Effect of inoculation density of bone marrow mesenchymal stem cells cultured on calcium phosphate cement scaffold on osteogenic differentiation

Abstract

BACKGROUND:

Calcium phosphate cements (CPCs) are biocompatible materials that have been evaluated as scaffolds in bone tissue engineering. At present, the stem cell density of inoculation on CPC scaffold varies.

OBJECTIVE:

The aim of this study is to analyze the effect of seeding densities on cell growth and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) on a calcium phosphate cements (CPCs) scaffold.

METHODS:

BMMSCs derived from minipigs were seeded onto a CPC scaffold at three densities [1 million/mL (1M), 5 million/mL (5M) and 25 million/mL 25M)], and cultured for osteogenic induction for 1, 4 and 8 days.

RESULTS:

Well adhered and extended BMMSCs on the CPC scaffold showed significantly different proliferation rates within each seeding density group at different time points (P < 0.05). The number of live cells per unit area in 1M, 5M and 25M increased by 3.5, 3.9 and 2.5 folds respectively. The expression of ALP peaked at 4 days post inoculation with the fold-change being 2.6 and 2.8 times higher in 5M and 25M respectively as compared to 1M. The expression levels of OC, Coll-1 and Runx-2 peaked at 8 days post inoculation.

CONCLUSIONS:

An optimal seeding density may be more conducive for cell proliferation, differentiation, and extracellular matrix synthesis on scaffolds. We suggest the optimal seeding density should be 5 million/mL.

Keywords

Bone marrow mesenchymal stem cells calcium phosphate cement seeding density osteogenic differentiation

1. Introduction

Calcium phosphate cements (CPCs) are self-setting bioactive and biocompatible materials that have been exploited in biomedical applications due to their unique properties [1–3]. Particularly, their use in dental, craniofacial, and orthopedic applications have been of recent research interest [4–6]. Therapeutic approaches based on stem/progenitor cells with biomaterials and scaffolds such as CPCs have gained popularity in tissue engineering and regenerative medicine [7–10].

Various studies have shown promising results for clinical applications for bone defect repair with mesenchymal stem cell seeding on CPCs. It has been shown that human umbilical cord mesenchymal stem cells (hUCMSCs) seeded CPC-fiber scaffold has increased osteodifferentiation and mineralization abilities at low cell densities [11]. Human embryonic stem cell-derived mesenchymal stem cells (hESCd-MSC) loaded on CPC-Chitosan-RGD scaffold showed good viability and osteogenic differentiation apart from increasing the cell attachment to the scaffold, enhanced proliferation and bone mineral synthesis [12]. Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) implanted on CPC have been shown to have higher gene expressions of osteogenic markers, four to five-fold higher bone mineralization synthesis in osteogenic medium compared to control medium [9] and greater cell proliferation on biofunctional CPCs [10]. Studies on bone regeneration for rat cranial defects have shown that hUCMSCs, hiPSCs and human bone marrow mesenchymal stem cells (hBMSCs) seeded on a CPC scaffold at a constant cell density had comparable bone mineral density, new bone amount and vessel density, and enhanced bone regeneration efficiency, more than doubling the new bone amount of cell-free CPC control [13,14].

Bone marrow mesenchymal stem cells (BMMSCs) have been widely used in tissue engineering, cell/gene therapy, clinical treatment of hematopoietic pathologies, cardiovascular diseases since possess strong differentiation potential, are easily isolated and cultured, and cause less immune rejection after transplantation in vivo; it is also in focus for treating other diseases of the musculoskeletal, digestive, integumentary, and nervous systems [15,16]. In spite of their biological importance, not many studies are dedicated to the cell seeding densities and delivery of BMMSCs through bioactive scaffolds for bone repair. It is known that cell seeding density is important for several physiological processes such as cell proliferation, differentiation, extracellular matrix synthesis [17], and also, for optimization of the cell–biomaterial microenvironments to increase the osteogenic signal expression as well as bone mineralization by enhanced osteogenic marker expression [18]; hence, it is imperative to study the influence of stem cell densities on CPC. Therefore, this study aimed to explore the effects of different inoculation densities of BMMSCs on a CPC scaffold on cell proliferation and osteogenic differentiation.

