Controlled Co-delivery of Growth Factors through Layer-by-Layer

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Controlled Co-delivery of Growth Factors through Layer-by-Layer Assembly of Core− Shell Nanofibers for Improving Bone Regeneration Gu Cheng,† Chengcheng Yin,† Hu Tu,‡ Shan Jiang,§ Qun Wang,∥ Xue Zhou,⊥ Xin Xing,† Congyong Xie,† Xiaowen Shi,‡ Yuming Du,‡ Hongbing Deng,*,‡ and Zubing Li*,† †

The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China ‡ Hubei International Scientific and Technological Cooperation Base of Sustainable Resource and Energy, Hubei Key Lab of Biomass Resource Chemistry and Environmental Biotechnology, School of Resource and Environmental Science, Wuhan University, Wuhan 430079, China § Department of Materials Science and Engineering, Iowa State University, Ames, Iowa 50011, United States ∥ Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States ⊥ School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China S Supporting Information *

ABSTRACT: The regeneration of bone tissue is regulated by both osteogenic and angiogenic growth factors which are expressed in a coordinated cascade of events. The aim of this study was to create a dual growth factor-release system that allows for time-controlled release to facilitate bone regeneration. We fabricated core−shell SF/PCL/PVA nanofibrous mats using coaxial electrospinning and layer-by-layer (LBL) techniques, where bone morphogenetic protein 2 (BMP2) was incorporated into the core of the nanofibers and connective tissue growth factor (CTGF) was attached onto the surface. Our study confirmed the sustained release of BMP2 and a rapid release of CTGF. Both in vitro and in vivo experiments demonstrated improvements in bone tissue recovery with the dual-drug release system. In vivo studies showed improvement in bone regeneration by 43% compared with single BMP2 release systems. Time-controlled release enabled by the core−shell nanofiber assembly provides a promising strategy to facilitate bone healing. KEYWORDS: growth factor, coaxial electrospinning, layer-by-layer, controlled release, bone defects

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Owing to its ability to enhance the migration of endothelial cells, CTGF also acted as an angiogenic regulator in endochondral ossification.12 Therefore, it is safe to conclude that BMP2 and CTGF all play vital roles in bone healing. The rationale of this study was guided by the results of our previous experiments investigating the endogenous synthesis of CTGF and BMP2 in the natural physiological microenvironment (Figure S1). We created animal models for bone trauma, and the results showed that CTGF was transiently expressed in the trauma sites of different bone tissues (Figure S1A−F)

one healing is a complex regenerative process and involves the synchronization of many growth factors.1 Among them, BMP2 and CTGF are secreted by osteoblasts and expressed at the region of the fracture site.2,3 BMP2 promotes osteoblastogenesis and new bone formation and has been widely used as a therapeutic growth factor for treatment of skeletal diseases.4,5 CTGF, a cysteine-rich protein belonging to the CCN family,6 plays a vital role in both endochondral and intramembranous ossification.7,8 It has been reported that CTGF promotes the attachment and migration of marrow stromal cells, which are necessary for cartilage and bone regeneration.9,10 The delayed maturation and mineralization of osteoblasts in culture caused skeletal abnormalities and a decrease in bone formation in CTGF-deficient mice.8,11 © 2019 American Chemical Society

Received: August 8, 2018 Accepted: June 11, 2019 Published: June 11, 2019 6372

DOI: 10.1021/acsnano.8b06032 ACS Nano 2019, 13, 6372−6382

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Figure 1. Characterization of the (SF/PCL)1:5/PVA-LBL20 electrospun nanofibers. TEM images illustrate the core−shell structure in the (SF/PCL)1:5/PVA-LBL20 electrospun nanofibers (A). SEM images of the (SF/PCL)1:5/PVA-LBL20 electrospun nanofibers (B). The frequency distribution of nanofibers diameters for the (SF/PCL)1:5/PVA-LBL20 electrospun nanofibrous mats (C). Confocal microscopy images of the coaxial nanofibers with rhodamine B-labeled shell and calcein-labeled core (D).

sustained release of BMP2, which is too short considering that bone healing typically takes 1−3 months. In this study, we incorporated BMP2 into an 8% aqueous poly(vinyl alcohol) (PVA) solution to fabricate core−shell composite nanofibrous mats using a coaxial electrospinning method. Subsequently, we immobilized CTGF onto the surface of the nanofibrous mats via an LBL self-assembly technique. Coaxial electrospinning is a highly versatile method recently developed to incorporate bioactive growth factors into composite nanofibers with a core−shell structure. This core− shell structure protects the biological growth factors and allows for their sustained release, maintaining their bioactivity. LBL assembly is a technique for preparing multiple functional coatings for a broad scope of therapeutic applications, including gene,18 drug,19 and protein delivery.20,21 Efficient methods for fabricating multilayer mats containing biological growth factors for biomedical applications have been widely studied, and it was shown that these growth factors were still active after they were incorporated into the LBL films.17,22,23 In a previous study, a multilayer coating was fabricated by LBL to release both PDGF-BB and BMP2 in a controlled fashion. As the scaffold degraded from the top down, PDGF-BB located on the surface of the scaffold eluted faster than BMP2 at the bottom.5 Using the technology of LBL, therefore, CTGF could be attached onto the surface of the coaxial electrospinning nanofibers and would be released from nanofibers in faster rate than BMP2 in the core of nanofibers.

during early stages of recovery. We also evaluated the expression of BMP2 and CTGF from mesenchymal stem cells (MSCs) cultured in vitro. The results indicated that BMP2 expression is stable during the whole period of osteogenic differentiation of MSCs, whereas CTGF is only expressed during the early period (Figure S1G−I). These results are consistent with reports of another early study13 in which CTGF was expressed mainly in chondrocytes during the early stages of long bone development. At the late stage of bone development, the number of CTGF-positive chondrocytes was dramatically reduced. In contrast to the transiently expression of CTGF, BMP2 was stably expressed throughout all phases of bone healing (from the soft callus to hard callus phase).4 Inspired by the above-mentioned results about endogenous synthesis of BMP2 and CTGF, we developed a dual-delivery release system with different release profiles for BMP2 and CTGF. A slow and sustained release was designed to ensure that BMP2 was present over the entire bone healing period, whereas a relatively fast and transient release was designed for CTGF during the early stage of bone healing. Certain dual-drug delivery systems not only minimize negative side effects but also improve therapeutic efficiency of drugs compared with single-drug delivery systems.14 For example, gelatin microparticles releasing both BMP2 and vascular endothelial growth factor (VEGF) were fabricated to treat cranial critical size defects and showed that the early bone regeneration was promoted by this dual delivery system.4 Another dual-release system with VEGF and platelet-derived growth factor-BB (PDGF-BB) loaded within nanofibers was fabricated using a blend electrospinning method.15 This system delivered PDGF-BB slowly and VEGF quickly and supported the growth of fibroblasts and wound healing of soft tissue. However, the burst release and reduced activity of growth factors may hinder its usage because this system directly disperses the growth factors into toxic polymer solution.16 Shah et al. fabricated a polyelectrolyte multilayer (PEM) film with both BMP2 and CTGF using LBL technology.17 Although this release system facilitated distinct release profiles for both growth factors, the film allowed for only 2 weeks of

