The synergetic effect of bioactive molecule–loaded electrospun core‐shell fibres for reconstruction of critical‐sized calvarial bone defect—The effect of synergetic release on bone Formation

Abstract Objectives Bone regeneration is a complex process modulated by multiple growth factors and hormones during long regeneration period; thus, designing biomaterials with the capacity to deliver multiple bioactive molecules and obtain sustained release has gained an increasing popularity in recent years. This study is aimed to evaluate the effect of a novel core‐shell electrospun fibre loaded with dexamethasone (DEX) and bone morphogenetic protein‐2 (BMP‐2) on bone regeneration. Materials and methods The core‐shell electrospun fibres were fabricated by coaxial electrospinning technology, which were composed of poly‐D, L‐lactide (PLA) shell and poly (ethylene glycol) (PEG) core embedded with BMP‐2 and DEX‐loaded micelles. Morphology, hydrophilicity, gradation, release profile of BMP‐2 and DEX, and cytological behaviour on bone marrow mesenchymal stem cells (BMSCs) were characterized. Furthermore, the effect on bone regeneration was evaluated via critical‐sized calvarial defect model. Results The electrospun fibres were featured by the core‐shell fibrous architecture and a suitable degradation rate. The sustained release of DEX and BMP‐2 was up to 562 hours. The osteogenic gene expression and calcium deposition of BMSCs were significantly enhanced, indicating the osteoinduction capacity of electrospun fibres. This core‐shell fibre could accelerate repair of calvarial defects in vivo via synergistic effect. Conclusions This core‐shell electrospun fibre loaded with DEX and BMP‐2 can act synergistically to enhance bone regeneration, which stands as a strong potential candidate for repairing bone defects.


| INTRODUC TI ON
The repair of bone defects generated by trauma, tumour and congenital malformation remains a long-standing challenge. It is reported that more than $2.5 billion is spent for over 2 million bone grafts in the United States each year. 1 Considering the shortcoming of the limited bone donors of autografts and the potential risk of immune reactions of allografts, 2,3 the bone tissue engineering emerges as a promising alternative. 2,4 An essential issue in bone tissue engineering is to construct the biomaterials with biofunction to regulate cellular function such as the migration, proliferation and differentiation. [5][6][7] However, conventional fabrication techniques, for example freeze-drying, 8 gas foaming 9 and salt leaching, 10 are incapable to fabricate the construct with various biofunction. 11,12 The bioactive molecule-loaded fibrous meshes fabricated through electrospinning technology, with fibre diameters ranging from several nanometres to a few micrometres and featured by an interconnected microporosity, [13][14][15] have exhibited a great potential in bone tissue engineering. 16 Nevertheless, traditional bioactive molecule-loaded electrospun fibres are faced with multi-deficiencies. The bioactive molecule cargos exposed to organic solvents are prone to denature during electrospinning procedure. 5 The lack of sustained release profile is another major shortcoming. 5,17 The coreshell fibres fabricated by the coaxial electrospinning have emerged as a potent candidate to overcome aforementioned limitations.
Attributed to the unique architecture, the bioactive molecules can be embedded into the polymer core under inorganic solutions, thus avoiding the potential denaturation. In addition, the burst release profile can be weakened to a great extent and the sustained release profile can be achieved via the shell structure. Moreover, through controlling the thickness and composition of the shell, the release rate of bioactive molecules can be further regulated. 12,18 The bone formation is a complex process involved plenty of growth factors and hormones in a spatiotemporal manner. Therefore, loading multiple bioactive molecules to the construct has been considered as an effective way to promote bone tissue regeneration.
The bone morphogenetic protein-2 (BMP-2) characterized by its superior osteoinductive capacity has been widely used in bone tissue engineering. 19,20 The dexamethasone (DEX), a hydrophobic bioactive molecule commonly used in bone tissue engineering, is able to promote the osteogenic differentiation of mesenchymal stem cells (MSCs). 21 26 However, owing to the lack of proper delivery carriers, employing high doses of BMP-2 and DEX is inevitable which results in the high cost and undesirable side effects. [27][28][29] To overcome the aforementioned shortcomings, fabricating dual-bioactive moleculeloaded construct has been strongly advocated. Nevertheless, the current construct in bone tissue engineering is still far from perfect, as the release of bioactive molecule cannot endure during the bone regeneration which usually requires a few weeks or months. 30 The superiorities of the core-shell electrospun fibre, characterized by controllable release barriers (shell) and a sustainable release profile of bioactive molecules, are supposed to be a strong candidate to simultaneously load both BMP-2 and DEX and achieve a long-term sustainable release. 12,18 In this study, a dual-bioactive molecule (BMP-2 and DEX)-loaded core-shell fibrous mesh was fabricated by the coaxial electrospinning technique. The shell consisted of poly-D, L-lactide (PLA), and the core was made up of poly (ethylene glycol) (PEG). Considering the hydrophobic nature of DEX, DEX was firstly encapsulated in micelles and then together with BMP-2 was loaded inside the PEG core. The morphology of core-shell fibrous mesh, degradation and release profile were investigated. The osteogenic differentiation and osteogenesis of this core-shell fibrous mesh were also evaluated both in vitro and in vivo.

