Enhancing the Osteogenic Capability of Core ... - ACS Publications

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Enhancing the Osteogenic Capability of Core−Shell Bilayered Bioceramic Microspheres with Adjustable Biodegradation Xiurong Ke,† Chen Zhuang,‡ Xianyan Yang,‡ Jia Fu,† Sanzhong Xu,§ Lijun Xie,∥ Zhongru Gou,*,‡ Juncheng Wang,† Lei Zhang,*,† and Guojing Yang*,† †

Rui’an People’s Hospital & The 3rd Hospital Affiliated to Wenzhou Medical University, Rui’an 325200, China Bio-Nanomaterials and Regenerative Medicine Research Division, Zhejiang-California International Nanosystem Institute, Zhejiang University, Hangzhou 310058, China § Department of Orthopaedic Surgery, The First Affiliated Hospital, School of Medicine of Zhejiang University, Hangzhou 310003, China ∥ Department of Orthopaedic Surgery, The Second Affiliated Hospital, School of Medicine of Zhejiang University, Hangzhou 310009, China ‡

S Supporting Information *

ABSTRACT: This study describes the fabrication and biological evaluation of core−shell bilayered bioceramic microspheres with adjustable compositional distribution via a coaxial bilayer capillary system. Beyond the homogeneous hybrid composites, varying the diameter of capillary nozzles and the composition of the bioceramic slurries makes it easy to create bilayered β-tricalcium phosphate (CaP)/β-calcium silicate (CaSi) microspheres with controllable compositional distribution in the core or shell layer. Primary investigations in vitro revealed that biodegradation could be adjusted by compositional distribution or shell thickness and that poorly soluble CaP located on the shell layer of CaP or CaSi@CaP microspheres was particularly beneficial for mesenchymal stem cell adhesion and growth in the early stage, but the ion release from the CaP@CaSi exhibited a potent stimulating effect on alkaline phosphatase expression of the cells at longer times. When the bilayered microspheres (CaSi@CaP, CaP@CaSi) and the monolayered microspheres (CaP, CaSi) were implanted into the critical-sized femoral bone defect in rabbit models, significant differences in osteogenic capacity over time were measured at 6−18 weeks post implantation. The CaP microspheres showed the lowest biodegradation rate and slow new bone regeneration, whereas the CaSi@CaP showed a fast degradation of the CaSi core through the porous CaP shell so that a significant osteogenic response was observed at 12−18 weeks. The CaP@CaSi microspheres possessed excellent surface bioactivity and osteogenic activity, whereas the CaSi microspheres group exhibited a poor bone augmentation in the later stage due to extreme biodegradation. These findings demonstrated that the bioactive response in such core−shell-structured bioceramic systems could be adjusted by compositional distribution, and this strategy can be used to fabricate a variety of bioceramic microspheres with adjustable biodegradation rates and enhanced biological response for bone regeneration applications in medicine. KEYWORDS: osteogenic capability, core−shell structure, bilayer bioceramic microspheres, biodegradation, bone regenerative medicine

1. INTRODUCTION

nonetheless, accelerated the development of innovative synthetic alternatives. It is widely accepted that an artificial porous scaffold provides the necessary three-dimensional (3D) structure for cell adhesion, proliferation, and secretion of extracellular matrix to guide the growth of new bony tissue. In this regard, successful approaches in scaffold design must be able to recreate the hierarchical organization and mass transport (i.e., permeability and diffusion) properties of bone and reproduce its porous architecture, and other biological functions, within arbitrary 3D

Currently, a significant increase in the number of bone trauma and pathological bone injuries has stimulated the development of new bone augmentation technologies.1 Artificial bone fillers that are used to stimulate skeletal healing have become more common in surgical practices. As a complex and wellorchestrated process, bone formation requires artificial grafts to provide high biocompatibility, osteogenic efficacy, and appropriate biodegradability. Many synthetic bone substitutes are currently available for clinical applications; however, only a few of them meet the requirements of bony tissue regeneration in situ. The delayed union, malunion, or nonunion caused by large bone defects still are formidable challenges,2,3 which have, © XXXX American Chemical Society

Received: May 14, 2017 Accepted: July 7, 2017

A

DOI: 10.1021/acsami.7b06798 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic of the preparation of core−shell microspheres (A), light images of the representative core−shell CaSi@CaP microspheres after undergoing different treatments (B−D), scanning electron microscopy (SEM) images of the fracture surface of the two groups of core−shell microspheres (E−H), and X-ray diffraction (XRD) patterns of the as-sintered bioceramic microspheres (I−L).

anatomical shapes.4,5 A number of architectural characteristics, including porosity, pore size, and permeability, play a significant role in the delivery of biological products and new tissue regeneration.6,7 Ideally, pores are fully interconnected and also the evolution of pore structures (i.e., material biodegradation) matches that of neotissue regeneration beyond the limits of passive diffusion. This is particularly important at early stages of neovascularization and later stages of new bone turnover when most of the young bony tissue has grown and become filled with the pore network.8,9 Accordingly, porous constructs with precision-controlled biodegradation and pore structure evolution over time are required to optimize the regeneration and repair of bony tissue; however, developing porous scaffolds with a degradation rate which readily matches that of physiological bone regeneration remains a challenge. To date, particulate fillers have been widely used to treat defective bones and thus developed into various shapes and used particularly in implantology due to their operational convenience.10−12 It is worthwhile mentioning that the interconnected pore network in closely packed microsphere systems can only benefit the bone tissue ingrowth.13,14 In this regard, it is beneficial to produce spherical particles out of bioactive ceramics to enhance bone repair. Dense and porous hydroxyapatite (HA) microspheres have been used for bone replacement due to their chemical similarity to the mineral part of bone.15 Unfortunately, some studies have shown that the high-temperature sintered HA cannot be resorbed.16 Recently, we have found that octacalcium phosphate (OCP) porous microspheres exhibited an excellent early stage osteogenic activity, whereas the phase conversion from OCP to HA affected the long-term bone augmentation.17 In addition, we