2. Materials and methods

2.1. Preparation of solid CPC powder and CPC disk

Calcium phosphate dibasic (DCPA) and calcium carbonate (CaCO₃) (Sigma, USA) were mixed (Ca/P ratio at about 1.9, range 1.5–2.0) and heated to 1500 °C to prepare tetra calcium phosphate (TTCP). The product was ground and sieved to obtain TTCP particles with a diameter of 1–80 μm. DCPA was treated in the same way to obtain DCPA particles with a diameter of 0.4–3 μm. Solid CPC powder was prepared by mixing TTCP (MW = 366.254 g/mol) with DCPA (MW = 136.057 g/mol) at a mole mass ratio of 1:1, and CPC powder was mixed with water at a mass ratio of 3:1. After that, the mixture was thoroughly stirred and poured into a circular mold with an inner diameter of 12 mm, an external diameter of 15 mm, and a height of 2 mm. Then, the mold was incubated at 37 °C for 4 h, and a CPC disk was demolded, with a round shape 12-mm in diameter and 2-mm in height. The scaffold was placed in a 37 °C water bath for 24 h, then dried and sterilized by ethylene oxide for future use.

2.2. BMMSCs isolation and culture

BMMSCs were obtained from the bone marrow of minipigs (obtained from an animal center at Southern Medical University) aged 12–18 months, weighted 30–40 kg. This research has been approved by the IRB of the authors’ affiliated institutions. Under sterile conditions, 5–10 mL of bone marrow was aspirated from the posterior superior iliac crest, and the Ficoll solution was added to isolate lymphocytes. After centrifugation at 1000 × g, the white layer in the middle was carefully aspirated, and adjusted to a concentration of 3 × 10⁵ cells/mL for primary culture. Cells were cultured in a cell culture flask containing low-glucose Dulbecco’s modified eagle medium (Gibco^® DMEM, ThermoFisher Scientific, USA) containing 10% fetal bovine serum (Gibco^® FBS, ThermoFisher Scientific, USA), 1% penicillin/streptomycin (10,000 U/mL, ThermoFisher Scientific, USA) and 1% non-essential amino acid (ThermoFisher Scientific, USA). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. When the cells reached a confluency of about 70–80%, the plates were washed with Gibco^® Trypsin-EDTA (0.05%; ThermoFisher Scientific, USA) and PBS in order to remove non-adhered cells and the medium was replaced. Osteogenesis was induced by adding dexamethasone (100 nM), b-phosphoglyceric acid (10 mM), ascorbic acid (0.05 mM) and 1, 25-dihydroxyvitamin D3 (10 nM) (Gibco) into the complete medium. BMMSCs within three passages were used for all the experiments. Three discs were made for each time frame and density.

2.3. BMMSCs inoculation onto CPC and SEM observation

Based on previously reported method [9], BMMSCs were diluted in 2 mL osteogenic inducing medium, adjusted to a concentration of 1 million cells/mL (1M), 5 million cells/mL (5M) and 25 million cells/mL (25M), and inoculated onto CPC disks placed in a 24-well plate. The medium was changed every 3 days, and cells cultured up to 1, 4 and 8 days were taken for observation. At 4 days after osteogenic culturing, cells were fixed by 2.5% glutaraldehyde, serially dehydrated by ethanol, sputter coated with platinum powder, and observed under a scanning electron microscope. CPC discs with medium but no cells were the controls.

2.4. EdU (5-ethynyl-2^′-deoxyuridine) labeling

The EdU solution (reagent A, Invitrogen) was diluted in the culture media (1:1000) to a final concentration of 50 μM; 2 × 10⁵ BMMSCs were incubated with 500 μL EdU for 2 h, washed with PBS, fixed using 3.7% formaldehyde-PBS at room temperature for 15 min, lysed by 0.5% Triton^® X-100 (dissolved in PBS), and stained by Click-iT (Click-iT^®-EdU; ThermoFisher, Waltham, MA, USA) at room temperature for 30 min in the dark. The cells were then stained with Hoechst 33321 (reagent G), for 30-min in the dark at room temperature. Cells were washed twice with PBS and observed using laser confocal microscope (Olympus, Japan).

2.5. Live/dead staining

Cells were added with 2 mL PBS containing 2 μM calcein-AM (labeling living cells) and 2 μM ethidium homodimer 1 (labeling dead cells), and were observed under a fluorescent microscope (Eclipse TE2000-S; Nikon, Melville, NY, USA). Cells from 1M, 5M and 25M cell densities on the CPC disks from 1, 4 and 8 days were checked for cell viability. The percentage of living cells (PLive) was calculated by (PLive) = NLive/(NLive+NDead), wherein NLive and NDead respectively represents the number of living and dead cells. The density of living cells (DLive) was calculated by (DLive) = NLive/A, wherein A represents the field area occupied by living cells [15].