RESULTS AND DISCUSSION The core−shell nanofibers were designed to release two growth factors at distinctly different rates. Previous studies showed that this specific design accelerated the healing process of bone defects compared to the results of single-growth factor release systems.24,25 The coaxial/LBL release system fabricated in this study was evaluated in vitro and in vivo for its efficacy in bone regeneration by a series of experiments, and the results are discussed in the following sections. 6373

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Figure 2. In vitro release kinetics of growth factors. BMP2 release profiles from the (SF/PCL)1:n nanofibrous mats with different ratios of SF and PCL (A). After treatment of LBL process, CTGF was released from the (SF/PCL)1:5 nanofibrous mats with no BMP2 incorporation (B). After treatment of the LBL process, BMP2 (C) and CTGF (D) were released from the (SF/PCL)1:5 nanofibrous mats with BMP2 located in the core of the nanofibers. CTGF and BMP2 release kinetics from the (SF/PCL)1:5/PVA-LBL20 electrospun nanofibers (E). In vivo tracking of the labeled BMP2 and CTGF in a same animal was detected by in vivo imaging (F). The release of BMP2 with a blue color lasted for a period of 30 days, while CTGF with a green color was undetectable at 15 days of implantation (F).

Release Profiles of the (SF/PCL)1:n/PVA−LBLn Nanofibrous Mats. The BMP2 release profile of the BMP2-loaded coaxial nanofibrous mats was determined for different ratios of SF to PCL (1:2, 1:3, 1:4, 1:5 and 1:6). As illustrated in Figure 2, five types of coaxial nanofibrous mats were fabricated to determine the effect of SF content on the release profile. The release rate of BMP2 increased with increasing content of SF (Figure 2A). Therefore, the degradation rate of the core−shell (SF/PCL)1:n/PVA mats can be tailored by adjusting the amount of SF in the shell solution. Each coaxial nanofibrous mat, independent of its SF-to-PCL ratio, exhibited a burst release of BMP2 during the first several days of incubation, followed by a linear release profile (Figure 2A). This suggests that the release mode for BMP2 was a combination of diffusion-based and degradation-based modes. In theory, degradation-based release should be dominant for coaxial electrospun nanofibers when drugs are embedded into the core of the nanofibers.26 However, only part of the nanofibers exhibits the core−shell structure because of the intrinsic defects caused by coaxial electrospinning, for example, unstable electrostatic forces and drifting environmental conditions.27,28 The nanofibers without a core−shell structure also contain some BMP2, and its release from these nanofibers is faster than that from those with a core−shell structure. This

may be responsible for the initial burst release of BMP2, as illustrated in Figure 2A. Nanofibrous mats with SF-to-PCL ratios of 1:2 and 1:3 showed an initial BMP2 burst release during the first 6 days of incubation, which leveled off over a period of 30 days. Nanofibrous mats with SF-to-PCL ratios of 1:4 showed a burst release of 42 ± 2% in the first 3 days, and the remaining BMP2 was released in a sustained fashion from day 6 to day 25. The nanofibrous mats with SF-to-PCL ratios of 1:5, however, showed a sustained, linear BMP2 release profile for the entire time period. Nanofibrous mats with SF-to-PCL ratios of 1:6 demonstrated a minimal burst of 13 ± 1%, and the cumulative release of BMP2 was the lowest (79 ± 4%). Because of the observed BMP2 expression in the osteogenic differentiation of MSCs, we chose the (SF/PCL)1:5/PVA nanofibrous mats for the subsequent studies to achieve a sustained and highly efficient release profile of BMP2 (Figure 2A). Subsequently, we evaluated the CTGF release kinetics of the coaxial (SF/PCL)1:5/PVA nanofibrous mats without BMP2. The mats were covered by 20, 30, and 40 layers (LBL20, LBL30, and LBL40, respectively) of chitosan (CS)-CTGF bilayers (Figure 2B). The release of CTGF from LBL20, LBL30, and LBL40 nanofibrous mats reached 90 ± 4%, 81 ± 4%, and 73 ± 3%, respectively, over a period of 40 days. The LBL20 6374

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Figure 3. Western blot analysis of ALP (A, D), Col-I (B, E), and VEGF (C, F) expression during the osteogenic differentiation of MSCs cultured on different nanofibrous mats.

fast rate, while BMP2 (embedded in the core) is released relatively slowly, as indicated in the Figure 2E. Characterization of the Core−Shell Nanofibrous Mats. TEM was used to visualize the core−shell structure of the (SF/PCL)1:5/PVA nanofibrous mats prior to the LBL process. Figure 1A shows the core−shell structure along the length of the (SF/PCL)1:5/PVA nanofiber. The coaxial (SF/ PCL)1:5/PVA nanofibers had overall diameters ranging from 175.2 to 1710.9 nm, with core diameters ranging from 134.8 to 729.2 nm (Figure 1A). Figure 1B shows the SEM images of coaxial (SF/PCL)1:5/ PVA-LBL20 nanofibrous mats created by the LBL process. The diameters of the nanofibers ranged from ∼300 nm to ∼1300 nm, and the most frequent nanofiber diameters ranged between 800 and 900 nm (Figure 1B,C). The pore size distribution shifted from 3.4 to 5.8 μm. Pores ranging in size from 3.6∼3.8 μm represented more than ∼70% of the total pores tested, confirming a relatively uniform pore distribution (Figure S3). After deposition of CS and CTGF, the (SF/ PCL)1:5/PVA-LBL20 nanofibrous mats still maintained a nanofibrous 3D structure. However, some conglutinations formed between bundles of adjacent nanofibers (white arrow, Figure 1B), which suggests that CS and CTGF successfully attached onto the surface of the mats. Compared with the smooth surface of the nanofibrous mats without coatings (Figure 1A), a coarser nanofibrous structure and more irregular protuberances were clearly observed in the nanofibrous mat with 20 coating layers (Figure 1B). The distribution of the solution at the core within the nanofibers was further investigated by confocal fluorescent microscopy. As shown in Figure 1D, the core−shell nanofibrous mats exhibited two different colors. The green PVA was located in the middle of the nanofiber and the red SF-PCL shell was distributed on the nanofiber surface surrounding the PVA core. This result demonstrated that the PVA solution was successfully encapsulated and uniformly distributed within the core of the nanofibers. After confirmation of a uniform PVA distribution within the nanofibers, the distribution of BMP2 and CTGF was also evaluated by confocal microscopy. As shown in Figure S2, the Alexa 647-labeled BMP2 (blue) and Alexa 488-labeled CTGF