developed a novel one-step technique for the fabrication of hollow or core−shell bioceramic microspheres on the basis of a coaxially aligned multilayer capillary system.18,19 Instead of homogeneous hybrid mixtures of biphasic bioceramics, it is possible to produce bilayered or multilayered microspheres with controllable shell-layer number and composition by varying the configuration of the capillary nozzles and the composition of the bioceramic slurries.18 Therefore, it is reasonable to hypothesize that the components that have significantly different biodegradability rates could be integrated into a core−shell microsphere and thus these bilayered composites could readily be endowed with time-dependent biodegradation in vivo. Here, we describe a straightforward one-step strategy to prepare wollastonite/β-tricalcium phosphate (β-TCP) microspheres with tunable core or shell compositions in addition to homogeneous hybrids (Figure 1A). Tricalcium phosphate (CaP) and calcium silicate (CaSi) were chosen as examples to demonstrate the effectiveness of this method to fabricate bioceramic microspheres with a compositional distribution, as their porous scaffolds show either a lower or higher biodegradation rate than the new bone tissue regeneration rate in vivo.20−23 On the basis of this hypothesis, the in vitro biodegradation test and cell culture experiment and a rabbit distal femur bone defect model were employed to evaluate the biodegradation and osteogenesis of the bilayer microspheres (CaSi@CaP, CaP@CaSi) with respect to their monolayer microspheres (CaP, CaSi). It is reasonable to say that this improvement in the compositional distribution of bioceramic composites, which is in line with the bone tissue regeneration efficiency of bone defects, opens the door for precise B


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ACS Applied Materials & Interfaces manufacturing of biomaterial fillers with interconnected 3D porous structures for highly efficient repair of bone defects.

culture medium was removed and the microspheres were rinsed three times in phosphate-buffered saline (PBS) and then treated with 1% osmium tetroxide for 2 h, followed by dehydration in a series of alcohol solutions for 4 min. Subsequently, the samples were placed in isoamyl acetate for 20 min, following vacuum-assisted drying at 40 °C for 4 h. The microspheres were sputter-coated with gold−palladium alloy before cell morphology and distribution analyses under SEM. The cell distribution on the microspheres was examined by laser scanning confocal microscopy (LSCM) (Leica, Germany). Briefly, the live cells were fixed in 4% paraformaldehyde for 20 min, followed by rinsing in PBS. The cells were under permeabilization with 0.1% Triton X-100 for 20 min and rinsed again with PBS. The samples were immersed for 30 min in PBS containing 1% bovine serum albumin (Sigma-Aldrich). Afterward, the solution was removed and the cell actin and nuclei were labeled with 600 μL of 5 μg/mL fluorescein isothiocyanate (FITC)-conjugated phalloidin (Invitrogen) in PBS for 40 min and 10 μg/mL 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen) separately for 5 min at room temperature. Finally, the labeled cells were observed with LSCM. 2.3.2. Cell Viability Assay. According to manufacturer’s protocol, the Cell Counting Kit-8 assay (CCK-8; Dojindo, Kumamoto) was used to count the cells for the examination of cell viability. In summary, 1 × 104 rBMSCs were cultured with the same sterilized samples in 24-well tissue culture plates. After cultured for 1, 4, and 7 days, the original culture medium was removed and replaced with 0.5 mL of DMEM containing 10% CCK-8 in every well before incubation at 37 °C for 2 h. Subsequently, 100 μL of the solution mentioned above was transferred to a 96-well plate. A microplate reader (Infinite F50; TECAN, Hombrechtikon) at 450 nm was used to analyze the optical density (OD) value of every well. The absorbance value of a mixture of 90 mL of DMEM and 10 mL of CCK-8 was used as a negative control, whereas the absorbance value of the rBMSC cultural system without the scaffolds was used as a positive control. Four specimens were used to measure the cell viability for every sample. 2.3.3. Analysis of Alkaline Phosphatase (ALP) Activity. The ALP activity assay was used to determine the osteogenic differentiation capacity of the rBMSCs seeded on the different bioceramic microspheres. For analysis, 5 × 104 cells were seeded on the microspheres of the four groups in a 24-well plate and incubated in DMEM (10% FBS). The culture medium of every well in the plate was aspirated on days 5 and 10. Nonidet P-40 solution (200 mL, 1%) was put into every well for incubation for 1 h. Next, the cells were allowed to lyse, and centrifugation was performed simultaneously to collect the lysate supernatant. The supernatant of every sample (50 mL) along with 2 mg/mL p-nitrophenyl phosphate (Beyotime, Shanghai, China) substrate solution (50 μL) was transferred to a 96-well plate and incubated for 30 min at room temperature. The OD value of every plate was quantified at a wavelength of 405 nm using the microplate reader. Using a protein assay kit (Pierce Biotechnology Inc.) based on the bicinchoninic acid method, the ALP levels were then normalized to the total protein content for activity standardization, and four specimens of each sample were used for measuring the ALP activity. 2.4. Surgical Procedure for Specimen Implantation and Harvesting. The repair of rabbit femoral bone defect was used to investigate the osteogenic efficacy and bioresorption behavior of the bioceramic microspheres. Approved by the Experimental Animals Ethics Committee of Zhejiang University (ZJU2015-9-09-095), 48 New Zealand white rabbits (male, ∼4.5 months; 3.0−3.5 kg) were randomly divided into four groups (Table 1). Before surgery, all rabbits were allowed to acclimate for 1 week in stainless steel cages singly. After general anesthesia by intravenous injection of 3% sodium phenobarbital at a dosage of 1 mg/kg, a 3 cm longitudinal skin incision was made on the lateral femoral condyle of each leg under rigorous aseptic conditions. A critical-sized defect (Ø 6 × 8 mm2) oriented vertically to the longitudinal and sagittal axes of the femur was generated by a dental drill before the implantation of the microspheres into the defects. After that, all rabbits were allowed to move freely in their cages and injected with penicillin (800 000 U) for 3 days. After 6, 12, and 18 weeks, four animals from each group were euthanized and the distal femurs containing the implants (n = 8) were harvested for

