Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential
© Yamaguchi et al.; licensee BioMed Central. 2014
Received: 7 September 2014
Accepted: 27 November 2014
Published: 6 December 2014
Mesenchymal stem cells (MSCs) are a favored cell source for regenerative medicine because of their multilinage potential. However, the conventional monolayer technique used to culture MSCs, inadequately overcomes their low differentiation capacity. Culture of MSCs in multicellular spheroids, more accurately mimics the in-vivo microenvironment; thus, resolving this problem. In this study, we assessed whether the osteoregenerative potential of MSC spheroids is greater than that of monolayer MSCs.
MSC spheroids were generated from rat MSCs (rMSCs) using low-binding plates. Real-time reverse transcription-polymerase chain reaction and immunocytochemical analysis indicated that osteogenic properties were accelerated in MSC spheroids compared with monolayer rMSCs when treated with an osteoblast-inducer reagent for 7 days. Moreover, increased calcium deposition was visualized in MSC spheroids using Alizarin red staining. In a rat calvarial defect model, micro-computed tomography and histological assays showed that MSC spheroid-engrafted defects experienced enhanced bone regeneration.
Our in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteoregenerative efficiency compared with monolayer MSCs.
KeywordsSpheroid Mesenchymal stem cell Osteogenesis Rat calvarial defect Bone regeneration
Adult stem cells are widely used as a cell source for regenerative medicine because of their multilineage potential. These cells are easily harvested from bone marrow ,, adipose tissue ,, and other sites -. Bone marrow contains hematopoietic stem cells, mesenchymal stem/stromal cells (MSCs), and multipotent adult progenitor cells -. MSCs are capable of self-renewal and differentiation into several mesenchymal lineages in-vitro and in-vivo, including bone, fat, cartilage, and skeletal muscles ,. These are considered suitable for use in tissue regeneration. As culture in the presence of osteogenic supplements facilitates MSCs to undergo differentiation into the osteogenic phenotypes, MSCs have been utilized as a cell source for osteogenic tissue regeneration ,. Bone marrow-derived MSCs have great potential for bone regeneration in future clinical applications.
MSCs are commonly cultured as a two-dimensional (2D) monolayer using conventional tissue-culture techniques. These 2D-monolayer techniques inadequately reproduce the in-vivo microenvironment of stem cells, established by extrinsic and intrinsic cellular signals and have a profound influence on their biological functions . Culturing multipotent MSCs in a 2D adherent monolayer can alter their normal physiological behavior, resulting in the loss of replicative ability, colony-forming efficiency, and the differentiation capabilities over time ,. Replication of this complex in-vivo microenvironment in-vitro requires highly sophisticated cell-culture systems.
To mimic the in-vivo microenvironment more accurately in-vitro, various three-dimensional (3D) culture systems have been developed. Spheroids, spherical clusters of cells formed by self-assembly, comprise one of the best models for the 3D culture ,. Our understanding of the spheroid cell biology mainly derives from the in-vitro culture of cancer cell lines. Many studies have highlighted significant differences between 2D and 3D cultures, with the latter better reflecting the in-vivo microenvironment in terms of cellular heterogeneity, nutrient and oxygen gradients, cell-cell interactions, matrix deposition, and gene expression profiles -. Previous studies have reported several methods of generating 3D MSC spheroids. Many of these methods involve the use of a cell-suspension system or nonadherent surface to induce the spheroid formation -. In general, these MSC spheroids possess a greater differentiation capacity.
In this study, we examined the osteogenerative potential of the spheroids of rat MSCs (rMSCs) isolated from bone marrow compared with monolayer rMSCs in both in-vitro assays and a rat calvarial defect model, to elucidate whether MSC spheroids exhibit enhanced bone regeneration.