nanofibrous mats exhibited an initial burst release of CTGF on day 1, which was maintained for the following 5 days before leveling off for the rest of time. The cumulative release of CTGF from the LBL20 nanofibrous mats was 90 ± 4%. The increase of layers up to 30 and 40 decreased the total release percentage by the nanofibrous mats to 81 ± 4% and 73 ± 3%, respectively, representing a waste of embedded growth factors. In addition, LBL30 and LBL40 nanofibrous mats continued to release CTGF for a period of 1−20 days of incubation (Figure 2B). This did not reflect CTGF in MSCs, where it was no longer expressed after 12 days of osteogenic differentiation (Figure S1G, I). We concluded that mats with 30 or 40 layers carried excessive CTGF loads, which did not represent the transient expression of CTGF. Next, we evaluated the release profile of both growth factors in BMP2/CTGF-loaded (SF/PCL)1:5/PVA nanofibrous mats covered by various bilayers (20, 30, and 40). Compared with the coaxial (SF/PCL)1:5/PVA mats loaded with BMP2 alone (Figure 2A), the (SF/PCL)1:5/PVA-LBL20−40 electrospun mats exhibited a linear and more sustainable release profile of BMP2 (Figure 2C). These results demonstrated that the burst release was bypassed in this dual release system, as the burst release of BMP2 already occurred in the process of LBL. On the other hand, the release kinetics of CTGF appeared to be unaffected by the LBL process, resulting in similar release profiles in (SF/ PCL)1:5/PVA-LBL20, (SF/PCL)1:5/PVA-LBL30, (SF/PCL)1:5/ PVA-LBL40, LBL20, LBL30, and LBL40 electrospun mats (Figure 2B,D). The (SF/PCL)1:5/PVA-LBL20 electrospun mats in particular maintained a sustainable BMP2 release rate during the whole process of incubation in phosphate buffered saline (PBS) (Figure 2C). CTGF was released from the nanofibers in the (SF/PCL)1:5/PVA-LBL20 group during the first 10 days and was not expressed during the remaining time (Figure 2D). We conclude that the release profile of (SF/ PCL)1:5/PVA nanofibrous mats with 20 layers reflects the expression of BMP2 and CTGF during bone healing. Based on these results, (SF/PCL)1:5/PVA-LBL20 nanofibrous mats were chosen for our subsequent experiments. When (SF/PCL)1:5/PVA-LBL20 mats degrade, the growth factors are released from the fiber outside to the core. CTGF (on the fiber surface) is released into the environment at a very 6375

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Figure 4. Immunofluorescence analysis of four kinds of nanofibrous mats. (SF/PCL)1:5/PVA-LBL20 loaded with BMP2 and CTGF (A, E), BMP2-loaded (SF/PCL)1:5/PVA (B, F), CTGF-loaded LBL20 (C, G), and the nonloaded coaxial nanofibrous mats (empty control) (D, H) at day 7 (A−D) and day 14 (E−H) were immunostained with anti-CD31 (red) and anti-ALP antibodies (green). The nuclei were counterstained with DAPI (blue). A strong CD-31 signal was observed in the (SF/PCL)1:5/PVA-LBL20 and LBL20 nanofibrous mats. Scale bars: 100 μm.

(green) were all uniformly distributed in the coaxial (SF/ PCL)1:5/PVA-LBL20 nanofibers (Figure S2A−C). In Vivo Tracking of Fluorescent Dye-Labeled BMP2 and CTGF. In vivo release profiles of BMP2 and CTGF were consistent with the results of in vitro release profiles. As shown in Figure 2F, strong blue signals were detected from the Alexa 647-labeled BMP2. These signals were confined to the implant site 1 h after implantation. Although BMP2 was transported from the implant site to more distant tissues, the strong fluorescence signals were still observed at days 6 and 15 of implantation (Figure 2F). Until day 30, fluorescence signals were still observed at the implant site, although the size of the fluorescence area decreased over time (Figure 2F). These results showed that BMP2 was released from the mats at an approximately constant rate, and the release was sustained over the entire period of 30 days. On the other hand, CTGF showed a strong initial signal at 1 h of implantation (Figure 2F) and then rapidly decreased to a minimal level within 6 days (Figure 2F), indicating a transient release of CTGF at an early stage. Dual Delivery of BMP2 and CTGF Promotes the Expression of Angiogenesis and Osteogenesis Markers by MSCs Loaded in the Coaxial Nanofibrous Mats. To confirm whether the dual release of BMP2 and CTGF promoted the angiogenic and osteogenic abilities of MSCs, Western blot analysis of alkaline phosphatase (ALP), type I collagen, and VEGF was performed.

As demonstrated in Figure 3, the BMP2-loaded (SF/ PCL)1:5/PVA nanofibrous mats exhibited higher ALP and col-I expression than those of the control independent of time (p < 0.05, Figure 3A,B,D,E). This result indicated that BMP2 induces osteogenic differentiation of MSCs. A transient release of CTGF tempered the BMP2-induced col-I expression in the early period, but did not influence the BMP2-induced col-I expression in the late period of osteogenic differentiation of MSCs. Col-I expression of MSCs in the (SF/ PCL)1:5/PVA-LBL20 group was low from day 1 to 5 of osteogenic differentiation. A burst release of CTGF may have inhibited BMP2-induced osteogenesis during the early period of osteogenic differentiation, which is consistent with the results of a previous study which reported that excessive expression of CTGF inhibits the osteogenic signal.29 However, this inhibition effect did not influence the entire process of osteogenic differentiation of MSCs. This is because the col-I expression in MSCs of (SF/PCL)1:5/PVA-LBL20 group gradually increased from day 10 to 15 and reached a comparable degree with those of the (SF/PCL)1:5/PVA group at day15 (p > 0.05, Figure 3B,E). The above results demonstrate that transient administration of CTGF does not hinder BMP2-induced osteogenesis in the late period of osteogenic differentiation of MSCs. The dual release of BMP2 and CTGF by (SF/PCL)1:5/PVALBL20 mats achieved a similar effect with regard to ALP expression compared to the single release of BMP2 by (SF/ PCL)1:5/PVA mats in the late stage of osteogenic differ6376

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Figure 5. Micro-CT angiography of the 3D architecture of microvasculature in cranial defects. Enhanced angiogenesis was observed in the CTGF-loaded LBL20 animals (C, H) compared to animals in other groups at week 4 and 8. Capillary area was smaller in animals receiving BMP2 and CTGF in the (SF/PCL)1:5/PVA-LBL20 group compared to those of the LBL20 group (A, C, F, H). Calculated total area of newly formed vessels in the defect site (K).

