Hydroxyapatite-containing silk fibroin nanofibrous scaffolds for tissue-engineered periosteum

Abstract

The periosteum plays an indispensable role in both bone formation and bone defect healing. The purpose of this study was to construct a functional periosteum in vitro. We developed a simple technology to generate a hydroxyapatite (HA)-containing silk fibroin nanofibrous scaffold as a potential substitute for periosteum. The chemical structural characteristics of the scaffold were evaluated and the results confirmed the presence of HA in the scaffolds. In addition, the Young's modulus of silk fibroin–hydroxyapatite (SF/HA) scaffolds increased with the increasing content of HA. Rat bone marrow derived mesenchymal stem cells (rBMSCs) were cultured on the scaffolds for 7, 14, and 21 days without adding any osteogenic factors. Cell proliferation assay and cell morphology observation indicated that 30% SF/HA scaffolds exhibited good cell attachment and proliferation. In addition, differentiation of rBMSCs into osteogenic lineage was more actively exhibited on 30% SF/HA scaffolds, as evident by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for osteoblast-related gene markers (e.g., COL1A1, ALP, and Runx2), ALP activities, mineral deposits and immunocytochemical evaluations of osteoblast-related extracellular matrix components (e.g., OPN, ONN, and OCN). All the data in this study suggested that 30% SF/HA scaffolds had great potential as osteogenesis promoting scaffolds for constructing tissue-engineered periosteum.

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1. Introduction

Bone is an active organ persistently undergoing remodeling to adapt to mechanical stress and to repair injuries. The use of an autograft is still the current ‘gold standard’ for repair of bone loss.^1,2 However, autografts are not readily available and are related to donor site morbidity, while other materials have poor integration with the host's own bone. This lack of integration is attributed largely to the absence of periosteum, the outer layer of bone that contains multipotent mesenchymal stem cells and osteoprogenitor cells, which is vital for the growth and remodeling of bone tissue.^3,4 Studies have demonstrated that the presence of a functional periosteum could accelerate bone healing^5,6 and periosteal tissue grafts have shown successful results for bone regeneration.^7,8 Strategies to recreate this tissue for long bone repair have been proposed due to the importance of periosteal integrity for fracture repair. Evidently, the studies of periosteum mimetics for bone regeneration are gathering pace and further study is eagerly anticipated. Therefore, the generation of an engineered, functional periosteum-like tissue may be a potential solution to the lack of osseointegration of bone substitutes.

The development of biomimetic materials is essential and has become one of the most important paradigms in today's tissue regeneration research.⁹ Silk fibroin from Bombyx mori has gained much interest as a scaffold material because of its favorable biocompatibility and remarkable mechanical properties.^10–13 Silk biomaterials have the high stiffness and toughness due to the unique β-sheet (crystalline)-rich structure, which makes it a useful biopolymer for bone engineering applications.¹⁴ Additionally, it can be processed easily into various structures, such as fiber meshes, membranes, hydrogels, three-dimensional (3D) porous scaffolds, and microspheres.^15–19 These unique behaviors of silk fibroin make it to be a promising material for bone regeneration. In addition, the materials for bone regeneration should also be osteoconductive so that the osteoprogenitor cells can adhere and migrate on the scaffolds and subsequently form new bone.²⁰ As the major mineral component of natural bone, hydroxyapatite (HA) has shown conducive to bone cell attachment and proliferation and has a high osteogenic potential (osteoconduction and osteointegration), making it a prospective one of inorganic biomaterials for bone regeneration. However, HA is brittle and their clinical applications are limited only for unloading bearing repair.²¹ Therefore, the incorporation of HA within silk fibroin network may provide a more favorable synthetic microenvironment to more closely mimic natural bone tissue physiology. While electrospinning of pure silk fibroin have been widely studied in the past few years, it is very attractive to incorporate those non-electrospinnable inorganic nanoparticles into nanofibers to form hybrid nanofibers realizing some specific functional applications, in particular, for bone tissue engineering.^22–25 However, little attention has been paid to develop HA-containing silk fibroin nanofibrous scaffolds which combine the physical properties of cortical bone and the mechanical and conductive properties of the periosteum. Thus, we hypothesize that electrospun silk fibroin–hydroxyapatite (SF/HA) nanofibers might work as a bioactive tissue-engineered bone scaffold by mimicking regenerative capacity of periosteum. Furthermore, the SF/HA scaffolds will also improve the differentiative capacity and extracellular cellular matrix (ECM) deposition of osteogenic rat bone marrow derived mesenchymal stem cells (rBMSCs) and then new bone can be formed.