2. MATERIAL AND METHODS 2.1. Preparation of the Microspheres. All of the chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd, China. A conventional chemical coprecipitation method was applied to synthesize β-CaSi and β-TCP powders. After calcination at 950 °C for 3 h, the powders were milled to a particle size of less than 5 μm. The microsized β-CaSi and β-TCP powders were dispersed separately in a 20 wt % sodium alginate (NaAlg) solution under gentle stirring, Then, the slurries were separately placed into two coaxially aligned nozzles (Ø 2.0 and Ø 1.2 mm) through different microtubes to form the core−shell droplets. The droplets were formed immediately after falling into the 0.5 mol/L Ca(NO3)2 solution, resulting in spherical particles. After hardening in the solution for 5 min, the CaSi@CaP or CaP@CaSi microspheres were washed with deionized water, dried at 85 °C overnight, and finally sintered at 1100 °C for 3 h. To evaluate how the shell thickness of the microspheres affected their biodegradation properties, the microspheres (CaSi-s@CaP, CaP-s@ CaSi) were also prepared using the coaxially aligned nozzles with a smaller internal nozzle diameter (Ø 0.8 mm) by keeping the other conditions same. The average size of the bioceramic microspheres before and after drying and sintering was measured using a Vernier caliper (n = 20). The phase composition of the bioceramic microspheres was analyzed by XRD (Rigaku, Japan) using Cu Kα radiation at a scanning rate of 2°/min. The fracture surface of the bioceramic microspheres was characterized by SEM (JEM-6700F, Tokyo) with energy-dispersive spectroscopy (EDX). The semiquantitative determination of the CaP/CaSi mass ratio in the bioceramic microspheres was based on the diameter of the sintered microspheres measured by SEM observation. 2.2. In Vitro Biodegradation Evaluation. The evolution of the surface microstructure and biodegradation was investigated by immersing the microspheres in Tris buffer at 37 °C, to simulate the environment in vivo. The bioceramic microspheres (1.0 g) prepared with different internal nozzles were incubated in 20 mL of Tris buffer water bath with an initial pH of 7.25 at 37 °C. At different time intervals, 2.0 mL of supernatant was centrifuged, diluted with a 5% HCl solution for Ca, P, and Si determination using inductively coupled plasma (ICP; Thermo), and an aliquot amount of fresh buffer was added. After immersing for 14 days, the microsphere samples were dried for SEM observation. To evaluate the biodegradation, the bioceramic microspheres (W0 = 1.0 g) were immersed in 20 mL of Tris buffer with an initial pH of 7.25 for 1, 4, 7, 10, and 14 days. At the preset time stage, the microspheres were rinsed with ethanol, then dried at 100 °C for 12 h, and weighed (Wt). Then, the microspheres were immersed in an equal volume of fresh Tris buffer to keep volume constant. The weight decrease at time t was expressed as Wt/W0 × 100%. 2.3. Isolation and Cell Culture of Rat-Derived Bone Marrow Mesenchymal Stem Cells (rBMSCs). The rBMSCs were harvested from the femoral bone marrow of a 12 week old male Fisher 344 rat. The surgery was approved by the Experimental Animals Ethics Committee of Zhejiang University (ZJU2015-9-09-095). Upon isolation, the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and 100 units/mL penicillin in an incubator at 37 °C and 5% CO2. The culture medium was refreshed every 2 days. The cells were expanded over two to four passages before use. 2.3.1. Cell Morphology and Distribution in Vitro. The bioceramic microspheres (1.0 g, i.e., CaP, CaSi@CaP, CaSi, and CaP@CaSi) were incubated in 10 mL of Tris buffer water bath with an initial pH of 7.25 at 37 °C. After immersing the bioceramic microspheres for 7 and 14 days, the samples were rinsed with ethanol and then dried. The 14 day immersed microspheres were placed in 24-well tissue culture plates and immersed in DMEM containing 10% FBS and 100 units/mL penicillin for 2 h. Afterward, 1 × 104 cultured rBMSCs were seeded on the surface of microspheres. After culturing them for 4 and 7 days, the C


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the slice thickness to be scanned was set to 15 μm. After scanning, a region of interest (Ø 5.5 × 8.0 mm2) in the femur was chosen to reconstruct 3D images for quantitative analysis using Inveon Acquisition Workplace (IAW; Siemens, Germany). For quantitative analysis, the newly formed bone (NB) volume-to-total volume (BV/ TV) ratio and (RV/TV) ratio were calculated using IAW. The stereological parameters of trabecular bone were also measured to evaluate the degree of maturity. 2.4.3. Histological Observation and Histomorphometric Analysis. The specimens were immersed in a 4% paraformaldehyde solution for 48 h and then decalcified in a 10% ethylenediaminetetraacetic acid solution (pH ∼ 7.2) for 2 weeks. Before being embedded in paraffin, the samples were dehydrated in ascending series of alcohol rinses (80, 90, 95, 98, 100%). For histological observation, the embedded samples were sectioned into a thickness of 5−7 μm through the midportion of the defect perpendicular to the longitudinal axis of the femur. Afterward, the sections were stained with hematoxylin/eosin (HE) and Masson’s trichrome separately. Finally, the stained sections were observed under a light microscope (DMLA; Leica, Germany) at different magnifications (40, 100, and 400×). For histomorphometric analysis, photographs of the HE-stained sections were taken with the light microscope. On the basis of five different photographs from the inner part of each sample with the magnification of 100, the area of the NB was evaluated quantitatively using IPP 6.0, and the percentage of NB (NB%) was then calculated as follows24