induced toward osteogenic differentiation by BMP2, the angiogenic differentiation of MSCs may have been compromised and VEGF production dramatically decreased.32 Compared with expression of BMP2 alone, the coadministration of BMP2 and CTGF stimulated the expression of VEGF (p < 0.05, Figure 3C,F). Considering the increased expression of ALP in BMP2/CTGF-loaded (SF/PCL)1:5/ PVA-LBL20 mats, these results demonstrated that timecontrolled release profiles of BMP2 and CTGF in the (SF/ PCL)1:5/PVA-LBL20 nanofibrous mats had a positive effect on both the angiogenesis and osteogenesis of MSCs. Moreover, the LBL20 mats exhibited the lowest expression of ALP and col-I of all mats between days 1 and 15 (p < 0.05, Figure 3A,B,D,E), which indicates that the controlled release of CTGF alone cannot induce osteogenic differentiation of MSCs. A previous study also suggested that the sustained release of CTGF induces angiogenic differentiation and thereby inhibits the osteogenic differentiation of MSCs.33 Here, CTGF may have a similar inhibitory effect on osteogenic differentiation of MSCs (Figure 3B,E), which further verifies the necessity of a transient release of CTGF. Dual Delivery of BMP2 and CTGF Promotes Vessel and Bone Formation. To investigate the effects of (SF/ PCL)1:5/PVA-LBL20, (SF/PCL)1:5/PVA, LBL20, and nonloaded nanofibrous mats (empty control) on vessel and bone

entiation of MSCs. Especially during the early stage (days 1− 3) of osteogenic differentiation of MSCs, ALP expression of the (SF/PCL)1:5/PVA-LBL20 mats was slightly higher than that of the (SF/PCL)1:5/PVA mats (p < 0.05, Figure 3A,D), which indicates that a transient administration of CTGF may play a beneficial role in BMP2-induced ALP expression during the early stage of osteogenic differentiation of MSCs. Moreover, ALP expression in the (SF/PCL)1:5/PVA-LBL20 group was mainly detected during the first 7 days of osteogenic differentiation of MSCs and subsequently gradually decreased (Figure 3A,D). Although a decreased expression of ALP was also found in the (SF/PCL)1:5/PVA group, there was still a certain level of ALP expression at the late stage (day15) of osteogenic differentiation of MSCs (Figure 3A,D). This was inconsistent with results of the previous study that reported that ALP only expressed in the early stage of MSCs osteogenic differentiation.30 In addition, BMP2 alone only resulted in a low expression of VEGF (Figure 3C,F), which is a marker of angiogenic differentiation of MSCs. The maturation of the newly formed bone and regression of microvessels may account for this observation. Other researchers also showed that BMP2 decreased the gene expression of VEGF in MSCs. It was claimed that increased bone formation may lead to limited vascular network formation.31 Another possible reason is the limited multidifferentiation ability of MSCs. When MSCs were 6377

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ACS Nano formation, we established both ectopic and in situ osteogenesis models. In the ectopic osteogenesis model, only a few newly formed CD31-positive blood vessels were observed with BMP2-loaded (SF/PCL)1:5/PVA nanofibrous mats. They also showed more ALP-positive tissue than both control and CTGF-loaded LBL20 mats after 7 days of implantation (Figure 4B−D,I,J). BMP2/ CTGF-loaded (SF/PCL)1:5/PVA-LBL20 nanofibrous mats exhibited similar blood vessel formation and more ALPpositive tissue compared to CTGF-loaded LBL20 nanofibrous mats (p > 0.05, Figure 4A,C,I,J) after 7 days of implantation. This result indicates that the release of BMP2 from the BMP2/ CTGF-loaded (SF/PCL)1:5/PVA-LBL20 nanofibrous mats has no negative effect on CTGF-induced angiogenesis in vivo. At day 14 after implantation, more new blood vessels within the cells/scaffold compounds in the CTGF-loaded LBL20 nanofibrous mats were strongly immunostained with the anti-CD31 antibody than in the control and BMP2-loaded (SF/PCL)1:5/ PVA mats (p < 0.05, Figure 4F−I). More importantly, the number of the CD31-positive newly formed blood vessels in the BMP2/CTGF-loaded (SF/PCL)1:5/PVA-LBL20 nanofibrous mats was slightly lower than in the CTGF-loaded LBL20 nanofibrous mats. This indicates that the release of CTGF alone stimulated the angiogenic responses of MSC, and the simultaneous release of BMP2 and CTGF had a similar effect on vessel formation (Figure 4E,G,I). The largest amount of ALP-positive tissue was detected with the BMP2-loaded (SF/PCL)1:5/PVA nanofibrous mats (p < 0.05, Figure 4E− H,J). This finding suggested that the sustained release of BMP2 significantly enhanced osteoblastic differentiation of MSCs. The number of microvessels in (SF/PCL)1:5/PVA nanofibrous mats was lower than in (SF/PCL)1:5/PVA-LBL20 and LBL20 mats, which may have been caused by a regression of microvessels as a result of bone maturation or reconstruction. This process may have been advanced by the excessive expression of BMP2 released from the (SF/PCL)1:5/ PVA nanofibrous mats.31 Therefore, it is safe to conclude that expression of BMP2 alone enhanced formation of new bone. Further proof of the beneficial effects of BMP2/CTGFloaded (SF/PCL)1:5/PVA-LBL20 mats on bone formation was provided by in situ osteogenesis. Figure S4 shows that at 4 weeks after implantation, the area percentage of newly formed bone in the BMP2/CTGF-loaded (SF/PCL)1:5/PVA-LBL20 mats was significantly higher (43%) than in the (SF/PCL)1:5/ PVA nanofibrous mats loaded with BMP2 alone (p < 0.001, Figure S4A,B,K). Although a similar bone volume was observed in the defect sites of (SF/PCL)1:5/PVA-LBL20 and (SF/PCL)1:5/PVA mats (p > 0.05, Figure S4A,B,L), residual material of the (SF/PCL)1:5/PVA-LBL20 mats had not been completely absorbed by the body. This material was gradually assimilated into autologous nanofibrous tissue at 4 weeks after implantation, as indicated by the results of histological observation (green asterisk, Figure 6A). The residual scaffolds of the (SF/PCL)1:5/PVA-LBL20 mats were infiltrated by some nanofibrous tissues (Figure 6A). At 8 weeks after implantation, a certain amount of cortical bone with the largest bone area and bone volume among all groups was observed in the BMP2/CTGF-loaded (SF/ PCL)1:5/PVA-LBL20 nanofibrous mats (p < 0.001, Figure S4F,K,L), although a complete bridging of the defects was not observed. Histological observations showed that the area of new bone gradually extended from the edges to the middle areas of the defects when (SF/PCL)1:5/PVA-LBL20 mats were

Figure 6. Masson’s trichrome staining of cranial defects in the (SF/ PCL)1:5/PVA-LBL20 (A, F), (SF/PCL)1:5/PVA (B, G), LBL20 (C, H), empty (D, I), and negative groups (E, J) at 4 and 8 weeks after implantation. Residual scaffolds are marked by a green asterisk, and newly formed bone is outlined by a yellow line. Bar represents 500 μm for all panels.

