SilkâHydroxyapatite Nanoscale Scaffolds with Programmable Growth

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Silk-Hydroxyapatite Nanoscale Scaffolds with Programmable Growth Factor Delivery for Bone Repair Zhaozhao Ding, Zhihai Fan, Xiaowei Huang, Qiang Lu, Weian Xu, and David L Kaplan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08180 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 2, 2016

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ACS Applied Materials & Interfaces

Silk-Hydroxyapatite Nanoscale Scaffolds with Programmable Growth Factor Delivery for Bone Repair Zhaozhao Dinga, Zhihai Fanb,#, Xiaowei Huangc, Qiang Lua, c, *, Weian Xua, David L Kaplan d a

School of Biology and Basic Medical Sciences & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China b

Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000, People’s Republic of China

c

National Engineering Laboratory for Modern Silk, Soochow University, Suzhou 215123, People’s Republic of China

d

Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States

# The author has same contribution with the first author *Address corresponding to [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Osteoinductive biomaterials are attractive for repairing a variety of bone defects and biomimetic strategies are useful towards developing bone scaffolds with such capacity. Here, a multiple biomimetic design was developed to improve the osteogenesis capacity of composite scaffolds consisting of hydroxyapatite nanoparticles (HA) and silk fibroin (SF). SF nanofibers and water-dispersible HA nanoparticles were blended to prepare the nanoscaled composite scaffolds with a uniform distribution of HA with a high HA content (40%), imitating the extracellular matrix (ECM) of bone. Bone morphogenetic protein-2 (BMP-2) was loaded in the SF scaffolds and HA, respectively, to tune BMP-2 release. In vitro studies showed the preservation of BMP-2 bioactivity in the composite scaffolds, and programmable sustained release was achieved through adjusting the ratio of BMP-2 loaded on SF and HA, respectively. In vitro and in vivo osteogenesis studies demonstrated that the composite scaffolds showed improved osteogenesis capacity under suitable BMP-2 release conditions, significantly better than that of BMP-2 loaded SF-HA composite scaffolds reported previously. Therefore, these biomimetic SF-HA nanoscaled

scaffolds

with

tunable

BMP-2

delivery

provide

microenvironments for bone regeneration.

KEYWORDS: Silk; Hydroxyapatite; Bone repair; Drug delivery; Biomimetic.

2


preferable

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1. INTRODUCTION Reconstruction of large bone defects remains a challenge worldwide, resulting in a significant cost to society.1 Traditional approaches for bone healing include autografts and allografts, both of which have inherent limitations such as low availability of suitable materials, donor site morbidity, immune responses, and possible infection and disease transmission.2,3 These shortcomings prompted the development of new bone tissue engineering strategies inspired by endogenous bone healing mechanisms.4 Due to the critical role of the extracellular matrix (ECM) in the maintenance and remodeling of bone, current bone tissue engineering strategies are centered on the formation of an osteoconductive microenvironment by mimicking the natural extracellular matrix (ECM).5-7 A preferred bone scaffold can provide osteo-inductive support for bone progenitor cells, and also act as a delivery system for different growth factors, providing a controlled environment with enhanced bone healing capacity.8 Therefore, both the selection of biomaterials, and the design and fabrication of the scaffold structure are crucial for successful development of the above preferred bone scaffolds.9 Silk fibroin (SF), a natural biomaterial, is a promising candidate for fabricating bone scaffolds due to its characteristics such as biocompatibility, suitable mechanical properties, low immunogenicity, and controllable degradation behavior. 10-12 SF also has potential as a carrier for controlled release of growth factors.13-15 Different fabrication processes have been developed to control microstructure, morphology, as well as the mechanical properties of SF-based scaffolds to mimic the native physiological structure and environment of bone tissue.16-18 Unfortunately, SF is not 3



