had a fracture toughness of 0.19 ± 0.01 MPa and compression
   strength of 8 ± 1 MPa (compression test results are shown in
   [165]Supporting Information Figure 2). l-(+)-Tar-treated
   collagen–brushite cements were also stiffer than control (plain
   brushite cement) ([166]Supporting Information Figure 2).

   The work until failure (area under the force–displacement curve) on the
   collagen-containing l-(+)-Tar-treated brushite cements was also
   significantly higher than on plain brushite cements, suggesting that
   the latter is more brittle and thus more prone to sudden failure
   ([167]Supporting Information Figure S3).

   Interestingly, when using gypsum powder, calcium sulfate, as the solid
   in the cement reaction instead of the brushite reagents, the fracture
   toughness of the collagen–bioceramic cements declined significantly
   ([168]Supporting Information Figure S5) despite the use of l-(+)-Tar,
   which has a similar effect on gypsum as brushite.^[169]52 This proposes
   that collagen has a unique interaction with calcium phosphates such as
   brushite, which is not the case for calcium sulfates. Researchers
   suggest^[170]53 that phosphate and sulfate both have similar amounts of
   binding sites on collagen as they likely interact with the same
   positively charged amino acids, namely, lysine and arginine. However,
   the study investigated phosphate and sulfate ions rather than their
   calcium salts. We could not find anything in the literature examining
   the interaction between gypsum and collagen, but it is well established
   that calcium phosphates such as hydroxyapatite (HA) bind strongly to
   collagen through the calcium ions in their lattice.^[171]54 This
   interaction is highly dependent on the calcium ion placement within the
   calcium phosphate lattice,^[172]55 which interact with aspartic and
   glutamic acid-rich motifs on collagen along the {100} crystal plane
   ([001] vector).^[173]56 Brushite shows the same Ca^2+ orientation as HA
   on the {100} plane^[174]57 and thus can interact with collagen on a
   plane other than {010}, which is occupied by chiral Tar. In contrast,
   gypsum has accessible calcium ions primarily along the [101] growing
   step ({010} plane),^[175]52 which is already occupied by chiral Tar.
   This could explain why the two bioceramics behave so differently
   mechanically when collagen is introduced.

   Addition of brushite into the l-(+)-Tar-treated gypsum–collagen cements
   restores its mechanical properties. The fracture toughness of
   l-(+)-Tar-treated gypsum–brushite–collagen cements was 0.32 ± 0.07 and
   0.33 ± 0.08 MPa·m^1/2 for 0.5 and 1.5% w/v collagen, respectively
   ([176]Supporting Information Figure S5). The production of this second
   composite bioceramic might be of interest as its in vivo resorbability
   could likely be finely tuned. Gypsum resorbs faster than
   brushite,^[177]58 suggesting that their composite could exhibit a
   spectrum of resorption times.

Improvement of Biological Response

   To test whether collagen incorporation into the l-(+)-Tar-treated
   brushite cements results in an improved hMSC response to the material,
   cell attachment and morphological analyses were carried out. Cells
   interact with collagen via the α1β1, α2β1, α11β1, or α12β1 integrin
   complexes.^[178]28 Integrin-mediated cell adhesions drive a complex
   process, which involves the formation of supramolecular structures,
   known as focal adhesions (FAs), involving proteins such as talin,
   vinculin, paxillin, FAK, and others.^[179]59 FAs are effectively the
   link between the cellular microenvironment and the extracellular matrix
   (ECM). This is essential for cell mechanosensing, i.e., allowing cells
   to respond to their environment based on forces they exert onto
   substrates (molecular clutch).^[180]59 Allowing hMSCs to respond to
   their environment can guide their fate and allows them to perform
   essential cell-related functions.^[181]59

   In this study, cell adhesion was significantly higher in all
   l-(+)-Tar-treated brushite cement groups containing collagen compared
   to the pure brushite control. After an initial seeding density of 5000,
   3000–5000 cells/cm^2 were counted on collagen-containing cements, while
   only 1800 cells/cm^2 were counted on pure brushite after 4 h of culture
   without FBS ([182]Supporting Information Figure S4). The addition of
   NCPs did not further affect cell attachment. Cell morphology studies
   further revealed that even at low collagen concentrations cells
   exhibited greater spreading, i.e., higher area and perimeter on
   l-(+)-Tar-treated collagen–brushite cements compared to pure brushite.
   Cell circularity also decreased. In the l-(+)-Tar-treated
   brushite–collagen cement groups, cells also formed more protrusions
   (lamellipodia and filopodia) away from the cell center compared to the
   control group (plain brushite cements) (compactness in collagen groups
   > compactness in brushite > 1 perfect circle, form factor in collagen
   groups < form factor in brushite < 1 perfect circle) ([183]Figure
   [184]5
   A,B). This is important as the ability of a cell to spread determines
   whether a cell proliferates, becomes quiescent, or dies.^[185]60

Figure 5.

