Rational Design and Fabrication of Porous Calcium–Magnesium

Article pubs.acs.org/journal/abseba

Rational Design and Fabrication of Porous Calcium−Magnesium Silicate Constructs That Enhance Angiogenesis and Improve Orbital Implantation Dongshuang He,† Chen Zhuang,† Cong Chen,‡ Sanzhong Xu,*,§ Xianyan Yang,† Chunlei Yao,∥ Juan Ye,∥ Changyou Gao,† and Zhongru Gou*,† †

Bio-Nanomaterials and Regenerative Medicine Research Division, ZhejiangCalifornia International Nanosystem Institute, Zhejiang University, Hangzhou 310058, China ‡ College of Material Science and Engineering, Zhejiang University, Hangzhou 310027, China § Department of Orthopaedic Surgery, The First Affiliated Hospital, School of Medicine of Zhejiang University, Hangzhou 310003, China ∥ Department of Ophthalmology, The Second Affiliated Hospital, College of Medicine of Zhejiang University, Hangzhou 310009, China ABSTRACT: Tissue integration of orbital implants, following orbital enucleation treatment, represents a challenge for rapid fibrovascularization, long-time stability, anti-infection, and even induction of vascule regeneration. The objective of this study was to develop porous calcium−magnesium silicate materials, with good stability, bioactivity, and antibacterial potential as new orbital fillers. Three-dimensional (3D) diopside scaffolds (low dissolvability) were fabricated by direct ceramic ink writing assembly and then followed by one-step sintering at 1150 °C for 3 h. The pore wall of the scaffold was modified by another calcium−magnesium silicate, such as bredigite or akermanite, which dissolves quickly but shows greater angiogenic potential. These two Ca−Mg-silicates can be coated onto the pore strut, and the coating layers were observed to slowly dissolve in Tris buffer. The vascularization-favorable Cu ions, which had been doped into the bredigite or akermanite coating, could also be measured in the immersion medium. A primary angiogenic test in a panniculus carnosus muscle model in rabbit indicated that the Cu-doped bredigite and akermanite coatings were significantly beneficial for the neovascularization in the early stages. These results suggest that the diopside-based porous materials modified with functional coatings hold great potential for application in orbital reconstruction. KEYWORDS: angiogenesis, calcium−magnesium-silicate, surface modification, copper doping, porous bioceramics, 3D robocasting release behavior in vivo.4,5 On the other hand, synthetic implant materials that can provide permanent integration and the ability to stimulate the fibrovascularization are of special significance for orbital reconstruction.6 A number of materials have historically been used for orbital filling repair, such as coralderived hydroxyapatite (HA), porous alumina, and polyethylene (Medpor). However, these materials are too inert, or may cause severe inflammatory reactions and secondary exposure if they result in delayed vascularization. However, previous studies and applications demonstrated that the bioinert nature of porous alumina, low bioactivity of porous HA and polymer orbital, and difficulties in pores enlargement of coral-derived HA constructs have raised some concerns and

1. INTRODUCTION Artificial orbital implantation is the most commonly used method to treat orbital enucleation and facial cosmesis. The success of such treatment critically depends upon, among other factors, achieving rapid angiogenesis and full integration of the embedded (porous) constructs. Conversely, inadequate integration will result in the risk of bacteria-laden bodily fluids penetrating into the orbital root, leading to chronic inflammation and secondary infection. As a consequence, orbital disease is one of the most important concerns for ophthalmologist, patients, and the public healthcare system.1,2 It is known that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) can be integrated into biomaterial matrixes to facilitate angiogenesis.3 However, this approach is restricted because of the high cost, complex techniques, short half-lives of angiogenic growth factors, and the potential safety problems caused by its uncontrollable © XXXX American Chemical Society