2. Experimental section

2.1 Preparation of silk fibroin

Raw Bombyx mori silk fibers were supplied from Rugao Chunqiu Textile Co. Ltd (Jiangsu, China). Silk fibroin solution was obtained from raw silk fibers that were degummed in a 0.1% (w/v) Na₂CO₃ solution, dissolved in CaCl₂–CH₃CH₂OH–H₂O (mole ratio = 1 : 2 : 8) at 78 ± 2 °C with continuous stirring and subsequently dialyzed against distilled water using a SnakeSkin Pleated Dialysis Tubing (PIERCE, MWCO 3500). Finally, the silk fibroin solution was freeze-dried for 24 h to form silk fibroin sponges and was kept in a vacuum drying desiccator for future use.

2.2 Electrospinning

The silk fibroin sponges were dissolved into hexafluoro-2-propanol (HFIP; Fluka Chemie GmBH, Germany) with stirring to generate a 10% (w/v) solution. The HA nanoparticles (Sigma) were introduced into the solutions and the mass ratio of HA to silk fibroin varied from 0 wt% to 30 wt%. All solutions were stirred at room temperature for at least 3 days. Electrospinning was performed with a glass syringe capped with a 0.9 mm inside diameter blunt needle. A constant volume flow rate of 0.8 mL h⁻¹ was maintained using a syringe pump. High voltage of 10 kV was applied when the solution was drawn into fibers and was collected on an aluminum foil covered collection plate kept at a distance of 13 cm from the needle tip. And then, the electrospun nanofibrous scaffolds were treated with 100% methanol for 10 min to induce a β-sheet conformational transition, which resulted in insolubility in water. To prepare scaffolds for cell culture, cover glasses were placed on the aluminum foil for depositing nanofibrous membranes onto them. All electrospun nanofibrous scaffolds were vacuumed for over 1 week in a vacuum oven to remove any potential residual solvents.

2.3 Scaffolds characterization

2.3.1 Scanning electron microscopy. The surface morphology of the HA/SF scaffolds was viewed under a scanning electron microscope (SEM; JEOL JSM-5600LV, Japan) with an accelerated voltage of 15 kV. The average diameters of SF/HA nanofibers were obtained by measuring 100 randomly selected nanofibers on the SEM images using Image J analysis software (National Institutes of Health, USA).

2.3.2 Energy dispersive X-ray spectroscopy (EDS). A field-emission scanning electron microscope (FESEM) equipped with an EDAX Energy Dispersive X-ray Spectroscopy system and was used to assess calcium and phosphorous contents and the element distribution of the scaffolds. X-ray spectra were taken at 10 kV using a 60 mm final aperture. EDS was performed using the FESEM at an acceleration voltage of 10 kV.

The element mapping of energy-dispersive X-ray spectroscopy was used to confirm the elements present on the electrospun fibres while assessing the distribution of these particles. Samples were first coated with a 3–5 nm thin layer of gold using a Edward Pirani 501 Scancoat six sputter coater and analyzed at an accelerated voltage of 10–20 kV.

2.3.3 ATR-FTIR spectrometry. The infrared spectra of HA/SF scaffolds were collected using attenuated total reflectance Fourier transform infrared spectrometry (ATR-FTIR, Nicolet 560, America). Every spectrum was acquired in transmission mode with a resolution of 4 cm⁻¹ and a spectral range of 4000–400 cm⁻¹.

2.3.4 XRD. Crystal structure was determined by X-ray diffraction (XRD). XRD was performed using an X'Pert-Pro MPD (PANalytical) diffractometer and Cu K_α radiation with a wavelength of 1.5406 Å. Data were collected for 2θ values of 5–50°, with a step width of 0.02° and a counting time of 1 s per step.

2.3.5 Water contact angle measurements. Water contact angle measurement was undertaken to evaluate the surface hydrophilicity of the HA/SF scaffolds. The water-in-air contact angles of the scaffolds were measured by a VCA optima surface analysis system (AST Products, Inc., USA). The scaffold was placed onto the work stage of the meter. The contact angle was measured within 10 s after a droplet of distilled water (5 μL) contacted the scaffold surface. Five samples for each group were measured.

2.3.6 Mechanical testing. The mechanical properties of the scaffolds were tested using a Universal Testing Machine (Instron 4505). The dimension of the specimen was 12 mm in width and 200 mm in height. The testing was done at the speed of 5 mm min⁻¹ at room temperature with a load cell capacity of 250 N. The tensile strength and Young's modulus were measured in this test. A minimum number of ten specimens were tested and the reported data were the average of ten independent samples.