   [186]Figure 5
   [187]Open in a new tab

   Human mesenchymal stem cell (hMSC) attachment studies carried out on
   brushite and brushite–collagen cements. Cells (5000) were seeded on
   each scaffold to allow for easier single-cell analysis. Cell morphology
   analysis (from 56 cells from triplicate material replicates) (A, B)
   revealed that collagen in the brushite cements treated with l-(+)-Tar
   significantly improved cell spreading and the formation of
   filopodia/lamellipodia (cells become less circular). This can be seen
   in the graphs comparing cell area, perimeter, form factor, and
   compactness on l-(+)-Tar-treated brushite cements with 0.5 and 1.5% w/v
   collagen content and plain brushite cement (control) samples. Although
   there is an increasing trend of cell spreading as the collagen content
   increases, that is not significant. Focal adhesion analysis (C) also
   reveals that introducing collagen to the l-(+)-Tar-treated brushite
   cements allows for the formation of integrin-mediated cell binding to
   the substrate (presence of distinct vinculin clusters). This is not the
   case when looking at the control group. Legend: graph shows mean ± SEM,
   n = 56 (cells), multiple t-tests with ***p < 0.001.

   A similar trend is observed on l-(+)-Tar-treated
   gypsum–brushite–collagen cements ([188]Supporting Information Figure
   S6A,B), suggesting that the differences in cell morphology due to
   collagen can be observed on different, independently studied composite
   bioceramics. Interestingly, no cells could be identified on pure gypsum
   samples ([189]Supporting Information Figure S6A,B). Comparatively,
   cells attached to pure brushite ([190]Figure [191]5
   A,B) as well as gypsum–brushite cements ([192]Supporting Information
   Figure S6A,B) despite the absence of dedicated cell-binding domains,
   which are present in collagen. Nonspecific cell binding can occur due
   to electrostatic forces between cells and the substrate.^[193]61 In the
   absence of serum proteins as the ζ-potential (measure of surface
   charge) approaches 0, cells find it increasingly hard to
   attach.^[194]62 Gypsum has a ζ-potential of ∼−15 mV^[195]63 while
   brushite has −30 to −45 mV at physiological pH,^[196]64,[197]65 likely
   explaining the lack of cells present on the former.

   The lack of specific cell-binding sites in the bioceramic-only samples
   is further shown through immunostaining of vinculin. Cement samples
   without collagen lack vinculin clusters overlapping with F-actin at the
   cell periphery ([198]Figure [199]5
   C and [200]Supporting Information Figure S6C). In contrast, these are
   present in the collagen-containing groups with and without NCPs,
   suggesting that cells interact with the material through integrin
   binding ([201]Figure [202]5C and [203]Supporting Information Figure
   S6C).

Conclusions

   This study demonstrates that chiral l-(+)-Tar can improve the
   mechanical properties of collagen–brushite cements in wet conditions
   via the inhibition of the [101] dynamic crystal growth step in contrast
   with d-(−)-Tar, which does not have the same effect. The novel
   composite bioceramics achieve a significantly enhanced fracture
   toughness as high as 0.27 MPa·m^1/2 and a compressive strength of 13
   MPa in wet conditions, which are well within the mechanical property
   range of cancellous bone (fracture toughness 0.1–0.8 MPa·m^1/2 and
   compressive strength 2–12 MPa).^[204]66 Cell attachment, morphology,
   and cell–material interaction were improved in the collagen–bioceramic
   composites treated with l-(+)-Tar compared to the currently used pure
   bioceramics. Our work suggests that the involvement of chiral
   molecules, namely, l-(+)-Tar, in the mineralization process allows the
   production of composite bioceramic materials with superior biological
   properties while still exhibiting impressive fracture toughness and
   compressive strength.

Acknowledgments