Received: May 24, 2016 Accepted: August 17, 2016

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DOI: 10.1021/acsbiomaterials.6b00282 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering resulted in limited use in many surgical situations.7−9 Thus, the ideal biomaterials for orbital implants should have the capability to induce rapid and adequate angiogenesis as well as very limited degradation. In this context, the desired features of an orbital filler are (1) a strong, nonbrittle construct that is easy to insert; (2) uniform interconnected pores and excellent bioactivity to promote the rapid and complete fibrovascularization; (3) good integration of materials and orbital tissues to facilitate sealing the orbital bed; and (4) slow biodegradation but good antibacterial properties to inhibit bacteria growth. Ca-silicates, as a group of recently introduced biomaterials, are considered promising biomaterials for hard tissue repair.10 It is known that wollastonite bioceramics can not only stimulate osteogenic cell differentiation, but also enhance angiogenesis of endothelial cells (ECs).11,12 Diba et al. have systematically reviewed the biological performance of Mg-containing Casilicate bioceramics such as diopside (CaMgSi2O6), bredigite (Ca7MgSi4O16), and akermanite (Ca2MgSi2O7).13 While previous studies have confirmed that diopside is bioactive and very stable in vivo,14,15 the later two bioceramics have the capability to guide the osteogenic differentiation of mesenchymal stem cells and to stimulate angiogenesis of ECs due to their appreciable biodegradation.16−19 Moreover, some biologically essential trace elements, copper in particular, are reported to suppress a range of bacterial pathogens (antibacterial activity).20,21 Meanwhile, it has been revealed that the Cu2+ ion is able to induce migration and proliferation of ECs during in vitro culture.22 Therefore, Cu-doped bioceramics have been developed to combine these beneficial biological properties, such as good antibacterial activity, and stimulate vascularization for improving bone, teeth, and even anophthalmic socket reconstruction.23,24 It is reasonable to assume that diopside is a good choice for the porous substrate of orbital implants, while the other Ca−Mg-silicates may be employed as multifunctional Cu-doped compositions, within the pore walls, to enhance the biological performance. The scaffold is an integral part of biological tissue engineering. Three-dimensional (3D) robocasting is an advanced additive manufacturing technique that facilitates scaling up complex constructs with periodic macropores and adjustable geometrical parameters.25 A fully interconnected macroporous structure can mimic the external cell matrix (ECM) properties, and provide a template for cell attachment and stimulate blood vessel formation in vivo.26 The pore parameters affect not only the size of the blood vessels growing into the porous structure but also the number of blood vessels growing into the macropores of bioceramic scaffolds. Furthermore, the size of the interconnections is more important for angiogenesis within the scaffold, rather than the pore size. The upper limit for pore size to facilitate vascularization is 400 μm.27 Herein, we demonstrate a diopside-based porous orbital that is endowed with fully interconnected macropores, adequate open porosity (>52%) and pore size (∼350 μm), and highbioactivity coatings that favor rapid angiogenesis. The diopside scaffolds were fabricated by ceramic ink writing assembly, with 0.5% copper-doped bredigite or akermanite paste coating posttreatment. Thus, the functionalized bioceramic scaffolds exhibited excellent bioactivity resulting in improved angiogenesis and antibacterial properties in vivo. It is reasonable to consider that such new bioactive porous orbital scaffolds, independent of conventional growth factors, are a promising treatment to enhance orbital integration.