2.4 Isolation and expansion of rBMSCs

rBMSCs were isolated from the femurs and tibias of 30 day-old male Sprague-Dawley rats (Peking University Laboratory Animal Center, Beijing, China) as previously described.^26,27 All experiments involving the use of animals were in compliance with Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation and were approved by Beijing Municipal Science & Technology Commission (Permit number: SCXK (Beijing) 2012-0001). In brief, density gradient centrifugation was performed using the percoll technique (Pharmacia, Uppsala, Sweden) to isolate cells from bone marrow. Cells were cultured in Dulbecco's Modified Eagle Medium-Low Glucose (DMEM-LG; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) in cell incubator at 37 °C with 5% CO₂. When primary rBMSCs became near confluent, they were detached from the flasks with trypsin–EDTA solution (Invitrogen, CA, USA), and expanded at a splitting rate of 1 : 3. Different scaffolds were co-cultured with rBMSCs in passages 2–4 in further study.

2.5 In vitro culture of rBMSCs on SF/HA scaffolds

All samples were sterilized in 75% ethanol, washed in PBS and incubated in expansion medium 24 h prior to seeding. rBMSCs were seeded at a density of 2.5 × 10⁵ cells per scaffolds onto 0% SF/HA and 30% SF/HA scaffolds placing in the wells of a non-treated 6-well culture plate. After pipetting 100 μL of cell medium suspension onto the scaffolds, the plates were incubated for 3 h to allow cell attachment. And then, cell-free medium was added to bring the total well volume to 3 mL. The cell-seeded scaffolds were grown in vitro in a 5% CO₂ incubator at 37 °C with the medium being replaced every 3 days.

2.6 Cell proliferation and cell morphology

The proliferation activity of rBMSCs cultured on the scaffolds was assayed by CCK-8 (Cell Counting Kit-8, Beyotime, China) according to the manufacturers' instructions. The optical density (OD) at 450 nm was determined via a microplate reader (Thermo-Labsystems, Multiskan Mk3, USA).

The cell morphology of the rBMSCs on the scaffolds was examined by confocal microscopy on day 7 and 14. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and then blocked in 1% bovine serum albumin. After washing with PBS, cells were incubated in phalloidin (Sigma-Aldrich) for 30 min to stain all filamentous actin (F-actin) filaments and with 4,6-diamino-2-phenyl indole (DAPI) (Sigma-Aldrich) for 3 min to label the nuclei at room temperature. Representative fluorescence images of stained samples were obtained using a confocal laser scanning microscope (Leica Microsystems GmbH, Germany).

The cell morphology on the scaffolds was confirmed by SEM on day 7 and 14. The cell-seeded scaffolds were fixed with 2.5% (v/v) glutaraldehyde solution in 0.1 M Na-cacodylate buffer (pH 7.2) for 1 h at 4 °C, washed with Na-cacodylate buffer, and then dehydrated at room temperature in a gradient ethanol series up to 100%. The samples were kept in 100% ethanol for 15 min and then critical point-dried with CO₂. The specimens were sputter-coated with gold by a sputter coater (Bal-Tec SCD 050, Bal-Tec AG, Liechtenstein) and observed using a scanning electron microscope (SEM, JEOL JSM-5600LV, Japan) with an accelerated voltage of 15 kV.

2.7 Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis

The mRNA levels of alpha l chain of type I collagen (COL1A1), alkaline phosphatase (ALP), and the Runt-related transcription factor 2 (Runx2) of rBMSCs cultured on the scaffolds were analyzed on day 7 and day 14 using quantitative real-time RT-PCR. Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions and quantified by optical density. Quantitative RT-PCR primers (synthesized by Sangon, Shanghai, China) were as follows: COL1A1, 5′-TTA CTA CCG GGC CGA TGA-3′ and 5′-CTG CGG ATG TTC TCA ATC TG-3′; ALP, 5′-CAT GTT CCT GGG AGA TGG TA-3′ and 5′-GTG TTG TAC GTC TTG GAG AGA-3′; Runx2, 5′-CAT GGC CGG GAA TGA TGA G-3′ and 5′-TGT GAA GAC CGT TAT GGT CAA ATT G-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TGT TCC TAC CCC CAA TGT ATC CG-3′ and 5′-TGC TTC ACC ACC TTC TTG ATG TCA T-3′. A 1 μL cDNA sample was added to each primer, 12.5 μL of 2× SYBR Green Supermix (Takara, Kyoto, Japan) and PCR-grade water to a volume of 25 μL. Real-time PCR was performed in an iCycler iQ real-time PCR detection system (Bio-Rad). Relative expression of genes was calculated by the comparative 2^−ΔΔCt method.²⁸ Each sample was assessed at least in triplicate.