Mesenchymal stem cell spheroid formation
Increased expression of osteogenic genes in mesenchymal stem cell spheroids treated with an osteoblast-inducer reagent
Immunocytochemical expression of osterix in differentiated mesenchymal stem cell spheroids
Increased alkaline phosphatase staining and induction of calcified deposits in mesenchymal stem cell spheroids treated with an osteoblast-inducer reagent
Mesenchymal stem cell spheroids enhance the healing of rat calvarial defects
We quantified these micro-CT findings to yield the percentage of defect healing by quantifying the pixels in these defects. These results are summarized in Figure 7B. MSC spheroid/ Matrigel™ -implanted rats (49.4 ± 8.7% at 2 weeks; 72.2 ± 7.0% at 4 weeks) showed higher percentages of new bones at 4 weeks compared with rats in the other groups. The area of regenerated bones in rats in the other three groups was less than 55% (blank: 25.6 ± 4.9% and 45.0% ± 8.1%, Matrigel™ alone: 26.3 ± 5.0% and 46.2 ± 5.6%, monolayer rMSC/ Matrigel™: 39.9 ± 6.0% and 52.3 ± 10.2% at 2 and 4 weeks, respectively]. Thus, the MSC spheroids group showed higher bone regeneration in the peripheral area surrounding the defect compared with other groups.
The spheroid culture system has some advantages over the standard monolayer culture. The methods of the spheroid culture, allows the cells to adapt to their native morphology, facilitating greater cell-cell contacts and interactions between the cells and the extracellular matrix. In this study, we present two lines of evidence to support the concept that MSC spheroids exhibit enhanced osteogenic potential compared with monolayer rMSCs cultured, using conventional techniques. First, in-vitro assays confirmed that MSC spheroids upregulate osteogenic genes and proteins and also exhibit increased calcium deposition. Second, the implantation of MSC spheroids in vivo enhanced bone regeneration in rat calvarial defects.
A standardized microplate method used in this study generated a single MSC spheroid in each well. The round-bottom, low-binding plates had the following desirable characteristics: (i) a single spheroid per well, centered for ease of optical imaging; (ii) high reproducibility; (iii) simple harvesting for further analysis . It was possible to form spheroids by altering the initial seeding density of rMSCs. Spheroids size must be important because of limitations in the length of nutrient transport by diffusion. Curcio et al. reported that the spheroid radii greater than 200 μm rendered cells in their core vulnerable to hypoxia and cell death . In both in-vitro and in-vivo experiments, we used the spheroids containing approximately 10,000 cells with a mean radius of approximately 200 μm. Conversely, delivery of a substantial number of cells is required for cell-based therapies to drive tissue formation, and larger spheroids facilitate the transplantation of fewer aggregates.
The results of our in-vitro assays indicate that osteogenic properties are accelerated in MSC spheroids treated with an OIR. We compared changes the osteogenic gene expression profiles of 3D MSC spheroids vs. 2D monolayer rMSCs by quantitative RT-PCR. Expression of the RUNX-2, OSX, OPN, and BSP genes were upregulated in MSC spheroids treated with an OIR compared with monolayer rMSCs. The process of osteoblastic differentiation is regulated by multiple factors and signaling pathways. Among the upregulated genes, RUNX-2, also called core-binding factor A1, and OSX are often referred to as the “master switch” of osteogenic differentiation ,. It is established that bone genes are crucial for the generation of a mineralized tissue, and are usually analyzed during the early phases of osteogenic differentiation. MSC spheroids treated with an OIR for 7 days appear to begin toward the osteogenic differentiation. Our immunocytochemical findings showing OSX expression in MSC spheroids support this speculation.
Interestingly, our PCR results also showed an upregulation of the intermediate to late markers of osteogenesis, OPN and BSP. OPN is a noncollagenous, secreted glycosylation phosphoprotein expressed during both the early and the intermediate stages of osteogenesis -. BSP is also a noncollagenous, acidic phosphoprotein normally expressed in mineralized tissues such as bone and dentin . Upregulation of these genes prompts us to suggest that they may serve as a matrix-associated signal directly promoting osteogenic differentiation and resulting in the increased production of a mineralized matrix in MSC spheroids. Using AR staining, we found increased calcium deposition in MSC spheroids treated with an OIR for 7 days. It is difficult to explain why the mixtures of various differentiation phases are contained within MSC spheroids. We believe that the multicellular spheroids consist of cells at various stages of osteogenic differentiation. Future work will address how osteogenic differentiation is regulated in the cells consisting of MSC spheroids.