2. MATERIALS AND METHODS 2.1. Chemicals and Materials. The reagent-grade Ca(NO3)2· 4H2O, Mg(NO3)2·6H2O, Na2SiO3·9H2O, NaCl, KCl, NaHCO3, MgCl2·6H2O, CaCl2, KH2PO4, trishydroxymethylaminomethane (Tris), and tetraethyl orthosilicate (TEOS) were bought from Sinopharm Reagent Co., Shanghai, and used without further purification. Poly(vinyl alcohol) (PVA) was purchased from SigmaAldrich. Tris was used to prepare to the 0.05 M Tris buffer (pH ∼ 7.25). 2.2. Synthesis of Ca−Mg Silicate Bioceramic Powders. The AKE-5Cu powders containing ∼4.54% Cu (i.e., 5 mol % Ca was substituted by Cu in akermanite) were synthesized by a conventional wet-chemical coprecipitation method. The BRE-5Cu powders containing ∼4.68% Cu (i.e., 5 mol % Ca was substituted by Cu in bredigite) were also synthesized by a similar coprecipitation method. The as-calcined bioceramic powders were ground using zirconia ball media in ethanol for 6 h. The particle size of the resulting powders was below 5 μm. The pure diopside, akermanite, and bredigite powders were synthesized in the absence of Cu2+ ions while the other conditions remained the same. 2.3. Three-Dimensional Robocasting Diopside Bioceramic Scaffolds. The diopside slurry for layer-by-layer printing of the scaffolds was prepared by mixing 4.5 g of diopside powders with 4.0 g of 10 wt % PVA solution. Then, the diopside scaffolds (8 mm × 8 mm × 4 mm) were prepared using 3D writing equipment (homemade precision three-axis positioning system and extruding device driven by the step motor, which is mounted on the X-axis). The diopside scaffolds using initial distance between green filaments were ∼420 μm. The moving speed of the dispensing unit was set to 6 mm/s, and the nozzle diameter was 400 μm. In order to make sure that the side-pore size of scaffolds was big enough, the two-wire writing was carried out for the bioceramic scaffolds. Then, the samples were dried at 80 °C overnight to remove excess water in the pore struts, followed by sintering in a microcontroller controlled temperature furnace at a target temperature of 1150 °C in air atmosphere using similar heating schedules (heating rate was 2 °C min−1 while maintaining at 500 °C for 60 min) and held at target temperature for 3 h, followed by cooling naturally. 2.4. Surface Modification of Bioceramic Scaffolds. The assintered diopside scaffolds were used for modification with pure akermanite (denoted as DIO/AKE), 5% Cu-doped akermanite (denoted as DIO/AKE-5Cu), pure bredigite (denoted as DIO/ BRE), and 5% Cu-doped bredigite (DIO/BRE-5Cu), respectively. The bioceramic slurries for the impregnation of these modified layers were prepared using the following procedure: 1.3 g bioceramic powder was added to 10.0 g PVA (5.0 wt %) solution, and stirred at room temperature (∼22 °C) to achieve homogeneous slurry. Then, the assintered scaffolds were immersed in the bioceramic slurries for 15 min, respectively. Subsequently, the scaffolds were kept under high vacuum for 1 h to make sure that the slurry had been coated homogeneously in the pore wall of the scaffolds. The samples were kept at 100 °C for 24 h to ensure the coating layer was compact and dry. Finally, the scaffolds were sintered at 1100 °C for 2 h with the heating rate of 2 °C min−1. 2.5. Ion Release and Weight Loss in Tris Buffer. In order to evaluate the weight loss (degradation) of the scaffolds with and without pore-wall modification (8 mm × 8 mm × 8 mm), the scaffolds (W0) were, respectively, immersed in Tris buffer with an initial pH 7.25 at 37 °C with a liquid/solid ratio of 50 mL/g. After immersion for 1, 3, 7, and 14 days, 0.5 mL of supernatant was extracted for inductively coupled plasma-optical emission spectrometry (ICP-OES; Thermo) measurement. An equal volume of fresh buffer was added. After immersion for 2, 4, 6, and 8 weeks, 20% of supernatant was exchanged with an equal volume of fresh buffer, the samples were rinsed with ethanol and then dried up to mass constancy (Wt) before weighing. The weight decrease was expressed as the following equation: weight decrease = Wt/W0 × 100%. 2.6. Animal Model. There were 24 male adult New Zealand rabbits (2.5−3.0 kg) used in this study. The animals were maintained B