Table 1. Composition Ratio, Strength of the Bioceramic Microspheres, and Grouping of the Animals group CaSi CaP CaSi@ CaP CaP@ CaSi

CaSi/CaP ratio (wt %)

crushing force (N)

animals (total)

survivors (%)

100:0 0:100 20.3:79.7

22.01 ± 0.18 25.33 ± 0.17 7.54 ± 0.45

4 (12) 4 (12) 4 (12)

100 100 100

75.1:24.9

8.82 ± 0.32

4 (12)

100

the following analyses. X-ray scan radiography, high-resolution microcomputer tomography (μCT; Inveon CT scanner, Siemens, Germany), and histological and immunohistochemical analyses were carried out on the specimens. 2.4.1. Radiological Examination by X-ray Scanning. After harvesting, the femoral specimens were scanned using a special imaging system (XPERT; Kubtec Co.) designed for small animals. For assessment of the new bone formation, the frontal and lateral X-ray films of each group (n = 5) were recorded at 45 kV and 100 μA. All of the films were recorded using a high-resolution camera (DMLA; Leica, Germany). The frontal films were quantitatively analyzed using the image analysis software Image-Pro Plus 6.0 (IPP 6.0, Media Cybernetics). The quantitative data of the residual materials in the defect were expressed as residual material volume-to-total volume (RV/TV). 2.4.2. μCT Analysis. μCT analysis was done on five specimens of each group at different time points using a high-resolution μCT system (Inveon μCT scanner; Siemens, Germany). All of the specimens were scanned vertically to the long bone axis covering the whole distal femur under exposure conditions of 80 kV and 100 mA. Meanwhile,

5

NB% = (∑ (newly formed bone area in each photograph n=1

/whole area of each photograph)/5) × 100%

Figure 2. Structural and physicochemical characterization of the bioceramic microspheres with different shell thickness. (A) SEM images of the fracture surface of the microspheres. (B) Diameter of microspheres before and after sintering and immersion in Tris buffer. (C) Weight decrease of the bioceramic microspheres during immersion in Tris buffer for 0−14 days. *p < 0.05, compared with the CaP microspheres; **p < 0.05, compared with the CaSi microspheres. D


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Figure 3. SEM observation of the surface morphology of the bioceramic microspheres before and after the Tris immersion and MSCs culture. (A) Surface microstructures of the bioceramic microspheres before and after immersion in Tris buffer for 0, 7, and 14 days. (B) SEM images of cell attachment and morphology on the microspheres after culturing for 4 and 7 days on the 14 day immersed bioceramics microspheres.

2.4.4. Immunohistochemistry Analysis. The expressions of type I collagen (Col-1) and osteocalcin (OCN) of the NB inside the defect were analyzed by immunohistochemical processing. In summary, the

deparaffinized and rehydrated sections of the harvested samples were first stained with antibodies Col-1 (90395, Abcam, U.K.; concentration = 1/100) and OCN (13420, Abcam, U.K.; concentration = 1/50), E


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Figure 4. Morphology and viability of MSC culture test on the bioceramic microspheres. (A) Observation of cytoskeleton on the microspheres stained with FITC-phalloidin (green) and nuclei stained with DAPI (blue) on the microspheres. (B) Cell viability on the microspheres after 1, 4, and 7 days determined by CCK-8 test (*p < 0.05, **p < 0.01). (C) ALP activity after 5 and 10 days of culture. respectively, and then incubated in a humidified chamber at room temperature overnight. After incubation, the sections were stained with labeled streptavidin−biotin reagents (SA1021; Boster, China). For further immunohistochemical study, all of the prepared sections were histomorphometrically analyzed under the light microscope. The 40× photographs were chosen to evaluate the amount of newborn bone around the residual microspheres, whereas the 400× photographs were used to estimate their maturity.25,26 2.5. Statistical Analysis. The quantitative data were transferred to the statistical software SPSS 19.0 (IBM). Although differences between the two groups were assessed using Student’s test, multiple comparisons were assessed using the one-way ANOVA test. The results were expressed as mean ± SD. The statistical outcomes were considered to be statistically significant when p < 0.05.

core−shell microspheres. The phase compositions after sintering were examined by XRD, as shown in Figure 1I−L. The definable crystalline phase for the CaSi microspheres was the highly crystalline β-phase of wollastonite (PDF #27-0088); however, biphasic calcium phosphates (BCPs) containing HA (PDF #09-0432) and β-TCP (PDF #09-0169) in the CaP, CaSi@CaP, and CaP@CaSi microspheres were confirmed by XRD analysis. Moreover, the core−shell bilayer bioceramic microspheres showed nearly one-third of the maximal crushing forces compared to those of the monolayer microspheres and the mass fraction in the core or shell layer of the core−shell microspheres was significantly different, according to the quantitative analysis (Table 1). The SEM observation in Figure 2A confirmed the different core or shell dimension in the core−shell bioceramic microspheres, which were prepared using the same shell nozzle (Ø 2.0 mm) but different core nozzles (Ø 0.8 or Ø 1.2 mm). Also, the pure CaSi and CaP microspheres were easily prepared using a single nozzle. According to the quantitative analysis, the as-formed microspheres in the Ca2+-rich solution showed some degree of shrinkage (from ∼4.0 to ∼2.6 and ∼2.0 mm) during the drying and sintering processes (Figure 2B). Particularly, the