In our in-vitro experiments, there were no differences in ALP gene expression and ALP activity between MSC spheroids and monolayer rMSCs. ALP is another important marker, as an effector protein responsible for osteoprogenitor markers and mineralization of the extracellular matrix during osteogenic differentiation . In this study, we found that the expression levels of ALP mRNA in monolayer rMSCs treated with an OIR for 7 days was identical to that in MSC spheroids. ALP staining was of identical intensity between monolayer rMSCs and MSC spheroids treated with an OIR for 7 days, but no AR deposits were observed in monolayer rMSCs. These results suggest that monolayer rMSCs can differentiate into a progenitor phase without the initiation of mineralization. Therefore, MSC spheroids treated with with an OIR exhibit enhanced the differentiation capabililties compared with monolayer rMSCs.
Micro-CT analysis of MSC spheroid-engrafted calvarial defects in rats indicates that these spheroids have a dramatic effect on bone regeneration. Histological findings complemented these micro-CT results, which showed that a bone bridge had almost covered the defect in rats in the MSC spheroids group, although the fibrous connective tissue remained in the defect in rats in the other groups. Based on the histological findings of engrafted defects in the control groups, we suggest that the bone healing process consistes of the extensions of new bone from the edges of the defect. This healing process is supported by our previous report . Our in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteogenic efficiency compared with those in the monolayer culture. However, the detailed mechanisms that enhanced in-vitro osteogenesis and in-vivo bone regeneration by which MSC spheroids occur remain unclear. Future studies will address the detailed effects of MSC spheroids on microenvironments during the induction of osteogeneis in bone defects.
Our in-vitro experiments on osteogenic induction showed that MSC spheroids possessed enhanced osteogenic potential compared with monolayer rMSCs. Moreover, we demonstrated that engrafted MSC spheroids induced efficient bone regeneration in calvarial defects in rats. Based on these results, we conclude that MSCs in the spheroid culture exhibit enhanced the osteogenic capabilities.
Bone marrow-derived rMSCs, isolated from Fischer 344 rats, were purchased from Lonza Group AG (Basel, Switzerland) and expanded according to the established protocols. Briefly, cryopreserved rMSCs were thawed and plated onto a 10-cm tissue-culture dish in 10 ml Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industiries,Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 1% (v/v) penicillin/ streptomycin (PS; Invitrogen/GIBCO, Carlsbad, CA, USA) for 7 days at 37°C in a humidified 5% CO2 atmosphere. The medium was changed every 3 days. After allowing the adherent cells to grow to approximately 80% confluence, they were detached from the tissue culture plate using 0.25% trypsin- 1 mM ethylenediamine tetraacetic acid (EDTA) solution (Sigma-Aldrich Corporation), counted, and either replated for monolayer cultures or used for spheroid formation.
Mesenchymal stem cell spheroid formation
Dissociated rMSC monolayers (Passage 3) were resuspended in medium to obtain a single cell suspension. MSCs (8 × 105 cells/ml, corresponding to approximately 100, 1,000, and 10,000 cells/well) were added to Nunc®Low Cell Binding Surface 96-well plates (Thermo Scientific Nunc A/S, Roskilde, Denmark) and incubated in DMEM medium supplemented with FBS and PS at 37°C for up to 7 days.
Osteogenic induction in mesenchymal stem cell spheroids
To generate osteogenic differentiation, MSC spheroids were cultured with DMEM in the presence of OIR (Takara Bio Inc., Otsu, Japan), which included ascorbic acid, hydrocortisone, and β-glycerophosphate. Culture of MSC spheroid was allowed to grow for 7 days with media exchanged every 2 days. For immunocytochemistry and, ALP and AR staining, MSC spheroids incubated with OIR were removed from the well and plated onto 13-mm cover glass for 2 hours. Parental rMSCs were seeded on to 13-mm cover glasses and cultured with DMEM containing 10% (v/v) FBS and an OIR for 7 days.