| Preparation of the DEX-loaded micelles
The blank and DEX-loaded micelles were fabricated by a solvent evaporation method. 32 Briefly, 0.1 g of mPEG-PCL copolymer was dissolved in 10 mL tetrahydrofuran (THF) prior to dissolving 100 μg of DEX. Then, the copolymer solution was added dropwise into 10 mL deionized water under violent stirring. The mixed solution was moderately stirred for 4 hours at room temperature to completely evaporate the THF and thereby forming polymeric micelle solution.
Subsequently, the DEX-loaded micelle solution was dialysed against the deionized water by a dialysis bag (MWCO 1000) to remove the unloaded DEX before freeze dried to obtain the micelle powder. Measured by UV-vis spectrophotometer (UV-2550, Shimadzu, Japan), DEX loading content (LC) was 45.7 ± 1.1% and encapsulation efficiency (EE) was 84.1 ± 3.4%.

| Coaxial electrospinning
The coaxial electrospinning process was conducted following the previous work. 33

| Characterization of different core-shell fibres
The morphologies of different fibres were characterized by scanning electron microscopy (SEM) (Quanta 200, Philips). The fibre diameter was measured from the SEM images using ImageJ software.

| Degradation test
The degradation of the electrospun fibres was performed in phosphate-buffered saline (PBS) at 37°C. The dried fibrous meshes were accurately weighed (20 mg) and then immersed in 25 mL PBS in test tubes. The tubes were kept in a thermostated shaking air bath maintained at 37°C and 120 cycles/min. The samples were taken at 4, 8 and 12 weeks, rinsed with deionized water to remove residual buffer salts, and dried in vacuum. The degradation percentage was determined by dividing the dry weight remained by initial weight.
The molecular weight was determined using gel permeation chromatography (GPC, Waters) analysis.

| In vitro drug release
To evaluate the drug release of PLA/PEG-DEX, PLA/PEG-BMP-2 and PLA/PEG-DEX-BMP-2 fibres, three samples (n = 3) weighing about 6 mg of each group were immersed in tubes containing 2 mL PBS. At the predetermined time, the release medium in the tubes was carefully collected and replaced by fresh PBS. The concentration of DEX in the release medium was tested using ultraviolet spectrophotometer at 242 nm, 34 and BMP-2 was measured by a BMP-2 ELISA kit (Boster Biological Engineering Co., Ltd).

| Cell culture and seeding on fibrous meshes
The bone mesenchymal stem cells (BMSCs) were harvested from 2-week-old SD rats using the whole bone marrow culture method.
All the electrospun fibrous meshes were sterilized with an ultraviolet lamp for 2 hours on each side. 2 × 10 4 cells were seeded on the surfaces of fibrous meshes in 24-well plates. The cells were cultured in osteogenic culture medium (DMEM supplemented with 50 μg/ mL ascorbic acid and 10 mmol/L glycerol phosphate), which was refreshed every 2 days.

| Alizarin red staining
Alizarin red staining was used to evaluate the extent of mineral deposition of BMSCs. The samples seeded with BMSCs were fixed with 4% paraformaldehyde and stained with 0.1% alizarin red after 7 and 14 days of culture.

| Osteogenic gene expression
The expression of the osteogenic differentiation marker genes of BMSCs was evaluated by real-time quantitative polymerase chain reaction (qPCR). After 7, 14 and 21 days, the total RNA was extracted using TRIzol reagent according to the manufacturer's instruction. Then, the RNA was reversely transcribed into cDNA with a PrimeScript RT Reagent Kit. qPCR was carried out with SYBR Premix Ex Taq. Alkaline phosphatase (ALP), osteocalcin (OCN) and osteopontin (OPN) gene were selected as target gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control.

| Analysis of new bone formation
After 12 weeks of implantation, the rats were sacrificed and the whole calvarium was collected and fixed in 4% paraformaldehyde solution for 24 hours. Then, the specimens were prepared for micro-computed tomography (micro-CT) analysis and histology observation.

| Statistical analysis
The data were described as the mean ± standard deviation. One-way ANOVA followed by the LSD test was used to assess statistical significance. The level of significance was set as P < .05.

| Characterization of different core-shell fibres
The microstructure of fibrous meshes was investigated by SEM. All the fibrous meshes consisted of interconnected and continuous fibres ( Figure 1A,B), whose diameter was almost comparable among them.  Figure 1D). Meanwhile, the microstructure of fibrous meshes was also tested by TEM (Figure 2A-D). The PLA/PEG fibres exhibited a high-contrast core and a low-contrast shell. This typical core-shell structure was presented in all bioactive molecule-loaded fibres, which indicated that embedding DEX and BMP-2 could not affect the core-shell structure.

