Calcium–Collagen Coupling is Vital for Biomineralization Schedule

Abstract Biomineralization is a chemical reaction that occurs in organisms in which collagen initiates and guides the growth and crystallization of matched apatite minerals. However, there is little known about the demand pattern for calcium salts and collagen needed by biomineralization. In this study, natural bone biomineralization is analyzed, and a novel interplay between calcium concentration and collagen production is observed. Any quantitative change in one of the entities causes a corresponding change in the other. Translocation‐associated membrane protein 2 (TRAM2) is identified as an intermediate factor whose silencing disrupts this relationship and causes poor mineralization. TRAM2 directly interacts with the sarcoplasmic/endoplasmic reticulum calcium ATPase 2b (SERCA2b) and modulates SERCA2b activity to couple calcium enrichment with collagen biosynthesis. Collectively, these findings indicate that osteoblasts can independently and directly regulate the process of biomineralization via this coupling. This knowledge has significant implications for the developmentally inspired design of biomaterials for bone regenerative applications.

. Histological analysis of cranium collected from different time points. Fig. S2. Correlation between Ca 2+ and Col1 during mineralization in vitro. Fig. S3. Characteristics of Ca 2+ changes after treatments of Ion or TG. Fig. S4. Effects of ER Ca 2+ changes on mineralization. Fig. S5. Characteristics of Ca 2+ concentrations after collagen inhibition and retrieval.  .  Table S1. The shRNA sequences used in study. Table S2. The primer sequences used in study.

Supplementary Materials and Methods
Mice and sample collection: To observe Ca 2+ deposition in the skull, calcein-AM (16 mg/kg body weight; C001, Dojindo, Japan) was injected intraperitoneally 4 days before sacrifice. To make paraffin sections, the whole calvaria tissues were fixed with 4% paraformaldehyde at 4 °C overnight. Tissues intended for ultrastructural studies were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB,pH = 7.4) at 4 °C overnight. Other samples intended for immuno-TEM were quickly fixed with a solution of 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PB (pH = 7.4) at 4 °C overnight. The same area of calvaria with supraorbital cranial sutures were fixed in these fixation fluids. To ensure that the ultrastructure of the same region was observed in different samples, the calvaria were observed at supraorbital sutures, consistent with the methods in previous studies. [1] Cell culture, transfection, and ALP and ARS staining: BMSCs were collected from the femurs and tibias of female 5-week-old C57BL/6 mice by digesting bone chips with collagenase type II (1 mg mL -1 , 17101015, Gibco, Life Technologies Corporation) as described previously. [2] The cells were cultured in alpha minimum essential medium (αMEM; Gibco, Life Technologies Corporation, USA) containing 20% fetal bovine serum (FBS; Gibco, Life Technologies Corporation, USA). Further purified cells (passage 3-10) were used in subsequent experiments. The osteogenic-inducing medium (OM) was composed of 10% FBS, 10 nM dexamethasone (D4902, Sigma-Aldrich, USA), 10 mM β-glycerophosphate (G9422, Sigma-Aldrich, USA), and 5 mM L-ascorbic acid (A4403, Sigma-Aldrich, USA) in αMEM.
The OM of each well was replaced with fresh OM every other day. BMSCs were induced to differentiating into osteoblasts by 3 days of OI. [2] A mouse TRAM2-specific shRNA lentivirus vector (pLVX, GeneChem, China) was used for knockdown of the TRAM2 and designated as Lv-shTRAM2#1 or #2. The sequences are listed in Table S1. An empty lentiviral vector only was used as a control (Lv-shCtrl). The cells were transfected with virus particles (35 particles per cell) with the help of 5 μg/mL hexadimethrine bromide (Polybrene, Millipore Sigma, USA).
Mineralized cells for ALP staining were prepared after incubation for 7 days. BMSCs were fixed and treated with an ALP staining kit (C3206, Beyotime, China), after with the enzyme activity was determined (P0321S, Beyotime, China). After incubation with working solution, the absorbance was tested immediately to determine the optical density (OD) of 405 nm. Additionally, total protein was extracted, and the concentration was quantified by the bicinchoninic acid (BCA) method (23250, Thermo Fisher Scientific, USA). ALP activity was normalized and calculated based on the OD405 value per milligram of total protein. After being induced for 14 days, cells in well plates were stained with 0.1% Alizarin red S (A5533, Sigma-Aldrich, USA) solution (pH = 4.6). After pictures were obtained, the Alizarin red was dissolved in 10% cetylpyridinium chloride (C9002, Sigma-Aldrich, USA) at room temperature for 4 h, and the absorbance value (OD value) was examined at 562 nm. In terms of the design of the ARS staining experiments, cells were treated with Ca 2+ flux regulators after OI for 3 days and 7 days. The treatment time was 6 h. The medium containing drug was then discarded, and fresh OM was added. Moreover, the OM was discarded at 3 or 7 days after OI and the cells were treated with 80 μM FT011 for 2h or transfected with pLVX-Col1 lentiviral vector (multiplicity of infection, MOI = 40). The medium of former group was then changed with αMEM (containing 10% FBS) to wait for the transfection of latter group. Then the transfection reagents were replaced with OM after transfection completion. The duration of actual osteogenic induction was 14 days.
Treatments: To properly regulate the Ca 2+ concentration in the ER, 5 μM Ion (S1672, Beyotime, China) and 100 nM TG (T9033, Sigma-Aldrich, USA) were added to the OM 3 days after OI based on our previous results. [2] After 6 h of treatment, cells were collected for subsequent experiments. To inhibit the expression of type I collagen, BMSCs were transfected with empty lentiviral vector and treated with the inhibitor, FT011 (HY-100495, MedChemExpress, USA) for 2 h. The concentrations of FT011 were as follows: 10 μM, 20 μM, 40 μM, 80 μM, 160 μM and 320 μM. [3] For rescue experiments, different virus particles expressing the lentiviral vector pLVX-Collagen1 (MOI: 5, 10, 20 or 40 particles per cell, GeneChem, China) were transfected into target cells and cells were purified by 6 μg mL -1 puromycin. After determination of rescue effectiveness, an MOI of 40 was used in the following experiments. When both viruses were transfected, the cells were first transfected with Lv-shTRAM2 and formed into a stable cell strain before transfection with the next kind of virus. To transiently evoke Ca 2+ flux, 10 μM Ion, 10 μM TG or 100 μM ATP was used in the indicated experiments. [4] To stimulate STIM1 redistribution, 10 μM TG was used for 10 min. [4a] To Quantification of total and intracellular collagen was performed using a Sircol soluble collagen assay kit (Biocolor, UK) according to the manufacturer's protocol and the methods used in a previous study. [5] Briefly, 10 9 mineralized cells or micro-dissected calvaria were treated with acid pepsin and mixed with Sircol dye. The absorbance was tested at 555 nm and a standard curve was made using rat tail collagen (354236, Corning, USA). To evaluate intracellular collagen production, single cells were first collected as described above. Then, the OD values were measured and analyzed. The data from each group were normalized according to the data of corresponding control group. Ultrastructural examination: Cells and calvaria were harvested, fixed, dehydrated, permeabilized, and embedded in epoxy resin as previously reported. [1b, 6] To measure the Ca 2+ distribution within the ER, 70-nm sections were observed by 100 kV high-resolution scanning TEM (STEM, HT-7700, Hitachi, Tokyo, Japan) coupled with energy-dispersive X-ray spectroscopy (EDX). [7] To quantify the chemical elements, regions of the same area that contained the ER were selected with an Aztec nanoanalysis system (Oxford Instruments, UK).

