Osteoinductivity of Porous Biphasic Calcium Phosphate Ceramic

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Biological and Medical Applications of Materials and Interfaces

Osteoinductivity of porous biphasic calcium phosphate ceramic spheres with nanocrystalline and their efficacy in guiding bone regeneration Xiangfeng Li, Tao Song, Xuening Chen, Menglu Wang, Xiao Yang, Yumei Xiao, and Xingdong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18525 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 13, 2019

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ACS Applied Materials & Interfaces

Osteoinductivity of porous biphasic calcium phosphate ceramic spheres with nanocrystalline and their efficacy in guiding bone regeneration Xiangfeng Li, Tao Song, Xuening Chen, Menglu Wang , Xiao Yang, Yumei Xiao*, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China * [email protected] (Y. Xiao) Abstract Conventional biphasic calcium phosphate (BCP) bioceramics are facing many challenges to meet the demands of regenerative medicine, their biological properties are limited to a large extent due to the large grain size in comparison with nanocrystalline of natural bone mineral. Herein, this study aimed to fabricate porous BCP ceramic spheres with nanocrystalline (BCP-N) by combining alginate gelatinizing with microwave hybrid sintering methods, and investigated their in vitro and in vivo combinational osteogenesis potential. For comparison, spherical BCP granules with microcrystalline (BCP-G) and commercially irregular BCP granules (BAM®, BCP-I) were selected as control. The obtained BCP-N with specific nanotopography could well initiate and regulate in vitro biological responds, such as degradation, protein adsorption, bone-like apatite formation, cell behaviors and osteogenic differentiation. In vivo canine intramuscular implantation and rabbit mandible critical-sized bone defect repair further confirmed that nanotopography in BCP-N might be responsible for the stronger osteoinductivity and bone regenerative ability than BCP-G and BCP-I. Collectedly, due to nanotopographic similarities with nature bone apatite, BCP-N has excellent efficacy in guiding bone regeneration, and hold great potential to become a potential alternative to standard bone grafts in bone defect filling applications. Keywords: Calcium phosphate ceramic spheres; Nanocrystalline; Biological effect; Osteoinductivity; Bone regeneration 1.

Introduction The development of regenerative medicine give rise to a revolution in the restoration and

replacement of damaged tissue1-2, a promising frontier is to endow biomaterials with the biological functions to induce host tissue regeneration3-5. Due to good biocompatibility, 1

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osteoconductivity and osteoinductivity, calcium phosphate (Ca-P) ceramics are extensively recognized as excellent bone grafts6-9. In the light of the osteoinductive Ca-P ceramics, more and more researchers come to realize that it’s possible to induce tissue regeneration by optimizing the material characteristics themselves rather than by adding cells and/or bioactive factors3, 10-11. However, present Ca-P ceramics still face many challenges to meet the demands of regenerative medicine, such as, the relatively low bioactivity as compared to nature bones, the unsuitable degradation kinetics to match new bone formation, the poor machining performance to process into specific shapes, and so on7, 12. Based on the point of regenerative medicine, an ideal implant should serve as a temporary scaffold to facilitate tissue regeneration, which should degrade gradually during the damaged tissue remodeling process and be replaced by the new forming tissue ultimately 2, 13.

For bone regeneration, the degradation rate of the bone grafts should match the growth of

new bone, that is important to increase the bone defect healing rate and reduce complications3, 12.

However, the degradation rate of present Ca-P ceramics is relatively slow. Generally, the

degradability of Ca-P ceramics, especially for biphasic calcium phosphate (BCP) ceramics, could be chemically controlled by adjusting the phase ratio of HA/β-TCP14-16, but the range of adjustment is limited. Recent researches have showed that the decrease of grain size could accelerate the degradation rate of Ca-P ceramics evidently7, 17. Moreover, in order to further enhance the bioactivity of present Ca-P ceramics, a possible approach is to mimic nature bone. Despite mimicking in part the porous structure and bony composition of spongy bone, the present Ca-P ceramics still have relatively large grain size as compared to the nanocrystalline of bone mineral, which is likely to debase their biological properties. It can be speculated that when the Ca-P ceramic grain size reduces to nanoscale, that is similar to nanocrystalline of nature bone apatite, their biological properties are expected to be improved evidently7, 18. Many in vitro findings have demonstrated that osteogenic protein and cells are inclined to interact with biomaterials possessing nanoscale surface, and the nanotopography of Ca-P materials could promote bone-related cells adhesion, spreading, proliferation, and differentiation19-23. Arnold M et al. have certified that small components of cells are mainly influenced by nanoscale substrate rather than microscale, due to cell adhering sites are within a range of 5~200 nm24. However, previous studies have mainly focused on the beneficial 2