2.8 ALP activity assays

The activity of alkaline phosphatase (ALP) of rBMSCs cultured on the scaffolds was analyzed on day 14. Cells were collected and treated with 0.05% Triton X-100 (Sigma, USA). ALP activity was detected by an ALP kit (Biosino, Beijing, China). Following manufacturer's instructions, 20 μL sample liquid was mixed with 1000 μL working liquid of the ALP kit, and then 200 μL mixing liquid was transferred into a new standard 96-well culture plate. After incubating at 37 °C for 60 s, the OD values were measured separately at 1 min, 2 min, and 3 min at 405 nm. And then, the ALP activity can be calculated according to the instruction.

2.9 Mineralization of rBMSCs

The amount of ECM secreted by rBMSCs can be both qualitatively and quantitatively analyzed by using Alizarin red staining. Alizarin Red S (ARS) is a dye that binds selectively to the calcium salts and hence can be used for mineral staining. On day 21, the scaffolds seeded with rBMSCs was washed thrice with PBS and fixed in 70% ice cold ethanol for 1 h. These constructs were then washed twice with distilled water and stained with ARS (40 mM) for 20 min at room temperature. After several washes with distilled water, the scaffolds were observed and photos were taken by an inverted fluorescence microscope (IX71, Olympus Inc., Japan). The stain was eluted by incubating the scaffold with 10% cetylpyridinium chloride for 1 h. The absorbance of the collected dye was then read at 540 nm in a microplate reader (Thermo-Labsystems, Multiskan Mk3, USA).

2.10 Immunocytochemistry analysis

Cells cultured on the scaffolds were fixed in 4% paraformaldehyde on day 21, permeabilized with 0.1% Triton X-100 in PBS and then blocked in 1% bovine serum albumin. After blocking, the cells were incubated overnight at 4 °C with the primary antibodies targeting the osteoblast specific marker protein osteopontin (OPN), osteonectin (ONN), and osteocalcin (OCN). After washing with PBS, fluorescein isothiocyanate (FITC) conjugated antibodies (Zhongshan, Beijing, China) at a dilution of 1 : 100 were added to the cells for 60 min and DAPI was used for nuclei staining. Representative fluorescence images of stained samples were obtained using a confocal laser scanning microscope (Leica Microsystems GmbH, Germany).

2.11 Statistical analysis

Each experiment was repeated independently for at least 3 times. All data were expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Turkey's multiple comparison test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1 Morphology of SF/HA scaffolds

Electrospinnability of the composite matrix plays an important role for the inorganic nanoparticles involving composite electrospinning. In this study, the SF/HA nanofibers with HA nanoparticles loading of 30 wt% could be readily electrospun into its fibrous form (Fig. 1d) due to the excellent electrospinnability of silk fibroin solution system. Moreover, the electrospun SF/HA nanofibers looked continuous and fairly uniform. In contrast to the smooth surface of silk fibroin fibers (Fig. 1i), HA particles were found on the surface of 10%, 20%, and 30% SF/HA nanofibers and more HA particles were observed as the HA content increased (Fig. 1j–l). From an image analysis, the average diameter of nanofibers gradually increased from 286 ± 41 to 471 ± 81 nm and its distribution became significantly broader (Fig. 1B).

Fig. 1 (A) SEM images of SF/HA scaffolds containing 0% (a, e and i), 10% (b, f and j), 20% (c, g and k), and 30% HA (d, h and l). (a–d) Scale bars = 30 μm; (e–h) scale bars = 5 μm; (i–l) scale bars = 1 μm. (B) The average diameters and diameter distributions of the different SF/HA nanofibers.

3.2 Chemical structural characteristics

XRD, ATR-FTIR, EDS, and EDS mapping of Ca element were used to further characterize the SF/HA scaffolds, specifically the nanoparticle deposits observed on the surface of the SF/HA nanofibers. As shown in Fig. 2B, two major 2θ reflection peaks at around 26° being the (002) diffraction, and around 32° being the overlapped diffractions of (211), (300), and (202) were observed in the 10%, 20%, and 30% SF/HA scaffolds, which indicates the introduction of crystalline properties into the amorphous nanostructure of the SF scaffolds due to the presence of HA.⁹ Fig. 2A showed the ATR-FTIR spectra of SF/HA scaffolds prepared with different content of HA. The phosphate groups in pure HA showed characteristic ATR-FTIR bands between 900–1100 cm⁻¹ and 500–600 cm⁻¹ (Fig. 2A).^3,29 ATR-FTIR spectra of the 10%, 20%, and 30% SF/HA scaffolds revealed bands at 900–1100 cm⁻¹ and 500–600 cm⁻¹ that did not appear in the 0% SF/HA scaffold spectra (Fig. 2A). Strong absorption bands at 1625 cm⁻¹ and 1528 cm⁻¹ were observed for all SF/HA scaffolds, which could be attributed to the silk-II structure.^30,31 In addition, EDS was employed to determine the elemental composition of the nanofibers. 10%, 20%, and 30% SF/HA scaffolds showed peaks for calcium and phosphorus (Fig. 3B–D), indicating that HA was incorporated into the scaffolds. Moreover, the quantitative results showed that more HA was incorporated into the scaffolds as the HA increased (insert in Fig. 3B–D). Moreover, as shown in Fig. 3E–G, EDS mapping of Ca element (a major component of HA) showed increasing amount of Ca element in the composite scaffolds electrospun from the SF/HA solution with higher HA contents.

