Influence of the mechanical environment on the engineering of mineralised tissues using human dental pulp stem cells and silk fibroin scaffolds

Teeth constitute a promising source of stem cells that can be used for tissue engineering and regenerative medicine purposes. Bone loss in the craniofacial complex due to pathological conditions and severe injuries could be treated with new materials combined with human dental pulp stem cells (hDPSCs) that have the same embryonic origin as craniofacial bones. Optimising combinations of scaffolds, cells, growth factors and culture conditions still remains a great challenge. In the present study, we evaluate the mineralisation potential of hDPSCs seeded on porous silk fibroin scaffolds in a mechanically dynamic environment provided by spinner flask bioreactors. Cell-seeded scaffolds were cultured in either standard or osteogenic media in both static and dynamic conditions for 47 days. Histological analysis and micro-computed tomography of the samples showed low levels of mineralisation when samples were cultured in static conditions (0.16±0.1 BV/TV%), while their culture in a dynamic environment with osteogenic medium and weekly µCT scans (4.9±1.6 BV/TV%) significantly increased the formation of homogeneously mineralised structures, which was also confirmed by the elevated calcium levels (4.5±1.0 vs. 8.8±1.7 mg/mL). Molecular analysis of the samples showed that the expression of tooth correlated genes such as Dentin Sialophosphoprotein and Nestin were downregulated by a factor of 6.7 and 7.4, respectively, in hDPSCs when cultured in presence of osteogenic medium. This finding indicates that hDPSCs are able to adopt a non-dental identity by changing the culture conditions only. Also an increased expression of Osteocalcin (1.4x) and Collagen type I (1.7x) was found after culture under mechanically dynamic conditions in control medium. In conclusion, the combination of hDPSCs and silk scaffolds cultured under mechanical loading in spinner flask bioreactors could offer a novel and promising approach for bone tissue engineering where appropriate and rapid bone regeneration in mechanically loaded tissues is required

Gene expression analysis.

<p>Images showing normalised fold expression against <i>GAPDH</i> for the genes <i>ALP</i> (<b>a</b>), <i>DSPP</i> (<b>b</b>), <i>Nestin</i> (<b>c</b>), <i>Collagen type I</i> (<b>d</b>) and <i>Osteocalcin</i> (<b>e</b>). Data is shown as average ± standard deviation of three samples. Asterisks indicate significant (<i>p</i><0.05) or highly significant (<i>p</i><0.01) difference between the groups. Abbreviations: Sp.O.W: spinner flask culture in osteogenic medium with weekly x-ray; Sp.C.W: spinner flask culture in control medium with weekly x-ray; Sp.O.E: spinner flask culture in osteogenic medium with x-ray at the end; St.O.E: static culture in osteogenic medium with x-ray at the end; St.C.E: static culture in control medium with x-ray at the end.</p

Histological analysis of hDPSCs seeded on silk fibroin scaffolds after 47 days of culture.

<p>(<b>a</b>) Haematoxylin and Eosin staining of histological sections from all groups. Extracellular matrix and silk scaffolds are stained red/pink. Cell nuclei are stained purple/violet. (<b>b</b>) Von Kossa staining. Silk scaffolds and mineralised nodules are stained in brown. Arrows indicate areas where phosphate is present. (<b>c</b>) Sirius Red staining showing the distribution of Collagen (red colour). Abbreviations: Sp.O.W: spinner flask culture in osteogenic medium with weekly x-ray; Sp.C.W: spinner flask culture in control medium with weekly x-ray; Sp.O.E: spinner flask culture in osteogenic medium with x-ray at the end; St.O.E: static culture in osteogenic medium with x-ray at the end; St.C.E: static culture in control medium with x-ray at the end. Scale bars: 500 µm.</p

Biochemical analysis after 47 days of culture.

<p>(<b>a</b>) Cell metabolic activity [activity/total DNA]. (<b>b</b>) DNA content per scaffold [ng/mL]. (<b>c</b>) Calcium content per scaffold [mg/mL]. (<b>d</b>) ALP activity normalised to cell number [µg/p-nitrophenol/total DNA]. Data is shown as average ± standard deviation (<i>n</i> = 5). Asterisks indicate significant (<i>p</i><0.05) or highly significant (<i>p</i><0.01) difference between the groups. Abbreviations: Sp.O.W: spinner flask culture in osteogenic medium with weekly x-ray; Sp.C.W: spinner flask culture in control medium with weekly x-ray; Sp.O.E: spinner flask culture in osteogenic medium with x-ray at the end; St.O.E: static culture in osteogenic medium with x-ray at the end; St.C.E: static culture in control medium with x-ray at the end.</p

Primers used in the study.

<p>Primers used in the study.</p

Mineralisation analysis using µCT.

<p>(<b>a</b>) Experimental groups. (<b>b</b>) Time-lapse µCT images of samples cultured in spinner flasks in either osteogenic (upper row) or control (lower row) medium. (<b>c</b>) µCT images of one representative sample per experimental group after 47 days of culture. (<b>d</b>) Time-dependent increase in bone-like tissue volume fraction as observed with weekly µCT scans. (<b>e</b>) Bone-like tissue volume fraction for all groups after 47 days of culture. Scale bars: 1 mm.</p