applied (Figure 6F). While BMP2 alone also resulted in a slight increase in new bone formation with (SF/PCL)1:5/PVA mats, the newly formed bone was thinner than the host bone (Figure 6G). In addition, most of the residual scaffolds were already replaced by host tissues with (SF/PCL)1:5/PVA-LBL20 mats (Figure 6F). However, with all other mats, a large amount of the residual scaffold was still attached to the defect sites with no sign of host fusion at 8 weeks after implantation (Figure 6G−J). These results indicate that co-delivery of BMP2 and CTGF induced better bone formation than delivery of BMP2 alone in the late stage of bone regeneration. CTGF-induced angiogenesis may have a beneficial role in the interaction of allograft materials with host tissue. As to the LBL20 nanofibrous mat, less newly formed bone was observed in the defect sites than in those of the (SF/ PCL)1:5/PVA-LBL20 and (SF/PCL)1:5/PVA groups (Figure S4C,H,K,L). Moreover, similar amounts of bone tissues formed in the defect sites with LBL20 and nonloaded mat (empty group) application as indicated by both bone area percentage and bone volume (Figure S4C,D,H,I,K,L). These results further showed that the transient release of CTGF had no negative effect on bone formation, but administration of CTGF alone was not sufficient to enhance new bone formation. In order to clearly depict the 3D network of newly formed vessels, in situ modeling and micro-CT angiography were utilized. The results showed that dual release of BMP2 and CTGF moderately enhanced microvasculature formation in vivo at 4 weeks after operation, as the capillary area was higher in animals treated with BMP2/CTGF-loaded (SF/PCL)1:5/ PVA-LBL20 mats compared to those exposed to BMP2-loaded (SF/PCL)1:5/PVA or nonloaded nanofibrous mats (empty control) (p < 0.05, Figure 5A,B,D). However, more microvessels were formed with CTGF-loaded LBL20 nanofibrous mats than with (SF/PCL)1:5/PVA-LBL20 mats (p < 0.05, Figure 5A,C). These results can be explained by the immunohistochemistry of CD-31, which showed that the angiogenesis of CS/β-TCP scaffolds covered by the (SF/ 6378

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Scheme 1. Schematic Illustration of the (SF/PCL)1:5/PVA-LBL20 Coaxial Fibers Loaded with BMP2 and CTGF for Bone Tissue Engineeringa

a

By combining the technique of coaxial electrospinning and LBL, BMP2 was incorporated into the core of the nanofibers, and CTGF was attached onto the surface of the nanofibers. Sustained release of BMP2 and transient release of CTGF could facilitate these proteins to play their respective roles at different stages of bone healing.

formation by inducing the expression of VEGF. With sufficient blood supply, the volume of newly formed bone in defects exposed to dual delivery nanofibrous mats was higher than that of defects exposed to single BMP2 release mats (Figure S4A,B,F,G,K,L). Therefore, this controlled-release system confirmed our assumption that dual delivery systems releasing BMP2 and CTGF in a time-controlled manner can result in faster and better bone formation.

PCL)1:5/PVA-LBL20 nanofibrous mats was higher than of those covered by (SF/PCL)1:5/PVA or nonloaded nanofibrous mats (empty group). However, angiogenesis for CTGF-loaded LBL20 nanofibrous mats was higher at any given time (p < 0.05, Figure 4A−H). At 8 weeks after operation, more microvessels were formed in defect sites covered by (SF/PCL)1:5/PVA mats (p < 0.05, Figure 5G) than after 4 weeks. However, the number of microvessels decreased in scaffolds of other mats, including (SF/PCL)1:5/PVA-LBL20 and LBL20 mats (Figure 5F,H−J) at 8 weeks after operation, indicating that newly formed blood vessels were gradually degraded at 8 weeks after surgery. Moreover, microvessel fragments merged into the functional blood vessels in the defect sites covered by LBL20 nanofibrous mats at 8 weeks after surgery (Figure 5H). It is worth mentioning that more vessels were still found in defect sites covered by (SF/PCL)1:5/PVA-LBL20 mats than in those covered by (SF/PCL)1:5/PVA and nonloaded nanofibrous mats (Figure 5F,G,I), suggesting that the time-controlled release of BMP2 and CTGF has an enhancing effect on vessel formation. These results verified that CTGF promoted vessel

CONCLUSION The coordinated temporal release of BMP2 and CTGF using core−shell nanofiber assembly and LBL technology showed excellent improvement in vessel formation and bone tissue recovery. The sustained release of BMP2 enhanced bone formation, while the transient release of CTGF had a proangiogenic effect on bone healing. Therefore, this study underscores the importance of temporal control of growth factor release for treatment of bone defects. 6379

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supernatant was collected and replenished with another 2 mL of fresh PBS solution. The average release profiles of BMP2 and CTGF in the collected supernatant were determined by the ELISA kits at specified time points. Each time point was measured in duplicate, and five different mats were used to evaluate the release profiles of BMP2 and CTGF. Expression of Osteogenesis and Angiogenesis Markers. To determine the osteogenic and angiogenic differentiation of MSCs cultured in the nanofibrous mats, the expressions of alkaline phosphatase (ALP), collagen type I (col-I), and vascular endothelial growth factor (VEGF) in each sample (n = 5) were analyzed by Western blot. Cells were washed with ice-cold PBS 3 times, lysed with a lysis buffer [20 mM Tris-HCl (pH 8.0), containing 1% Triton X100, 150 mM NaCl, 1 mM Na3VO4, 1 mM EDTA, 5% glycerol, 1 mM methanol, and 40 mM ammonium molybdate], homogenized, centrifuged at 14,000 g for 15 min, and the supernatant collected. The protein concentrations were determined by BCA assay. Equal amounts of protein samples in all groups were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% degreased milk in PBS (w/ v) for 1 h, each membrane was incubated at 4 °C for 24 h with one of the following antibodies: anti-VEGF (1:800), anti-Col-I (1:2000), and anti-ALP (1:800). The blots were incubated with antirabbit IgG conjugated with horseradish peroxidase for 0.5 h. The signals of washed blots were detected by enhanced chemiluminescence and exposed to X-ray film. A housekeeping gene (GAPDH) was used as control. In Vivo Osteogenesis and Angiogenesis Assay. To understand the combined effects of CTGF and BMP2 on bone healing, we implanted the nanofibrous mats/cells/CS/β-TCP scaffold compounds subcutaneously in the abdominal midline of nude mice and evaluated the capacity of the transplanted MSCs to form bone and vessel-like tissues in vivo. Before implantation, MSCs were seeded onto the CS/β-TCP scaffolds. After 24 h of seeding, the MSCs/ scaffold compounds were implanted into nude mice subcutaneously and covered by BMP2/CTGF-loaded ((SF/PCL)1:5/PVA-LBL20), BMP2-loaded ((SF/PCL)1:5/PVA), and CTGF-loaded (SF/PCL)1:5/ PVA core−shell nanofibrous mats. The (SF/PCL)1:5/PVA core−shell nanofibrous mats without growth factor loads were used as the empty control (nonloaded mats). Implants were harvested after 7 and 14 days, fixed in 4% formaldehyde at 4 °C for 30 min, embedded in an optimum cutting temperature compound at −20 °C, and sectioned. Immunofluorescent staining for ALP and CD31 was performed on 4 μm-thick sections to identify the new bone and blood vessels, respectively. The sections were incubated with 3% H2O2 for 10 min to inhibit endogenous peroxidase activity after being rinsed in PBS for 5 min, followed by incubating in 1.5% horse serum at room temperature for 20 min to inhibit the nonspecific binding site of the primary antibody. After incubated with goat anti-CD31 and rabbit anti-ALP antibody diluted at 1:50 at 4 °C overnight, the sections were subsequently incubated with secondary antibodies against goat and rabbit IgG at 37 °C for 1 h in the dark, followed by 3 rinses with PBS. The nucleus was counterstained with DAPI. After being visualized and photographed by a confocal laser microscope (Leica LCS Sp2 AOBS MP, Leica), angiogenesis of the cells/scaffold compounds was determined by counting the total number of blood vessels, and CD31positive cells formed in the implants using Image-Pro Plus 6.0. At least five images of nanofibrous mats in each group were taken and analyzed. The neovascularization of the scaffolds was evaluated using the methods described in a previous study:35 the total numbers of vessels on each slide divided by the area of the scaffold as a measure of mean vessel density. Critical-Sized Calvarial Bone Defect Model. To investigate the effects of dual delivery of BMP2 and CTGF on vessel formation and bone regeneration in a critical-size cranial defect, 60 C57BL/6 mice aged 8 weeks were randomly divided into 5 groups. Group 1: (SF/ PCL)1:5/PVA core−shell nanofibrous mats loaded with BMP2 and CTGF ((SF/PCL)1:5/PVA-LBL20 group); group 2: (SF/PCL)1:5/ PVA core−shell structure loaded with BMP2 only ((SF/PCL)1:5/PVA