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Figure 1. Primary SEM (A1−E1) and XRD (A2−E2) characterizations of the as-milled bioceramic powders. Bar: 10 μm. singly in stainless-steel cages and fed and watered. The study protocol was reviewed and approved by the Ethics Committee for Experimental Animals, Zhejiang University (No. 866), and in accordance with the ARRIVE guidelines. All the rabbits were implanted with a construct from five different groups: DIO/AKE, DIO/AKE-5Cu, DIO/BRE, DIO/BRE-5Cu, and DIO (control). The rabbits were immobilized after administration of sodium pentobarbital solution (w/v, 3%) through intraperitoneal injection at a dosage of 1 mg kg−1. Under isoflurane anesthesia, midsagittal incisions were made on the dorsa of the rabbits. Each implant construct was inserted beneath the panniculus carnosus muscle on the back of the rabbits, so that vascular formation could be observed around the implant. Five blocks were placed in each animal. The rabbits were euthanized with pentobarbital sodium (100 mg kg−1), and finally, the implants were harvested at 6 and 12 weeks postoperatively. Digital images of the macroscopic views of the harvested implants were captured with a Canon D1200 camera. 2.7. Histomorphology. The samples, with soft tissue cleaned, were fixed in 10% neutral buffered formaldehyde (pH 7.2) for 10 days, and then rinsed in distilled water for 2 h. The fixed samples were then dehydrated in successive alcohol concentrations, cleared with xylene, and embedded in poly(methyl methacrylate). After hardening, the samples were cut into 800-μm-thick sections perpendicular to the implants, under cooling water with a sawing microtome (Germany, Leica SP600). The sections then were glued onto a plastic support and polished to 150 μm in thickness and finally were stained with hematoxylin and eosin (H&E) observed using an optical microscope (Olympus). 2.8. Statistical Analysis. The results were expressed as mean ± standard deviation (mean ± SD). Statistical analysis was carried out using one-way ANOVA, and a p-value of less than 0.05 (p < 0.05) was considered statistically significant.

akermanite and bredigite, which suggests there was no phase transformation after doping a minor amount of Cu ions. Additionally, the ICP-OES analysis showed that the replacement amount of Ca by Cu was 4.54 mol % in akermanite, and 4.68 mol % in bredigite, though these data were slightly lower than the theoretical value (Table 1). Evaluation of the size Table 1. Ionic Percentage (mol %) Detected by ICP sample

Cu (mol %)

Mg (mol %)

Si (mol %)

Cu replace Ca (mol %)

AKE-Cu0 AKE-Cu5 BRE-Cu0 BRE-Cu5

40.00 47.04 58.33 52.47

20 23.65 8.33 7.35

40 29.31 33.33 40.18

0 4.54 0 4.68

distribution of the bioceramic powders conducted using dynamic light scattering revealed a size distribution of 200− 2600 nm, aside from the smaller particle size distribution of pure diopside powders (data not shown). These were also consistent with the SEM observation. 3.2. Structural Evaluation. Figure 2 shows the SEM micrographs and EDX spectra of the five groups of bioceramic scaffolds before and after pore-wall modification. The outward appearance (top view) of the representative scaffolds with precisely defined pore size after undergoing the pore-wall modification and secondary sintering was characterized by SEM observation (Figure 2A1−A5). It was observed that nearly rectangular pores occurred throughout the whole horizontal direction and not only in the vertical direction in the scaffolds (Figure 2B1−B5). The constructs retained their shape with no noticeable deformation of the total structure, except for thicker struts and decreased pore size after modification. Line-scanning EDX spectra across the pore struts (Figure 2C1−C5) showed the changes in peak strength for Si, Ca, and Mg (Figure 2D1− D5), which strongly demonstrated that the physical modification approach was successful. Characterization of the coating layer around the sintered filament and pore parameters by optical microscopy clearly showed similar shrinkage of the pore dimension (Figure 3); that is, modification of different