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inherently osteoconductive and therefore it is difficult to induce osteogenic behavior of bone marrow derived mesenchymal stem cells (BMSCs) unless additional osteoinductive factors are added. Thus, the design of SF-hydroxyapatite (HA) composite scaffold systems was pursued, with the advantage that the components in combination could improve osteogenic differentiation of BMSCs.19,20 Further studies have been developed to prepare SF-based delivery systems for growth factors as well as SF-based scaffolds for bone regeneration, suggesting their promising clinical future as bone scaffolds.21-23 Few studies, however, have focused on the fabrication of silk-based scaffolds with a combination of complex bone niche which could achieve ECM-biomimetic nanofibrous structures, similar levels and types of organic/inorganic components, and a suitable release behavior of growth factor. In our recent study, water-dispersible HA nanoparticles were prepared with SF as template and surface stabilizer.24 The HA nanoparticles were coated by SF to form core-shell structures suitable for drug delivery. Then a facile approach was developed to load BMP-2 in the nanoparticles, achieving improved loading efficiency and better sustained release when compared with pure SF or pure HA nanocarriers.25 Furthermore, we developed porous SF scaffolds with extracellular matrix (ECM) mimetic nanofibrous structures via a modified lyophilization method and further induced the SF-coated HA nanoparticles into the nanofibrous scaffolds with a homogeneous distribution at the nanometer scale.26 Both the drug delivery system and the nanofibrous SF/HA composite scaffolds showed improved osteogenic differentiation of BMSCs when compared to the SF-based bone matrices reported previously.27 4


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The goal in the present study was to exploit the nanofibrous SF/HA scaffolds and the water-dispersible HA nanoparticles to build biomaterial systems for optimized bone repair. Further, bioactivity could be endowed to the system through tuning the delivery of BMP-2. We suggest that the current system provides an improved osteogenic microenvironment due to the multiple points of simulation of natural bone ECM. A significant promotion in the osteogenesis of BMSCs was demonstrated in vitro and in vivo using these SF/HA composite systems with optimized BMP-2 delivery behavior.

2. EXPERIMENTIAL SECTION: 2.1 Preparation of aqueous SF solutions. Bombyx mori silk solutions were prepared based on previously published procedures.28-30 First, sericin proteins were extracted when silks were boiled in 0.02 M Na2CO3 solution for 20 min. Then the aqueous solutions were prepared through dissolving the extracted silks in 9.3 M LiBr solution at 60oC for 4 h and dialyzing against distilled water for 72 h. The solution was further centrifuged at 9,000 rpm for 20 min at 4oC to remove silk aggregates and to generate optically clear SF solutions with final concentration of about 6 wt%. 2.2 Fabrication of SF-coated HA nanoparticles An aqueous precipitation reaction was reported previously and used to prepare SF-coated HA nanoparticles with SF as template and surface stabilizer.31 Homogeneous silk nanoparticles were generated when incubating the fresh SF solution (6 wt%) at 60oC for 24 h (Fig S1). Then 20 mL of SF nanoparticle 5



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solution was mixed with 20 mL H3PO4 solutions (0.06 M). The mixed solution was drop-wise added to 100 mL Ca(OH)2 solution (0.02 M) at 90 mL h-1 with vigorous stirring. A suitable content of 0.1 M NaOH was added to adjust the pH of the reaction system. The reaction was maintained in a water bath at 70oC and emulsion solutions were obtained. The SF-coated HA nanoparticles were obtained after centrifugation at 9,000 rpm for 20 min and gently washing treatment with distilled water. 2.3 Fabrication of SF nanofibers The