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effects of Ca-P nanomaterials on their in vitro biological properties, including protein adsorption and cell behaviors25-28. To the best of our knowledge, limited attention has been paid to the efficacy of nanocrystalline in affecting the in vivo biological properties of Ca-P ceramics, such as osteoinductivity and bone regeneration ability (especially the ability of repairing large bone defects), which will prevent us from assessing its potential clinical performances. In clinical applications, Ca-P granules are generally utilized to fill irregular bone cavity defects causing by trauma, tumor, osteoporosis and so on7, 29. Generally, spherical granules are more suitable for implanting the irregular sites than irregular granules, but most of the existing commercial Ca-P granules in clinic (i.g., BAM®, Algipore®, Endobone®) present irregular or cubic shapes29-32. Although several previous studies have successfully fabricated Ca-P spherical granules via centrifugal granulation technology33-34, the porosity of the obtained granules was quite low. It is generally recognized that the interconnected pore structure plays an vital role in determining the bioactivity of Ca-P ceramics31, 35. Our previous studies have employed gas foaming and alginate gelatinizing methods to fabricate porous spherical Ca-P ceramic granules conveniently30-31. The addition of alginate could decrease the Ca-P ceramic grain size to some extent, but the grain size is still on submicro scale10, 30. Nowadays, progress in nanotechnology allows the fabrication of Ca-P nanoceramics. Microwave sintering is one of the most attractive techniques for fabricating Ca-P nanoceramics, due to its high efficient, rapid, and low energy cost23, 36-37. But the dielectric absorption of Ca-P materials for microwave is quite low, which lead to the relatively low heating efficiency7, 38. Many studies have suggested to introduce radiant heating in microwave heating, thereby the low hearing efficiency and thermal gradients can be overcome substantially3, 31, 39. Our previous work selected active carbon as radiant material to process BCP nanoceramics by microwave sintering method23, 40, but the heating efficiency remained not high, and the addition of active carbon had some influences on the phase composition of the ceramic body23. Therefore, it is urgent to develop a suitable microwave sintering method to fabricate Ca-P porous nanoceramics. On the basic of the above descriptions, the study herein attempt to develop a novel strategy to fabricate porous BCP ceramic spheres with nanocrystalline by combining the 3



alginate gelatinizing technology with a novel microwave hybrid sintering method (Scheme 1). The physicochemical properties of BCP granules as well as the in vitro biological responds (i.e. Tris-HCl buffer degradation, protein adsorption, bone-like apatite formation and cellular responses) within the nanotopography of BCP granules were characterized. Subsequently, the in-vivo canine intramuscular implantation experiment was also performed to evaluate the osteoinductivity of the obtained BCP spheres, and their ability of inducing bone regeneration was assessed by means of a mandible critical-size defect model in rabbits. 2.

Materials and methods 2.1 Fabrication and characterization of BCP granules BCP powder (HA/β-TCP=40/60) was prepared by a liquid-phase precipitation method as

described before23, 41. First of all, 10 g BCP powder was dispersed uniformly in 25 mL sodium alginate solution (6 wt%) by a homogenizer, and the slurry was foamed via H2O2 foaming method. Subsequently, the bubbly slurry was dripped and gelled in CaCl2 solution with the concentration of 0.45 mol L-1, wherein hydrogel spheres were formed instantaneously, and the spheres were then hardened in CaCl2 solution, washed, and dried. The dried spheres were pre-sintered at 750 oC for 2 h to decompose most organic residue, and then sintered at 1050 oC

for 6 min via a novel microwave hybrid sintering method (heating rate of 150 oC min-1) to

obtain BCP ceramic spheres with nanocrystalline (denoted as BCP-N). The sketch map of fabricating BCP-N was shown in Scheme 1. A novel microwave workstation (Tangshan Nayuan Microwave co. LTD, China) was employed in this study, there is a microwave-adsorbed layer in the heating chamber to enhance the heating efficiency of ceramics without the help of radiant materials. The operating parameters (i.e. heating rate, sintering temperature, sintering duration, and so on) of the microwave hybrid sintering program was optimized via a number of trial runs. For comparison, BCP ceramic spheres with microcrystalline sintered by conventional muffle sintering method (1050 oC for 2 h, heating rate of 5 oC min-1) were also fabricated(denoted as BCP-G). In addition, commercial BCP irregular granules (BAM®, National Engineering Research Center for Biomaterials of Sichuan University, China) were also chosen as control, which were fabricated by crushing BCP porous bioceramic blocks into irregular granules (denoted as BCP-I). Scanning electron microscopy (SEM; JSE-5900LV, Japan) was used to observe the 4


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morphology of three kinds of BCP granules. The phase composition was certified by X-ray diffractometry (XRD; Philips X’Pert 1 X-ray diffractometer, Netherlands) with CuKα radiation at voltage of 30 kV and current of 20 mA. The specific surface area (SSA) of BCP granules were tested by a surface area analyzer (Gemini VII 2390t, Micromeritics), and their porosities and pore size distributions were characterized by a mercury porosimetry (AutoPore IV 9500, Micromeritics). Moreover, the concentrations of Ca2+ and PO43- were measured by an inductively coupled plasma optimal emission spectrometer (ICP-OES, SPECTRO ARCOS). 2.2 In-vitro biological performances 2.2.1 Bone-like apatite formation Bone-like apatite forming ability of the three kinds of BCP granules was carried out by immersing them in the simulated body fluid (SBF) for 3 days at 37 oC. SBF was prepared according to previous literatures42-43. After immersing, the bone-like apatite formation on their surfaces were visualized by SEM. 2.2.2 Protein adsorption Protein adsorption abilities of BCP granules were carried out with the protein concentrations of 10 mg mL-1, and the detailed process was similar to our previous work31, 44. In short, the samples were socked in the protein solution with the concentration of 10 mg mL-1 at 37 °C for 4 h. Then the adsorbed protein was extracted by SDS solution (1%) and quantified by a BCATM Protein Assay Kit (Pierce, USA). The test was carried out with triplicate samples and each sample was tested three times. 2.2.3 Tris-HCl degradation The samples were also immersed in Tris-HCl buffer solution (solid-liquid ratio is 1:100) for 120 days to evaluate their degradation abilities. The procedure was carried out in a thermostatic oscillator (37 oC, 2 Hz). During the first seven days (day 0.5, 1, 3, 5 and 7), a certain amount of immersed solution (0.5 mL) was taken out to measure Ca2+ and PO43- ions concentrations, and equivoluminal fresh Tris-HCl buffer solution was replenished. At each determining point (day 7, 14, 28, 42, 56, 70, 90, 120), the samples were taken out, dried to constant weight and weighted, then putted back in fresh Tris-HCl solution. 2.2.4 Cell spreading and proliferation 5