EXPERIMENTAL SECTION Fabrication of Core−shell Nanofibers by Coaxial Electrospinning and LBL Coating Process. Coaxial electrospinning and an LBL process were performed in sequence shown schematically in Scheme 1. Growth factors were incorporated into nanofibers in three forms: (1) (SF/PCL)/PVA core−shell structure with BMP2 loaded in the core and CTGF attached onto the surface using LBL ((SF/ PCL)1:n/PVA-LBLn), (2) (SF/PCL)/PVA core−shell structure with BMP2 loaded in the core ((SF/PCL)1:n/PVA), and (3) (SF/PCL)/ PVA core−shell nanofibrous mats without core loading and CTGF attached to the surface by LBL (LBLn). To fabricate the core−shell nanofibers, SF and PCL with ratios of 1:2, 1:3, 1:4, 1:5, and 1:6 were dissolved in hexafluoroisopropanol (HFIP) solution and delivered to the outer coaxial needle with a syringe pump. In addition, BMP2 was incorporated into 8% of PVA aqueous solution (10 μg/mL), and the BMP2/PVA solution was transported to the inner needle with another syringe pump. Flow rates in the capillaries were controlled by these two separate pumps. The fluid flow rates of the core and shell solutions were 0.6 and 1.8 mL/h, respectively. A DC voltage of 22 kV and 16 cm distance was applied between the spinneret and the collector (aluminum mesh). The specific parameters defining the electrospinning system were identical for the coaxial electrospinning process. All electrospinning processes were performed at 25 °C and 60% humidity. The LBL process of coating multilayers on the nanofibrous mats was performed according to our previous reports.34 A CS solution (1 mg/mL, pH = 4.5) was selected as the positively charged and a CTGF solution (10 μg/mL) as the negatively charged material. The negatively charged (SF/PCL)1:n/PVA mats were alternatively soaked in the two above-mentioned solutions for 15 min each to form CTGF- and CS-coatings on the surface of the nanofibrous mats. After each layer deposition, the nanofibrous mats were washed 3 times with NaCl solutions (1 mg/mL) for 3 min to remove the residual solution. These coating steps were repeated until the designed number of coating layers was obtained. LBLn labels the number of layers, where n is the number of CS-CTGF bilayers. The outermost layer was CTGF. Characterization of the Coaxial Nanofibrous Mats. The morphology of the nanofibrous mats was analyzed by field emission scanning electron microscopy (FE-SEM, Zeiss Sigma, Germany). The average diameter of nanofibers was determined by image analysis software (Nano Measurer 1.2). The mean pore size of nanofibers was measured by a capillary flow porometer (CFP-1100AI, Porous Materials Inc., USA). Transmission electron microscopy (TEM, JEM-2100, JEOL, Japan) was performed to determine the core−shell structure of the nanofibers. To observe the distribution of the BMP2/ PVA solution in the core−shell electrospun nanofibers, confocal microscopy images (LEICA SP8 STED) were taken to visualize the calcein-labeled BMP2/PVA solutions (green) and the rhodamine Blabeled SF/PCL solution (red). The samples for confocal microscopy analysis were prepared by adding 0.01g rhodamine B into 10 mL of SF-PCL solution and 0.01g calcein into 10 mL of 8% PVA. TEM samples were prepared by placing copper grids between the spinneret and the collector (aluminum mesh) and then depositing a thin layer of coaxial electrospun nanofibers onto the copper grids. To examine the hydrophilicity of the fabricated nanofibrous mats, the dynamic water contact angle was measured by a contact angle analyzer (JGW360B, China). In Vitro Drug Release Study. To determine the most suitable amount of SF in the composite nanofibrous mats, the release profiles of (SF/PCL)1:n/PVA core−shell nanofibrous mats with different ratios of SF and PCL were evaluated by ELISA. After preparation, the electrospun nanofibrous mats were immersed in 2 mL of PBS in 6well tissue culture plates. The cumulative release curve of BMP2 was established by measuring the amount of BMP2 in the collected BMP2-PBS solutions using the ELISA kits following the manufacturer’s instruction. Nanofibrous mat samples with a diameter of 2 cm were immersed in 5 mL of PBS and incubated at 37 °C in a shaking incubator for 1, 3, 6, 10, 15, 20, 25, 30, and 40 days. At predetermined time intervals, the 6380