CCK
The boundary of the ER was identified by the membrane. The average percentages of calcium content were statistically analyzed.
Immuno-TEM was used to identify collagen molecules within ER lumen. Cells were embedded with LR-White resin (Ted Pella, USA). Grids were treated with 1% H 2 O 2 in phosphate buffer, neutralized with ammonium chloride, blocked with 5% bovine serum albumin, and incubated with a primary antibody against Col 1 (1:100, A16891, ABclonal, USA) and then conjugated with 1.4 nm nanogold (Nanoprobes, USA). The samples were examined with a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) at 120 kV. Equal areas were randomly selected in each figure, and the number of positive dots per cell was calculated.
To describe ER activity, dilated ER elements were defined as ribosome-studded organelles with expanded lumina (with cross-sectional areas of more than 0.05 μm 2 ). The proportion of cells with a dilated ER and the averaged luminal size of each dilated ER-containing cell were determined, and statistical comparisons were performed. anti-collagen 1 antibody used recognizes the procollagen alpha1(I) chain with the N-and C-propeptides, the pC-propeptide chain (pC-alpha1(I)), and the alpha1(I) chain with both the N-and C-propeptides cleaved. [9] To separate cells and extracellular matrix (ECM) fractions, a method using deoxycholate (DOC) was modified from a previous study. [9b] After treatment, plates were washed with PBS.
Cells and ECM were scraped with 300 μL of 4% DOC solution containing 20 mM Tris-HCl were resuspended with IB cells -2 buffer and considered the ER compartment. The lysates were added to loading buffer for subsequent western blotting assay. The quality of ER protein was confirmed with different markers (with calnexin as an ER marker).
To collect calvaria proteins, cranial bones were carefully isolated and micro-dissected, followed by washing with PBS three times and lysed with RIPA lysis buffer containing 1 mM PMSF for 30 min on ice. Subsequently, mixtures were centrifuged and performed BCA assay, then denatured for subsequent western blotting process. Images were acquired by confocal microscopy (InSIGHT Plus-IQ, Meridian, USA).

Cytosolic ([Ca 2+ ] cyto ) and ER ([Ca 2+ ] ER ) Ca 2+ measurements:
To visualize cytosolic Ca 2+ , the Fura-2-AM fluorescent Ca 2+ sensor (S1052, Beyotime, China) was used. After treated by drugs, cells were loaded with 5 μM Fura-2 for 30 min and stained with DAPI. Images were acquired by confocal microscopy (InSIGHT Plus-IQ, Meridian, USA) by alternating 340 nm and 380 nm wavelengths. NIS-elements software (Nikon) was used to process and analyze 340/380 ratiometric images. An increase in 340/380 ratio of 10% or more from baseline levels was considered a positive response to a ligand. Intensity was calculated based on ratiometric data. [11] To evaluate Ca 2+ changes in the cytoplasm after treatment, the Fluo-4/AM (S1060, Beyotime, China) fluorescent Ca 2+ sensor was used according to previous study. [4]  inflow and was then replaced with 100 μM ATP·2Na·3H 2 O or 10 μM TG in Ca 2+ -free HBSS.