SF

nanofibers

were

assembled

from

fresh

SF

solution

via

a

concentration-dilution process as reported in our recent study.32,33 Metastable nanoparticles were assembled after slow concentration from 6 wt% to about 20 wt% from the solution (6 wt%) over 24 h at 60oC. The nanoparticle solutions were diluted to 0.5 wt% with distilled water and incubated for above 24 h at 60oC to induce the nanofiber formation. 2.4 Preparation of BMP-2 loaded SF/HA composite scaffolds Porous SF/HA composite scaffolds were prepared using a modified freeze-drying method.26 The fresh SF solution and the SF nanofiber solution were blended at a dry weight ratio (SF: SF nanofiber) of 88:12, and then adjusted to 2 wt% with distilled water. The HA nanoparticles at a dry weight of ratio (silk: HA) of 60:40 were added to the blend SF solution under stirring to get a uniform mixture. After transferred into plastic dishes, the mixture was frozen in a freezer at -20oC 6


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overnight to solidify the solvent, and then lyophilized for 48 h. The water-insoluble

composite

scaffolds

were

directly

prepared

after

the

lyophilization without post-treatment. The preparation process of BMP-2-loaded scaffolds is shown in Scheme 1. BMP-2 was directly added to the SF/SF nanofiber blend solution. The loading of BMP-2 on HA particles was processed according to our previous method.25 After incubating 1 mL 15 µg/mL BMP-2 solution (Ruibang Company, Shanghai, China) with 1.5 mg of SF-coated HA nanoparticles at 4°C for 24 h, and then centrifuging the mixture at 12,000 rpm for 3 min, BMP-loaded nanoparticles with a loading efficiency of 99.6% was achieved. Then, the BMP-2-loaded SF solution, BMP-2-loaded HA particles and BMP-2 free HA particles were blended at a dry weight of ratio (silk: HA) of 60:40 to prepare BMP-2-loaded scaffolds according to the above scaffold fabrication process. When all the BMP-2 was loaded on the SF matrices and HA particles, respectively, the scaffolds formed were termed S-100, and H-100. If 50% of BMP-2 was loaded on the silk matrices while the rest was loaded on HA particles, the scaffolds formed were termed S-50. As a control, BMP-2 free SF/HA composite scaffolds were also prepared through the same process, and termed as S-0. 2.5 Characterization The morphology of the scaffolds was observed with a scanning electron microscope (SEM) (S-4800, Hitach, Tokyo, Japan) at a voltage of 3.0 kV. The

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samples were measured after gold sputter coating treatment. Fourier transform infrared spectroscopy (FTIR, Nicolet 5700, Thermo Scientific, FL, America) was used to analyze the structural properties of the scaffolds. X-ray diffraction (XRD, Nano ZS90, Malvern instruments, Malvern, UK) was conducted to analyze the crystal structure. The X-ray generation conditions were 30 mA and 40 kV, and data were obtained at diffraction angles from 10 to 60o, with a step size of 0.02o and a scanning speed of 6o/min. 2.6 BMP-2 release. For BMP-2 release studies, 5 mg of BMP-2 loaded scaffolds were placed into 1 mL PBS solution (pH 7.4) and cultured in a thermostatic shaker at 37oC. At predetermined time points, the supernatant was taken out and measured using a BMP-2 ELISA kit (Biovision, San Francisco, CA). Fresh PBS buffer (1 mL) was replenished into the release medium. The measurement was repeated in triplicate with different samples. 2.7. In vitro cytocompatibility of the scaffolds. The in vitro cytocompatibility of the scaffolds was assessed with BMSCs derived from Sprague-Dawley (SD) rats. Small scaffold disks with diameter of 5 mm and height of 2 mm were prepared and sterilized through 60Co γ-irradiation at the dose of 25 kGy. BMSCs were cultured in Dulbecco’s modified Eagle medium (DMEM, low glucose)

supplemented

with

10%

fetal

bovine

serum

(FBS),

100U/ml

penicillin-streptomycin (all from Invitrogen, Carlsbad, CA) with 5% CO2, at 37℃ 8


