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Impact of Four Protein Additives in Cryogels on Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells

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Impact of Four Protein Additives in Cryogels on Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells

Victor Häussling et al. Bioengineering (Basel).

Abstract

Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet-rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds' porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad-MSCs were osteogenically differentiated on these scaffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients' PRP.

Keywords: 3D-culture; RGD; adipose-derived mesenchymal stem/stromal cells (Ad-MSCs); bone tissue engineering; collagen; cryogel; immune-cell conditioned medium; platelet-rich plasma (PRP); scaffold.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Pore size and stiffness of cryogels with different composition. (A) Variation of the pHEMA concentrations (10%, 20%, 30%, and 40% pHEMA with constant bisacrylamide ratio) in cryogels containing 5% hydroxyapatite. (B) Variation of the bisacrylamide concentration in 16% pHEMA cryogels containing 5% hydroxyapatite. (C,D) Variation of the hydroxyapatite concentration in 16% pHEMA (0.3% bisacrylamide) cryogels. (C) Representative photograph of the cryogel. (A,B,D) In the graphics pore diameter is represented with the blue line (right scale) and cryogel stiffness is represented with the red line (left scale). Single data points represent mean ± 95% C.I. * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the lowest concentration presented.
Figure 2
Figure 2
Pore size and stiffness of cryogels with or without hydroxyapatite. Cryogels containing 16% pHEMA (0.3% bisacrylamide, PRP) were supplemented with insoluble hydroxyapatite or with crystallized calcium–phosphate. (A) Stiffness of the resulting cryogels in kPa. (B) Pore diameter of the resulting cryogels in µm. Data are presented as violin plots (mean ± 95% C.I. is marked) of N = 5 (n = 3) individual experiments. (C) Scanning electron microscopy (SEM) images of the resulting cryogels.
Figure 3
Figure 3
Pore size and stiffness of cryogels with different protein additives. Cryogels containing 16% pHEMA and crystallized calcium–phosphate were supplemented with different protein components: collagen (Coll), platelet-rich plasma (PRP), immune-cell conditioned medium (CM), and RGD peptides (RGD). (A) Stiffness of the resulting cryogels in kPa. (B) Pore diameter of the resulting cryogels in µm. (C) scanning electron microscopy (SEM) images of the resulting cryogels. (D) Scaffold porosity in %. (E) Water-uptake capacity of the scaffolds (fold of dry weight). Data are presented as violin plots (mean ± 95% C.I. is marked) of N = 5 (n = 4) individual experiments.
Figure 4
Figure 4
Cell attachment and survival on the four different cryogels. Cryogels containing 16% pHEMA, crystallized calcium–phosphate, and collagen (Coll), platelet-rich plasma (PRP), immune-cell conditioned medium (CM), or RGD peptides (RGD) were seeded with adipose-derived mesenchymal stem cells (Ad-MSCs). On days 0, 7, and 14 of osteogenic differentiation (A) total DNA content in ng/µl, (B) mitochondrial activity (resazurin conversion) in fluorescent intensities, and (C) glucose consumption (within 2 days) in ng/ml were determined. Data are presented as floating signs (mean ± 95% C.I.) of N = 5 (n ≤ 3) individual experiments. (D) Calcein–AM staining showing living cells in bright green on the cryogels. Hoechst was used as nuclear counterstain.
Figure 5
Figure 5
Osteogenic characteristics of Ad-MSCS differentiated for 14 days on the four different cryogels. Adipose-derived mesenchymal stem cells (Ad-MSCs) were osteogenically differentiated for 14 days on cryogels containing 16% pHEMA, crystallized calcium–phosphate, and collagen (Coll), platelet-rich plasma (PRP), immune-cell conditioned medium (CM), or RGD peptides (RGD). On days 0, 7, and 14 intracellular (A) Runt-related transcription factor 2 (Runx2) and (B) and osterix were determined by dot blot. (C) Alkaline phosphatase (ALP) activity on days 0, 7, and 14 of differentiation normalized to the total DNA contents. On days 0, 7, and 14 secreted levels of (D) osteocalcin, (E) osteoprotegerin (OPG), and (F) receptor activator of nuclear factor kappa-Β ligand (RANKL) were detected in the culture supernatants by dot blot. Data are presented as floating signs (mean ± 95% C.I.) of N ≥ 3 (n ≤ 3) individual experiments.

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