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. Author manuscript; available in PMC: 2026 Jul 13.
Published in final edited form as: Macromol Biosci. 2025 Jul 13;25(11):e00636. doi: 10.1002/mabi.202400636

Orai1 is Required for Osteogenic Differentiation on Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

Meiwand Bedar 1,2,3,4, Wei Chen 1,2,3,4, Xiaoyan Ren 1,2,3,4, Shahrzad Moghadam 1,2,3,4, Youngnam Kang 1,2,3,4, Kaavian Shariati 1,2,3,4, Kelly X Huang 1,2,3,4, Grace Rubino 5, Brendan AC Harley 5,6, Justine C Lee 1,2,3,4,*
PMCID: PMC12371709  NIHMSID: NIHMS2095847  PMID: 40653607

Abstract

The role of the extracellular matrix (ECM) in guiding cell fate has spurred the development of synthetic, ECM-inspired regenerative biomaterials. We previously described a porous, open cell foam composed of nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG), capable of inducing in vitro osteogenesis of primary human mesenchymal stem cells (hMSCs) and in vivo skull regeneration without adding growth factors or expanded progenitor cells. This study investigated the role of store-operated calcium entry (SOCE) in MC-GAG activity by evaluating the impact of Orai1, the pore-forming subunit of the calcium release activated channel. Compared to nonmineralized collagen glycosaminoglycan (Col-GAG), hMSCs cultured on MC-GAG exhibited increased Orai1 expression. Chemical inhibition of SOCE using MRS1845 reduced expression of both gene and protein expression of osteogenic markers as well as mineralization on MC-GAG, while MRS1845 exerted no effects on Col-GAG materials. Orai1 knockdown similarly inhibited matrix mineralization and expression of the late osteogenic marker, bone sialoprotein-2 (BSP2), on MC-GAG, whereas no effects were notable on Col-GAG. Orai1 knockdown reduced ERK1/2 phosphorylation, suggesting that the osteogenic effects of Orai1 functioned downstream of ERK1/2 pathways. These findings suggest Orai1 is necessary for osteoprogenitor maturation and matrix mineralization in MC-GAG-mediated osteogenic differentiation.

Keywords: bone regeneration, nanoparticulate mineralized collagen glycosaminoglycan scaffold, calcium signaling, Orai1

Graphical Abstract

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Understanding how regenerative materials engage signaling pathways can guide the design of more effective biomaterials. This study demonstrates that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) materials activate store-operated calcium entry through Orai1 to drive osteogenic differentiation and matrix mineralization. These findings reveal a previously underexplored mechanism that may inform the development of next-generation materials for skeletal regeneration.

Introduction

The extracellular matrix (ECM) plays a pivotal role in directing cell fate, inspiring the design of biomaterials that mimic its dynamic functions.1–4 Over the past decade, our collaborative groups have detailed the cellular mechanisms modulated by a novel, ECM-inspired scaffold composed of nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) capable of partially regenerating biparietal skull defects in vivo without the need for growth factor supplementation or pre-seeding with progenitor cells.5–17 In primary osteoprogenitor cells, MC-GAG activates osteogenic differentiation via the canonical bone morphogenetic protein receptor (BMPR) and small mothers against decapentaplegic-1/5 (Smad1/5) pathway,7,10 heterodimers of the type III sodium phosphate symporters PiT-1 and PiT-2,6,18 canonical Wnt/β-catenin,11,18 and YAP/TAZ-mediated mechanotransduction.9,18 Unlike non-mineralized collagen glycosaminoglycan (Col-GAG), MC-GAG also exhibits anti-osteoclastogenic and anti-resorptive properties,19–21 suggesting that the mineral content of MC-GAG is fundamental to its capabilities.

Although Col-GAG and MC-GAG share similar compositions in collagen, chondroitin-6-sulfate, porosity, and synthesis methods, only MC-GAG contains calcium and phosphate ions in its cell-free state.6–8,10,22,23 MC-GAG with a stoichiometric Ca:P ratio of 1:1, consistent with brushite, and shifts to a hydroxyapatite-like composition (Ca:P ~1.7–1.8:1), consistent with physiologic bone mineral content, after inducing osteogenic differentiation of primary human mesenchymal stem cells (hMSCs).6 This differentiation occurs in the absence of any osteogenic additives and depends on signaling through PiT-1/PiT-2 as well as the inherent stiffness of MC-GAG.6,18 In contrast, Col-GAG lacks the capacity to independently differentiate hMSCs without osteogenic additives and ultimately produces amorphous mineral deposition (Ca:P ~2.5:1). Thus, the ability of MC-GAG to serve as a local depot of inorganic ions with temporal control of release is one of the main drivers of its osteogenic activity.

Beyond the direct effects of phosphate ion on osteogenic differentiation,24–26 phosphate ion also upregulates store-operated calcium ion entry (SOCE), a major pathway for calcium influx in non-excitable cells.27,28 While the most widely studied mechanisms involving calcium ions in bone homeostasis have involved the voltage-gated calcium channels and calcium-sensing receptors,29–31 SOCE has recently emerged as a promising target for skeletal regeneration.32–36 SOCE is triggered upon sensing of calcium ion depletion within the endoplasmic reticulum by stromal interaction molecules (STIM)-1 and STIM-2.37 STIM-1/2 then oligomerizes and activates Orai1, the pore-forming subunit of the calcium release-activated channel (CRAC).38 Using murine genetic models, several groups have demonstrated that Orai1−/− mice have impaired skeletal development due to disruptions in both osteoblast and osteoclast differentiation.39,40 In relationship to osteogenic signaling, Orai1 has been reported to be upstream of both the canonical BMPR signaling pathway through Smad1/5 phosphorylation and well as the ERK1/2-mediated pathways.41–45

Given the importance of SOCE in bone biology, as well as its major role in calcium ion entry for non-excitable cells, we aimed to investigate the potential for targeting Orai1 for maximizing the regenerative potential of MC-GAG materials.

Materials and Methods

Fabrication and chemical crosslinking of non-mineralized and mineralized collagen scaffolds

We fabricated non-mineralized (Col-GAG) and mineralized (MC-GAG) collagen scaffolds through lyophilization, following our previously described methods.12–14 In summary, a suspension of microfibrillar type I collagen (Collagen Matrix, Oakland, NJ) and chondroitin-6-sulfate (Sigma-Aldrich, St. Louis, MO) was combined in an acetic acid solution for Col-GAG scaffolds, while a phosphoric acid solution containing calcium salts (calcium nitrate hydrate: Ca(NO3)2·4H2O; calcium hydroxide: Ca(OH)2, Sigma-Aldrich) was used for MC-GAG scaffolds. The solution was then frozen at a constant cooling rate of 1°C/min using a freeze dryer (Genesis, VirTis, Gardiner, NY), going from room temperature to −10°C. After sublimation, the scaffolds were sterilized with ethylene oxide and stored at −20°C. For culture preparation, the scaffolds were cut into 6–8 mm disks and rehydrated with ethanol, followed by phosphate buffered saline (PBS) overnight. Subsequently, the scaffolds underwent crosslinking for four hours using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, Sigma-Aldrich) and N-hydroxysuccinimide (NHS, Sigma-Aldrich) following established protocols.46 To remove residual chemicals, the scaffolds were washed with PBS and then incubated in culture media overnight prior to cell seeding.

Mechanical properties of Col-GAG and MC-GAG have been reported previously. Briefly, the materials are open cell foams with a porosity of 85±3%, pore size of 156±6 μm, and hydrated elastic modulus ranging from 4–16 kPa depending on the molar ratios of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma Aldrich) to N-hydroxysuccinimide (NHS, Sigma Aldrich) for collagen crosslinking.47–50

Cell Cultures

Primary bone marrow-derived human mesenchymal stem cells (hMSCs, CD105+CD166+CD29+CD44+CD14−CD34−CD45−) (Lonza, Inc., Allendale, NJ) were cultured and expanded in Corning Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), 2 mM L-glutamine (Life Technologies, Carlsbad, CA), 100 IU/mL penicillin/100 mg/mL streptomycin (Life Technologies). Inhibition of store-operated calcium entry was performed using 25 μM of MRS1845 (Sigma-Aldrich). MRS1845 was dissolved at 2000X concentration in Dimethylsulfoxide (DMSO), filtered, and stored at −80°C. At passage 3, 3 × 105 hMSCs were seeded onto 8-mm Col-GAG and MC-GAG scaffolds in growth media. After 24 hours, growth media was replaced with osteogenic differentiation media (OGM) consisting of 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid and 0.1 μM dexamethasone. Scaffolds were either left untreated (DMSO only) or treated with MRS1845 (25 μM), also known as N-propargylnifrendipine. Fresh MRS1845 was added to each media change every 72 hours.

RNA Interference

For RNA interference experiments, 2.5 × 104 hMSCs were seeded per well in 24-well plates for 24 hours. Transfection was performed using DharmaFECT 1 transfection reagent (Dharmacon, LaFayette, CO) according to the manufacturer’s protocol. Briefly, 2 μL of reagent was diluted in 48 μL of Opti-MEM and combined with 100 nM of scrambled small interfering RNA (siRNA) for the control group (siControl, ON-TARGETplus Non-targeting Control siRNA, Dharmacon), siRNA targeting Orai1 (siOrai1, ON-TARGETplus Human Orai1 SMARTpool, targeting sequences 5′-GGCCUGAUCUUUAUCGUCU-3’, 5’-GCACCUGUUUGCGCUCAUG-’3, 5’-GGAGUUUGCCCGCUUACAG-3’, and 5’-UCAACGAGCACUCCAUGCA-3’, Dharmacon) for the siOrai1 group, and siRNA targeting CACNA1C (siCACNA1C, ON-TARGETplus Human CACNA1C SMARTpool, targeting sequences 5′-GGAGGAGCACAUUCGAUAA-3’, 5’-GGAUGUUAGUCUGUAUUUA-’3, 5’-GGGUAGCAUUGUUGAUAUA-3’, and 5’-GAAGAUGACUGCUUAUGGG-3’, Dharmacon) in 45 μL of Opti-MEM, and incubated at room temperature for 30 min. Subsequently, each well was incubated with the transfection reagent and siRNA solution in 400 μL of growth medium. After 24 hours of transfection, cells were harvested, and 1 × 105 transfected hMSCs were seeded onto 6 mm Col-GAG and MC-GAG scaffolds.

Quantitative Real-Time Reverse-Transcriptase Polymerase Chain Reaction

Total RNA was extracted at 0, 3, 7, and 14 days of culture using the RNeasy kit (Qiagen, Valencia, CA). Gene sequences were obtained from the National Center for Biotechnology Information gene database, and primers (Eurofins Genomics, Louisville, KY) were designed accordingly (Supplemental Table 1). Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was conducted on the Opticon Continuous Fluorescence System (Bio-Rad Laboratories, Inc., Hercules, CA) utilizing the QuantiTect SYBR Green RT-PCR kit (Qiagen). The cycling conditions consisted of reverse transcription at 50°C for 30 minutes, activation of HotStarTaq DNA polymerase and inactivation of reverse transcriptase at 95°C for 15 minutes, followed by 45 cycles of 94°C for 15 seconds, 58°C for 30 seconds, and 72°C for 45 seconds. The results were analyzed and presented as representative graphs of triplicate experiments.

Water Soluble Tetrazolium-8 (WST-8) Assay

Cell proliferation and viability were assessed using the Cell Counting Kit-8 colorimetric assay (Sigma-Aldrich) at a 1:15 concentration. Scaffolds were incubated at 37°C with 5% CO2 for 2 h. Absorbance of the incubation medium was subsequently measured at 450 and 690 nm using an Epoch spectrophotometer (BioTek, Winooski, VT).

Quantitative Alizarin Red Staining

At 56 days of culture, scaffolds were fixed in 10% formalin overnight, embedded in paraffin, and cut into 4-micron sections. Following deparaffinization with xylene and graded alcohol, sections were stained with 1 mL of 1 mg/mL of Alizarin Red Stain solution (Acros Organics, Fair Lawn, NJ) for 30 minutes. After multiple washing steps with distilled water, images were acquired with a Zeiss Axio Observer 3 inverted microscope using the ZEN 2.3 Pro software (Zeiss, Oberkochen, Germany). Image J software (NIH, Bethesda, MD) was used for image quantification.

Western Blot

Scaffolds at 0, 7, and 14 days of culture were morselized using scissors in 3x SDS reducing sample buffer to create total protein lysates. Lysates were subsequently incubated at 95°C for 5 minutes and centrifuged in 0.2 μm Spin-X filters (Corning, Costar, Corning, NY) at 14,000 rpm for 5 minutes. Equal amounts of protein lysates were subjected to 4 to 20% SDS-polyacrylamide gel electrophoresis (Bio-Rad). Western blot analysis was performed with antibodies against, phosphorylated Smad1/5 (p-Smad1/5), total Smad5, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, Runx2, and Orai1 followed by 1:4000 dilutions of HRP-conjugated immunoglobulin G antibodies (Bio-Rad). Detection was carried out using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL). Primary antibody against Orai1 was obtained from Thermo Fisher Scientific, and all other primary antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Imaging was carried out with the Amersham ImageQuant™ 800 western blot imaging system (Cytiva Life Sciences, Amersham, United Kingdom) and quantified using Image J (NIH).

Statistical Analysis

Independent samples t tests were performed for comparisons between two groups. Analyses of variance (ANOVA) followed by post hoc tests using the Tukey criterion were performed for all quantitative data with 3 or more groups. A p-value less than 0.05 was considered significant. All statistical analyses were performed using SPSS Version 28 (Chicago, IL).

Results

Differential Orai1 Expression at Baseline on Col-GAG Versus MC-GAG

To define the effect of scaffold composition on Orai1 expression, we evaluated the baseline expression of Orai1 in non-mineralized Col-GAG versus MC-GAG biomaterials (Figure 1). Orai1 was found to be elevated on MC-GAG scaffolds compared to Col-GAG in both gene expression (particularly days 3 and 14) as well as protein expression. scaffolds compared to Col-GAG on days 7 and 14. We did not observe any differences in gene expression for Orai1 homologs, Orai2 and Orai3, suggesting the specific impact of scaffold mineral content on the Orai1 subunit. We also evaluated other calcium channels known to be involved in bone metabolism,31,51–54 including the calcium sensing receptor (CaSR) and L-type voltage-gated calcium channel subunits Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), which did not demonstrate differences in expression between Col-GAG and MC-GAG. These data suggest that of the major calcium channels and receptors known to contribute to osteogenic differentiation, mineralized MC-GAG scaffolds preferentially upregulate Orai1 gene and protein expression. Moreover, Orai1 expression is scaffold-dependent, and is significantly more profound upon MC-GAG compared to Col-GAG.

Figure 1. Differential Expression of Calcium Release Activated Channel Subunits on Col-GAG and MC-GAG materials.

Figure 1.

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(A) Primary hMSCs cultured on non-mineralized collagen glycosaminoglycan (Col-GAG) and mineralized collagen glycosaminoglycan (MC-GAG) in growth media for 3, 7, and 14 days were subjected to qRT-PCR analysis for Calcium Release Activated Channel Subunits Orai1, Orai2, and Orai3 expression (n=3). (B) hMSCs cultured on Col-GAG and MC-GAG in growth media for 7 days subjected to qRT-PCR analysis for calcium sensing receptor and L-type voltage-gated calcium channel subunits Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), and Cav1.3 (CACNA1D) expression (n=3). (C) Representative western blots and densitometric quantification for Orai1 and GAPDH expression at days 7 and 14 are shown. Bars represent means, error bars represent standard error. Asterisks denote significant comparisons. *, p<0.05; **, p<0.01.

Inhibition of SOCE Through MRS1845 reduces Osteogenesis and Mineralization Upon MC-GAG

Given the Orai1 upregulation observed in hMSCs on MC-GAG materials, we next sought to understand whether store-operated calcium entry would impact MC-GAG-induced osteogenic differentiation. Using MRS1845, a chemical inhibitor of store-operated calcium channels, we first evaluated cell viability and proliferation in primary hMSC cultures using WST-8 assays (Figure 2). No differences were found between control and MRS1845-treated cells, confirming the inhibitor did not impact viability. Next, we evaluated the expression of osteogenic genes by hMSCs cultured on the two materials with and without exposure to MRS1845. Similar to our prior reports,7,8 Similar to our prior reports, hMSCs cultured on Col-GAG scaffolds demonstrated decreased expression of osteogenic markers compared to MC-GAG. In the presence of MRS1845, expression of both early (alkaline phosphatase, ALP) and late (osteocalcin, OCN) were reduced in hMSCs on MC-GAG scaffolds, an effect not seen in hMSCs on non-mineralized Col-GAG materials.

Figure 2. Effects of Store-Operated Calcium Channel Inhibition on MC-GAG-mediated Osteogenic Differentiation and Mineralization.

Figure 2.

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(A) Schematic of chemical inhibition of Orai1 with N-propargylnifrendipine (MRS1845). (B) WST-8 assay of hMSCs cultured for 7 days in the presence of growth media with or without MRS1845. (C) qRT-PCR for alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) expression in primary hMSCs cultured on either Col-GAG or MC-GAG for 7 days in the presence of OGM alone or with the addition of MRS1845 (n=3). (D) Representative images of Alizarin red staining and (E) quantitative analysis for empty Col-GAG or MC-GAG scaffolds (Empty) or hMSCs cultured for 8 weeks in the presence or absence of MRS1845 on respective scaffolds. (F) Representative images and (G) densitometric quantification of Western blots for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated Smad1/5 (p-Smad1/5), total Smad5, Runx2, and GAPDH in hMSCs cultured on Col-GAG or MC-GAG for 7 days with or without MRS1845. Bars represent means, errors bars represent standard error. Asterisks denote significant comparisons. *, p<0.05; **, p<0.01; ***, p<0.001.

Next, we evaluated matrix mineralization on Col-GAG and MC-GAG scaffolds via Alizarin red staining following 8 weeks of culture. Compared to empty (cell-free) control scaffolds, MC-GAG seeded with hMSCs demonstrated increased mineralization on Alizarin Red staining, while minimal changes were observed on Col-GAG materials. In the presence of MRS1845, a significant decrease in mineralization in MC-GAG scaffolds was observed compared to untreated controls (p<0.001). Although the overall reduction was quantitatively similar to that seen in cell-free MC-GAG, qualitatively, Alizarin Red staining to be greater than cell-free MC-GAG, indicating incomplete inhibition. In contrast, no significant Alizarin Red staining was observed on the non-mineralized Col-GAG scaffolds.

To investigate the influence of MRS1845 on intracellular signaling pathways, we next evaluated phosphorylation of ERK1/2 and Smad1/5, both of which have been reported as downstream effectors of Orai1-mediated osteogenic signaling.7,41,45,55 In the presence of MRS1845, p-ERK1/2 was significantly reduced in hMSCs differentiated within mineralized MC-GAG scaffolds, while p-Smad1/5 trended towards increased expression. In contrast, hMSCs differentiated within non-mineralized Col-GAG scaffolds demonstrated no significant differences in p-ERK1/2 or p-Smad1/5 with MRS1845 treatment. No difference in expression of Runx2 protein was found on either scaffold with MRS1845 treatment. Collectively, these data suggested that MRS1845 selectively inhibited osteogenic differentiation of hMSCs cultured on MC-GAG in a manner correlating to a reduction in ERK1/2 phosphorylation. The slightly increased p-Smad1/5 may suggest potential compensatory activation when ERK1/2 is suppressed. Overall, SOCE is essential for full osteogenic function of MC-GAG materials.

Orai1 Knockdown on Col-GAG and MC-GAG Did Not Impact Cell Viability or Expression of Other Calcium Channels and Receptors

The inhibition of mineralization by MRS1845 underscored the significance of store-operated calcium channels in MC-GAG-mediated differentiation. Although MRS1845 is a potent SOCE channel blocker, it also exhibits inhibitory effects on L-type voltage-gated calcium channels such as Cav1.2 (CACNA1C). This prompted us to further investigate the role of Orai1 using siRNA transfection (Figure 3).56

Figure 3. Effects of siOrai1 on Orai1 Expression, Cell Viability, and Osteogenic Differentiation in Two-Dimensional Cultures.

Figure 3.

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(A) Schematic of Orai1 siRNA target. (B) QPCR of primary hMSCs transfected with siOrai1 or control siRNA (siControl) for 3, 7, and 14 days for Orai1 expression (n=3). (C) Representative images and densitometric quantification of Western blots for Orai1 and GAPDH in primary hMSCs transfected with siOrai1 or siControl for 7 and 14 days. (D) WST-8 assay of hMSCs transfected with siOrai1 or siControl after 7 days. (E) Alizarin red staining of hMSCs cultured for 14 days in growth media, osteogenic differentiation media (OGM), or osteogenic differentiation media after transfection with siOrai1 or siControl. Bars represent means, error bars represent SE. Asterisks denote significant comparisons. *, p<0.05; **, p<0.01; ***, p<0.001.

After transient transfection of primary hMSCs with Orai1 siRNA, both gene and protein expression were evaluated over a 14 day period. Orai1 gene expression was significantly diminished compared to scrambled control siRNA (siControl) at day 3 and 7 with near significance at day 14. At the protein level, Orai1 expression was also found to be reduced compared to siControl over 7 days, albeit protein expression recovered by day 14. Based on these data, we confirm both gene and protein expression of Orai1 were significantly diminished for at least one week of culture after siRNA transfection.

We then evaluated the effect of Orai1 knockdown on cell viability and osteogenic differentiation of hMSCs. WST-8 assays demonstrated no differences in cell viability or proliferation between siOrai1 and siControl. When we evaluated mineralization after 14 days using Alizarin red staining, siOrai1-transfected hMSCs cultured in osteogenic growth media (growth media supplemented with β-glycerophosphate, dexamethasone, and ascorbic acid) demonstrated significantly less mineralization compared to siControl-treated hMSCs.

Next, we evaluated the impact of siOrai1 on hMSCs on MC-GAG and Col-GAG materials. As seen in the two-dimensional cultures, knockdown of Orai1 was efficient on both scaffolds compared to siControl (Figure 4). With respect to cell viability and proliferation, WST-8 assays demonstrated no significant differences between siControl and siOrai1-transfected cells on either material. To evaluate the specificity of the Orai1 siRNA transfection and potential compensatory effects, we next examined the gene expression of other calcium channels potentially relevant to bone metabolism in cells transfected with siOrai1 and siControl on both Col-GAG and MC-GAG scaffolds (Figure 4E).31,51–54 On both materials, there were no observed effects of Orai1 knockdown in the expression of other channels.

Figure 4. Effects of siOrai1 on Orai1 Expression, Cell Viability, and Calcium Channel Gene Expression on MC-GAG.

Figure 4.

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Primary hMSCs transfected with siOrai1 or control siRNA (siControl) and cultured on either Col-GAG or MC-GAG for 7 days were subjected to (A) qRT-PCR for Orai1 expression (n=3) or (B) western blots for Orai1 and GAPDH. Representative images are shown. (C) WST-8 assay of hMSCs transfected with siControl or hMSCs transfected with siOrai1 cultured on Col-GAG or MC-GAG. (D) Schematic depicting the effects of siOrai1 on pertinent calcium channels. (E) qRT-PCR in hMSCs transfected with siOrai1 or siControl cultured on Col-GAG or MC-GAG for 7 days for Calcium Release Activated Channel Subunits Orai2 and Orai3, calcium sensing receptor (CaSR) and L-type voltage-gated calcium channel (VGCC) subunits Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), and Cav1.3 (CACNA1D), N-type VGCC Cav2.1 (CACNA1A) and Cav2.2 (CACNA1B), and T-type VGCC Cav3.2 (CACNA1H) expression (n=3). Bars represent means, error bars represent standard error. Asterisks denote significant comparisons. ***, p<0.001.

siOrai1 Inhibits MC-GAG-Induced Osteoblast Maturation and Mineralization

To understand the impact of Orai1 on MC-GAG-mediated osteogenic differentiation, osteogenic gene expression was evaluated in siOrai1 and siControl-transfected hMSCs differentiated on Col-GAG and MC-GAG scaffolds (Figure 5). In contrast to MRS1845, siOrai1 increased the expression of the early osteogenic marker ALP on mineralized MC-GAG scaffolds. Expression of ALP was also increased on non-mineralized Col-GAG with siOrai1, however, this difference did not reach statistical significance. Similar to MRS1845, RUNX2 expression did not seem to be affected by siOrai1 on either scaffold. siOrai1 resulted in a significant reduction of the late osteogenic marker, BSP2, in mineralized MC-GAG scaffolds, which was also noted to trend lower in Col-GAG. These data suggest that siOrai1 downregulates late, but not early, osteogenic gene expression.

Figure 5. Relationship Between Orai1 and MC-GAG Induced Osteogenic Differentiation and Mineralization.

Figure 5.

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(A) qRT-PCR of primary hMSCs transfected with siOrai1 or control siRNA (siControl) and cultured on Col-GAG or MC-GAG for 7 days for alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and bone sialoprotein II (BSP2) expression (n=3). (B) Alizarin red staining (C) and quantitative analysis of empty scaffolds (Empty), hMSCs (Control), hMCSs transfected with siOrai1, or hMSCs transfected with siControl after 8 weeks of culture on Col-GAG or MC-GAG. (D-E) hMSCs transfected with siOrai1 or siControl cultured on Col-GAG or MC-GAG for 7 days were subjected to Western blot analysis for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated Smad1/5 (p-Smad1/5), total Smad5, Runx2, and GAPDH. (D) Representative images and (E) densitometric quantification are shown. Bars represent means, errors bars represent standard error. Asterisks denote significant comparisons. *, p<0.05; **, p<0.01; ***, p<0.001.

To evaluate whether this reduction translated to reduced matrix mineralization, we evaluated the impact of knockdown on Alizarin Red staining. While no differences were noted on non-mineralized Col-GAG materials in the presence of siOrai1, mineralization on MC-GAG scaffolds was significantly reduced in hMSCs expressing siOrai1 compared to siControl.

Given that the inhibition Cav1.2 (CACNA1C) may have contributed to the reduction in osteogenesis with MRS1845, we evaluated the effect of siRNA transfection of CACNA1C, as this is the dominant isoform of L-type VGCC in osteoblasts (Supplementary Figure 1).57,58 While the knockdown of CACNA1C was effective on both materials, it did not impact cell viability, osteogenic gene expression (ALP, RUNX2, and BSP2), or mineralization as assessed by Alizarin red staining. Moreover, CACNA1C knockdown had no effect on Orai1 expression on either material.

To assess the intracellular signaling pathways affected by siOrai1, we next evaluated phosphorylation of ERK1/2 and Smad1/5 (Figure 5D–E). Similar to MRS1845, downregulation of Orai1 in cells on mineralized MC-GAG scaffolds resulted in a decrease in ERK1/2 phosphorylation whereas siOrai1 did not affect expression on non-mineralized Col-GAG. Additionally, p-Smad1/5 trended up with Orai1 knockdown on MC-GAG, also consistent with our MRS1845 results. While no effect was seen on Col-GAG, expression of RUNX2 protein was reduced in siOrai1-transfected hMSCs within mineralized MC-GAG scaffolds. These data were consistent with the findings of our results in the presence of MRS1845 with the exception of a reduction in RUNX2 expression. The combination of these data suggested that Orai1 knockdown prevented osteogenic maturation and matrix mineralization of hMSCs within mineralized MC-GAG scaffolds, coinciding with a reduction in ERK1/2 signaling.

Discussion

In this work, we investigated the impact of Orai1 and store-operated calcium entry on the osteogenic activities of MC-GAG. We found that MC-GAG upregulates expression of Orai1 at the gene and protein level contrasting with Col-GAG. Inhibition of SOCE with MRS1845 significantly reduced osteogenic gene expression and mineralization on MC-GAG, but not on Col-GAG. Given the potential for non-specific inhibition by MRS1845, we then performed Orai1 knockdown. siOrai1 demonstrated a reduction in late osteogenic gene expression and mineralization on MC-GAG, but not expression of the early osteogenic gene, ALP. It is of note that neither the material type nor siOrai1 affected the expression of other calcium channels known for their relevance in bone metabolism,31,51–54 including Orai1 homologs, calcium sensing receptor, and L-type VGCCs. In contrast to homologs Orai2 and Orai3, Orai1 is considered the primary channel responsible for mediating SOCE.59–62 The upregulation solely of Orai1 upon MC-GAG and lack of upregulation of Orai2 and Orai3 further emphasizes the purported relevance of SOCE to the capabilities of MC-GAG. An investigation of the intracellular signaling pathways involved showed that the addition of MRS1845 or Orai1 knockdown led to a decrease in the phosphorylation of ERK1/2 on MC-GAG materials suggesting the potential involvement of the ERK1/2 pathways in Orai1-induced osteogenesis on MC-GAG. Taken together, these data indicated that mineralized MC-GAG scaffold-induced osteogenic differentiation and function is partially dependent on the activity of Orai1.

While MC-GAG serves as an excellent starting material with the capability of regenerating up to 60% the mineralization and strength of native calvarium in in vivo rabbit models,8 further material refinement is necessary for clinical translation. The current work suggests that targeting Orai1 may be a potential future avenue for improving the skeletal regenerative capacity of MC-GAG. For example, Azimi and colleagues recently reported 4-((5-Phenyl-4-(trifluoromethyl)thiazol-2-yl)amino)benzoic acid (IA65) as a selective pharmacologic agonist for Orai1.63 In their investigation in HEK293 cells expressing either Orai1 or Orai3, IA65 potentiated CRAC currents only in cells expressing Orai1. However, this agonist has never been tested in the context of osteogenic differentiation and represents a potential method of augmenting MC-GAG activity.

Interestingly, inhibiting SOCE with MRS1845 resulted in reduced expression of both early and late osteogenic markers on MC-GAG materials, whereas siRNA-mediated Orai1 knockdown led to an upregulation of the early osteogenic marker ALP and a downregulation of the late osteogenic marker BSP2. This discrepancy suggests that while Orai1 may be necessary for osteoblast maturation and function, early differentiation is likely regulated through other osteogenic pathways. Similar to our MRS1845 studies, Robinson et al. demonstrated that human osteoprogenitor cells treated with the SOCE inhibitor dimethyl tetrachloroterephthalate (DCPA) showed diminished differentiation and function.39 Also, parallelling our findings, Hwang and colleagues noted that overexpression of a pore-dead Orai1 in MC3T3-E1 pre-osteoblastic cell line did not affect alkaline phosphatase expression but substantially inhibited mineralization.40 These data align with our findings to suggest that SOCE is an obligate process for osteoblast differentiation, however, the action of Orai1 is specifically important for late stage maturation rather than initiation of osteogenic differentiation.

While both MRS1845 and Orai1 knockdown preferentially decreased ERK1/2 phosphorylation for cells within mineralized MC-GAG scaffolds, it is unclear whether ERK1/2 serves a causal role for Orai1-induced osteogenic differentiation. Li and colleagues demonstrated that overexpression of Orai1 in MC3T3-E1 osteoblast precursor cells both increased osteogenic differentiation as well as p-ERK1/2 expression on textured titanium substrates.45 Conversely, expression of an Orai1 dominant negative diminished both, suggesting that ERK1/2 activation was necessary for Orai1-mediated osteogenic effects. While our results suggested no impact on Smad1/5 phosphorylation, Lee et al. suggested that murine Orai1 knockout bone marrow-derived mesenchymal stem cells were unresponsive to recombinant BMP2 treatment with absence of Smad1/5/8 phosphorylation.41 These discrepancies may be due to the use of different species, cell lines, methods of inducing knockdown, and exogenous growth factor treatment.

Our previous work demonstrated that MC-GAG functions through multiple pathways, of which one of the most important is governed by phosphate ion.6,18 A key characteristic of MC-GAG is the elution of phosphate ions in physiological fluids during the first 9–12 days of culture in both cell-free and cell-laden materials likely due to the hydrolysis of nanoparticulate brushite. This spatiotemporally controlled elevation in phosphate ion concentration induces osteogenic differentiation downstream of the PiT-1/PiT-2 heterodimers. Given that phosphate ion upregulates Orai1 expression,27,28 it is likely that the intrinsic phosphate elution of MC-GAG also triggers SOCE. This assertion is further supported by the observation that Orai1 knockdown reduces mineralization in two-dimensional cultures which are induced to undergo osteogenic differentiation by β-glycerophosphate (Figure 3). Alternatively, other material features such as nanoscale topographical cues and substrate topology have been demonstrated to contribute to variability in hMSC fate.64,65 Of particular relevance to this study, biomaterial nanotopography has been reported to induced SOCE, suggesting that alteration of topography itself may be considered in the design of second-generation MC-GAG materials to enhance osteogenic activities.66

Supplementary Material

Analysis
Supinfo

Supplementary Figure 1. Relationship Between CACNA1C and MC-GAG Induced Osteogenic Differentiation and Mineralization

(A) Schematic of CACNA1C siRNA target. Primary hMSCs transfected with siCACNA1C or control siRNA (siControl) and cultured on either Col-GAG or MC-GAG for 7 days were subjected to (B) qRT-PCR for CACNA1C expression, (C) WST-8 assays, and (D) Orai1 expression (n=3). (E) qRT-PCR of primary hMSCs transfected with siCACNA1C or control siRNA (siControl) and cultured on Col-GAG or MC-GAG for 7 days for alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and bone sialoprotein II (BSP2) expression (n=3). (F) Alizarin red staining (G) and quantitative analysis of empty scaffolds (Empty), hMSCs (Control), hMCSs transfected with siCACNA1C, or hMSCs transfected with siControl after 8 weeks of culture on Col-GAG or MC-GAG. Bars represent means, errors bars represent standard error. Asterisks denote significant comparisons. ***, p<0.001.

Acknowledgements:

This work was supported by the National Institutes of Health/National Institute of Dental and Craniofacial Research under award numbers R01 DE029234 (JCL), R01 DE028098 (JCL), R01 DE030491 (BACH), the National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases under award number R01 AR077858 (BACH), the Bernard G. Sarnat Endowment for Craniofacial Biology (JCL), and the Jean Perkins Foundation (JCL).

Footnotes

Conflict of Interest Disclosures: JCL is a medical education consultant for Stryker.

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Associated Data

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Supplementary Materials

Analysis
NIHMS2095847-supplement-Analysis.docx (19.8KB, docx)
Supinfo

Supplementary Figure 1. Relationship Between CACNA1C and MC-GAG Induced Osteogenic Differentiation and Mineralization

(A) Schematic of CACNA1C siRNA target. Primary hMSCs transfected with siCACNA1C or control siRNA (siControl) and cultured on either Col-GAG or MC-GAG for 7 days were subjected to (B) qRT-PCR for CACNA1C expression, (C) WST-8 assays, and (D) Orai1 expression (n=3). (E) qRT-PCR of primary hMSCs transfected with siCACNA1C or control siRNA (siControl) and cultured on Col-GAG or MC-GAG for 7 days for alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and bone sialoprotein II (BSP2) expression (n=3). (F) Alizarin red staining (G) and quantitative analysis of empty scaffolds (Empty), hMSCs (Control), hMCSs transfected with siCACNA1C, or hMSCs transfected with siControl after 8 weeks of culture on Col-GAG or MC-GAG. Bars represent means, errors bars represent standard error. Asterisks denote significant comparisons. ***, p<0.001.

NIHMS2095847-supplement-Supinfo.docx (2.5MB, docx)

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