Fig. 2 ATR-FTIR (A) and XRD (B) curves for SF/HA scaffolds with different content of HA. (C) Water contact angles of SF/HA scaffolds with different content of HA. * indicates significant difference of p < 0.05. (D) Photographs of water droplets on the SF/HA scaffolds.

Fig. 3 (A–D) Elemental analysis of the different SF/HA scaffolds shows presence of phosphorus (P), calcium (Ca) with oxygen (O) and nitrogen (N). EDS mapping of Ca element in SF/HA scaffolds with increasing concentrations of HA from (E) 10, (F) 20, and (G) 30 wt%.

3.3 Mechanical properties

The tensile strength of SF/HA scaffolds containing 0%, 10%, 20%, and 30% HA were 0.57 ± 0.08, 0.59 ± 0.08, 0.60 ± 0.08, and 0.60 ± 0.04 MPa, respectively (Table 1). There was no significant difference between the tensile strength values when the content of HA was increased from 0% to 30%. The Young's modulus of scaffolds containing 0%, 10%, 20% and 30% HA were 13.53 ± 3.34, 15.61 ± 4.16, 22.48 ± 5.99 and 24.78 ± 7.06 MPa, respectively (Table 1). Increasing the HA content from 0 to 30% increased the Young's modulus of SF/HA scaffolds from 0% to 30%.

Table 1 The tensile properties of SF/HA nanofibers scaffolds

HA contents (%)	Tensile strength (MPa)	Young's modulus (MPa)
0	0.57 ± 0.08	13.53 ± 3.34
10	0.59 ± 0.08	15.61 ± 4.16
20	0.60 ± 0.08	22.48 ± 5.99
30	0.60 ± 0.04	24.78 ± 7.06

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3.4 Hydrophilicity

Surface hydrophilicity is one of the important aspects that affect the biocompatibility of the materials in a biological environment.³² As shown in Fig. 2C, it was observed that the induction of HA into the scaffolds significantly increased the hydrophilicity of pure SF nanofibers (p < 0.05). The reduction in the contact angles suggested that an improvement in surface hydrophilicity was achieved with the incorporation of HA. Moreover, 30% SF/HA scaffolds showed the highest hydrophilicity, which should benefit cell attachment and growth on the surface of the scaffolds.³³ In addition, while the static water surface contact angle was measured, water droplets on the surface of the membranes were photographed (Fig. 2D).

3.5 Cell proliferation

The growth and metabolic behavior of cells cultured on the scaffolds is a critical issue for clinical transplantation. The viability and proliferation of rBMSCs cultured on the scaffolds were determined by CCK-8 assay in this study. As shown in Fig. 4A, after 4 days of culture, the OD values of cells cultured on 30% HA/SF scaffolds were significantly higher than those of the cells cultured on 0% HA/SF scaffolds (p < 0.05), which indicated that 30% SF/HA scaffolds could promote cell proliferation. Notably, after an initial increase, the OD values of cells on 30% SF/HA scaffolds remained relatively stable over the experimental time course, which indicated that the cells were undergoing differentiation.

Fig. 4 (A) Cell proliferation study of rBMSCs cultured on SF/HA scaffolds. * indicates significant difference of p < 0.05. (B) Immunocytochemistry staining of rBMSCs cultured on 0% and 30% SF/HA scaffolds for 7 and 14 days, respectively. (a–d) Scale bars = 250 μm.

3.6 Cell morphologies

Confocal microscopy analysis of rBMSCs cultured on the scaffolds revealed their complex morphological features. The cells were stained with phalloidin specific for F-actin filaments, and DAPI to visualize the nuclei. The confocal micrographs indicated that rBMSCs were of more spindle shape on 30% SF/HA scaffolds (Fig. 4b), while most of rBMSCs exhibited a more stellate-patterned phenotype on 0% SF/HA scaffolds (Fig. 4a). It means that cells spread widely on the 30% SF/HA scaffolds with distinct spread actin filaments compared to those on the 0% SF/HA scaffolds. After 7 and 14 days culturing, there were more rBMSCs on 30% SF/HA scaffolds as compared with the 0% SF/HA scaffolds (Fig. 4b and d).