3. RESULTS 3.1. Physicochemical Characterization the Core−Shell Microspheres. Figure 1A−D describes the preparation method and structure of the core−shell microspheres. The SEM images (Figure 1E−H) confirmed the core−shell structures and appropriate sizes in the core and shell layers, which match the two coaxially aligned capillaries. The facescanning EDX spectra in the core and shell layers also confirmed the definite distribution of the CaSi and CaP in the F


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Figure 5. Progress of implanting operation and primary characterization. (A) Material implantation in the critical-sized lateral femoral defect and the femoral specimen. (B) X-ray radiographic analysis of the femoral bone specimens harvested from the rabbits. (C) Quantitative analysis of residual material in the defect areas. *p < 0.05.

the microspheres with a CaSi external shell but increased mildly for the microspheres with a CaP external shell. Moreover, the SEM observation for the microspheres after immersing for 14 days indicated that the microspheres with a CaSi external shell showed a rougher surface layer than those with a CaP external shell (Figure S2). 3.2. Surface Biodissolution and Cytocompatibility in Vitro. Figure 3A shows changes in the surface morphology of microspheres with prolonged immersion time, ranging from 7 to 14 days, in Tris buffer. The grain boundaries of the CaP surface layer in the CaP and CaSi@CaP microspheres underwent slow dissolution, whereas the CaSi surface layer in the CaSi and CaP@CaSi underwent fast dissolution, as indicated by an increased roughness. SEM images of the grown rBMSCs on the microspheres at 4 and 7 days are shown in Figure 2B. The images show that cells on the CaP surface layer were in polygonal and widespread forms and distributed densely with time. Meanwhile, the cells also presented abundant prominent filopodia and unidirectional lamellipodia extensions. In contrast, the cells on the CaSi surface layer possessed fewer filopodia, although it could be seen that some biomimetic apatite-like deposits occurred on the CaSi substrate. The cell distribution and CCK-8 assay after incubation for 1, 4, and 7 days was used to characterize the cell density and viability (Figure 4A,B). Compared to that on the CaSi microspheres, the density of rBMSCs on the CaP after 1−7

pure CaP and CaSi bioceramic microspheres after sintering showed the shortest and longest diameters, respectively. The in vitro degradation behavior of the bioceramic microspheres is shown in Figure 2C. The microspheres with a poorly soluble CaP external shell showed a much slower weight decrease than those with a highly soluble CaSi external shell at the early stage. The pure CaSi and CaP showed the fastest and slowest biodissolution, respectively, and the former was degraded by over 40% at 10 days, which was 4-fold higher than that of the later. Meanwhile, the microspheres with a thicker CaSi shell (i.e., CaP-s@CaSi) or CaSi core (i.e., CaSi@ CaP) showed a significantly faster dissolution than the those with a thinner CaSi shell (i.e., CaP@CaSi) or CaSi core (i.e., CaSi-s@CaP) during the 7−14 days. The changes in the ion concentrations of Tris buffer during soaking of the bioceramic microspheres were also measured by ICP analysis. It is seen that appreciable differences in Ca and Si concentrations were observed for the two groups of microspheres with CaP and CaSi external shells, respectively (Figure S1 in the Supporting Information). For the microspheres with a CaP external shell, the Ca concentrations for the CaSi-s@CaP and CaSi@CaP microspheres showed a fast increase compared to those for the pure CaP microspheres. Meanwhile, the Si concentration for the CaSi@CaP microspheres increased faster than that for the CaSi-s@CaP. For the microspheres with a CaSi external shell, the Ca and Si concentrations increased rapidly. Overall, the P concentration changed very slowly for G


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Figure 6. μCT-reconstructed 2D images of the bone defects implanted with bioceramic microspheres (A) and quantitative data of BV/TV (B), RV/ TV (C), and Tb.N (D). Yellow: new bone; blue: bioceramic spheres. *p < 0.05.

Figure 7. HE straining images (40, 100×) of the bone defects implanted with CaSi (A1−A6), CaP (B1−B6), CaSi@CaP (C1−C6), and CaP@CaSi (D1−D6) bioceramic microspheres at 6 weeks (A1−D1, A2−D2), 12 weeks (A3−D3, A4−D4), and 18 weeks (A5−D5, A6−D6). N: NB; F: fibrous tissue; V: vessel; Si: CaSi; P: CaP.

H


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In contrast, as a more biologically stable material, a poor bone regeneration was observed in the pure CaP microsphere group at 6 weeks, but more new bone tissue was observed after 18 weeks. As for the core−shell CaSi@CaP group, the CaSi core layer in CaSi@CaP showed a fast biodegradation and thus the bioactive-ion products may activate a sustained new bone ingrowth in the microsphere system. In the case of the CaP@ CaSi group, conversely, the CaSi shell layer was degraded rapidly, but the CaP core remained for a long time. This group showed more new osteoid-like tissue formation homogeneously extending throughout the microsphere during 6−12 weeks. And more matured bone tissues were formed compared to those of the other groups after 18 weeks. The CaP cores were reduced to small islands that appeared to be completely embedded in the new bone tissue. The quantitative morphometric analysis of BV/TV confirmed that the CaP@CaSi showed the highest osteogenic