3. RESULTS 3.1. Primary Characterization of the Bioceramic Powders. Figure 1 represents the SEM images and XRD patterns of the as-milled bioceramic powders. The SEM observation reveals the superfine particle of the powders after undergoing a ball milling treatment. X-ray diffraction of the Ca−Mg silicate precursor powders following calcining at 1000 °C indicated that the product was highly crystalline, according to the strong peaks at 10−50°/2θ. The XRD patterns of the AKE-5Cu and BRE-5Cu powders were similar to the pure C

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Figure 2. SEM images of the surface (A1−E1) and fracture surface (A2−E2) of the bioceramic scaffolds and line-scanning EDX spectra (A3−E3) showing the changes of ionic strength of the fracture surface across the pore strut and wall (dashed line showing in A2−E2).

bioceramic coatings (i.e., DIO/AKE-5Cu, DIO/BRE-5Cu). The medium from DIO/BRE-5Cu showed a more rapid increase in Cu concentration. As for the Ca and Si concentrations, a similar trend was observed, with a large increase within 7 days (Figure 4C,D), but the doping of Cu into the akermanite or bredigite did not significantly change the release profile of SiO44− ions. Figure 5 shows that the weight decreases of the bioceramic scaffolds with AKE and AKE-5Cu modification were similar to each other after 2 weeks (∼2.6 ± 0.2%) and 8 weeks (∼4.5 ± 0.2%) of immersion, whereas the weight loss of the scaffolds modified with BRE or BRE-5Cu in Tris buffer increased significantly with increasing time, probably due to a less dense structure or improved dissolution of the coating layers in the scaffolds. The pure DIO scaffold showed a weight loss of ∼1.7% and ∼3.5% after immersion for 2 and 8 weeks, respectively. 3.4. In Vivo Implantation of the Porous Bioceramic Constructs. Figure 6 illustrates the process of bioceramic scaffolds implanted beneath the panniculus carnosus muscle on the back of rabbits. At weeks 6 and 12, the implants together with the ingrew tissue were carefully harvested to compare the vascular formation (Figure 7). It is seen that the DIO group showed limited vascular networks and fibrous tissue encapsulating the scaffolds, even after 12 weeks. In contrast, the modified scaffolds, especially the DIO/AKE-5Cu and DIO/BRE-5Cu groups, showed plenty of vascular tissue that readily infiltrated into the porous constructs. Figure 8 showed the representative optical image of the fracture surface of the DIO/BRE-5Cu scaffold at 6 weeks postimplantation. Vascularization of the porous constructs was revealed by H&E staining (Figures 8 and 9) and was quantified by assessing the new blood vessel density (Figure 10). Figure 8 gives the vascular longitudinal section of the vessels grown in the scaffolds, which shows rich red blood cell aggregates. As shown in Figure 9, the vascularization of the different bioceramic constructs was revealed by H&E staining, and the DIO group showed significantly lower newly formed blood

Figure 3. Changes in porosity of the diopside scaffolds before and after Ca−Mg silicate modification.

Ca−Mg silicate bioceramics did affect the surface morphology and composition in the pore wall, but all the bioceramic scaffolds after undergoing the secondary sintering at 1000 °C showed tight bonding between the coating layer and diopside substrates. 3.3. Ion Release and Degradation in Vitro. Figure 4A,B shows the changes of Mg and Cu concentrations in Tris buffer during soaking of the bioceramic scaffolds. Appreciable differences in Mg concentrations were observed for the five groups within 3 days, but thereafter followed a similarly increasing trend in Mg concentration. Overall, the Mg concentration changed very slowly for the pure DIO scaffolds, but increased rapidly for the DIO/BRE scaffolds. For the DIO/ AKE and DIO/AKE-5Cu scaffolds, the Mg concentrations in Tris buffer increased mildly within 3 days and then slowly increased. As for the scaffolds modified with bredigite coatings (i.e., DIO/BRE, DIO/BRE-5Cu), the increase of Mg concentration was significantly influenced by the Cu doping in bredigite. The Mg concentration in the medium increased abruptly within the initial 7 days for the Cu-free scaffolds. ICP analyses only detected Cu in the scaffolds with Cu-doped D

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Figure 4. Changes in Mg (A), Cu (B), Ca (C), and Si (D) concentration in the Tris buffer during immersion of the bioceramic scaffolds.

