Ultrastructural changes during osteogenic differentiation in mesenchymal stromal cells cultured in alginate hydrogel
© The Author(s) 2017
Received: 8 September 2016
Accepted: 9 December 2016
Published: 3 January 2017
Osteogenic differentiation of mesenchymal stem cells has been extensively investigated with regards to different aspects, including the analysis of cell intracellular and extracellular proteome, cell gene expression pattern, and morphology. During the osteogenic differentiation, osteoblasts produce and release specific proteins, like osteocalcin and osteopontin. Simultaneously, cells produce the extracellular matrix (ECM) that resembles the bone ECM, with high quantity of calcium and phosphorus. We focused on the ultrastructural changes occurring during the osteogenic differentiation of MSC cultured in alginate hydrogel.
The analysis revealed that during the osteogenic differentiation the most of cells become dead, and these dead cells contain large quantities of calcium and deposition is strictly connected with the cellular death and small membrane vesicles released by cells. Cell organelles were not present within differentiated cells, while in cells from non-osteogenic group the cellular ultrastructure was proper, with single nuclei, endoplasmic reticulum and numerous mitochondria.
The ECM synthesis and deposition during the osteogenic differentiation of MSC involves cellular programmed death. The small membrane vesicles become the mineralization sites of formed bone ECM.
KeywordsMesenchymal stromal cells Alginate hydrogel Osteogenic differentiation Mineralizing vesicles Apoptosis Ultrastructure
Osteogenic differentiation in vitro by mesenchymal stromal cells (MSC) is the process involving the change in cell behavior, protein secretion profile and cell ultrastructure. While this process is well analyzed in monolayer cell cultures , the osteogenic differentiation in 3D hydrogel culture model needs to be enclosed, especially concerning the ultrastructural features of cells undergoing the differentiation, and the mechanisms of ECM deposition.
Mesenchymal stem cells (MSC), which have been extensively used in experiments and clinical trials are characterized by their multilineage differentiation potential, which means that they are able to differentiate into the specialized cells, like osteoblasts, adipoblasts or chondroblasts, among others . During the osteogenic differentiation, MSC change their behavior, morphology, gene and proteome expression patterns. For example, in MSC cultured in osteogenic conditions the alkaline phosphatase becomes active, which is followed by the synthesis and secretion of osteocalcin protein. Simultaneously, the process of biomineralization occurs . Nonetheless, the exact mechanism of matrix synthesis and calcification is still not clear enough. Anderson  showed with electron microscopic techniques that during the biomineralization occurring in the cartilage, bone and dentin tissues, the abundant small vesicles—called matrix vesicles (MV), could be found at the initial calcification sites .
Matrix vesicles, also known as microvesicles are small extracellular organelles which arise for example by budding from cell surface, therefore they are composed of small cytoplasm fragment surrounded by the double layered plasma membrane. They have been identified in all tissues, but their role is still not sufficiently explained. They constitute the one of intercellular communication mechanisms, as it was shown in previous studies . Moreover, numerous observations of Anderson [4, 6–12] and others revealed that similar submicron organelles may be also involved in the process of biomineralization [4, 6–12].
In 2009 Golub described the three hypothetical mechanisms on which the calcification of extracellular matrix might be based . One of the hypothesis assumes, that MV released by osteoblasts only regulate the concentrations of ions outside the MV, which leads to the formation of soluble molecular species, followed by the initiation of mineral formation in collagen. Second hypothesis states that the apatite crystals are formed within the MV, which is followed by their release and deposition over the collagen fibers. Third hypothesis assumes that besides the accumulation of calcium phosphate within the MV, its further propagation is facilitated by the interaction between MV and collagen fibrils. These hypotheses has not been proven nor excluded yet. Moreover, it was noticed by Proudfoot et al. that during the osteogenic differentiation, the osteoblasts loss their viability and undergo apoptosis, and the apoptotic bodies establish the nucleation sites for newly formed mineralized matrix .
In this work we analyzed if the process of osteogenic differentiation of MSC cultured in calcium alginate beads occurs also by means of similar mechanisms to in vivo conditions, with the major attention put on ultrastructure of cells and the process of biomineralization at the nano-scale level—the features that have not been analyzed at this scale yet. We showed that indeed the microvesicles may become the mineralization sites for forming hydroxyapatite crystals. Moreover, we showed that the ultimately differentiated cell become dead and filled with hydroxyapatite.