days was higher. The rBMSCs on the surface of the CaSi@CaP microspheres presented polygonal spreading and higher viability at 1 and 7 days (p < 0.01), which were also evidently higher than those of the CaP@CaSi group after 4 and 7 days (p < 0.05), respectively. The ALP activity analysis was subsequently conducted to monitor how the core−shell bioceramic microspheres affected the osteogenic differentiation capacity of the cells (Figure 4C). The results indicated that the CaP@CaSi group at 5 days presented the lowest ALP activity but showed the highest ALP activity at 10 days. Furthermore, a significant difference was noticed between the CaP and CaSi@CaP groups (p < 0.05). 3.3. Primary Evaluation and Radiographic Analysis of the Animal Models. The composition ratio, strength of the bioceramic microspheres, and the animal groups are listed in Table 1. All of the rabbits were awake at 1 h postoperatively and then kept in cages for feeding. No rabbits showed obvious infection symptoms, and all of them survived until the time of specimen harvesting. Figure 5A shows a representative bone defect wound (Ø 6 × 8 mm) filled with microspheres and the femoral specimen harvested at 6 weeks. According to the X-ray radiographic analysis (Figure 5B), it could be seen that the microspheres showed significantly different bioresorption rates between 6 and 18 weeks postoperatively. The pure CaSi displayed the fastest biodegradation, whereas the CaP remained in the defects after 18 weeks. The CaP@CaSi group showed a faster bioresorption than the CaSi@CaP group in the early stage (6 weeks), but the later showed a faster bioresorption at 12−18 weeks. This was supported by the quantitative data of the X-ray radiographic analysis, which showed that the CaSi and CaP groups had the lowest and highest RV/TV values, respectively, during 6−18 weeks; however, the CaP@CaSi group showed a higher RV/TV value than the CaSi@CaP group at 6 weeks but a lower value after 12 and 18 weeks (Figure 5C). 3.4. μCT Reconstruction Analysis. Figure 6A shows the μCT-reconstructed two-dimensional (2D) images of the four groups of animal models at 6−18 weeks postoperatively. It was seen that the CaP microsphere group showed the lowest material biodegradation and new bone tissue ingrowth; however, the pure CaSi group showed an overfast biodegradation within 12 weeks, which led to a very low bone repair after 18 weeks. Although the CaSi@CaP microspheres had a stable CaP shell layer, this group showed an appreciable bone tissue ingrowth in the closely packed microsphere system at 6 weeks. In contrast, the CaP@CaSi group, with a bioresorbable shell layer, showed appreciable new bone formation after 18 weeks. These results were consistent with the quantitative BV/ TV and RV/TV data, which responded with the new bone formation and material residual in the bone defects (Figure 6B,C). Moreover, the CaSi@CaP and CaP@CaSi groups possessed much higher trabecular number (Tb.N) values, but the pure CaSi groups showed significantly lower Tb.N data (Figure 6D). 3.5. Histological and Histomorphometric Analysis. The histological straining analysis was used to determine whether the bone regeneration could be activated and sustained through core−shell material biodegradation and tissue− materials interactions. As shown in Figure 7, the new bone tissue was regenerated into the pore network of the closely packed CaSi microspheres after 6 weeks. However, the CaSi microspheres were nearly fully biodegraded at 12−18 weeks so that they could not maintain a stable new bone tissue growth.

Figure 8. Osteogenesis of the ceramic scaffolds in vivo. Morphometric analysis of the volume of the NB (BV/TV) in the skull defect area at 6−18 weeks post operation (*p < 0.05).

capacity, whereas pure CaSi had the lowest osteogenic capacity (Figure 8). Similarly, the CaP group exhibited a low bone regeneration rate compared to that of the CaSi@CaP group, which displayed an improved osteogenic capacity at 6−18 weeks postoperatively. Masson’s trichrome staining images showed a clearer new bone tissue, tissue/material interface, and material residual, as shown in Figure 9. Evidently, the CaSi was degraded rapidly so that there was scarce newly mineralized tissue in the CaSi microsphere group after 18 weeks. However, the CaP group showed very limited osteogenesis in the early stage but then appreciable bone tissue formed in the interconnected gap between the microspheres. On the other hand, the CaSi@CaP microspheres exhibited a hollow structure in the CaP shell after 12 weeks, whereas the CaP@CaSi microspheres kept a residual core of CaP material during CaSi shell degradation and new bone regeneration. 3.6. Immunohistochemical Analysis. Immunohistochemical staining was employed to measure new bone mineralization (Figure 10). The expression levels of Col-1 and OC (two representative markers of ossification) were significantly higher in the two bilayered microsphere groups compared to those in the two monolayered microsphere groups I


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Figure 9. Masson’s trichrome staining images (40×) of the bone defects implanted with CaSi (A1−A3), CaP (B1−B3), CaSi@CaP (C1−C3), and CaP@CaSi (D1−D3) bioceramic microspheres at 6 weeks (A1−D1), 12 weeks (A2−D2), and 18 weeks (A3−D3). N: NB; F: fibrous tissue; Si: CaSi; P: CaP.

strength, biodegradation rate, and osteogenesis of uniform CaSi/CaP hybrids;31,32 however, to our knowledge, very few reports concerning gradient distribution design are available for tailoring biodegradation to stimulated bone regeneration.33 Some recent studies have shown that the CaP bioceramics with specified surface microporous or hierarchical micro− nanohybrid structure gave the best simultaneous enhancement of protein adsorption, osteoblast proliferation, differentiation, and even osteoinductivity.34,35 However, an irregular and rough surface usually reduces cytocompatibility. Our in vitro Tris buffer immersion test shows that a fast biodissolution does occur in the CaSi surface layer of the CaSi and CaP@CaSi microspheres. This results in a very irregular and rough sphere surface, which could potentially deteriorate osteogenic cell adhesion and growth (Figure 3). It is interesting to note that the release of bioactive-ion from the CaSi phase will benefit osteogenic differentiation of the cells. Thus, these preliminary in vitro results support the different osteogenic capabilities of the four groups of microspheres in vivo. On the other hand, the in vivo results show that the femoral bone defects filled with pure CaSi microspheres remain unhealed at 18 weeks postoperatively due to the fast biodegradation of CaSi; however, when the CaP@CaSi microspheres (with lowdegradation CaP core) are implanted in similar bone defects, new bone tissue regeneration appears higher than that in the pure CaSi-implanted defects. This suggests that the poorly degradable CaP core probably retards the structural collapse of the closely packed microsphere system and thus benefits bone tissue regeneration and ossification. Similarly, the bilayered

at 6 and 18 weeks. In particular, compared to that of the pure CaSi group, the CaP@CaSi group showed significantly increased matrix Col-1 and OCN levels at 6 weeks. On the other hand, the CaSi@CaP system enhanced the expression of ossifying makers compared to that of the pure CaP system at 6 weeks, possibly due to a higher bioactive stimulation triggered by the ions released from the CaSi core.