The purified mouse bone marrow-derived mesenchymal stem cells (BMSCs) were purchased from Cyagen Co., Ltd., (Guangzhou, China). Following the similar procedures reported previously31, BMSCs were trypsinized and seeded on the sterilized BCP granules (G-ray irradiation sterilization) in 24-well cell culture plate (Corning, USA). Cell density was 2×104 cells per well. At each specified point (day 1, 3, 5), The survival status of BMSCs attached on BCP granules were stained with FDA/PI and examined by a confocal laser scanning microscopy (CLSM, TCS SP5, Leica). Besides, cell proliferation was further quantified by MTT assay14. Moreover, the attached BMSCs were also observed by SEM, which dehydrated through gradient ethanol solution and dried by a critical-point drier (HCP-2, Hitachi, Japan). 2.2.5 Osteogenic gene expression Gene expressions of osteogenesis-related markers (ALP, OSX, OPN, BSP) were measured using qRT-PCR method, and primer sequences were listed in supplementary Table S1. BCP granules were placed into the ultralow attachment 24-well plates (Corning, USA), and cell density was 2×105 cells per well. At each time point (day 3, 7, 14), total RNA of BMSCs grown on the samples was extracted using Rneasy Mini Kit (Qiagen, Germany) and transcribed into complementary DNA (cDNA) using iScript cDNA Synthesis Kit (Bio-Rad, USA). The CFX96TM real-time PCR detection system (CFX960, Bio-Rad, USA) with SoFastTM EvaGreen® Supremix (Bio-Rad, USA) were used to perform q-PCR. The relative expression value for each interested gene was calculated using the Ct-value method, and GAPDH was selected as the housekeeping gene to normalize the expression levels of the target genes. 2.2.6 Quantification of ALP and OCN protein ALP activity and OCN production of BMSCs were also measured. At each determining point, the samples were washed with PBS, and lysed by Pierce® RIPA Lysis and Extraction Buffer (Themofisher Scientific, USA). ALP activity of BMSCs cultured on each group was tested colorimetrically by Alkaline Phosphatase Assay Kit (Beyotime, China) following the manufacturer’s instruction. The results were normalized to the total protein content, which was measured by Pierce® BCA protein assay kit (Themofisher Scientific, USA). To quantify the OCN production of BMSCs cultured on samples, a Enzyme-linked Immunosorbent Assay 6


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(ELISA) Kit for OCN (Cloud-clone Corp, USA) was performed according to the specifications, and the results were also normalized to the total protein content. 2.3 Animal experiment The animal experiment was approved by the Institutional Animal Care and Use Committee of Sichuan University. The animals were supplied from Sichuan Province Medical Experimental Animal Center of China. The surgical procedure was carried out under general sterile conditions. The animals were anesthetized with pentobarbital sodium (1 mg Kg-1). After surgery, each animal was received gentamicin (20,000 U Kg-1) daily by intramuscular injection for three consecutive days. At certain time points, animals were sacrificed by lethal intravenous injection of sodium pentobarbital. The retrieving tissues were immediately fixed in 4% neutral buffed formalin for 7 days before further tissue analysis. 2.3.1 Canine intramuscular implantation The intramuscular implantation was performed on four healthy adult dogs (10~15 kg). After shaving and disinfecting the skin, the longitudinal skin incision (5 mm) was made along the line of spine, and six incisions were made on each side. Each sample was implanted in each pouch separately with >10 mm distance from neighboring ones. Subsequently, the muscle and skin of the dogs were sutured in layers. At specific time points (day 45 and 90), dogs were killed and the samples with their surrounding tissue were retrieved for tissue processing. 2.3.2 Rabbit mandible critical-sized defect repair A total of thirty-six rabbits (New Zealand White, about 2.5 Kg of weight) were used for the mandible defect implantation. Rabbits were divided randomly into four groups: BCP-G group; BCP-N group; BCP-I group and untreated group. Five parallel defects were performed for each BCP group and three parallel defects were performed for the untreated group at each time point. The bone of jaw angle was exposed followed the procedure, shaving the skin, disinfecting the surgical site, making a parallel skin incision, elevating the masseter muscle and detaching the periosteum. The circular defects with the diameter of 8 mm in the region anterior to the jaw angles were created by a slowly rotating trephine burr, and then implanted with materials. The defect was treated sham surgery as the untreated group. Subsequently, the muscle and skin were sutured in layers. At specific time points (day 45 and 90), rabbits were 7



sacrificed and the total left mandible were retrieved for tissue processing. 2.3.3 Micro-computed tomography (μ-CT) measurement μ-CT 80 (Scanco Medical AG, Switzerland) at a voltage of 70 kV and current of 114 μA was used for scanning, and the spatial resolution was set to 22 μm. The obtained radiographic images were reconstructed and analyzed referring to the previous works’ methods45-46. For canine intramuscular implanting samples, the percentage changes of material density, porosity, mean wall thickness, mean pore radius before and after implantation were analyzed. In the rabbit mandible critical-size defect repairing experiment, bone volume fraction (BV/TV) at 45 and 90 days post implantation were assessed. All the analysis were performed by an expert (see the acknowledgement) who was blind to material groups. 2.3.4 Histological examination The implants harvested from the dorsal muscle of dogs were performed by soft-tissue slice method. The samples were firstly fixed in 4% paraformaldehyde and embedded in paraffin after decalcification by EDTA. Then they were cut into 3~5 μm sections and stained with hematoxylin and eosin (H&E). The mandibles containing implants harvested from rabbits were performed by hard-tissue slice method. The tissue was dehydrated by gradient alcohol and embedded in PMMA. The sections were cut and polished to 10~30 μm by a microtome (EXAKT 300, Germany) and stained with toluidine blue (TB). The stained sections were scanned using a digital scanner (BA600, MOTIC CHINA GROUP CO., LTD.) to obtain an overview, and representative images were used for histomorphometrical analysis by Image-Pro Plus (IPP, Media Cybernetic, USA) 6.0 software. 2.3.5 Immunohistochemistry (IHC) IHC staining was performed to detect the protein expression of BMP-2 and OCN for specimen obtained from canine intramuscular implantation, and a streptavidin-biotin complex (SABC) method was employed. Briefly, following a series of pretreatments as mentioned before44,