Observation of rBMSCs on the scaffolds by SEM showed cell attachment and co-existence of cells in different stages of spreading over a 3 weeks period (Fig. 5). As shown in Fig. 5G, cells on 30% SF/HA scaffolds spread out better and nearly grew to confluence after being cultured for 14 days. Moreover, the cells showed uniform distribution on the scaffolds and exhibited fibriform shape morphology (Fig. 5G). The surface of the 30% SF/HA scaffolds was covered with cell/ECM and globular accretions were also observed at day 21 (Fig. 5K), indicative of calcification. The results suggested that 30% SF/HA scaffolds could provide a more favorable environment for cell proliferation and mineral deposits.

Fig. 5 SEM photomicrographs showing adherence and proliferation of rBMSCs cultured on 0% and 30% SF/HA scaffolds for 7, 14 and 21 days. (A, C, E, G, I, and K) Scale bars = 100 μm; (B, D, F, H, J, and L) scale bars = 50 μm.

3.7 Osteoblast-related gene expressions

Comparisons of mRNA transcript levels of osteoblast-related ECM genes were made among rBMSCs cultured on 30% SF/HA scaffolds and 0% SF/HA scaffolds. mRNA transcript expressions of COL1A1, ALP and Runx2 (ref. 27 and 34–36) were evaluated by real-time RT-PCR after 7 and 14 days in culture (Fig. 6A–C). As shown in Fig. 6A, expression of COL1A1 was significantly higher on 30% SF/HA scaffolds than that on 0% SF/HA scaffolds at day 7 (p < 0.05). Two osteogenic markers (ALP and Runx2) were upregulated in rBMSCs cultured on 30% SF/HA scaffolds compared with the other group at day 14 (Fig. 6B and C), suggesting 30% SF/HA scaffolds promoted the osteogenic differentiation of rBMSCs.

Fig. 6 COL1A1 (A), ALP (B) and Runx2 (C) mRNA expression of rBMSCs on 0% and 30% SF/HA scaffolds. * indicates significant difference of p < 0.05. (D) ALP activity showing the osteogenic differentiation of rBMSCs on SF/HA scaffolds at day 14. * indicates significant difference of p < 0.05. (E) Mineral deposition staining of rBMSCs cultured on 0% and 30% SF/HA scaffolds at day 21. (a) Macroscopic view of ARS staining of rBMSCs on 0% and 30% SF/HA scaffolds. (b) Quantification of mineral deposition by detecting absorbance of ARS extracts. * indicates significant difference of p < 0.01. (c and d) Microscopic view of ARS staining of rBMSCs on 0% and 30% SF/HA scaffolds respectively. (c and d) Scale bars = 200 μm.

3.8 ALP activity

To further determine the effects of SF/HA scaffolds on the osteogenic differentiation of rBMSCs, the expressions of ALP were assessed by ALP kit at day 14 (Fig. 6D). rBMSCs cultured on 30% SF/HA scaffolds exhibited significantly higher ALP activity than those on the 0% SF/HA scaffolds (p < 0.05). The results showed that rBMSCs differentiated towards osteogenic lineage better on 30% SF/HA scaffolds than on the 0% SF/HA scaffolds.

3.9 The mineral deposition of rBMSCs

Upon undergoing osteogenic differentiation the cells begin to secrete mineral matrix, called the mineralization phase. The mineral deposition was determined both qualitatively and quantitatively as shown in Fig. 6E. ARS staining was performed and revealed that there was more calcium deposition on the 30% SF/HA scaffolds compared to the 0% SF/HA scaffolds at day 21 (Fig. 6a, c and d). Quantification analysis of calcium deposition further confirmed the significant difference between the scaffolds at day 21 (p < 0.01). Since the ability to deposit minerals is a marker for mature osteoblasts, it could be confirmed that rBMSCs seeded onto 30% SF/HA scaffolds differentiated and led to mineralization phase to deposit mineralized ECM.

3.10 Osteoblast-related protein expressions of rBMSCs

The expressions of osteoblast-related proteins of rBMSCs cultured on the SF/HA scaffolds for 21 days were detected by immunocytochemistry with protein-specific antibodies (Fig. 7A and B). Confocal microscopy images showed that OPN, ONN, and OCN were all expressed on the scaffolds at day 21. However, the expressions of OPN, ONN, and OCN on 30% SF/HA scaffolds were much higher than those on the 0% SF/HA scaffolds (Fig. 7A and B).