Figure 6. Schematic of implantation in rabbits with the bioceramic scaffolds and treatment after 6 and 12 weeks, respectively.

Figure 5. Weight decrease of the diopside scaffolds with and without Ca−Mg silicate modification during immersion in the Tris buffer for 0−8 weeks.

orbital volume loss and as a substitute of an internal orbital growth stimulus, both resulting in improvement of cosmetic appearance. Our study, for the first time, describes a bifunctional inorganic coating mode in 3D robocasting orbital implants. The Cu doping of a Ca−Mg silicate coating layer is of specific significance in enhancing the angiogenesic and antibacterial potential of diopside scaffolds. This new strategy may be of benefit for preventing implant-related infections or retardation in angiogenesis, and it may reduce the risk of secondary exposure and implant removal. In general, the risk of complications associated with orbital implants, such as implant extrusion, implant exposure, and infection, has prompted investigation of novel materials, new pore-making techniques, and improved operative technique.28 In most cases, the low bioactivity, suboptimal pore size, and

vessel density than in the DIO/BRE-5Cu and DIO/BRE-5Cu groups. To further confirm these results, the quantitative analysis at two time points demonstrated (Figure 10) that the DIO/BRE5sCu group exhibited the highest vessel density, which was 101.6 ± 3.0/mm2, 138.6 ± 4.3/mm2 at weeks 6 and 12, respectively. Moreover, the vessel density in the akermaniteor bredigite-modified scaffolds confirmed the significant differences (p < 0.01) in comparison with the pure diopside scaffolds.

4. DISCUSSION Orbital defects usually result from tumor, birth anomalies, or trauma leading to disfunction of the patient. The orbital implant is usually inserted for the purpose of compensating E

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Figure 7. Outward appearance of the bioceramic scaffolds after embedding for 6 and 12 weeks, respectively.

investigation indicated that the dissolution rate of diopside under physiological conditions is less than that of pure HA ceramic sintered at high temperature. Also, it is known that the 3D robocasting technique is flexible in tailoring the pore size and pore interconnectivity of porous constructs. Accordingly, the diopside-based porous orbital implants with optimal pore architectures favorable for angiogenesis could be easily obtained by adjustment of the printing parameters (e.g., filament diameter, distance between filaments). Ca−Mg-silicates, including akermanite and bredigite, are highly bioactive in vitro and in vivo. Zhai et al. have reported that akermanite and bredigite showed excellent potential in stimulating angiogenesis.19 According to the SEM/EDX analysis, our studies have shown that these two Ca−Mgsilicates can readily modify the pore-wall composition of 3D robocasting diopside scaffolds. It is likely that, thus, the suboptimal bioactivity of diopside scaffolds can be enhanced by a conventional sol−gel modification post-treatment. As a result, the porous construct may be more resistant to the intraocular microenvironment concordant with rapid angiogenesis in the pore networks. On the other hand, our immersion test in vitro showed that the diopside-based scaffolds, irrespective of pore-wall modification with other Ca−Mg-silicates, showed limited mass loss (3.9−4.8 wt %) in Tris buffer within 8 weeks. This mass loss is far less than that for the compact akermanite ceramic (∼11 wt %) after immersion for 8 weeks or the dilute Mg-doped wollastonite scaffolds (over 15 wt %) after immersion for 6 weeks in vitro, in our previous studies.31,32 Thus, these results suggest the fully interconnected diopside-based porous bioceramics may be a good candidate to maintain a longterm stability in the intraocular environment. Clinically, the presoaking treatment of the orbital implants in antibiotics has been performed to avoid possible introduction of bacteria at the time of implantation. Although some new approaches can be used to improve the drug penetration in the porous orbital implants,33 the drug loading capacity and the following release is burst (uncontrollable). In this regard, the present study describes a Cu doping of akermanite or bredigite coating approach which may produce multifunctional orbital implants with enhanced antibacterial properties. According to ICP analysis, it was confirmed that Cu ions were released from the AKE-5Cu or BRE-5Cu coating into the immersion medium. In addition, the doping of copper into the Ca−Mg silicate crystal latter framework might slow the release of ions in the