47,

the sections were exposed to each diluted primary antibody against BMP-2

(BeacomBio, UK) or OCN (BeacomBio, UK) at 4 oC overnight. And then the sections was incubated with the biotinlabeled secondary antibody (BeacomBio, UK) at 37 oC for 30 min and SABC for 30 min. Finally, they were stained by DAB reagent kit (DAKO, Germany) for 10 min and slightly counterstained with hematoxylin for the nucleus. A light microscope 8


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(Olympus Bx60) was employed to observe the positive expression of BMP-2 and OCN (brown staining) inside the implants. And the quantitative analysis was measured by IPP 6.0 software, and the mean optical density (MOD) was used as the evaluation index. 2.4 Statistical analysis Group data were expressed as mean ± SD (standard deviation). One-way analysis of variance (ANOVA) by SPSS 11.0 software (SPSS Inc, USA) was used to perform statistical analysis, and statistical significance was assumed at having p < 0.05. 3.

Results 3.1 Material characterization 3.1.1 SEM observation BCP-G and BCP-N (shown in Fig. 1a-b) had well spherical shape with diameter of about

3 mm, while, BCP-I (Fig. 1c) had randomly irregular shape with size of 2~4 mm. All of them had the interconnected pore structure, but the pore sizes of BCP-G and BCP-N were smaller than that of BCP-I, partly due to the relatively large shrinkage of BCP-G and BCP-N during the drying and sintering process, which could be observed in Scheme 1. The grain sizes of three kinds of BCP granules were different (Fig. 1d-f), BCP-N owned nanocystalline with the average grain size of 105.4±22.17 nm (Fig. 1e), while both BCP-G (383.6±89.13 nm, Fig. 1d) and BCP-I (824.8±112.82 nm, Fig. 1f) had microcrystalline. 3.1.2 XRD patterns XRD patterns of BCP-G, BCP-N and BCP-I were compared in Fig. 1g, all of them were composed of β-TCP phase and HA phase from determining their diffraction peaks. By calculating their phase ratios, it could be observed that HA phase ratios in BCP-G (81.8%, listed in Table 1) and BCP-N (80.3%) were similar, which were much higher than that in BCP-I (39.1%), indicating that the addition of alginate salt could increase the HA phase ratio of BCP ceramics. 3.1.3 Pore structure and specific surface area (SSA) The pore size distribution and pore quantity distribution BCP-G, BCP-N and BCP-I were presented in Fig. 1h and Fig. 1i, respectively. As shown in Fig. 1h, BCP-G and BCP-N possessed similar pore size distribution of macro-pores (200~800 μm) and minor-pores (10~100 μm), which were smaller than those of BCP-I. But the amount of micro-pores 9



ranging of 50~500 nm in BCP-N was significantly more than BCP-G and BCP-I (Fig. 1i). Besides, three kinds of BCP granules had relatively high porosity (>70%, listed in Table 1), but the micro-porosities (< 10 μm) of BCP-N (24.32%) took significantly advantages on BCP-G (19.22%) and BCP-I (11.24%). Moreover, BCP-N with nanocrystalline had relatively higher specific surface area (5.625 m2 g-1, listed in Table 1) than BCP-G (2.730 m2 g-1) and BCP-I (0.556 m2 g-1). 3.2 In-vitro biological performances 3.2.1 Bone-like apatite formation and protein adsorption The surface morphology of BCP-G, BCP-N and BCP-I after immersing in SBF for 3 days was shown in Fig. 2a. Al the three kinds of BCP granules could facilitate bone-like apatite deposition on their surfaces after immersing. It was found that many reticulated crystals grew on the surface of BCP-G and BCP-N, the surface of BCP-N was totally covered by crystals, while, partial region of BCP-G surface was still uncoated. For BCP-I, although some crystals deposited on its surface, most areas were not covered. The bone-like apatite formation ability could be ordered as BCP-N > BCP-G >BCP-I. The results of serum protein adsorption (Fig. 2b) showed that BCP-N with nanocrystalline could adsorb the most protein (3.245 ± 0.037 mg g-1), followed by BCP-G (2.867 ± 0.196 mg g-1), and BCP-I adsorbed the least protein (2.331 ± 0.281 mg g-1). 3.2.2 Tris-HCl degradation Tris-HCl buffer solution (pH=7.4) was employed to evaluate the degradation abilities of the three kinds of BCP granules. Fig. 2c showed Ca2+ and PO43- concentrations of Tris-HCl solution immersed by BCP-G, BCP-N and BCP-C for different durations. It was found that c(Ca2+) and c(PO43-) of the three groups increased as the degradation process progressed, and c(Ca2+) and c(PO43-) releasing from BCP-N were significantly higher than BCP-G and BCP-I. Moreover, c(Ca2+) releasing from BCP-G was lower than BCP-I, but c(PO43-) releasing from the two groups was similar. Fig. 2d showed weight loss of samples during the 120-day degradation process, all the three groups exhibited a sustained weight loss in the degradation process. At the end of degradation (120 d), The weight loss of BCP-N was the highest (10.606 ± 0.422%), followed by BCP-G (9.571 ± 1.117%), and BCP-I had the lowest weight loss (8.241 ± 0.507%) . 10