Preparation of alginate solution
Sodium alginate solution was prepared from low viscosity sodium alginate (Sigma Aldrich) dissolved in sterile 0.9% NaCl at a final concentration of 2% w/v. Obtained solution was sterile filtered using 0.45 and 0.22 µm syringe filters and stored in 4 °C for further usage.
Bone marrow mesenchymal stem cells (BMSC) were isolated from adult rat of Wistar bred, using method described before . Briefly, the femurs and tibias were collected directly after the euthanasia of the donor and placed in sterile Hank’s balanced salt solution (HBSS). For cell collection, bone heads were cut and the marrow was flushed out from the bone with Dulbecco modified eagle medium (DMEM) to a falcon tube, followed by centrifugation (300×g/4 min) and resuspension in DMEM containing the 10% of fetal bovine serum (FBS) and 1% of penicillin/streptomycin (standard medium). Cells were plated in T-75 culture flasks for primary culture. Cells were propagated for 5–7 days until they reach full confluence.
At this stage of culture, cells positive for specific antigens of mesenchymal stem cell population, i.e. CD44+/CD90+ were isolated, and the remaining cells discarded, including cells positive for hematopoietic cell marker CD45+ by means of magnetic, immuno-based separation technique (rabbit anti-rat CD44, 3 µg/ml, Sigma Aldrich; mouse anti-rat CD90, 5 µg/ml, Sigma Aldrich; anti-rabbit/anti-mouse IgG-magnetic microbeads labeled, MACS, Miltenyi Biotec, Germany). Purified BMSC were centrifuged 300×g/4 min and resuspended in 2% sodium alginate solution at a concentration of 5 × 105 cells/ml as the secondary cultures. To obtain calcium alginate gels with BMSC embedded within, the 50 µl-droplets of cell suspension in alginate solution were dropped in 0.1 M CaCl2 and kept in for 5 min at room temperature. Materials were washed three times in DMEM and placed in 96-well plate (every hydrogel sample in separate well) for osteogenic differentiation assay. Prior to assay, two samples were taken for evaluation of cell viability, using calcein AM and propidium iodide fluorescent staining and microscope, which resulted in over 95% of cell viability. Cultures were maintained in osteogenic differentiation medium (StemPro Osteogenesis Differentiation Kit, Life Technologies), and in standard medium (DMEM with l-glutamine, 10% of fetal bovine serum). Cell culture media were changed twice a week, cell differentiation was maintained for 3 weeks.
After the three weeks of culture, hydrogel samples were stained for viability using calcein AM and propidium iodide, using standard protocol. Other samples were fixed in 2.5% glutaraldehyde overnight in 4 °C for electron microscopic analysis, in 4% neutral buffered formalin for 1 h for H&E and histochemistry, and in 4% paraformaldehyde for immunofluorescence. Prior to histological staining, hydrogels were dehydrated in graded series of ethanol, embedded in paraffin, cut on 5 µm sections and collected on microscopic slides. Sections were stained with hematoxylin (3 min) and eosin (2 min), washed with water and covered with coverslips using DPX. Preparations were analyzed with light microscope (AxioImager, Zeiss). Documentation was made with Canon PowerShot digital camera.
To analyze the presence of apoptotic processes during the differentiation, immunofluorescent staining for caspase-3 protein was performed. Paraformaldehyde-fixed hydrogels were washed three times with PBS containing 2% of FBS. Next, samples were incubated in 0.05% Triton x-100 in PBS for 15 min, followed by triple washing and blocking in 5% normal goat serum in PBS for 1 h. After blocking, samples were washed three times in PBS and incubated overnight with primary antibody (mouse anti-casp3, Thermo Scientific, cat. no. 437,800, concentration 1:100). Next, samples were washed three times with PBS and incubated with secondary antibody solution (goat anti-mouse IgG conjugated to Atto-629, Thermo Scientific, concentration 1:500) for 1 h at 37 °C. After triple washing in PBS, samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 5 µg/ml) and observed in confocal microscope as a whole mount (Zeiss Axio LiveImager). To prepare a simple 3D reconstruction of the signals, the z-stacked images were processed using Imaris software.