Figure 8. Longitudinal cutting of the vessels in the pore struts of the DIO/BRE-5Cu scaffold after embedding in panniculus carnosus muscle for 6 weeks. (A) Fracture surface of the scaffold. (B) Vessel wall and a great amount of red cells in the vessel.

limited pore interconnectivity of the clinically available orbital implants are potentially the most critical factors leading to retardation in angiogenesis and secondary exposure. In some cases antibacterial treatment implants are a deciding factor in the prevention of infection and a chronic inflammatory reaction.29,30 Alternatively, it is possible that 3D robocasted diopside porous constructs are preferable candidates as an orbital substrate as a consequence of inherent tissue/material biocompatibility and appropriate bioactivity in vitro and in vivo.11,12 Diopside is very stable in vivo according to Nonami and Tsutsumi’s study in bone defect environment.11 Their F

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Figure 9. H&E staining of sections of the bioceramic implants after embedding in panniculus carnosus muscle for 12 weeks. The arrows showing vessels in the pore struts.

transport movement of atoms. This was reflected by the Mg2+ and Ca2+ ion concentration in the Tris buffer immersion (Figure 4). It is evident that although the doping of Cu into the Ca−Mg silicate ceramic coating may suppress the release profile of Mg2+ ions from BRE-5Cu coating (Figure 4A) and Ca2+ ions from the Ake-5Cu coating (Figure 4C), the release profile of biologically active Mg2+ and Ca2+ ions was improved, in which Ca−Mg silicate modification exhibited a faster increase in ion concentrations than those from the pure diopside scaffolds. On the other hand, although the resulting concentration profile in vivo is difficult to predict, Cu doping into Ca−Mg silicate coatings offers a promising strategy to ultimately provide an appreciable copper concentration, as they allow tailored ion release rates through modification of the Cu doping ratio in the coating layers. It is well-known that rapid angiogenesis in artificial orbital constructs is favorable for improving the integration with the host tissue; otherwise, complications may arise, such as slow inflammation, infection, and exposure.34 Angiogenic induction by a biomaterial itself may be a simple and effective neovascularization strategy. Many reports have shown the

Figure 10. Number of newly formed blood vessels in tissue sections of the bioceramic implants at 6 and 12 weeks postimplantation.

lattice because of the smaller interplanar distance and crystal volume of AKE-5Cu or BRE-5Cu, which could inhibit the G

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through which the Ca−Mg silicate biomaterials scaffolds without any growth factors stimulated vascularization in a panniculus carnosus muscle model can be summarized as follows. Diopside acts as the main body of the scaffolds while akermanite, bredgite, and copper ions served as angiogenic stimulating factors via surface modification. During this process, each Ca−Mg silicate affected the coating efficacy and, collectively, behaved as an appropriate scaffold for orbital implantation.