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3.2.3 Cell spreading and proliferation BMSCs cultured on BCP-G, BCP-N and BCP-I all spread well with few dead cells from CLSM observations. The initial cell attachment on the surface of three BCP granules were observed at day 1, showing that most cells exhibited spherical morphology and some cells began spreading. At day 3, BMSCs spread well with elongated-spindle morphology and began to migrate into the pores of BCP granules. As time went on, the cells grew gradually and formed extensive cell-cell interactions at day 5. From the MTT results (Fig. 3c), there was no significant difference among three kinds of BCP granules in promoting BMSC proliferation, which was consistent with CLSM observations. SEM images further confirmed that cells attached and spread well on BCP granules after two days culturing (Fig. 3b), as the BMSCs exhibited a typical spindle-like morphology, and abundant filopodia were outstretched to tightly grasp the crystal grains. BCP-G and BCP-N seemed more likely to promote cell spreading at early stage, due to BMSCs attached on BCP-G and BCP-N had larger spreading area (calculating from the above SEM images) than BCP-I (Fig. 3d). 3.2.4 Osteogenic gene expression Osteogenic genes expressions of ALP, OSX, OCN and BSP in BMSCs culturing on three kinds of BCP granules for 3, 7 and 14 days was detected by qRT-PCR analysis. As shown in Fig. 4a, ALP expression was up-regulated by BCP-N, followed by BCP-G, and BMSCs on BCP-I owned the lowest ALP expression among the three samples at day 3 and day 14, but there was no significant difference between BCP-G and BCP-N at day 14. BMSCs on BCP-G and BCP-N owned higher expression of OSX gene than BCP-I, and BCP-N exhibited the highest expression at day 3. OCN expressions were also up-regulated by BCP-G and BCP-N comparing with BCP-I, and BCP-N exhibited the highest expression at day 3 and day 14. Moreover, BCP-G and BCP-N also performed better regarding of BSP expression than BCP-I, and BCP-G exhibited the highest expression at day 7. Overall, the BCP-G and BCP-N spheres could up-regulate the osteogenic gene expressions of BMSCs comparing with the irregular BCP-I, BCP-N with nanocrystalline exhibited the strongest ability of the osteogenic gene expression among the three kinds of BCP granules. 3.2.5 ALP activity and OCN production Fig. 4b showed the ALP activity and OCN production in BMSCs cultured on BCP-G, 11



BCP-N and BCP-I. BCP-G exhibited relatively low ALP activity as comparing to BCP-N and BCP-I at day 3. But at day 7, BMSCs cultured on BCP-G exhibited higher ALP activity than BCP-I, and BCP-N with nanocrystalline owed the highest ALP activity. Moreover, the amount of OCN produced by BMSCs cultured on BCP-N could be also dramatically promoted as compared with BCP-G and BCP-I at day 7 and day 14, while, there was no significant difference between BCP-G and BCP-I . 3.3 In vivo osteoinductivity 3.3.1 μ-CT measurement μ-CT rendered 3D visualizations and the quantitative micro-architectural changes of BCP-G, BCP-N and BCP-I before and after canine intramuscular implantation were shown in Fig. 5. After 90-day implantation (Fig. 5a), material degradation occurred in all the three kinds of BCP granules, their outlines were partly disintegrated and some small particles were degraded from BCP granules. Fig 5b showed the micro-architectural changes of three kinds of BCP granules after 90-day implantation. It was found that the density and mean wall thickness of all the three kinds of BCP granules were dramatically increased after implantation. And the increment in BCP-G and BCP-N were significantly higher than that in BCP-I, and the increase of mean wall thickness in BCP-N was the highest among the three BCP granules. On the contrary, the porosity of the BCP granules decreased after implantation, BCP-N had the lowest porosity among the three BCP granules. It was worth noted that the mean pore radius of BCP-G and BCP-N decreased after implantation, while the one of BCP-I increased, BCP-I had the highest mean pore radius of among the three BCP granules. 3.3.2 Histological analysis Fig. 6 showed histological analysis of BCP-G, BCP-N and BCP-I after canine intramuscular implantation for 90 days. H&E staining demonstrated that obvious ectopic bone formation could be occurred in all the three kinds of BCP granules after implantations, accompanied with many osteoblasts and blood vessels (Fig. 6a). Among the eight specimens, ectopic bone formation was detected in five samples in BCP-I group, six samples in BCP-G group, and seven samples in BCP-N group at day 45 (Fig. 6b). Quantitatively, the new bone forming in BCP-N was most (23.60 ± 1.06%), which was followed by BCP-G group (16.04 ± 0.65%), BCP-I group had lest new bone formation (6.48 ± 0.60%) among the three groups 12