For electron microscopic observations, samples were prepared as critical point-dried (CPD) fractures of whole hydrogels with cells, and as heavy metal-stained (HM) samples embedded in resin. Prior to observations, CPD samples were undertaken for conduction-providing preparation (OTOTO protocol) . Briefly, fixed samples were washed in cacodylate buffer (pH = 6.8) for glutaraldehyde removal and incubated in cacodylate-buffered 1% osmium tetroxide with 1.5% of potassium ferricyanide for 1 h. After washing three times in buffer and two times in ultrapure water (UPW), samples were incubated in 1% thiocarbohydrazide (TCH) aqueous solution for 30 min. Next, samples were washed 5 times in UPW and incubated in 2% osmium tetroxide in cacodylate buffer for 30 min, followed by another five washings in UPW and incubation in TCH solution for 30 min. After another five washings in UPW, samples were incubated in 2% osmium tetroxide in cacodylate buffer for 20 min, washed 5 times in UPW and dehydrated using graded series of acetone (from 10% up to 100% of acetone concentration, change every 10%). At this stage, hydrogels were sectioned using surgical blade, and dried using automatic critical point dryer (CPD300, Leica). Dried samples were mounted on microscope stub and placed in scanning electron microscope (Auriga 60, Zeiss) for observations and elemental composition analysis. Observations were made at 2 kV of filament tension. The focused ion beam milling was performed to reveal the internal structure of cells (FIB, Cobra, Auriga 60, Zeiss).
HM samples were fixed in 2.5% glutaraldehyde in cacodylate buffer (pH = 6.8) for 24 h, followed by their triple washing in buffer and incubation in buffered 1% osmium tetroxide with 1.5% of potassium ferricyanide for 1 h on ice. Next, samples were washed three times in buffer and three times in UPW. Material was then incubated with 1% uranyl acetate aqueous solution overnight at 4 °C. After five washings in UPW, samples were dehydrated using graded acetone series, incubated in agar resin (1:3, 1:1, 3:1 of resin:acetone and pure resin, 2 h each). Resin blocks were polymerized for 48 h at 60 °C. Samples were then trimmed with glass knife using ultramicrotome (AC7, Leica) to prepare the surface for FIB milling. Trimmed samples were mounted on microscope stub with carbon tape, covered with 20 nm layer of gold with high vacuum coater (EM ACE600, Leica), and placed in microscope chamber (Auriga 60, Zeiss). The cells were found by observations of polished surface at 20 kV of beam acceleration voltage, where the single cells were visible. Prior to FIB milling, milled surface was covered with protective 5 nm layer of platinum using gas injection system. Materials were milled using 30 kV/16 nA aperture to obtain the trench, and 30 kV/2 nA to polish the imaged surface. Cells were then observed using SE2 detector at 2 kV of acceleration voltage and LUT display set on inverted mode.
Markers of osteogenic differentiation
Alizarin Red S staining
To analyze the presence of calcium ECM in hydrogels, Alizarin Red S staining was applied. Briefly, fixed hydrogels were washed in distilled water, incubated for 10 min in Alizarin Red S working solution, washed three times in water and observed with inverted microscope as a whole mount. Additionally, fixed hydrogels were embedded in paraffin, cut for 5 µm sections and stained for 5 min. Documentation was made with Canon PowerShot digital camera.
Energy dispersive X-ray (EDX) analysis of calcium and phosphorus content within the hydrogels was performed using Oxford detector and Inca software combined with SEM at 20 kV of accelerating voltage. For this analysis, critical point-dried, fractured samples were taken. Three repeats from every group were made, and the results were averaged and statistically verified using Student’s t test. The distribution of calcium and phosphorus was determined at the FIB-milled area to show the concentration of these elements around the single cells.
Measurements of alkaline phosphatase, osteocalcin and osteopontin
Extracellular activity of ALP was determined in the supernatants collected during the last day of experimental culture. The assay was performed using the alkaline phosphatase colorimetric assay kit (Abcam, Cambridge, UK), according to protocol provided by the manufacturer. The experimental samples were prepared in duplicates and were diluted twofold. Sample background control was included and the background was corrected by subtracting the value derived from the zero standards from all standards, samples, and sample background control. As a phosphatase substrate, the p-nitrophenyl phosphate (pNPP) was used. The substrate was hydrolyzed into p-nitrophenol by alkaline phosphatase. The reaction product was measured at 405 nm of wavelength using microplate reader (BMG Labtech). Sample readings were applied to the standard curve to obtain the amount of pNP generated by the ALP sample. The enzymatic activity was determined using the following formula:
ALP activity (U/ml) = A/V/T, where: (i) A is the amount of pNP generated by the samples (in nmol); (ii) V is the volume of sample added to the assay well (in ml) and (iii) T is the reaction time.