Real-time reverse transcription-polymerase chain reaction
Primers and probes used in this study
Forward and reverse primers (5′ → 3′)
Immunocytochemical detection of osterix in mesenchymal stem cell spheroids
After culturing for the indicated time, spheroids and parental cells on 13-mm cover glass were fixed with 4% paraformaldehyde for 10 min and then washed in 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 15 min. They were incubated with a rabbit polyclonal antibody to Sp7/OSX (1:100; Abcam, Cambridge, UK), at 4°C overnight. After washing with PBS, both spheroids and cells were incubated in a mixture of anti-rabbit Immunoglobulin G antibody conjugated with Alexa Fluor® 568 (1:200; Molecular Probes, Eugene, OR, USA). To visualize the nuclei, immunostained spheroids and cells were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA).
Alkaline phosphatase and Alizarin red staining
ALP and AR staining were assessed in both MSC spheroids and monolayer rMSCs induced after exposure to the OIR for 7 days. ALP staining was performed using an ALP staining kit (Takara Bio Inc., Otsu, Japan), according to the manufacturer’s instructions. Calcified deposits were detected by AR staining. Both MSC spheroids and monolayer rMSCs were fixed for 10 min in 4% paraformaldehyde in PBS and was rinsed twice with distilled water. The cells were stained at room temperature for 5 min with AR.
The rat calvarial bone defect model
Ten-week-old male Fischer rats (weight approximately 250 g; n = 20) were anesthetized with 2% isoflurane (Abbott Laboratories, Abbott Park, IL, USA) and an air mixture gas flow of 1 l/min using an anesthesia gas machine (Anesthesia Machine SF-B01; MR Technology, Inc., Tsukuba, Japan). After shaving the skin, an incision was made in the skull, and the periosteum was opened, exposing the surface of the calvarial bones. A circular bone defect (full-thickness, 5 mm diameter) was created in the left parietal bone with a trephine drill and was irrigated with saline to remove bone debris. MSC spheroids were then implanted into the defects. Five MSC spheroids or monolayer rMSCs (5 × 104 cells), which had been cultured in medium containing the OIR for 7 days, were loaded into 20 μl Matrigel™ on ice and transplanted into the defects at room temperature. Controls for the transplant experiments included calvarial defects implanted with or without Matrigel™. Our animal experimentation protocols were approved by the Animal Care and Use Committee of Fukuoka Dental College, Fukuoka, Japan (No. 13006).
Evaluation of bone regeneration
Bone regeneration was evaluated using an in-vivo micro-CT system (Skyscan-1176 Micro-CT Scanner; Burker MicroCT, Kontich, Belgium) at 50 kVp and 500 μA on rats under anesthesia (as described above), 4 weeks after implantation. Each image data set consisted of a scan size of approximately 35 mm. The percentage of newly formed bone in each calvarial bone defect was calculated as previously described ,.
After micro-CT scanning, rats were sacrificed by injecting an overdose of isoflurane. Subsequently, the cranial tissues containing MSC spheroids, monolayer rMSCs, or control substances were immediately excised. Tissue specimens were fixed in 4% paraformaldehyde in PBS, decalcified in 10% EDTA for 4 weeks at 4°C, and then was embedded in paraffin. Paraffin sections were then stained with hematoxylin and eosin and Azan Mallory to visualize any histological changes. In histomorphometric analysis, the percentage of the volume of the newly formed bone per the original volume of the defect was calculated.
The results are presented as means ± standard deviation. Group results were compared using one-way analysis of variance and Scheffe’s multiple comparison tests. A p-value of <0.05 was considered statistically significant.
The present study was supported in part by a Grant-in-aid for Scientific Research (B) (23390455) and by a Grant-in-aid for strategic study base formation support business (S1001059) form the Japan Society for the Promotion of Science. The authors would like to thank Enago (http://www.enago.jp) for the English language review.
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