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(Fig. 6b). At day 90, ectopic bone formation was detected in seven out of eight parallel samples in BCP-I group, while, all of BCP-G and BCP-N specimens had new bone formation (Fig. 6b). Similarly, the new bone forming area in the three kinds of BCP granules at day 90 was followed by: BCP-I < BCP-G < BCP-N (Fig. 6b). It could be also observed that new bone in BCP-I seemed relatively independent, which grew along the pore walls and formed “doughnut-like” structure; but the newly formed bone in BCP-G and BCP-N (especially for BCP-N) could integrate and interconnect together to infiltrate the ceramic body (Fig. 6a). IHC staining was used to detect the BMP-2 and OCN expressions of the cells in the implanted samples. As shown in Fig. 7a-b, the positive expressions of BMP-2 and OCN were obviously identified in the cells growth into the pores of all the three kinds of BCP granules at day 45 and 90. From the quantitative analysis (Fig. 7c), it could be found that the cells in BCP-G and BCP-N groups had significantly higher BMP-2 and OCN expressions than thiose in BCP-I at day 45 and 90, and BCP-N owed highest expressions of BMP-2 among the three kinds of BCP granules at day 45. 3.4 In vivo bone regeneration in rabbit mandible defects 3.4.1 μ-CT reconstruction The representative 3D visualizations of repaired mandible critical sized defects reconstructed by μ-CT were exhibited in Fig. 8a. Qualitatively, abundant newly formed bone was observed in all the three kinds of BCP granules at day 45, while, the mandible defect was left with an apparent introcession in the untreated group. At 90 days, the regenerated bone tissue had been integrated into the surrounding normal bone for all the treatment groups, and surface appearances of the healed defects, especially in BCP-G and BCP-N groups, were exactly similar to that of the surrounding host bone tissue. From quantitative analysis of new bone forming in the defect sites (Fig. 8b), the bone volume fraction (BV/TV) followed the order of BCP-N > BCP-G > BCP-I > untreated group at day 45, significantly greater BV/TV was observed in BCP-N, followed by BCP-G and BCP-I at day 90. 3.4.2 Histological analysis The details of bone regeneration in rabbit mandible defects after implantation for 45 and 90 days were shown in Fig. 9, respectively. No inflammation was observed around the wound from the panorama of the mandible defect. After 45-day implantation, the osteogenic 13



process occurred in all the three kinds of BCP granules, and many blood vessels appeared in the defects. Fig. 9a-b showed that BCP-G and BCP-N had been inosculated with the newly formed bone tissue, even many internal pores, were filled with new bone. In contrast, the growth of bone was relatively slow in BCP-I (Fig. 9c), as only little new bone was observed in the areas contacting with host bone. The quantitative analysis of the newly formed bone (Fig. 9g) found that the amount of new bone in BCP-N was the highest (44.29 ± 1.09%), followed by BCP-G (41.12 ± 1.12%), and BCP-I was the lowest (33.31 ± 2.81%). After 90 day-post-surgery, the osteogenic process continued, and all the three kinds of BCP granules presented good osteoconductive property. Most of pores in BCP-G (Fig. 9d) and BCP-N (Fig. 9e) were filled with the new bone and the outline of pores was hardly observed. The growth of bone in BCP-I (Fig. 9f) was still relatively slow, as only the part of pores that contacted host bone tissue were filled with new bone, while, many un-contacted parts were filled with fibrous tissue. The amount of new bone area in three groups (Fig. 9g) were still accorded with the order as BCP-N (62.24 ± 2.95%) > BCP-G (53.34 ± 3.25%) > BCP-I (42.54 ± 2.36%). Moreover, it could be also observed that BCP-N presented the fastest degradation rate, as many parts of substrates were absorbed, which was in accordance with the in-vitro Tris-HCl degradation results (Fig. 2). 4.

Discussion Ca-P bioceramics have been widely used in various orthopaedic treatments for damaged

or diseased bones, and many commercial products have been put into market. The excellent work processed by Yuan HP et al. has demonstrated that Ca-P bioceramics with certain composition and structure are equally valid in bone repair as autologous bone grafts in animal implantation6. To meet the demands of regenerative medicine, further improving the bioactivity of present Ca-P ceramics seems important and urgent. In bionic terms, it can be speculated that when the Ca-P grain size falls into the nanoscale similar to that of bony mineral, their biological performances should be improved evidently. Several in vitro studies have demonstrated that Ca-P ceramics with nanocrystalline could be an effective strategy to enhance their bioactivity25-28, but little is known about their in vivo biological properties. In the present study, we develop a novel strategy to fabricate BCP ceramic spheres with nanocrystalline by combining alginate gelatinizing technology with microwave hybrid 14


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sintering method, and their in vitro and in vivo biological properties, especially their efficacy in guiding bone regeneration, were evaluated. The obtained BCP bioceramic spheres possess well spherical shape, which should be suitable for filling the bone cavity defects as comparing to the irregular granules. Compared with the microcrystalline in BCP-G and BCP-I (Fig. 1d, f), BCP-N have nanocrystaline of 105.4±22.17 nm (Fig. 1e). All the three kinds of BCP granules are composed of β-TCP phase and HA phase, but HA phase ratios in BCP-G and BCP-N (about 80%, list in Table 1) are significantly higher than that in BCP-I (39.1%). Our previous studies have certified that the addition of calcium alginate could stabilize Ca-deficient hydroxyapatite (CDHA, its diffraction peaks are similar to HA phase) in BCP ceramics, and the abundant CDHA phase further enhance their biological performances10. Moreover, the decomposition of calcium alginate could also reduce the grain size of BCP ceramics10, 30, and abundant micropores (< 10 μm) could be generated in its decomposition process. It is well known that the interconnected pore structure and abundant micropores play important roles in determining the biological performances of Ca-P ceramics3, 48. Furthermore, BCP-N with nanocrystalline also have the highest microporosity and largest specific surface area (SSA) among the three kinds of BCP granules. The nanocrsytlline, high content of CDHA phase, abundant micropores and large surface area of BCP-N maybe efficient in enhancing its biological performances. All the three kinds of BCP granules have good cytocompatibility, which can well promote BMSCs adhesion, spreading and proliferation. However, BCP-G and BCP-N seemed more beneficial to cell adhesion and spreading than BCP-I at early stage (day 2), they have significantly larger cell area than BCP-I. BCP ceramics with small cystalline have increased grain boundaries on their surface, which favors to cell adhesion and proliferation. Webster T et al have proved that the adhesion of osteoblasts appears at the grain boundaries primarily49. Moreover, when the grain size in BCP ceramics decreases into nanoscale, cells may generate more focal adhesion and filopodia with nano-sized protrusions to grasp nanocrystalline. In the study of Bello DG et al50, they demonstrated that a nanoporous titanium surface could promote the focal adhesion maturation and filopodia formation, thereby trigger cellar cascades that regular cell behavior. Furthermore, bioactive ions (i.e Ca2+ and PO43-) releasing from the degradation of BCP granules (Fig. 2c) can generate a local ionic microenvironment 15