The osteocalcin (OCL) and osteopontin (OPN) levels were determined in culture supernatants collected after the last day of culture, from three plate wells (repeats) for each analyzed group. Analyzed protein content was determined using Rat Gla-Osteocalcin High Sensitive ELISA Kit (Takara) and Mouse/Rat Osteopontin Quantikine ELISA Kit (R&D Systems). The quantitative determination of OCL and OPN was performed according to manufacturers’ instructions. The amount of detected proteins was expressed as protein weight/supernatant volume ratio (w/v). Statistical significance of obtained results was verified using OneWay Anova test.
Measurements of alkaline phosphatase, osteocalcin and osteopontin
The osteogenic differentiation of MSC is a complex process involving the change of gene expression pattern, protein synthesis and the ultrastructure of cells after the process is completed. There are two distinct paths of cell development after its osteogenic differentiation described in the literature. In the first one osteoblast gets on the surface of bone and becomes the bone lining cell after the synthesis of ECM. In the second path osteoblast stays within the ECM and becomes an osteocyte. However, Jilka et al. described, that the number of osteoblasts in the bone growth zone is not equal to the number of cells present in this area after the tissue development is finished, which suggests that some part of cells undergo cellular death . Our results showed that the significant number of cells undergo apoptosis, and this process is probably connected to the bone ECM mineralization process. The apoptotic bodies released from dead cells become the nucleation sites of hydroxyapatite crystallization. Such process occurs not only in bone tissues, but also can be observed for example in the blood vessels where it is connected with the apoptosis of vascular smooth muscle cells .
Our results confirm these observations, while the majority of MSC underwent osteogenic differentiation have lost their viability during the process via the apoptosis. Despite the occurrence of cellular death, MSC deposited significant amount of calcium ions within the hydrogel, which was confirmed by the Alizarin Red staining. It should be mentioned however, that in control culture cells also produced and deposited calcium ions, but the histochemical reaction was observed only at the single cell sites, while the majority of hydrogels volume was not stained. This suggest that calcium alginate hydrogel might exert pro-osteogenic effect even without the presence of osteogenic culture medium, which is consistent with observations of other groups, though it is known that particular biomaterials may induce osteogenesis of MSC [18–21]. In the control group the Alizarin Red staining showed no reaction at the hydrogels interior, because the calcium bounded with alginate carbohydrates do not induce the precipitation of a dye. We assume that prominent staining reaction noticed within the superficial zone of hydrogel was caused by its slow dissolution in culture medium and release of calcium ions.
Microscopic observations of BMSC during osteogenic differentiation in alginate hydrogel showed, that the structure of biomaterial did not allow cells for migration and formation of close intercellular contact. Thus in our experiment, cells did not develop multicellular clusters, typical for osteogenic differentiation of cellular monolayer as observed by us and others [1, 22, 23], but were evenly dispersed. We decided to immobilize cells at low concentration to avoid the cell clustering process, so we could analyze the osteogenic differentiation of single cells. As it was reported before, the cell–cell interaction plays significant role during osteogenic differentiation . Our results demonstrate however, that this feature is not crucial for initializing the deposition of bone-like ECM. The Alizarin Red S showed strong reaction of single cells with no developed, multicellular clusters. Therefore, the signals derived only from osteogenic medium, enhanced probably by paracrine cellular communication were sufficient enough for inducing the osteogenic differentiation in mesenchymal stem cells.