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ACS Nano group); group 3, (SF/PCL)1:5/PVA core−shell nanofibrous mats loaded with CTGF only (LBL20 group); group 4, nonloaded (SF/ PCL)1:5/PVA core−shell nanofibrous mats (empty control group); and group 5, no implantation (negative control group) (n = 6 for each group). Before implantation, all nanofibrous mats were inoculated with MSCs and cultured for 1 week. After general anesthesia with isoflurane, an incision was made along the midline of the mice skull. After raising the full-thickness skin and the periosteum to expose the calvarial bone, a 4 mm-diameter round defect was created on the left side of the calvarial bone using trephine bur. During drilling, the surgical site was irrigated with 0.9% saline solution, and the integrity of the underlying dura mater was maintained during the whole operation process. After implantation of the cells/mat compounds on each defect, the skin and periosteum were closed in layers with 4−0 prolene sutures. After 4 and 8 weeks of surgery, all animals were perfused with gold nanoparticle (200 mg Au/mice, AuroVist-15 nm, Nanoprobes, USA) according to the manufacturer’s instruction and evaluated by microcomputed tomography (micro-CT) (Scanco Medical AG, Bassersdorf, Switzerland) to quantify blood vessel formation.36 In addition, tissues in defect sites were retrieved and evaluated by Masson’s trichrome staining. The protocol of in vivo experimental procedures was approved by the Wuhan University’s Animal Care and Use Committee.

and immunochemistry. G.C. analyzed all the data and wrote the manuscript. S.J., Q.W., and X.Z revised the manuscript and commented on it. Z.L., H.D., X.S., and Y.D. supervised the project. All the authors participated in the discussion of this study. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China (nos. 51873157, 81800943, and 81771051) and was partially supported by the Natural Science Foundation of Hubei Province of China (no. 2018CFB497) and the Fundamental Research Funds for the Central Universities of China (no. 2042017kf0063). REFERENCES (1) Vo, T. N.; Kasper, F. K.; Mikos, A. G. Strategies for Controlled Delivery of Growth Factors and Cells for Bone Regeneration. Adv. Drug Delivery Rev. 2012, 64, 1292−1309. (2) Yu, Y. Y.; Lieu, S.; Lu, C.; Miclau, T.; Marcucio, R. S.; Colnot, C. Immunolocalization of BMPs, BMP Antagonists, Receptors, and Effectors During Fracture Repair. Bone. 2010, 46, 841−851. (3) Kloen, P.; Lauzier, D.; Hamdy, R. C. Co-Expression of BMPs and BMP-Inhibitors in Human Fractures and Non-Unions. Bone. 2012, 51, 59. (4) Patel, Z. S.; Young, S.; Tabata, Y.; Jansen, J. A.; Wong, M. E. K.; Mikos, A. G. Dual Delivery of An Angiogenic and An Osteogenic Growth Factor for Bone Regeneration in a Critical Size Defect Model. Bone. 2008, 43, 931−940. (5) Shah, N. J.; Hyder, M. N.; Quadir, M. A.; Courchesne, N.-M. D.; Seeherman, H. J.; Nevins, M.; Spector, M.; Hammond, P. T. Adaptive Growth Factor Delivery from a Polyelectrolyte Coating Promotes Synergistic Bone Tissue Repair and Reconstruction. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 12847−12852. (6) Shi-Wen, X.; Leask, A.; Abraham, D. Regulation and Function of Connective Tissue Growth Factor/CCN2 in Tissue Repair, Scarring and Fibrosis. Cytokine Growth Factor Rev. 2008, 19, 133−144. (7) Takigawa, M.; Nakanishi, T.; Kubota, S.; Nishida, T. Role of CTGF/HCS24/Ecogenin in Skeletal Growth Control. J. Cell. Physiol. 2003, 194, 256−266. (8) Kawaki, H.; Kubota, S.; Suzuki, A.; Yamada, T.; Matsumura, T.; Mandai, T.; Yao, M.; Maeda, T.; Lyons, K. M.; Takigawa, M. Functional Requirement of CCN2 for Intramembranous Bone Formation in Embryonic Mice. Biochem. Biophys. Res. Commun. 2008, 366, 450−456. (9) Ono, M.; Kubota, S.; Fujisawa, T.; Sonoyama, W.; Kawaki, H.; Akiyama, K.; Oshima, M.; Nishida, T.; Yoshida, Y.; Suzuki, K.; et al. Promotion of Attachment of Human Bone Marrow Stromal Cells by CCN2. Biochem. Biophys. Res. Commun. 2007, 357, 20−25. (10) Fahmy-Garcia, S.; van Driel, M.; Witte-Buoma, J.; Walles, H.; van Leeuwen, J. P.; van Osch, G. J.; Farrell, E. NELL-1, HMGB1, and CCN2 Enhance Migration and Vasculogenesis, But Not Osteogenic Differentiation Compared to BMP2. Tissue Eng., Part A 2018, 24, 207−218. (11) Ivkovic, S.; Yoon, B. S.; Popoff, S. N.; Safadi, F. F.; Libuda, D. E.; Stephenson, R. C.; Daluiski, A.; Lyons, K. M. Connective Tissue Growth Factor Coordinates Chondrogenesis and Angiogenesis During Skeletal Development. Development 2003, 130, 2779−2791. (12) Babic, A. M.; Chen, C.-C.; Lau, L. F. Fisp12/Mouse Connective Tissue Growth Factor Mediates Endothelial Cell Adhesion and Migration through Integrin αvβ3, Promotes Endothelial Cell Survival, and Induces Angiogenesis In Vivo. Mol. Cell. Biol. 1999, 19, 2958−2966. (13) Kubota, S.; Takigawa, M. Role of CCN2/CTGF/Hcs24 in Bone Growth. Int. Rev. Cytol. 2007, 257, 1−41.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b06032. Figures showing transient expression of CTGF in different kinds of bone defects and expression of BMP2 and CTGF during the osteogenic differentiation of MSCs (Figure S1), the distribution of the fluorescent dyes-labeled growth factors within the coaxial (SF/ PCL)1:5/PVA-LBL20 nanofibers (Figure S2), pore size distribution of the (SF/PCL)1:5/PVA-LBL20 nanofibrous mats (Figure S3), and micro-CT images of cranial defects attached by different kinds of nanofibrous mats (Figure S4). Additional information about experimental materials, isolation method of mouse MSCs, fabrication method of condylar or tibial fracture model, fabrication method of cranial defect model, Western blot analysis, in vivo tracking of the labeled proteins, and results about transient expression of CTGF in bone defects (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]; [email protected]. *E-mail: [email protected]. ORCID

Gu Cheng: 0000-0003-3203-3096 Shan Jiang: 0000-0001-8119-9012 Qun Wang: 0000-0002-5660-5602 Xue Zhou: 0000-0002-8254-9341 Xiaowen Shi: 0000-0001-8294-2920 Hongbing Deng: 0000-0002-9784-6138 Zubing Li: 0000-0002-3499-7684 Author Contributions

Z.L. and H.D. designed the idea of this work. G.C., H.T., and C.X. fabricated the nanofibrous mats and evaluated the characteristics of the mats. C.Y. and X.X. performed in vitro experiments of cells culture, and Western blot. G.C. and C.Y performed in vivo experiments of micro-CT, angiography, Masson’s trichrome staining, immunofluorescence staining, 6381