to trigger the osteoprogenitors osteogenic differentiation, which are quite important in the osteogenesis process44, 46. Thereby, BCP-N spheres with nanocrystalline could up-regulate the osteogenic gene expressions of OSX, ALP, OCN and BSP (Fig. 4a) as comparing to BCP-G and BCP-I with microcrystalline. Moreover, protein adsorption is also related to the surface topography of implants, the small grain size, abundant micropores, high specific surface area could provide plentiful active sites and crystal defects on the surface of biomaterials. BCP-N with nanocrystalline could adsorb the most protein, followed by BCP-G, while, BCP-I adsorbed the least (Fig. 2b). Moreover, BCP-N with nanocrystalline also could promote the expression of osteogenic protein (ALP and OCN, Fig. 4a) as compared to BCP-G and BCP-I. The strong adsorption affinity for serum protein may favor cell adhesion and mediate cell surface receptor-integrin expressions, and then induce MSCs osteogenic differentiation by triggering the MAPK signaling cascade51. Generally, the new bone formation in porous scaffolds can cause the increase of density and wall thickness, and the decrease of porosity and pore radius; in contrast, the material degradation and biological resorption process have opposite influences on these parameters. After 90-day implantation, BCP-N had the highest density and mean wall thickness, and the lowest porosity and mean wall radius among the three groups from μ-CT analysis, indicating that new bone formation in BCP-N was more than that in BCP-G and BCP-I. It was consistent with H&E staining (Fig. 6) and immunohistochemical staining (for BMP-2 and OCN) results (Fig. 7), BCP-N with nanocrystalline had the strongest osteoinductivity among three kinds of BCP granules. Moreover, the area of new forming bone in BCP-N was much larger than those in the CaP ceramics fabricated by our previous work7. The results might be attributed to the nanocrystalline and abundant micropores of BCP-N. Study on bioactive glass nanoceramics showed that their enhanced osteointegrative functions were attributed to topographical features52. Furthermore, micropores distributing on the pore walls could increase the surface roughness and showed a propensity to induce new bone formation6. The abundant micropores also increase the connectivity of pore structure in ceramics, the new bone could interconnect together and integrate into a large area in BCP-G and BCP-N, but the new bone was relatively independent in BCP-I, which was consistent with our previous study10. The ideal biomaterials for regenerative medicine need to work as primary scaffolds to 16


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guide tissue regeneration, and then degrade gradually, and finally be replaced by newly formed tissue. In the rabbit mandible critical-sized bone defect repair experiment, there was no obvious gap between the BCP ceramic body and host bone tissue (Fig. 8a), indicating a satisfactory bone growth rate after implantation for 90 days. μ-CT analysis (Fig. 8b) revealed an infiltrated new bone formation pattern in the BCP-N group with a significantly higher bone volume fraction than that of BCP-G and BCP-I groups. Histological staining further certified that the presumed abundant bone tissue in the pores of BCP-N was truly new forming bone. From TB staining, it could be found that the newly formed bone tissue had inosculated with BCP-G and BCP-N, and many inner pores were filled with new bone. Due to the nanostalline of BCP-N was similar to that of natural bone apatite, which could absorb more bone-related proteins and better regulate a cascade of osteogenic gene activities of cells, thereby resulting in stronger osteoinductivity than BCP-G and BCP-I. In the study of Rezaei M.53, they evaluated the effect of nanonization on BCP ceramic in repairing canine mandible cavities, and found that the rate of resorption increased significantly after nanonization, but the bone regeneration of nano-sized BCP materials was comparable with the commercial BCP. That partly might due to that there was no porous architecture in their materials, and the nano-sized BCP materials were not truly nanoceramics. Our recent work also constructed a nanoparticles hybrid-structured surface on BCP ceramics, with respect to the local integration at the nanoscale, the ceramics showed enhanced bone regenerative capability based a long bone defect model of dogs54. Moreover, abundant micropores in BCP-N may also facilitate the nutrients transport and the capillaries growth, thus further accelerating the new bone formation. The study of Chen Y et al. have proved that porous Ca-P ceramics own the angiogenic inducing ability, and the neovascularization plays a critical role in the osteoinductivity of Ca-P ceramics55. Furthermore, BCP-N with nanocrystalline presented the fastest degradation rate among the three groups (Fig. 2d and Fig. 9), the relatively high degradation and local ion micro-environment (Ca2+ and PO43- ions releasing) could be beneficial to the ingrowth of tissues into implants, deposition of bone-like apatite (Fig. 2a) and the osteogenic differentiation of MSCs, thereby resulting the high bone regeneration ability of BCP-N. Therefore, BCP-N holds great potential to serve as a kind of bioactive scaffold or bone defect filling materials in orthopedic applications. 17