Microscopic observations of cellular ultrastructure revealed that BMSC during osteogenic differentiation lost their normal morphology and became filled with hydroxyapatite crystals. Moreover, the alginate fibers were not visible at the cell proximity, which suggest that the calcium composing the forming hydroxyapatite could derive from cross-linking sites of alginate chains, resulting in alginate dispersion. The numerous vesicles of size between 100 and 500 nm and diverse surface morphology were observed at the cell surface and its proximity. This observation corresponds with results obtained by Li et al., where osteoblasts cultured on chitosan/alginate scaffolds were covered with microvesicles containing the calcium-rich minerals . The connection between vesicles and biomineralization has been found by other groups [8–13, 26]. This hypothesis can now be supported by our observations. There were no organelles like nucleus or mitochondria visible within the cell, instead the cell interior was filled with hydroxyapatite crystals of various sizes. We assume that the cellular components become the mineralization sites for growing hydroxyapatite crystals, probably by certain autophagocytic processes. Our observations indicated that the caspase-3 signals were present outside the nuclear area, while the shape of the signal volume might resemble the mineralized microvesicles present around cells. Therefore, it is an another clue connecting the process of apoptosis and biomineralization. As we have shown recently, the macro autophagy is connected to the process of chondrogenic differentiation . Therefore, the similar processes may be involved in the osteogenesis at the cellular level, while these two lineages are relatively similar. In control cells, the three weeks of culture in alginate hydrogel did not alter the proper morphology of cells, leaving numerous mitochondria, endosomes and prominent endoplasmatic reticulum within the cytoplasm. The mineralizing microvesicles were absent on cellular surface, which presented more fibrous structure.
The surface of cells from osteogenic group was covered with round, vesicular objects, with single nanometer-scale fibers. Observations of the inner ultrastructure using ESB detector revealed high heterogeneity in the distribution of areas composed of heavier and lighter elements within the cell. While the calcium and phosphorus have higher mass than carbon, they give higher contrast when observed with backscattered electron detectors. Therefore we concluded that areas of higher contrast seen with ESB detector are in fact the calcium and phosphorus deposits, which was confirmed by SEM–EDX mapping. These observations supports the hypothesis that during the osteogenic differentiation, matrix vesicles accumulate the calcium within their interior . Though in non-osteogenic group the vesicular structures inside cells were numerous, they did not show the presence of heavier elements inside. Observations performed with block-face low voltage imaging technique revealed the presence of expanded apatite-like structures of irregular shape concentrating in the round, membrane vesicles, supporting the theory of mineralizing role of these structures.
In non-osteogenic group the majority of cells were alive and of normal morphology and ultrastructure. Despite the lack of adhesion sites in the pure calcium alginate, cells survived three weeks of culture and did not change their appearance. The mineralized microvesicles were absent in cells from control group, and the alginate fibers were tightly packed around the cell.
The effectiveness of osteogenic stimulation was also reflected by the increased concentrations of osteocalcin secreted to the supernatants and the activity of ALP. As it should be expected, the quantity of osteopontin in media was comparable in both groups. Osteopontin, beside the role in osteogenesis, is also considered as important factor in cell proliferation, and it is actively synthesized also in not differentiated MSC [28, 29]. Analysis of calcium and phosphorus content using EDX showed higher concentrations of both elements in hydrogels from osteogenic conditions. Moreover, the calcium-to-phosphorus ratio in experimental group was almost exact 2-to-1, like in natural bone . All observations confirmed the positively induced osteogenic differentiation in experimental group.
The mechanisms responsible for production and deposition of ECM in the 3D-culture in vitro model using mesenchymal stromal cells seems to be similar to natural osteogenesis occurring in vivo. We showed at the ultrastructural level that the deposition of mineralized extracellular matrix during osteogenic differentiation is connected with cell dead, where cellular remnants become the nucleation sites of mineralized matrix. This process probably involves the cell apoptotis and autophagocytosis, however the exact physiological mechanisms directing the cell death still need to be investigated.
mesenchymal stem cells
Hank’s balanced salt solution
Dulbecco’s modified eagle medium
bone marrow-derived mesenchymal stem cells
magnetic-activated cell sorting
critical point drying
focused ion beam
energy dispersive X-ray spectrometry
JG designed the study and performed the in vitro cultures and microscopic analysis, and wrote the manuscript. AŚ performed the proteomic analysis. KM provided the expertize, helped in interpreting the results and wrote the manuscript. All authors read and approved the final manuscript.
We would like to thank Dr. Grzegorz Chodaczek for his great help in preparing the 3D-reconstruction of confocal microscopic images.
The authors declare that they have no competing interests.
Data availability statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The experiment was approved by the Second Local Ethics Committee by the Wroclaw University of Environmental and Life Sciences, address: Wroclaw Chelmonskiego 38C (Dec. No. 177/2010 from 11.15.2010).
The cost of this study was covered by the University of Environmental and Life Sciences (design of the study, in vitro experiments, publication fee coverage) and Wroclaw Research Centre EIT+ (measurements and analysis). The cost of the publication was supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018.
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