4. DISCUSSION To date, third-generation biomaterials are being designed with the aim that once implanted they will help the body to heal by itself. One desirable characteristic of these materials in bone is the ability to be biodegraded and be replaced by NB through promoting the activation of osteogeneous cells in a controlled time frame.27 Usually, the rationale for developing CaP-based biomaterials is mainly their similarity to bone mineral, and intrinsic biocompatibility and osteoconductivity. However, in spite of these desirable properties, biodegradability, bioactivity, and especially the osteostimulation ability of stoichiometric CaPs are ambiguous according to the clinical applications of the past two decades.28,29 On the other hand, the Ca-silicate porous bioceramics exhibit enhanced fibrovascularization, bone tissue ingrowth, and appropriate degradation comparable to CaPs.30 We have succeeded in fabricating the binary core−shellstructured CaSi−CaP (porous) microspheres using a coaxially aligned capillary system.18 This is a particularly time-saving process to yield enough spherical products with tuned compositional distribution for bone regeneration and repair in situ. A few studies have shown improved mechanical J


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Figure 10. Immunohistochemical analysis of the bone defects implanted with CaSi (A1−A4), CaP (B1−B4), CaSi@CaP (C1−C4), and CaP@CaSi (D1−D4) bioceramic microspheres using antibodies against Col-1 (A1−D1, A2−D2) and OCN (A3−D3, A4−D4). N: NB; F: fibrous tissue; Si: CaSi; P: CaP.

immersion or implantation and that such microparticles could be at the origin of local inflammation and cell damage.40 That is, the osteogenic capability of bioceramics would be heavily affected by the surface microstructural features in vivo.41 This suggests that, conversely, the pore-wall microstructure evolution in the CaSi@CaP or CaP@CaSi microspheres with the CaP or CaSi degradation would favorably mediate the osteogenic cell activity and osteogenesis in the early stage. In general, pore size and pore evolution with time has always been implicated in osteogenesis.42 These structural parameters are not only able to adjust the cell migration and fibrovascularization in scaffold43 but can also influence the osteogenic efficacy when they change dynamically.44,45 As for the closely packed bioceramic microsphere system, indeed, the primary porosity is very low (∼30%) and the pore size is directly associated with the diameter of the microspheres. Therefore, the low-biodegradation pure CaP microspheres could not trigger a fast new bone tissue ingrowth and complete repair due to the slow pore size magnification.15,46 In our study, the in vitro immersion experiments confirmed that the core− shell-structured CaSi−CaP porous microspheres with different compositional distribution and shell thickness showed a significantly different weight loss in the early stage (14 days). The weight decrease of CaP@CaSi microspheres due to biodissolution in Tris buffer was over 1.7-fold higher than that of the CaSi@CaP microspheres at 14 days (Figure 2C). Furthermore, the increase in CaSi shell thickness (CaP@CaSi vs CaP-s@CaSi) and the decrease in CaP shell thickness (CaSi@CaP vs CaSi-s@CaP) are both beneficial for enhancing the biodegradation rate of the core−shell microspheres in the

CaSi@CaP microspheres (with low-degradation CaP shell) also display a significantly faster osteogenesis than the monolayer CaP microspheres. This may be attributed to the porous nature of the CaP shell layer through which the calcium and silicon are released from the CaSi core and stimulate osteogenesis. Accordingly, this study, for the first time, provides evidence that bioceramic microspheres with gradient compositional distribution and time-dependent biodegradation properties enhance bone tissue regeneration. It is well known that the typical CaP and CaSi biomaterials, for example, β-TCP and wollastonite, show significantly different biodegradation rates in vivo; especially, the latter, which has been confirmed to have good degradability and excellent bioactivity in the osteogenic cell culture medium.20,21 Thus, the rationale for developing wollastonite-containing biomaterials is mainly due to its bioactive potential superior to synthetic TCP and its good biodegradability. The conventional mechanical mixing approach usually results in a uniform hybrid of the CaP or CaSi component in bioceramic composites and thus the physicochemical and biological properties of the composites are difficult to tailor and must be compromised.20−22 It is reasonable to postulate that the lowbiodegradation ceramic grains readily separate from the pore wall due to the fast biodegradation of the highly biodegradable ceramic phase; thus, it may facilitate a chronic local inflammation and could perhaps modify osteogenesis.36−38 In previous in vitro experiments, Lu et al. observed a cytotoxic effect of BCP, although no cytotoxic effect was observed for HA or TCP.39 Lu et al. also found that the HA microparticles in BCP were incompletely sintered and released easily after K


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operatively, implying a strong dependence of the compositional distribution on the high- and low-biodegradation ceramic phases in the core−shell microspheres. It should be noted, however, that because the sparingly dissoluble BCP in the core layer of the CaP@CaSi microspheres was more difficult to biodegrade than that located on the shell layer in the CaSi@ CaP microspheres, the latter only underwent an initial slow biodegradation but a fast biodegradation at the later stage due to the high biodegradation of CaSi cores through the porous CaP shell. This was confirmed by the RV/TV data (Figures 5C and 6C) and the histological observation (Figures 7 and 9). However, because of the faster CaSi shell biodegradation and pore-size increase in the CaP@CaSi microsphere scaffold, there is a higher osteogenic capacity (BV/TV) in the CaP@CaSi group than that in the CaSi@CaP group. This increase is attributed to both porous network evolution and bioactive effects of the Ca2+ and SiO32− release that stimulate new bone tissue ingrowth. Therefore, this is possibly helpful to develop new bioactive scaffold systems with adjustable time-dependent bioactivity and pore structure, to enhance bone regeneration applications, especially for arbitrary 3D anatomical bone defects.