2.6. Real time PCR

Cells from 1M, 5M and 25M cell densities on the CPC disks from 1, 4 and 8 days were checked for mRNA expression. Total RNA extraction was extracted using TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan, Code No. 9767), quantitated and reverse transcription was carried out using PrimeScript^{^TM} RT Master Mix (TaKaRa, Code No. RR036A) and real-time PCR was carried out using SYBR^® Green I (TaKaRa, Code No. RR820A) on an ABIPRISM 7500 (USA). The expression of ALP (Alkalinephosphatase,), OC (Osteocalcin), Coll-1 (collagen type I) and Runx2 (runt-related transcription factor 2) was examined using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as control. The Ct value of target genes was normalized by GAPDH to obtain the ΔCt value, and the relative expression of each target gene was calculated by 2^−ΔΔCT. The Ct value of target genes was normalized by the housekeeping gene GAPDH (glutaraldehyde 3 phosphate dehydrogenase) to obtain ^Ct value. The relative expression level of each target gene was calculated by the 2^−ΔΔCt method for data analysis. The primers are listed in Table 1.

Table 1
Primers for RT-PCR

Gene Forward (5^′–3^′) Reverse (5^′ –3^′)

ALP 5^′-CCCCAATCACTGCAAGACAGA-3^′ 5^′ -ATTCCCAGACAGCACAAAGTAGG-3^′

RUNX2 5^′-CTGGGAGGGAGAAGGGAAA-3^′ 5^′ -TGGGGTCCAAGTCCTCAAA-3^′

Collagen type I 5^′-TCGCACATGCCGTGACTT-3^′ 5^′ -GCTTCTTGACCTTGGAGTTTCTGT-3^′

Osteocalcin 5^′-CCTCACACTGCTTGCCCTAC-3^′ 5^′ -TGCCAGAATCCGCACCA-3^′

GAPDH

Gene	Forward (5^′–3^′)	Reverse (5^′ –3^′)
ALP	5^′-CCCCAATCACTGCAAGACAGA-3^′	5^′ -ATTCCCAGACAGCACAAAGTAGG-3^′
RUNX2	5^′-CTGGGAGGGAGAAGGGAAA-3^′	5^′ -TGGGGTCCAAGTCCTCAAA-3^′
Collagen type I	5^′-TCGCACATGCCGTGACTT-3^′	5^′ -GCTTCTTGACCTTGGAGTTTCTGT-3^′
Osteocalcin	5^′-CCTCACACTGCTTGCCCTAC-3^′	5^′ -TGCCAGAATCCGCACCA-3^′
GAPDH

2.7. Statistical analysis

Statistical analysis was performed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± SD. One-way ANOVA was used for determination of statistical significance between groups and Least Significant Difference (LSD) was performed for multiple comparisons between groups. Statistical significance was defined at P < 0.05.

3. Results

3.1. Adherence of BMMSCs to CPC scaffolds

Representative SEM images of cells adhered on CPC are shown in Fig. 1A, B and C, at high and low magnification respectively. It can be seen that CPC could form apatite crystals after solidification, and BMMSCs could adhere to the CPC surface and extend into pores. This was found in all cell seeding concentrations.

Fig. 1.

Scanning electron micrographs of BMMSCs adhered to the surface of the CPC scaffold at high (1A: 5M, 4 days) and low (1B: 25M, 8 days) resolution, and control (1C: 1 day). C: cell; E: cell extension.

3.2. Cell proliferation assay

Cell proliferation was analyzed by EdU assay. As shown in Fig. 2A, at 1 day after inoculation, the proliferation rates in three groups with different cell seeding concentrations of 1M, 5M and 25M were significantly different (F = 56.262, P = 0.000). Further LSD method showed that there was no significant difference between the 5M and 25M groups (P = 0.090), and similar results also appeared at day 4 and day 8 after inoculation. Figure 2B shows a graphical representation of these results. However, the proliferation rates detected at three time points in the 1M group were significantly different (0.544 ± 0.030, 0.428 ± 0.022, 0.176 ± 0.016, P = 0.027), and similar results were obtained from the 5M and 25M groups (P < 0.05).

Fig. 2.

EdU staining of proliferating cells. Representative images of the proliferating cells of 1M, 5M and 25M seeding densities on the CPC scaffold at 1, 4 and 8 days (A); graphical representation of the percentage of proliferating BMMSCs (B). All the images are magnified at 40×.