| In vitro degradation test and bioactive molecules release
The change in molecular weight of electrospun fibrous meshes during degradation test was shown in Figure 2E. The molecular weight of all fibres gradually decreased. Generally, the PLA/PEG fibrous meshes exhibited the highest degradation rate in comparison with the bioactive molecule-loaded fibrous meshes, and the PLA/PEG-DEX fibres revealed the lowest degradation rate.
The release profiles of BMP-2 and/or DEX in different fibres were presented in Figure 2F. A biphasic release profile was observed for all the bioactive molecule-loaded fibrous meshes, which was characterized by a short burst release phase and a relatively long sustained release phase.
For the BMP-2, approximately 28.82% and 27.02% of cargo was, respectively, released from PLA/PEG-BMP-2 and PLA/PEG-DEX-BMP-2 fibrous meshes after the initial burst release (around 22 hours). Subsequently, a sustained release emerged, which continued for more than 274 h. As for DEX, a rather weak initial burst (around 22 hours) of the DEX was detected,

| Proliferation and morphology of BMSCs on core-shell fibrous meshes
The live/dead images of BMSCs seeded on fibrous meshes were displayed in Figure 3A-D. The overwhelming majority of cells was alive

| Alizarin red staining
The calcium depositions of BMSCs on the fibrous meshes were investi-

| Osteogenic genes expression
The expression of osteogenic genes, including ALP, OCN and OPN, was determined by qPCR ( Figure 5)

| Micro-CT
The repair of bone defect of different fibrous meshes was assessed in critical-sized calvarial defect rat model. The 3D reconstructed images were presented in Figure 6A

| D ISCUSS I ON
The bone regeneration is a complex process modulated by multiple growth factors and hormones. 37,38 Therefore, engineered constructs loaded with multiple and therapeutically relevant molecules are considered to be the potent candidates in promoting bone regeneration. 39,40 Although many studies have employed the electrospun fibrous meshes to load the bioactive molecule to promote the bone regeneration, most of them only loaded a single kind of biomolecule. Therefore, an electrospun fibrous mesh with the capacity to load the BMP-2 and DEX was developed in this study.
To obtain the sustained release of both biomolecules, a core-shell fibrous mesh containing biomolecules was fabricated via coaxial electrospinning and employed to exert a synergistic effect during osteogenesis.
To control the sustained release of the two bioactive molecules, DEX-loaded micelles and BMP-2 were incorporated into the core.
The release of bioactive molecules involved two mechanisms: the passive diffusion and the degradation of the polymers which was donated as the erosion-coupled mechanism. 30 Initially, the bioactive molecules were released from fibres due to passive diffusion. With the proceeding of polymer degradation, the passive diffusion-driven release gradually changed into the diffusion/erosion-coupled release. Notably, BMP-2 was released faster than DEX in the initial stage. Since the DEX was encapsulated inside the micelles, there might be two paths for its release from fibres before polymer degradation. One was that DEX-loaded micelles were first released from the fibre shell prior to DEX release; the other one was that DEX successively passed through the micelles and the shell in sequence. As for BMP-2, it could directly efflux through the shell owing to the lacking of micelles. Therefore, BMP-2 exhibited a faster release rate than DEX.
Proliferation, maturation and mineralization of extracellular matrix are three major stages during the growth and differentiation of MSCs. 41 The enhancement of cell proliferation in PLA/ PEG-DEX, PLA/PEG-BMP-2 and PLA/PEG-DEX-BMP-2 fibrous meshes was attributed to the encapsulated bioactive molecules, which have been reported to stimulate cellular proliferation. 42,43 The enhanced proliferation also indirectly indicated that the bioactivity of DEX and BMP-2 maintained during the coaxial electrospinning process. Due to more BMP-2 was released from fibrous mesh than DEX at initial stage, the cell proliferation was higher in intracellular glucocorticoid receptors, which activates transcription through glucocorticoid receptor-responsive elements located in the promoters of glucocorticoid receptor target genes. 24 It has been reported that DEX can enhance the effect of BMP-2 on cell differentiation. 26 The binding of BMP-2 to its receptor activates Janus kinases

| CON CLUS IONS
A novel core-shell fibrous mesh loaded with DEX and BMP-2 was fabricated through coaxial electrospinning to enhance bone regeneration. Through embedding BMP-2 and DEX-loaded micelles into the core of fibres, a relatively long sustainable release of both bioactive molecules was obtained. Based on the synergistic effect of BMP-2 and DEX, an enhanced bone regeneration can be achieved.
This core-shell fibrous mesh loaded with DEX and BMP-2 stands as a strong potential candidate for repairing bone defects.

ACK N OWLED G EM ENTS
The work was supported by National Natural Science Foundation of

CO N FLI C T O F I NTE R E S T S
There are no conflicts to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.