Fig. 7 Confocal microscopy images showing the expression of OPN, ONN, and OCN of rBMSCs on 0% and 30% SF/HA scaffolds. Cell nuclei were stained by DAPI (blue) and OPN, ONN, and OCN were stained by FITC-labeled antibody (green). (A) Scale bars = 100 μm; (B) scale bars = 50 μm.

4. Discussion

The treatment of large bone defects is still challenging. Studies have demonstrated that the recruitment of stem cells by functional periosteum could give rise to the relative success of autografts in the repair of large bone defects.³⁷ The osteogenic function of periosteum has shown to determine the rate of postnatal periosteal bone formation and the bone strength during the process of growth or defect healing.^38,39 The development of an in vitro tissue engineered periosteum to mimic the anatomical structure of natural periosteum would be a novel approach in the treatment of large bone defects. Our aim was to construct a functional periosteum in vitro and evaluated the physicochemical characteristics of the SF/HA scaffolds as well as their ability to promote adhesion, proliferation, and osteogenic differentiation of BMSCs.

To generate engineered bone tissue, it is vital to design scaffolds with micro- and nano-sized architectures similar to that of native bone.⁴⁰ In this study, silk fibroin was used to fabricate biomimetic HA-containing nanofibers to resemble the native ECM inducing cells to function naturally. HA particles were found on the surface of 10%, 20%, and 30% SF/HA nanofibers and more HA particles present on the surface of the composite nanofibers as the content of HA increased (Fig. 1j–l). Furthermore, the average diameters of nanofibers gradually increased from 286 ± 41 to 471 ± 81 nm and its distribution became significantly broader (Fig. 1B). Some HA aggregates are found on the surfaces or the interconnection points of the SF/HA nanofibers (Fig. 1A), which may contribute to the increase in the diameter of 10%, 20%, and 30% SF/HA nanofibers.⁴¹ HA particles were present on the fiber surface as well as embedded in depth, allowing a slow and constantly growing exposure of ceramic surface to the cells, while the polymer is degraded.⁴² While other studies tried to achieve composite fibers by orienting the particles parallel to the longitudinal axis of the nanofibers,⁴³ here the HA distribution made the inorganic phase potentially available for the cellular microenvironment in a continuous and consistent way.⁴²

To evaluate the chemical structure characteristics of HA-containing electrospun nanofibers, we used 4 independent material characterization techniques (XRD, ATR-FTIR, EDS, and EDS mapping). The XRD spectra of the 10%, 20%, and 30% SF/HA scaffolds showed distinct peaks that specifically matched those found in pure HA samples (Fig. 2B). However, the diffraction reflection seems to be broadened with respect to the HA samples. This indicated that the HA crystallites were partially demineralized under current condition and then led to decreased crystallinity, which suggested that it was hard to completely reduce mineral loss.⁹ ATR-FTIR spectrum was carried out to confirm the functional groups in the electrospinning SF/HA nanofibers (Fig. 2A). The results demonstrated that both phosphate and carbonate groups were obviously presented in 10%, 20%, and 30% SF/HA nanofibers. When the content of HA increased up to 30%, the peak position of the characteristic bands of silk fibroin was not shifted, demonstrating that the HA content was not significantly affected the secondary structure of silk fibroin.²³ EDS characterization ascertained that the electrospun 10%, 20%, and 30% SF/HA nanofibers contained small amounts of calcium and phosphorous. Moreover, the quantitative results showed that more HA were presented within the scaffolds as HA increased (insert in Fig. 3B–D). In addition, EDS mapping of Ca element showed increasing amount of Ca element in the composite scaffolds with higher HA contents. All the data demonstrated that HA was presented within the scaffolds and more HA was incorporated into the scaffolds as the HA content increased.

The static water contact angle assay indicated that 30% SF/HA scaffolds were more hydrophilic compared with the other groups (Fig. 2C and D), which should benefit cell attachment and growth on the scaffolds. In addition, as shown in Table 1, the Young's modulus of SF/HA scaffolds increased with the increasing content of HA, while the tensile strength was only marginally increased. Since the mechanical properties of our scaffolds increased with HA concentrations up to 30%, we focused on 30% HA as a working concentration for the subsequent studies.