early stage. This suggests that the rate of biodissolution occurring in the shell layer in the early stage and then in the core layer is changed dynamically, consistent with the biodegradation nature of the components, and thus it is reasonable to assume that a “fast-to-slow” or “slow-to-fast” biodegradation behavior would result for the CaP@CaSi and CaSi@CaP microspheres in vivo. Indeed, this was confirmed by the animal model experiment results in this study (Figures 5 and 6). Because of the high biodegradation rate of the pure CaSi, the macroporous architecture in the CaSi microsphere system was hardly retained for enough time and thus the new bone tissue ingrowth may not have been maintained via osteoconduction (Figure 7). In this regard, the core−shell CaSi@CaP or CaP@CaSi microspheres provided a timedependent dynamical biodegradation of the shell and core layers and thus the pore size magnification was readily controlled by the phase composition and distribution in the core−shell composites (Figures 5−7). Furthermore, comparing the phase distribution of CaSi and CaP resulted in biodegradation of the core−shell microspheres in the bone defects. It should be pointed out that, according to the μCT and histological analyses, the CaSi phase showed a significant bioresorption from the core layer of CaSi@CaP microspheres. This was predominantly associated with the porous nature of the CaP shell layer. Actually, the core−shell microspheres were primarily prepared through a chelation reaction of powder/NaAlg slurry beads with Ca2+ ions and then followed by a sintering post treatment. It is reasonable to postulate that the bioceramic phase could not be completely densified although the alginate was volatilized by thermolysis. This is consistent with the quantitative analysis of the material residues (RV/TV) in the bone defects that the CaSi@CaP group showed a much faster decrease in RV/TV than that in the pure CaP group in the early stage (6−12 weeks; Figures 5C and 6C). On the basis of this abnormal but expected structural property, it could be concluded that the bioactive-ion dissolution products from the CaSi cores in CaSi@CaP may help stimulate bone tissue regeneration and contribute to a higher osteogenic capability than that of the pure CaP microsphere scaffolds (Figures 6B and 8). More interestingly, a clear distinction between material degradation and osteogenic capability of the bioceramic composites via core−shell design could be confirmed by a variety of characterization approaches. On the one hand, the XRD analysis showed that there is a phase transformation from TCP to TCP/HA during the sintering process. It is known that the BCPs are a family of two-phase ceramics that combine the low solubility of HA with the soluble phase, such as β-TCP. BCPs may be produced physically, by mixing HA and β-TCP, or chemically, by sintering Ca-deficient HA.47 In this study, it was assumed that the presence of Ca-Alg in the CaP@CaSi or CaP@CaSi@CaP green beads possibly induced the abnormal phase transformation of β-TCP during a high-temperature treatment. In this regard, our primary in vitro cell culture analysis on the Tris-immersed microspheres demonstrated that the cytocompatibility of such in situ phase-transformed BCP is acceptable (Figures 3 and4). On the other hand, the biodegradation of CaSi shell increased drastically within the initial 6 weeks after implantation (Figures 5C and 6C), which was predominantly associated with the increase in pore size in the CaP@CaSi microsphere scaffolds. In contrast, the material residues of the CaSi@CaP microspheres changed slowly at 6 weeks post-

5. CONCLUSIONS In summary, this study systematically evaluated the in vitro biodegradation behavior and osteogenic stem cell responses and in vivo bone regeneration efficacy of bilayer microspheres (CaSi@CaP, CaP@CaSi) and monolayer microspheres (CaP or CaSi). In all of the groups, the materials fused with the host bone but different amounts of osseous callus formed around the microspheres in the early stage. This reflects the different osteostimulation of the spherical biomaterials, where the host osteogenic cells migrated into the porous architecture with different microenvironment responses. The most prominent difference in osteogenic capacity between the CaP@CaSi and CaSi groups appeared in the later stage due to the fast degradation of the CaSi microspheres. In the CaP group, the new bone regeneration rate was the lowest, but the porous shell nature, together with the interconnected porous architecture (∼300−500 μm) of the CaSi@CaP microsphere system, facilitated the high bioactivity and time-dependent pore evolution, thereby opening up void spaces for cellular ingrowth and bone regeneration. The core−shell structural design of the bioceramic composites was an added advantage that could adjust the gradient phase distribution, leading to a faster bone regeneration and an adjustable material degradation. It is believed that, therefore, this straightforward, highly efficient, but miraculous process of manipulating the compositional distribution in biphasic hybrid bioceramics provides a simple and convenient route to manufacture more interesting bioceramics with specific interior architecture and novel functions for new biomaterial technologies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06798. ICP analysis for the ion concentration in Tris buffer and SEM observation of the bioceramic microspheres (PDF) L


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: 86 571 8820 8353. Fax: 86 571 8697 1539 (Z.G.). *E-mail: [email protected]. Tel: 86 577 6586 6003. Fax: 86 577 6586 6586 (L.Z.). *E-mail: [email protected]. Tel: 86 577 6586 6003. Fax: 86 577 6586 6586 (G.Y.). ORCID

Zhongru Gou: 0000-0001-6718-0585 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (51372218, 81601881) and the Zhejiang Provincial Natural Science Foundation of China (LY15H180006, LY15H140004).

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