Hypoxia and amino acid supplementation synergistically promote the osteogenesis of human mesenchymal stem cells on silk protein scaffolds

Abstract

Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and oxygen level. The cells were assessed to probe how the microenvironment impacted metabolic pathways and thus osteogenesis. The most favorable osteogenesis outcomes were found in the presence of low (5%) oxygen combined with high lysine and proline concentrations during in vitro cultivation. This same set of culture conditions also showed the highest glucose consumption, lactate synthesis, and certain amino acid consumption rates. On the basis of these results and known pathways, a holistic metabolic model was derived which shows that lysine and proline supplements as well as low (5%) oxygen levels regulate collagen matrix synthesis and thereby rates of osteogenesis. This study establishes early steps toward a foundation for patient- and tissue-specific matches between metabolism, repair site, and tissue engineering approaches toward optimized bone regeneration.

FIG. 1.

Scanning electron microscopy images of the different treatment groups cultured with human mesenchymal stem cells at week 7. (a), (b), (c), and (d) shows matrix formation by cells in groups 1 (control), 2 (5 × lysine and proline), 3 (low oxygen), and 4 (combined effect of low oxygen, 5 × lysine and proline), respectively, at 200 × (scale bars = 100 μm). (e) shows the presence of mineralized nodules, marked by black arrows, in group 4 (combined effect of low oxygen, 5 × lysine and proline) at 600 × (scale bar = 10 μm).

FIG. 2.

Images of (a) ColI immunostaining, (b) hematoxylin and eosin staining, and (c) von Kossa staining of the different treatment groups at week 7, under light microscope at 20 × . All scale bars = 50 μm. The presence of collagen and calcium is shown by brown and black staining respectively, both of which are marked by black arrows. ColI, collagen type I. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Picogreen assay was done to assess the DNA content within scaffolds from each group. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Chemical analysis tests studied (a) ALP activity, (b) collagen content, and (c) calcium content, which are important indicators of osteogenesis. The figure shows the combined effect of low oxygen and 5 × lysine and proline on these osteogenesis outcomes. Statistical significance was assigned as * denoting p < 0.05. ALP, alkaline phosphatase. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Semi-quantitative reverse transcriptase polymerase chain reaction measured gene expression of certain bone markers and hypoxia marker. The figure shows the effect of low oxygen and 5 × lysine and proline on the gene expression levels of osteogenic markers: (a) ALP, (b) Runx2, and (c) ColI. Expression of the gene (d) HIF-1α was also studied to assess the effect of hypoxic condition on gene expression. Statistical significance was assigned as * denoting p < 0.05. HIF, hypoxia inducible factor; Runx2, runt-related transcription factor 2. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Analysis of (a) glucose and (b) lactate content of the media demonstrated the effect of low oxygen and high proline and lysine concentration on the metabolic activity of the cells. Statistical significance was assigned as * denoting p < 0.05. Color images available online at www.liebertonline.com/ten.

FIG. 7.

High performance liquid chromatography results show the consumption of amino acids, (a) glutamate, (b) glutamine, (c) proline, and (d) lysine, during osteogenesis under the effect of low oxygen and high lysine and proline concentration. Statistical significance was assigned as * denoting p < 0.05. Color images available online at www.liebertonline.com/ten.

FIG. 8.

On the basis of the results of this study and known pathways relating energy metabolism and matrix metabolism, the given metabolic model flowchart was derived. It describes the mechanism behind increased osteogenesis rate in the presence of low oxygen as well as high proline and lysine concentration. Low oxygen triggers expression of HIF-1α, which in turn upregulates expression of a number of target genes that directly or indirectly help in osteogenesis. The glucose consumption rate as well as lactate synthesis rate increases in the presence of low oxygen, while Kreb's cycle and thereby gutamine consumption is downregulated. Lactate accumulation, in turn, has been reported to activate prolyl hydroxylase proenzyme. This enzyme catalyses hydroxylation of proline residues and thus plays an important role in collagen biosynthesis. High lysine and proline concentration increases lysine and proline consumption and thereby increases glutamate consumption, the net result being an upregulation in collagen synthesis and bone formation. Color images available online at www.liebertonline.com/ten.