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. 2018 Apr 16;2018:4854619.
doi: 10.1155/2018/4854619. eCollection 2018.

The Effects of Hyperacute Serum on the Elements of the Human Subchondral Bone Marrow Niche

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Free PMC article

The Effects of Hyperacute Serum on the Elements of the Human Subchondral Bone Marrow Niche

Melinda Simon et al. Stem Cells Int. .
Free PMC article

Abstract

Mesenchymal stem cells (MSCs) are widely used in laboratory experiments as well as in human cell therapy. Their culture requires animal sera like fetal calf serum (FCS) as essential supplementation; however, animal sera pose a risk for clinical applications. Human blood derivatives, for example, platelet-rich plasma (PRP) releasates, are potential replacements of FCS; however, it is unclear which serum variant has the best effect on the given cell or tissue type. Additionally, blood derivatives are commonly used in musculoskeletal diseases like osteoarthritis (OA) or osteonecrosis as "proliferative agents" for the topical MSC pool. Hyperacute serum (HAS), a new serum derivative, has been designed to approximate the natural coagulation cascade with a single-step, additive-free preparation method. We investigated the effects of HAS on monolayer MSC cultures and in their natural niche, in 3D subchondral bone and marrow explants. Viability measurements, RT-qPCR evaluation for gene expression and flow cytometry for cell surface marker analysis were performed to compare the effects of FCS-, PRP-, or HAS-supplemented culture media. Monolayer MSCs showed significantly higher metabolic activity following 5 days' incubation in HAS, and osteoblast-specific mRNA expression was markedly increased, while cells also retained their MSC-specific cell surface markers. A similar effect was observed on bone and marrow explants, which was further confirmed with confocal microscopy analysis. Moreover, markedly higher bone marrow preservation was observed with histology in case of HAS supplementation compared to FCS. These findings indicate possible application of HAS in regenerative solutions of skeletal diseases like OA or osteonecrosis.

Figures

Figure 1
Figure 1
Bone marrow tissue explantation model. During total hip replacement surgery, the femoral head was discarded (a) and replaced with an endoprosthesis. For experimental reasons, small bone pieces from the cut surface of the femoral head were isolated with a sharp chisel (b). μCT image shows the structure of the BMEs (c). (d) The experimental setup of the BMEs' and MSC cell cultures.
Figure 2
Figure 2
Time-course effect of serum supplements on MSCs. (a) Subconfluent MSCs were cultured in DMEM in the absence of supplement (serum free (white bar)), or supplemented with FCS (light-gray bar), FCS + bFGF (dark-gray bar), PRP (black bar), or HAS (red bar). (b) Cell morphology of MSCs using phase-contrast microscopy. ∗∗∗Confidence interval P-value: 0.0001 to 0.001, extremely significant.
Figure 3
Figure 3
Cell immunophenotypes of MSCs cultured in differently supplemented media. MSCs were cultured in FCS (light-gray shading), FCS + bFGF (dark-gray shading), PRP (black shading), or HAS (red shading); stained with specific antibodies; and analyzed with flow cytometry. Unstained control cells are represented by dotted lines. Representative images of MSC and hematopoietic marker expression.
Figure 4
Figure 4
Gene expression analysis of MSCs cultured in serum-supplemented media. Following 5 days' culture in FCS + bFGF (gray bar), PRP (black bar), or HAS (red bar) and FCS as control, mRNA was isolated for RT-qPCR analysis of MSC- (a), adipocyte- (b), osteoblast- (c), apoptosis-, and antiapoptosis-specific (d) genes. Data are presented as fold change values to the expression of MSCs cultured in FCS-supplemented medium that was considered as the standard growth medium. Confidence interval P-value 0.05 to 0.01, significant; ∗∗ P-value 0.001 to 0.01, very significant; ∗∗∗ P-value 0.0001 to 0.001, extremely significant.
Figure 5
Figure 5
Culture of BMEs. Viability of BMEs cultured in serum-supplemented media (a). Serum-free medium (white bard) and medium supplemented with FCS (gray bar) or HAS (red bar). (b) Representative confocal microscopy images stained with the live/dead dye combination Calcein-AM/ethidium homodimer. Nuclei were counterstained with Hoechst. Coloured terms indicate the colour of the representative dye. Confidence interval P-value 0.05 to 0.01, significant; ∗∗∗ P-value 0.0001 to 0.001, extremely significant.
Figure 6
Figure 6
MSC-specific and hematopoietic stem cell-specific gene expression profiles of BMEs as determined by RT-qPCR. Total RNA extraction was performed from BMEs cultured either in serum-free medium (white circles) or in medium supplemented with FCS (gray triangles) or HAS (red diamonds) after 5 days' incubation. ∗∗Confidence interval P-value 0.001 to 0.01, very significant.
Figure 7
Figure 7
Adipocyte-, osteoblast-, and osteocyte-specific gene expression profiles of BMEs as determined by RT-qPCR. Total RNA extraction was performed from BMEs cultured either in serum-free medium (white circles) or in medium supplemented with FCS (gray triangles) or HAS (red diamonds) after 5 days' incubation.
Figure 8
Figure 8
Histological analysis of BMEs. (a) Representative images of HAS- and FCS-supplemented samples stained with hematoxylin and eosin, Masson's trichrome, and von Kossa. (b) Histological scores of bone marrow integrity.

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