Abstract
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.
Highlights
Hard tissues of the craniofacial complex are subjected to physical, chemical and biological health risk factors of the surrounding environment
Mineralised tissue formation by human dental pulp stem cells (hDPSCs) Mineralisation was monitored by time-lapse Micro-computed tomography (mCT) starting from day 19 of in vitro culture in two of the five experimental groups, which were both cultured in a mechanically dynamic system in either osteogenic (Sp.O.W) or control medium (Sp.C.W) (Fig. 1b)
Mineralisation followed a linear pattern (Fig. 1d) with correlation coefficients of 0.99 and 0.98 for samples cultured in osteogenic and control medium, respectively. Both qualitative and quantitative analyses of the bone-like tissue volume fraction for all groups demonstrated that mineralisation was significantly higher in dental pulp stem cells (DPSCs)-seeded scaffolds cultured in spinner flasks at 120 rpm in osteogenic medium with weekly mCT scans (Fig. 1c1, 1e) when compared to the static control samples (Fig. 1c4, 1c5, 1e) (p, 0.01)
Summary
Hard tissues of the craniofacial complex are subjected to physical, chemical and biological health risk factors of the surrounding environment. Examples are high mechanical impact resulting in fractures, smoking as a risk factor for osteoporosis and bone fracture [1], as well as microbiological infections of the periodontal tissues resulting in alveolar bone and subsequent tooth loss [2]. Repair of these bones requires the reconstruction of their anatomical, physiological, and functional properties, which cannot yet be fully accomplished by current treatment strategies [3]. Mesenchymal cells forming craniofacial bones and dental tissues (e.g. dentin) have the same embryonic origin and share many biochemical and molecular properties [4,5,6]. These cells are characterised by their ability to self-renew without losing their potency to differentiate into various cell types [11]
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