Nevertheless, this study has some limitations. Firstly, it would be interesting to investigate the vascularization process of BCP-N with nanocrystalline as compared to BCP-G and BCP-I. There are signs that BCP-N with nanocrystalline implants were beneficial to neovascularization from the light microscopy images. Secondly, the further genome and proteome explaining the advanced bone regeneration ability of BCP-N with nanocrystalline should be carried out. The nice thing here is that the new generation of spherical Ca-P bone filling granules with nanocrystalline disclosed in this study will be under pilot-scale production56. 5. Conclusion As discussed, the present study introduced a new kind of porous BCP ceramic spheres with nanocrystalline (BCP-N). Due to its nanotopography was similar to that of natural bone apatite, BCP-N was more conducive to protein adsorption, bone-like apatite formation and BMSCs osteogenic differentiation than BCP ceramic spheres (BCP-G) and commercial BCP irregular granules (BAM®, BCP-I) with microcrystalline. Further in vivo intramuscular implantation and critical-sized bone defect repair confirmed the superior osteoinductivity and bone regenerative abilities of BCP-N as compared with BCP-G and BCP-I. These findings highlight that BCP ceramics with nanocrystalline can enhance their bioactivity, and a better understanding of its efficacy in guiding bone regeneration will be fundamental for further applications of BCP nanoceramics in bone defect repair. Although further long-term evaluations are required, we believe that the porous BCP ceramic spheres with nanocrystalline fabricated in this research can be potential alternatives to standard osseous grafts in bone defect filling applications. Acknowledgements The authors would like to thank Dr. Li Chen from Analytical &Testing Center Sichuan University for her help with μ-CT. This work was financially supported by the National Key Research and Development Program of China (2016YFB0700804), the National Science Foundation of China (31670984, 51873116), and China Postdoctoral Innovation Talent Support program (BX20180204).

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Tables Table 1 Summary of physicochemical data of BCP-G, BCP-N and BCP-I Samples BCP-G BCP-N BCP-I

Phase ratio Porosity Micro-porosity* SSA (HA/β-TCP) (%) (%) (m2 g-1) 81.8/18.2 80.3/19.7 39.1/60.9

70.92 75.04 77.97

19.22 24.32 11.24

2.730 5.625 0.556

*Volume percentage of micro-pores smaller than 10 μm within the BCP granules.

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Figure captions Scheme 1 The sketch map illustrating the fabrication process of the spherical BCP ceramic spheres with nanocrystalline Fig. 1 SEM images (a, d, BCP-G; b, e, BCP-N; c, f, BCP-I), XRD patterns (g), pore size distributions (h) and pore quantity distributions (i) of three kinds of BCP granules. Fig. 2 Bone-like apatite forming (a), protein adsorption (b), Ca2+ and PO43- ions releasing (c), weight loss (d) of BCP-G, BCP-N and BCP-I after degrading in Tris-HCl for 120 days. Fig. 3 CLSM observations (a), SEM images (b), MTT results (c) and cell spreading area (d) for BMSCs growing on BCP granules (BCP-G, BCP-N and BCP-I). Values are expressed as the mean ± SD (n = 3), * refers to p < 0.05, and ** refers to p < 0.01. Fig. 4 Expression of osteogenic genes - ALP, OSX, OCN and BSP (a), and intracellular ALP activity and OCN production (b) in BMSCs cultured on BCP-G, BCP-N and BCP-I. Values are expressed as the mean ± SD (n = 3), * refers to p < 0.05, and ** refers to p < 0.01. Fig. 5 Images and parameters rendered by μ-CT: (a) 3D visualizations of BCP-G, BCP-N and BCP-I before and after implantation; (b) percentage changes of density, porosity, mean wall thickness and mean pore radius in the three kinds of samples after implantation for 90 days. Values are expressed as the mean ± SD (n = 3), * refers to p < 0.05, and ** refers to p < 0.01. Fig. 6 Histological analysis of bone formation after intramuscular implantation in dogs for 45 and 90 days: (a) H&E staining (M = material, Ft = fibrous tissue, V=new capillary vessel, B = new bone); (b) quantitative analysis of bone incident, new bone area. Values are expressed as the mean ± SD (n = 8), * refers to p < 0.05, and ** refers to p < 0.01. Fig. 7 Immunohistochemical staining of BMP-2 (a) and OCN (b), and quantitative analysis (c) in the decalcified sections of BCP-G, BCP-N and BCP-I after canine intramuscular implantation for 45 and 90 days. Values are expressed as the mean ± SD (n = 8), * refers to p < 0.05, and ** refers to p < 0.01. Fig. 8 Images and parameters of rabbit mandibular bone defects rendered by μ-CT: (a) 3D visualization of rabbit mandibular bone defects at 45 and 90 days post implantation; (b) quantitative comparison of the changes in bone volume/total volume ratio (BV/TV). Values are expressed as the mean ± SD (n = 3), * refers to p < 0.05, and ** refers to p < 0.01. Fig. 9 Histological analysis of BCP-G, BCP-N and BCP-I after implanting into the rabbit 24


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mandible defects for 45 days and 90 days. (a-f) TB staining (B = new bone, M = material residue); (g) quantitative analysis of the new bone area forming in defects. Values are expressed as the mean ± SD (n = 5), * refers to p < 0.05, and ** refers to p < 0.01.

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Table of Contents (TOC)

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Scheme 1

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

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Fig. 9

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Osteoinductivity of Porous Biphasic Calcium Phosphate Ceramic

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