In terms of functional tissue engineering, our aim was to develop a bioactive scaffold capable of inducing osteogenic differentiation similar to what occurs when osteoprogenitor cells from the periosteum migrate to the damaged bone tissue. The periosteum plays an indispensable role in both bone formation and bone defect healing because it is the source for the recruitment of osteoprogenitor cells used for the initiation of repair and regeneration at sites of injury.^4,44,45 The most commonly used osteogenic cells are BMSCs derived from bone marrow aspirates. It has been demonstrated that HA is also osteoinductive in the absence of osteogenic factors.^41,42 Thus, the osteogenic properties of the SF/HA fibrous scaffolds were assessed in vitro using rBMSCs without adding any osteogenic factors. As shown in Fig. 4A, the metabolism activities of rBMSCs on 30% SF/HA scaffolds were significantly higher than those on 0% SF/HA scaffolds after 4 days of culture (p < 0.05). This result indicated that 30% SF/HA scaffolds could promote the proliferation of rBMSCs. The confocal micrographs and the SEM results illustrated that 30% SF/HA scaffolds could support cell adhesion and spreading, which was consistent with the findings of previous studies.⁴⁶

Differentiation is most often judged in terms of the upregulation of markers indicative of a mature, differentiated osteoblast phenotype. COL1A1 is essential for the development of the bone cell phenotype, being related to the formation of the ECM. COL1A1 is actively expressed in the first proliferation period and then gradually down-regulated during subsequent osteoblast differentiation.⁴⁷ ALP cleaves organic phosphate esters and is a crucial part of bone matrix vesicles because of its role in the formation of apatite calcium phosphate.⁴⁸ Runx2 significantly influences the differentiation process of BMSCs into osteogenesis in the early stage, regulating bone formation by G protein-coupled signaling pathway and enhancing an up-regulation of ALP, OPN, OCN and bone sialoprotein.^42,49 In this study, the mRNA transcript levels of ALP and Runx2 of rBMSCs cultured on 30% SF/HA scaffolds were upregulated compared with the other group (Fig. 6B and C). The ALP activity analysis also revealed that rBMSCs exhibited higher expression of ALP on 30% SF/HA scaffolds than on 0% SF/HA scaffolds (Fig. 6D). Furthermore, calcium nodule creation was detected by ARS staining. 30% SF/HA scaffolds contained significantly higher level of calcium deposition than those on the 0% SF/HA scaffolds (p < 0.01). As higher amount of mineral deposits implies a higher degree of differentiation of the cells, 30% SF/HA scaffolds are expected to enhance bone formation ability.⁹

Production of minerals is principally bioapatites in the form of globular accretions, as reported in previous studies.^9,50,51 High magnification SEM micrographs showed that the calcified globular accretions were associated with ECM. Protein levels were determined by immunocytochemical staining, and the results revealed that the deposition of the ECM rich in OPN, ONN, and OCN on the 30% SF/HA scaffolds (Fig. 7). OPN, an extracellular structural protein responsible for mineralization of the ECM.⁵² OCN, a calcium binding protein being considered as the most specific marker of mature osteoblasts, gets accumulated in mineralized bone and binds actively to hydroxyapatite crystals promoting bone crystal growth.^53,54 ONN, a bone-specific protein that binds selectively to both collagen and hydroxyapatite, is expressed to promote bone formation and initiation of bone mineralization. It was apparent that the upregulation of ONN would contribute to the accelerated formation of bone tissue and mineralization.⁵⁵ The protein expression of OPN, ONN and OCN further demonstrated that the rBMSCs on 30% SF/HA scaffolds were more prone to differentiate into the osteoblast lineage. Based on the above-mentioned results, 30% SF/HA scaffolds displayed much stronger osteoinductive potential than 0% SF/HA scaffolds in vitro. Our data are in line with previous studies demonstrating that HA-containing scaffolds support the osteogenic differentiation of BMSCs without adding any osteogenic factors.^41,42

5. Conclusions

The present study developed a simple technology to generate HA-containing silk fibroin nanofibrous scaffolds as potential substitutes for periosteum. The chemical structural characteristics of the scaffold were evaluated and the results confirmed the presence of HA in the scaffolds. The Young's modulus of SF/HA scaffolds increased with the increasing content of HA. The cell proliferation and cell morphology assays indicated that 30% SF/HA scaffolds could promote cell attachment and growth. The results of osteoblast-related gene expressions, ALP activity, mineral deposits, and osteoblast-related protein expressions showed that 30% SF/HA scaffolds could support the osteogenic differentiation of rBMSCs. Overall, the data in this study suggested that 30% SF/HA scaffolds might be a suitable candidate material for constructing tissue-engineered periosteum.

Acknowledgements

This work was supported by the National Key Technology R&D Program (2012BAI18B06, 2014BAI11B02, 2014BAI11B03), National Natural Science Foundation of China (31470938, 11421202, 61227902, and 11120101001), International Joint Research Center of Aerospace Biotechnology and Medical Engineering from Ministry of Science and Technology of China, 111 Project (B13003), Research Fund for the Doctoral Program of Higher Education of China (20131102130004), The transformation project for major achievements of Central Universities in Beijing (ZDZH20141000601), Fundamental Research Funds for the Central Universities, and Innovation Foundation of BUAA for PhD Graduates.

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