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Formation in the Rat Arterio-Venous Loop Model. Tissue Eng., Part A 2015, 21, 96−105. (32) Noel, D.; Gazit, D.; Bouquet, C.; Apparailly, F.; Bony, C.; Plence, P.; Millet, V.; Turgeman, G.; Perricaudet, M.; Sany, J. ShortTerm BMP-2 Expression is Sufficient for In Vivo Osteochondral Differentiation of Mesenchymal Stem Cells. Stem Cells. 2004, 22, 74− 85. (33) Lee, C. H.; et al. CTGF Directs Fibroblast Differentiation from Human Mesenchymal Stem/Stromal Cells and Defines Connective Tissue Healing in a Rodent Injury Model. J. Clin. Invest. 2010, 120, 3340−3349. (34) Huang, R.; Li, W.; Lv, X.; Lei, Z.; Bian, Y.; Deng, H.; Wang, H.; Li, J.; Li, X. Biomimetic LBL Structured Nanofibrous Matrices Assembled by Chitosan/Collagen for Promoting Wound Healing. Biomaterials 2015, 53, 58−75. (35) Laschke, M. W.; Schank, T. E.; Scheuer, C.; Kleer, S.; Shadmanov, T.; Eglin, D.; Alini, M.; Menger, M. D. In Vitro Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cell Spheroids Impairs Their In Vivo Vascularization Capacity Inside Implanted Porous Polyurethane Scaffolds. Acta Biomater. 2014, 10, 4226. (36) Hainfeld, J. F.; Smilowitz, H. M.; O’Connor, M. J.; Dilmanian, F. A.; Slatkin, D. N. Gold Nanoparticle Imaging and Radiotherapy of Brain Tumors in Mice. Nanomedicine 2013, 8, 1601.

(14) Konishi, M.; Tabata, Y.; Kariya, M.; Hosseinkhani, H.; Suzuki, A.; Fukuhara, K.; Mandai, M.; Takakura, K.; Fujii, S. In Vivo AntiTumor Effect of Dual Release of Cisplatin and Adriamycin from Biodegradable Gelatin Hydrogel. J. Controlled Release 2005, 103, 7− 19. (15) Xie, Z.; Paras, C. B.; Weng, H.; Punnakitikashem, P.; Su, L. C.; Vu, K.; Tang, L.; Yang, J.; Nguyen, K. T. Dual Growth Factor Releasing Multi-Functional Nanofibers for Wound Healing. Acta Biomater. 2013, 9, 9351. (16) Kim, T. G.; Lee, D. S.; Park, T. G. Controlled Protein Release from Electrospun Biodegradable Fiber Mesh Composed of Poly(Epsilon-Caprolactone) and Poly(Ethylene Oxide). Int. J. Pharm. (Amsterdam, Neth.) 2007, 338, 276−283. (17) Shah, N. J.; Macdonald, M. L.; Beben, Y. M.; Padera, R. F.; Samuel, R. E.; Hammond, P. T. Tunable Dual Growth Factor Delivery from Polyelectrolyte Multilayer Films. Biomaterials 2011, 32, 6183−6193. (18) Deng, Z. J.; Morton, S. W.; Ben-Akiva, E.; Dreaden, E. C.; Shopsowitz, K. E.; Hammond, P. T. Layer-By-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment. ACS Nano 2013, 7, 9571− 9584. (19) He, Y.; Hong, C.; Li, J.; Howard, M. T.; Li, Y.; Turvey, M. E.; Uppu, D. S. S. M.; Martin, J. R.; Zhang, K.; Irvine, D. J.; et al. Synthetic Charge-Invertible Polymer for Rapid and Complete Implantation of Layer-by-Layer Microneedle Drug Films for Enhanced Transdermal Vaccination. ACS Nano 2018, 12, 10272− 10280. (20) Oliveira, S. M.; Santo, V. E.; Gomes, M. E.; Reis, R. L.; Mano, J. F. Layer-By-Layer Assembled Cell Instructive Nanocoatings Containing Platelet Lysate. Biomaterials 2015, 48, 56−65. (21) Morton, S. W.; Poon, Z.; Hammond, P. T. The Architecture and Biological Performance of Drug-Loaded LbL Nanoparticles. Biomaterials 2013, 34, 5328−5335. (22) Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Biomedical Applications of Layer-By-Layer Assembly: from Biomimetics to Tissue Engineering. Adv. Mater. 2006, 18, 3203−3224. (23) Crouzier, T.; Szarpak, A.; Boudou, T.; Auzély-Velty, R.; Picart, C. Polysaccharide-Blend Multilayers Containing Hyaluronan and Heparin as a Delivery System for rhBMP-2. Small 2010, 6, 651. (24) Wang, Y.; Wang, B.; Qiao, W.; Yin, T. A Novel Controlled Release Drug Delivery System for Multiple Drugs Based on Electrospun Nanofibers Containing Nanoparticles. J. Pharm. Sci. 2010, 99, 4805. (25) Mickova, A.; Buzgo, M.; Benada, O.; Rampichova, M.; Fisar, Z.; Filova, E.; Tesarova, M.; Lukas, D.; Amler, E. Core/Shell Nanofibers with Embedded Liposomes as a Drug Delivery System. Biomacromolecules 2012, 13, 952. (26) Rambhia, K. J.; Ma, P. X. Controlled Drug Release for Tissue Engineering. J. Controlled Release 2015, 219, 119−128. (27) Zhang, Y.; Wang, X.; Feng, Y.; Li, J.; Lim, C.; Ramakrishna, S. Coaxial Electrospinning of (Fluorescein Iisothiocyanate-Conjugated Bovine Serum Albumin)-Encapsulated Poly (ε-Caprolactone) Nanofibers for Sustained Release. Biomacromolecules 2006, 7, 1049−1057. (28) Yang, Y.; Xia, T.; Zhi, W.; Wei, L.; Weng, J.; Zhang, C.; Li, X. Promotion of Skin Regeneration in Diabetic Rats by Electrospun Core-Sheath Fibers Loaded with Basic Fibroblast Growth Factor. Biomaterials 2011, 32, 4243−4254. (29) Canalis, E.; Zanotti, S.; Smerdel-Ramoya, A. Connective Tissue growth Factor is a Target of Notch Signaling in Cells of the Osteoblastic Lineage. Bone. 2014, 64, 273−280. (30) Huang, J.; Wang, D.; Chen, J.; Liu, W.; Duan, L.; You, W.; Zhu, W.; Xiong, J.; Wang, D. Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Magnetic Nanoparticle Composite Scaffolds Under a Pulsed Electromagnetic Field. Saudi Pharm. J. 2017, 25, 575−579. (31) Buehrer, G.; Balzer, A.; Arnold, I.; Beier, J. P.; Koerner, C.; Bleiziffer, O.; Brandl, A.; Weis, C.; Horch, R. E.; Kneser, U.; et al. Combination of BMP2 and MSCs Significantly Increases Bone 6382

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