3.3. Cell viability assay

Live/dead assay was used to determine cell viability. As depicted in Fig. 3(A–I), with the increase of incubation time and cell seeding densities, cells were able to attach as well as thrive well on the CPC scaffold. The number of live cells (stained green) were maximum at 8 days in the 25M group (Fig. 3I) due to cell proliferation. The percentage of live cells increased from 70% to 85% when the seeding density increased from 1M to 25M at 1 day. At 8 days, the percentage of live cells in the 25M group reached above 93% (Fig. 4A). The number of live cells per unit area also increased with the increasing seeding densities and culture time. From 1 to 8 days, the number of live cells per unit area in cell seeding densities of 1M and 5M increased by 3.5 and 3.9 fold respectively, whereas in 25M seeding density, increased by 2.5 folds since the cells became confluent with time (Fig. 4B). At 8 days, the dead cells (stained red) were relatively few.

Fig. 3.

Live/Dead staining assay. Representative images of live cells observed on 1, 4 and 8 days after inoculation on CPC scaffold with seeding densities of 1M, 5M and 25M (A–I). At 8 days after inoculation, the number of dead cells seen at all seeding 3 densities were minimal (J–L). All the images are magnified at 100×. Samples n = 3.

3.4. Expression of osteogenic differentiation genes

The gene expression profile of the four osteogenic differentiation genes (ALP, OC Coll-1 and Runx-2) as measured by RT-PCR are plotted in Fig. 5. As shown in Fig. 5A, the peak of ALP expression occurred at 4 days for all seeding densities. At this time point, the expression of ALP in group 5M and 25M was 2.6 times and 2.8 times higher than that in group 1M (P < 0.05). However, no significant difference was identified between group 5M and 25M. The expression of OC peaked at 8 days, and seeding density of 5M showed no significant difference from that of 25M (F = 1.131, P = 0.383, P > 0.05, Fig. 5B). The expression of Coll-1 increased significantly at 8 days, and there was no significant difference in the expression profile at seeding densities of 5M and 25M (F = 3.940, P = 0.081, P > 0.05, Fig. 5C). A rapid increase in Runx-2 expression was seen at 8 days; the 5M and 25M groups showed no significant differences in the level of expression (LSD method, P = 0.211, P > 0.05, Fig. 5D).

Fig. 4.

Graphical representation of the percentage of live cells (A) and number of live cells per unit area (B). Samples n = 3.

Fig. 5.

Graphical representation of the RT-PCR results for osteogenic differentiation of BMMSCs on CPC scaffold: ALP (A), OC (B), Coll-1 (C) and Runx-2 (D). Samples n = 3.

4. Discussion

In the current study, the effect of BMMSCs cell seeding densities on cell proliferation, osteogenic differentiation, and mineral synthesis on CPC was evaluated. CPC had good affinity for cell attachment, and no negative effects on cell viability. The rate of cell proliferation was significantly different for all the three seeding densities between the three time points. The expression of the osteogenic differentiation markers showed that ALP expression peaked at 4 days of seeding while the other genes were upregulated by 8 days.

CPC has been shown to be a durable biomaterial for stem cell adhesion in various studies [9,11]. Our study confirmed these findings and proved that BMMSCs have a good affinity for CPC since cell extension could also be observed under electron microscopy. Therefore, CPC can be safely used in clinical applications with BMMSCs.

Though the rate of cell proliferation was similar across the different cell seeding densities, there was a difference in it when compared between the three different time points. BMMSCs had good proliferative activity after being seeded onto CPC, but the activity decreased with time. By the 8th day after seeding, the proliferation was reduced from 52% to 16% in group 25M. This might be due to the influence of cell accumulation because of cell extension and proliferation. Since the in vitro conditions do not entirely correspond to in vivo environment, when cell density is too high, limited nutrients, hypoxia, inadequate waste removal, contact inhibition, and other factors might inhibit cell growth, proliferation, or expression of functional activities [11,19].

In this study, the percentage of live cells at 1 day after seeding was 67% and 86% in group 1M and 25M, respectively. With increasing culture time, cell proliferation led to an increased percentage of live cells, and the difference between three groups was also reduced. From 1 to 8 days, the number of live cells per unit area in group 1M and 5M more than that in the 25M group, which can be explained by contact inhibition [18].