combination of biomaterials, specifically scaffolds, and proangiogenic growth factors play a key role in angiogenesis. 35−37 However, little research on blood vessel proliferation achieved by biomaterials alone has been reported.11,19 We compared the angiogenesis of five types of matrixes, based on the 3D robocasting diopside-based bioceramic porous constructs. From a macroscopic view, the DIO group without surface modification showed weakest angiogenesis in the pore networks, while the DIO/AKE, DIO/ AKE-5Cu, DIO/BRE, and DIO/BRE-5Cu showed increased blood vessel formation. Within these groups, the DIO/BRE5Cu group was a better stimulator of new vessel growth than the DIO/AKE-5Cu, possibly due to the synergistic effect of copper ions and bredigite in the formation of blood vessels. According to the quantitative analysis, the number of newly formed blood vessels in tissue sections of the modified scaffolds further confirmed the trend. On the other hand, the employed model was based on the following main components: (1) the panniculus carnosus muscle flap, which was sufficiently extended to entirely cover the large 3D porous scaffold constructs and which included an abundant vascular network supplied by defined axial vessels and a vascular pedicle;38 (2) the semipermeable membrane, which was wrapped around the prefabricated material flap in order to prevent adherence to the surrounding tissue and thus potentially allow angiogenic transfer through the vascular pedicle at a later stage;39 and (3) the porous bioceramic material, previously modified with highly bioactive layer to generate soft tissue and establish that the process of vascular tissue formation in the scaffold is effectively regulated by the functional layer, and thus to exclude that the pattern of any connective tissue formation was possibly related to that of initial cell distribution. Thus, it is reasonable to assume that the panniculus carnosus muscle model in rabbit is effective to evaluate the angiogenic potential which occurs in the orbital microenvironment. For the purpose of developing ideal orbital implants independent of growth factors, it is necessary to clarify the underlying molecular mechanisms directing the angiogenic response. In this regard, Chang’s group reported that the ion extracts from akermanite and bredegite could stimulate the proliferation of human umbilical endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) and in vitro angiogenesis, with improved angiogenic gene expression (KDR, FGFR1, ACVRL1, and NOS3).11,19 They also demonstrated that the ion extracts from the bredegite bioceramic showed the highest angiogenic potential in vitro, which is consistent with our observation in vivo. More recently, they also demonstrated that the ionic products with specific Si ion concentration dissolution from Ca-silicates could stimulate the proliferation of HUVECs, and up-regulated the expression of VEGF, bFGF, and their receptors, and stimulated angiogenesis.40,41 Also, Cudoped inorganic biomaterials have been confirmed to effectively stimulate angiogenesis-related gene expression and to inhibit bacterial viability.21,24 On the other hand, it is agreed that an insufficient level of oxygen, a condition known as hypoxia, plays a critical role in blood vessel formation, and hypoxia can be artificially mimicked by stabilizing an HIF-1 expression (e.g., Cu2+ ions application). Therefore, Cu2+ ions have been doped into the bioactive glasses42,43 or mesoporous silica spheres44 to stimulate the angiogenesis or to promote wound healing. Our animal experiments demonstrate that modified scaffolds have greater bioactivity in promoting cellular infiltration and capillary invasion in scaffolds in vivo. Therefore, the entire mechanism

5. CONCLUSIONS In summary, mechanically stable and fully interconnective porous diopside ceramic scaffolds coated with other biodegradable Ca−Mg silicate bioceramics were successfully fabricated (DIO/AKE, DIO/AKE-5Cu, DIO/BRE, DIO/ BRE-5Cu, and diopside), and their physicochemical and biological performance was evaluated in vitro and in vivo. After incubation in vivo for 12 weeks, the diopside scaffolds remained intact with minimal degradation, while the coating layer (e.g., BRE-5Cu) produced significant angiogenesic effects. Thus, the use of highly bioactive Ca−Mg silicate modified diopside porous implants in retaining orbital prosthesis is expected to improve patient acceptance of the prosthesis as a result of improved retention and stability.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: 86 571 8820 8353. Fax: 86 571 8697 1539. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Science and Technology Department of Zhejiang Province Foundation (2015C33119, 2014C33202), the Zhejiang Provincial Natural Science Foundation of China (LZ14E020001, LQ14H060003), and the National Science Foundation of China (51372218, 81271956, 81301326).

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DOI: 10.1021/acsbiomaterials.6b00282 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX