Enhanced proliferation and differentiation effects of a CGRP- and Sr-enriched calcium phosphate cement on bone mesenchymal stem cells

Preparation of materials

To make an implant of CPC, dicalcium phosphate anhydrous (DCPA; Alfa Aesar) together with tetracalcium phosphate (TTCP; Wako, Japan) were mixed as previously described (31, 32). For the Sr-CPC group, strontium hydrogen phosphate (DSPA; Sigma) was used to partially replace DCPA to make a total molar ratio for Sr/(Sr.Ca) of 10%. The liquid phase consisted of a mixture of 12 wt% polyvinylpyrrolidone K-30 (PVP; Wako, Japan) and 20 wt% citric acid (Wako, Japan). Then, the cement paste was made in a mixture with 2 different phases: the liquid and solid phases. For the CGRP-CPC and CGRP-Sr-CPC groups, the CPC or CPC-Sr was immersed with 0.5% CGRP buffer for 24 hours to make the materials absorb the CGRP completely. For the CGRP/Sr-CPC group (addition of free CGRP to the Sr-CPC group), 200 pg/mL of CGRP was added together with the Sr-CPC group to BMSC cultures daily.

Measurement of compressive strength

The cement samples, which had a diameter of 6 mm and a height of 12 mm, were incubated with simulated body fluid (SBF) at a temperature of 37°C (33), for 1, 7, 14 or 28 days. Then the compressive strength of CPC, Sr-CPC, CGRP-CPC or CGRP-Sr-CPC was measured by a MTS 858 Bionix testing machine (MTS Systems, Minneapolis, MN, USA). In particular, continuous loads were applied to the samples, which were placed between the plates of the machine with the axis along the center. The compressive strength was measured when the sample broke down. In detail, a 1-kN load cell at a rate of 0.1 mm/min was continuously exerted on the samples until breakdown occurred. Five samples were tested in each group at 1 time point.

Measurement of composition and observations of morphology

The samples were incubated in SBF at 37°C for 1, 14 and 28 days, followed by immersion in liquid nitrogen for 30 minutes to stop the reaction, as described before (31). Next, the samples were dried out with ModulyoD-230 (Thermo Electron Corp., Marietta, OH, USA) and ground into small powders. Then, X-ray diffraction (XRD; Rigaku, Tokyo, Japan) and Fourier transform infrared (FTIR) spectroscopy (Perkin-Elmer, Waltham, MA, USA) were used to measure the constitution and structure of the samples. Meanwhile, electron microscopy (LEO 1530 VP; LEO 1530 FESEM, Oxford, UK) was used to scan the morphology of the surface of the samples. The images were collected with a secondary electron detector under high vacuum (30 Pa) at an acceleration of 15 kV.

Measurement of pore size distribution

The pore size distribution of cements was determined using micro-computed tomography (micro-CT; Skyscan1076; Skyscan, Kontich, Belgium). The micro-CT was tuned at 70 kV and 140 μA, with a resolution of 9 μm. Micro-CT shadow projection images were converted into a 3-dimensional (3D) reconstruction of cross-sectional images in bitmap files using volumetric reconstruction software (Nrecon software; Skyscan, Belgium). The pore size was calculated from these 3D reconstructions using CTAn software (SkyScan, Kontich, Belgium).

Measurement of degradation of CPC samples in vitro

The CPC (CPC or CGRP-Sr-CPC) samples were immersed in SBF (mass to volume ratio = 0.2 g/L) for 28 days. Each day, the samples were taken out, dried and weighed. Then, the samples were immersed again in fresh SBF. The percentage of residual mass of each sample was calculated as (Wt × 100)/Wo, where Wt was the weight of the sample after immersion in SBF, and Wo was the initial weight of the sample before immersion in SBF.

Measurement of CGRP and Sr concentrations

CGRP-Sr-CPC was incubated in SBF (mass/volume = 1.25 cm²/mL) for 1 to 28 days. The cement samples immersed in SBF were gently shaken at 37°C. A portion of the immersed SBF buffer was removed each day for ELISA analysis to measure CGRP concentration as described in a previous report (11). The amount of Sr was measured using inductively coupled plasma–mass spectrometry (ICP-MS) assay according to the method previously published (34). The experiments were repeated 3 times.

Isolation and culture of BMSCs

BMSCs were generated from femoral and tibial bone marrow of OVX female mice as reported previously (11). BMSCs were plated with 6 × 10⁵ cells/60-mm dish and cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% fetal bovine serum (FBS) in an incubator at 37°C with 5% CO₂. Culture medium was replaced every 3 days, and the cells were passaged with 0.25% trypsin (#25200-072; Gibco) when they reached 80% confluence.

Cytotoxicity assay

We performed an indirect cytotoxicity assay to measure the cytotoxicity of the cement samples. To do that, extracts from cell culture medium with immersed cement samples were prepared. Phenol solution (0.02%) in phosphate-buffered saline (PBS) diluted with culture medium was used as a positive control extract. The negative control was collected by incubating an innocuous pure titanium sample in culture medium for 24 hours at 37°C. BMSCs were diluted at 1 × 10⁴ cells/mL in culture medium, and then 100 μL of BMSCs was added to each well in a 96-well plate, followed by incubation of BMSCs for 24 hours at 37°C. Then the cell medium was replaced by the extracts from cell culture medium with immersed cement samples as mentioned above. Next, the plates were incubated for 4 hours. After that, cell viability was measured using a WST-1 assay kit according to protocol (Cat. no. 05015944001; Roche). Triplicated experiments were performed.

Cell morphology

BMSCs with a density of 2 × 10⁴ cells/mL were added to each well in a 24-well plate. In each well, there was a cement disk with a diameter of 12 mm and a thickness of 2 mm. After culture for 1 or 14 days, the sample cell constructs were carefully washed twice with PBS, followed by fixation with 4% formalin. Next, the sample was dehydrated with a gradient of ethanol: 50%, 70%, 90%, 95% and 100%. The samples were then dried using liquid CO₂ and coated with a layer of gold of 20-nm thickness. The morphology of the cells, which were in contact with the samples was observed with a scanning electron microscope.

Cell proliferation assay

BMSCs at passage 3 (P3) were dissociated and cocultured with the cement samples 6 mm in diameter and 2 mm in height in a 96-well plate at 2.5 × 10⁴ cells per well. After culture for 1, 3, 5 or 7 days, a MTT assay was performed according to protocol using a commercial kit (#V13154; Life Technologies). Three experiments were performed at each time point for each cement sample.

Measurement of ALP activity

Cells were plated to 24-well plate at 5 × 10⁴ cells per well and co-cultured with the cement samples with a diameter of 12 mm and a height of 2 mm. After culture for 1, 3, 7 or 14 days, ALP activity was performed using an ALP assay kit according to the manufacturer’s protocol (#ab83369; Abcam). Triplicate experiments were performed.

Real-time PCR

BMSCs cultured on the cement samples for 1, 3, 7 or 14 days were subjected to RNA extraction using TRIzol reagent (#15596-066; Invitrogen). Quantitative PCR was performed in accordance with a previous report (35). PCR primers are listed in Table I.

Statistical analysis

All experiments were repeated at least 3 times independently. Graphs were generated with SPSS statistical software, version 19.0 (SPSS, Chicago, IL, USA). Two-way analysis of variance (ANOVA) followed by Tukey multiple comparisons was performed to determine the significance of the different treatments of implants at different time points. Data are expressed as means ± SD. A value of p less than 0.05 was considered significant.

Composition of the cement samples

To analyze the composition of the CPC, Sr-CPC, CGRP-CPC and CGRP-Sr-CPC, the XRD and FTIR techniques were used. With immersion in SBF for 1 day, the cement samples were analyzed with XRD. The data showed that there were intensive peaks for TTCP and DCPA (CPC and CGRP-CPC) or DSPA (Sr-CPC and CGRP-Sr-CPC), which are characteristic of the starting materials (Fig. 1A, B). We noted that there was an absence of any peaks of hydroxyapatite (HA), suggesting that the composition of the cements was in the original solid phase at this stage. Then, FTIR spectroscopy was performed, and it showed that the symmetric COO band was present at around 1,430 cm-1 and the antisymmetric COO band was present at 1,600 cm-1 in all CPC samples (Fig. 1C), demonstrating an occurrence of a chelated reaction in the body of the cements. The results also showed that there were no obvious differences between CPC and CGRP-CPC or between Sr-CPC and CGRP-Sr-CPC samples, suggesting that CGRP did not interfere with the initial chemical reactions in the cement body.

Then we examined the composition of cement samples that were immersed in SBF for 28 days. We found that the intensity of the original peaks of DSPA and TTCP remained but decreased gradually, and the intensive peaks of HA were observed, suggesting that most of the initial phases were converted into apatite (Fig. 1D). This result was consistent with our observations that there were typical characteristic peaks of PO₄ at around 1,033 cm^-1 and around 560-600 cm^-1, respectively (Fig. 1E). We also noted a deviation of the FTIR peaks of around 850 cm^-1 and 1,400 cm^-1 on the right side (Fig. 1E), which may have been due to the incorporation of carbonate. In addition, we assayed the degradation of CPC and CGRP-Sr-CPC in SBF buffer. Overall, both cements slightly degraded after immersion in SBF in vitro, and CGRP-Sr-CPC (degraded by ~12% on day 28) showed a rapid degradation as compared with CPC (degraded by ~5.9% on day 28) (Suppl Fig. 1 available online as supplementary material at www.jab-fm.com).

Morphology of the cement samples

To confirm the results of XRD and FTIR, we used scanning electron microscopy to scan the fractured surfaces of the samples. The data showed that the fractured surfaces were composed of entangled structures and irregular patterns after 1-day immersion of cements in SBF (Fig. 2A-B-C-D). The fractured surface that was exposed to the SBF solution was covered by a precipitation of apatite after 28 days of immersion of cements in SBF (Fig. 2E-F-G-H). These observations were consistent with the observations with XRD and FTIR that the precipitation of apatite was present after cements had been immersed in SBF for 28 days. In addition, we did not observe any differences between Sr-CPC and CGRP-Sr-CPC after immersion in SBF for 1 or 28 days (Fig. 2B, D, F and H).

Pore size distribution and compressive strength of samples

To further characterize the cement samples, the pore size and compressive strength of CPC, Sr-CPC, CGRP-CPC and CGRP-Sr-CPC were examined. These cements have similar pore size distributions and mass median pore sizes in a range from 45 to 64 μm (Fig. 3A-B-C-D). The results of compressive strength tests showed there was no significant difference detected in compressive strength for cement samples with the enrichment of Sr or CGRP after immersion in SBF for a day. All cement samples had a mean value of compressive strength of around 15 to 20 MPa (Fig. 3E). The compressive strength increased gradually with immersion in SBF for longer time periods (Fig. 3E). In addition, no significant difference was observed in compressive strength among cement samples with or without Sr or CGRP incorporation (Fig. 3E).

Next, we examined the release of CGRP after CGRP-Sr-CPC was immersed in SBF continuously for 28 days. Initially, CGRP had a concentration of 225.9 pg/mL (34.02% released) after CGRP-Sr-CPC had been immersed in SBF for 1 day (Fig. 3F). Then, the release of CGRP gradually increased after CGRP-Sr-CPC had been immersed in SBF for 4 days (97.49% released) and 7 days (99% released) (Fig. 3F). Thereafter, the concentration of CGRP was maintained at a high level of around 620 pg/mL. The release of Sr gradually increased from about 32 mg/L on day 1 to 54 mg/L on day 28 after being immersed in SBF (Suppl Fig. 2 available online as supplementary material at www.jab-fm.com). These results suggest that CGRP and Sr are gradually released, accompanied by degradation of CGRP-Sr-CPC (Suppl Fig. 1).

CGRP and Sr released from CGRP-Sr-CPC enhance proliferation of BMSCs

To investigate the effects of cements on BMSCs derived from pathological conditions, BMSCs isolated from osteoporotic rat tissues were cocultured with cement samples. The BMSCs were grown on a flat surface and spread on the surfaces of the samples with coculture for 1 day (Fig. 4A-B-C-D). After coculture for 14 days, the cement surface was almost covered with a confluence of cells in all groups, suggesting a good compatibility between cells and cement samples (Fig.4E-F-G-H). Consistent with the morphological observations, the cytotoxicity tests showed that all extracts from cocultures of BMSCs with the cement samples had a relative cell viability of 100%, compared with the negative control group, indicating there was no cytotoxicity for these cement samples (Fig. 5A).

Next we measured the effects of cement samples on proliferation of BMSCs. After culture for 1 day, there was no significant difference among all groups. After culture for 3 days, there was no significant difference between the CPC and Sr-CPC groups or between the CGRP-CPC and CGRP-Sr-CPC groups, but a significantly higher proliferation rate was detected in the CGRP-CPC and CGRP-Sr-CPC groups compared with the CPC and Sr-CPC groups (Fig. 5B). After culture for 5 and 7 days, the Sr-CPC and CGRP-CPC groups a significantly higher proliferation rates compared with the CPC group (Fig. 5B), suggesting that both Sr and CGRP promote proliferation of BMSCs. Notably, the CGRP-Sr-CPC group enriched by both CGRP and Sr showed enhanced effects on proliferation of BMSCs as compared with CPCs with CGRP or Sr alone (Fig. 5B), indicating that CGRP together with Sr strengthened and optimized the efficiency of the promotion of proliferation of BMSCs. In addition, adding free CGRP to Sr-CPC (CGRP/Sr-CPC) had a similar effect on proliferation of BMSCs compared with CGRP-Sr-CPC (Fig. 5B), which confirmed the role of CGRP and Sr on BMSCs.

CGRP-Sr-CPC has enhanced effects on induction of ALP activity

To further evaluate the effects of cements on regulation of differentiation, we measured the ALP activity. After culture for 1 day, the ALP activity was relatively low, and there were no significant differences among BMSC cultures with different cement groups (Fig. 6). After culture for 3 days, there were no significant differences among the Sr-CPC, CGRP-CPC, CGRP-Sr-CPC and CGRP/Sr-CPC groups, but there was a significantly higher ALP activity for these groups compared with the CPC group (Fig. 6). On days 7 and 14, the Sr-CPC and CGRP-CPC groups showed a significantly higher level of ALP activity compared with the CPC group (Fig. 6), suggesting that both Sr and CGRP induce ALP activity during coculture with BMSCs. The CGRP-Sr-CPC and CGRP/Sr-CPC groups showed enhanced effects on induction of ALP activity compared with the CGRP-CPC and Sr-CPC groups (Fig. 6). Altogether, the data suggest that either CGRP or Sr could increase ALP activity during differentiation of BMSCs, while the combination of CGRP and Sr has an enhanced effect on ALP activity.

CGRP-Sr-CPC increases expression of osteogenic differentiation–related genes

To validate the inductive role of CPC samples during osteogenic differentiation, genes associated with osteoblast differentiation were examined. Cells cocultured with CPC, Sr-CPC, CGRP-CPC or CGRP-Sr-CPC for 1, 7 and 14 days were collected to generate mRNA. The real-time PCR results showed that Sr or CGRP alone could significantly increase expression levels of genes involved in osteogenic differentiation, including Alp, Bmp2, Osteonectin and Runx2 during differentiation for 7 and 14 days (Fig. 7). With the combination of CGRP and Sr, the CGRP-Sr-CPC group showed significantly increased expression of these 4 genes compared with the CGRP-CPC and Sr-CPC groups (Fig. 7). Therefore, CPC with enriched CGRP and Sr has enhanced inductive effects to promote osteogenic differentiation–related gene expression.