ALP, OC, Coll-1, and Runx2 have important roles in the osteogenic differentiation of MSCs [18]. ALP and OC are well-established markers for osteogenic differentiation [20]. In the present study, though 25M group had the highest expression of ALP and OC genes, the difference was insignificant when compared to 5M group. Yet, the expression of ALP and OC in group 5M and 25M was significantly different from that in group 1M. Due to the cascade effect of differentiation, other marker genes for osteogenesis, OC for example, would be up-regulated, while ALP would be down-regulated as shown in previous studies [11,21]. In this study, ALP expression peaked at 4 days and reduced at 8 days, whereas the expression of OC peaked at 8 days. Many studies confirm that the expression of ALP gene would increase first and decrease later, and the peak of ALP expression might occur within 4–16 days. According to one study, ALP had the lowest expression at 1 day, the highest at 4 days and the expression level decreased at 8 days and the expression of OC peaked at 8 days [18]. According to another study, ALP expression in MSCs under osteogenic induction peaked at 8 days, and reduced at 16 days [22]. Our study results concur with the earlier reports, where ALP expression peaked at 4 d and lowered at 8 days, and the levels of OC, Coll-1 and Runx2 peaked at 8 days.

There have been varying views related to the initial density of cell inoculation on bioactive scaffolds. While some report a high seeding density would promote cellular synthesis, high expression of ALP and OC and produce more bone minerals [18,23,24], others opine that a high seeding density does not necessarily have more advantages; an optimal seeding density would help to form better extracellular matrix and achieve the best tissue regeneration effects [17,25,26]. When cells were seeded and cultured at a low concentration, the function and biosynthesis could be enhanced during cell proliferation. Endogenous signaling molecules would promote cell-to-cell interactions, and extracellular matrix secretion would improve adhesion of adjacent cells. Such intra- and extra-cellular interactions would proceed until cells reach the optimal density, and once the optimal density is exceeded, cells would be limited by insufficient nutrients and space. This might further lead to hypoxia and incomplete removal of metabolic waste, and inhibit cell proliferation via contact inhibition produced by gap junctions of adjacent cells [11].

5. Conclusion

Our results showed that cells which inoculated at a low density of 1M show limited viable and lower capacity of both osteogenic differentiation and mineral production. By rising the initial seeding density or prolonging the time of culture of cells on the scaffold, an increased percentage of live cells were observed suggesting that neighboring cells depend on each other to enhance adherence and viability for growth. However, when cell density is too high, cell growth and expression of functional activities stagnates due to the constrained environment. In this study, though 25M group had an initial seeding density 5 times higher than that of 5M group, the percentage of live cells, as well as the expression of osteogenesis markers (ALP or OC), was slightly higher, but insignificantly different from those of group 5M. Therefore, we suggest that the optimal seeding density should be 5 million/Ml when cells are diluted to 2 mL and inoculated onto a CPC disk (12 mm diameter and 2 mm thickness) and cultured in a 24-well plate. In conclusion, a higher inoculation density does not necessarily promote BMMSCs growth; an optimal seeding density should be more conducive for cell proliferation, differentiation, and extracellular matrix synthesis on a specific stem cell scaffold. Further studies are warranted to evaluate the in vivo regeneration ability of BMMSCs inoculated at different concentrations on CPC scaffolds. Thus, BMMSCs construct on CPC scaffold could be a promising prospect of clinical treatment for bone repair and regeneration in plastic, maxillofacial and orthopedic surgeries.

Footnotes

Acknowledgements

Not applicable.

Ethical statement

This research was approved by the EC Review Certificate of Deqing County Wukangjianbao Group (LL2016-09).

Conflict of interest

All authors declare that they have no conflict of interests.

Informed consent

The patients and/or their families were informed that data from the research would be submitted for publication, and they provided consent.

Funding

This study was supported by the Natural Science Capital of Huzhou (2016YZ09) and National Natural Science Foundation of China (31328008).

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Effect of inoculation density of bone marrow mesenchymal stem cells cultured on calcium phosphate cement scaffold on osteogenic differentiation

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1. Preparation of solid CPC powder and CPC disk

2.2. BMMSCs isolation and culture

2.3. BMMSCs inoculation onto CPC and SEM observation

2.4. EdU (5-ethynyl-2 ′ -deoxyuridine) labeling

2.5. Live/dead staining

2.6. Real time PCR

3. Results

3.1. Adherence of BMMSCs to CPC scaffolds

5. Conclusion

Footnotes

Acknowledgements

Ethical statement

Conflict of interest

Informed consent

Funding

References

2.4. EdU (5-ethynyl-2^′-deoxyuridine) labeling