Electrospun Materials and cell culture for bone research

There are numerous studies on using electrospun fibers to facilitate bone regeneration. In the early 2000s, biodegradable polymers have been electrospun and shown to be effective in supporting adhesion and proliferation bone marrow derived mesenchymal stem cells [Yoshimoto et al 2003]. The natural extracellular matrix (ECM) of bone is made out mainly of hydroxyapatite (HA) and collagen. The important role of calcium and in particular HA, has motivated studies to support or replicate that composition [Fujihara et al 2005]. Various methods of incorporating HA into electrospun fibers such as blending with nano-HA [Jose et al 2009; Venugopal et al 2007] and post-electrospinning mineralization was used to enhance the response of bone cells. Collagen and gelatin was also added to synthetic polymers for electrospinning to introduce biological cues to promote better cell response [Ekaputra et al 2009, Kim et al 2005].

The diversity of polymers and additives that can be incorporated into the electrospun scaffold has allowed researchers to experiment with different materials and additives to improve treatment outcome. Kim et al (2006) was able to electrospin precusors of bioactive glass (58SiO₂38CaO4P₂O₅) with poly-vinyl-butyral (PVB) to form nanofibers. Sintering was carried out to remove the organic compounds to stabilize the resulting bioactive glass nanofibers. The bioactive glasses nanofiber membrane was subsequently surface coated with collagen. Scaffolds comprising of bioactive glass and collagen showed higher ALP levels from human osteoblastic cells compared with scaffolds containing collagen alone. Immersing the bioactive glasses/collagen nanofiber membrane in simulated body fluid also showed better induction of apatite minerals on it surface.

Core-shell fiber is another form that can be used for encapsulation of bioactive molecules. In this fiber form, the bioactive molecules may be encapsulated either in the core for slow release or on the sheath for faster release. Where the bioactive molecules are encapsulated at the sheath, the core functions as a support to prevent full disintegration of the scaffold. Huang et al (2021) showed the benefits of using citrate-stabilized gold-nanoparticles (GNPs) to promote osteogenesis. In their core-shell fiber, the GNPs were blended into water soluble polyvinylpyrrolidone (PVP) as the sheath material. The core was made of ethylcellulose (EC) material. A coaxial nozzle was used to produce the core-shell fiber by electrospinning. The electrospun core-shell fibers containing GNPs were found to significantly increase osteogenic bioactivities by mouse calvarial pre-osteoblast cell line MC3T3-E1 compared to fibers without GNPs. In a rat full thickness skull defect model, the group containing GNPs loaded PVP/EC scaffold showed almost complete bone coverage of the defect site after 4 weeks. In the same period, the PVP/EC group showed partial coverage with new bone and the group without any scaffolds showed a thin fibrous layer across the defect. Examination of other vital organs showed no observable tissue damage or inflammation hence demonstrating non-toxicity to metabolic and excretory organs.

Dexamethasone (DEX) and bone morphogenic proteins (BMP) are widely known to drive osteogenic differentiation in mesenchymal stem cells (MSCS). Zhao et al (2019) investigated the delivery of both biomolecules in electrospun core-shell fibers and their influence on MSC differentiation. The shell material is made of Zein with DEX and the core material is made of poly-L-lactic acid (PLLA) and BMP2. The extent of cell differentiation was evaluated by alkaline phosphatase (ALP) activity and calcium deposits. Compared to fibers without loaded biomolecules and fibers loaded with just one of the biomolecules, the presence of both DEX and BMP2 has a synergistic effect in enhancing MSC differentiation.

Beyond the addition of biomolecules into electrospun fibers, researchers are exploring the effect of adding novel substances into electrospun fibers and the response of cells in bone regeneration. MXene which is made of 2-dimension transition metal carbides and carbonitrides, has been examined for use in biomedical applications, such as tissue engineering scaffolds. Lee et al (2022) blended MXene nanoparticles into a solution of poly(L-lactide-co-ε-caprolactone, PLCL) and collagen (Col) and electrospun into PLCL/Col/MXene scaffolds for bone tissue engineering. MC3T3-E1 preosteoblasts cultured on the scaffold without osteogenic medium showed significantly enhanced cellular behavior with increased ALP activity compared to scaffolds without MXene. However, the presence of collagen is also important as the PLCL/MXene combination without collagen showed lower ALP activity than the PLCL/Col combination. Mineralized bone nodules were also found on cells cultured on PLCL/Col/MXene scaffolds after 14 days but no mineralization was observed on PLCL and PLCL/Col samples. The osteogenic effect of MXene on preosteoblasts may be attributed to the functional groups of MXene nanoparticles introducing negative charges and the hydroxy groups of MXene forming hydrogen bonds with serum proteins. Carbon elements present in MXene and the nanofiber topography may also contribute to osteogenic effects.