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temperature. A density of 1.4×105 cells per well was used to seed the cells into the scaffolds. Cell proliferation was analyzed by DNA content assay on days 1, 3, 6, 9 and 12. Samples were cultured with proteinase K overnight at 56oC to digest the scaffolds.29 The DNA content was achieved using the PicoGreenTM DNA assay (Invitrogen, Carlsbad, CA) and measured with a BioTeK Synergy 4 spectrofluorometer (BioTeK, Winooski, VT). The excitation wavelength and emission wavelength were 480 nm and 530 nm, respectively. A standard curve prepared with λ-phage DNA in 10×10-3 M Tris-HCl (pH 7.4), 5×10-3 M NaCl, 0.1×10-3 M EDTA over a range of concentrations was also obtained to calculate the amount of DNA. Confocal laser scanning microscope (CLSM, Olympus FV10 inverted microscope, Nagano, Japan) and SEM were used to observe cell morphology on the scaffolds. The samples for CLSM and SEM measurement were prepared according to the protocols reported previously in our group.25, 34 Different areas of the samples were randomly examined by SEM and CLSM. Representative results are presented. 2.8 In vitro cell differentiation on the scaffolds. Alkaline phosphatase (ALP) assays, bone-related gene expression, calcium content, and immune-fluorescence staining were performed to evaluate the osteogenic differentiation of BMSCs on the scaffolds after the seeded BMSCs (5×105 cells per well) cultured in osteogenic differentiation medium (low

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glucose-DMEM, 10% FBS, 1% streptomycin-penicillin, 10 nM dexamethasone, 10 mM sodium-β-glycerophosphate, and 0.05 mM ascorbic acid-2-phosphate) for 3, 7, 14, and 21 d, respectively. Two different bone-related gene expression markers, Runx2 (an early marker of osteogenic maturation)

35-37

and osteocalcin (later

marker of osteogenic differentiation and mineralization), 38,39 were measured to assess the various differentiation states of BMSCs. All the measurements were processed according to the protocols that have been reported in our recent studies.24, 25 2.9 In vivo study The use of SD rats in this experiment was approved and granted by the animal ethics committee of Soochow University. Twelve 8-week old male rats (Animal Resource Center, Soochow University) were used. Four subcutaneous pockets were created along the central line of the shaved dorsal area with approximately 2 cm apart. Two different scaffolds (S-100 scaffold and H-100 scaffold) were used in the in vivo study. After seeded with cells and cultured for 7 days, the scaffolds were implanted into the pockets. Each individual pocket had one scaffold. At weeks 4 and 8 post-implantation, animals were euthanized and the implanted scaffolds along with the adjacent tissues were collected. For further examination, the specimens were fixed in 4% paraformaldehyde in PBS at pH 7.4.40 2.10 Histology and immunohistochemistry

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After washing in PBS and fixing in 10% formalin, constructs were decalcified in 10% EDTA at pH 7.4 for 1 month at 4oC. The samples were then embedded in paraffin, sectioned into 4µm slices and stained with haematoxylin and eosin (H＆ E) for further observation. Immunohistochemistry staining for osteocalcin (OCN) was used and prepared as previously described.41 2.11 Statistical analysis. SPSS v.16.0 software was used for statistical analyses. Two-way ANOVA followed by the Student-Newman-Keuls test (SNK q-test) was used to compare the mean values of the data. Unless otherwise specified, measures are shown as means ± standard deviations, and p< 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1 Design and fabrication of SF/HA scaffolds for tunable BMP-2 delivery Porous SF scaffolds have promising potential toward bone tissue regeneration due to their biocompatibility, maintenance of nutrient transport and mechanical properties.42 However, SF alone lacks osteoinductivity, thus the addition of osteoinductive features is preferred to improve bone regeneration. Unlike silk, HA, the mineral phase of bone, has excellent osteoconductivity and was incorporated together with SF to achieve composite scaffolds with porous structure and improved osteoinduction.43-45 Another efficient method of enhancing repair of bone defect is to load BMP-2 inside SF-based scaffolds.46 Although the BMP-2 could be released in a sustained manner, it remains a challenge to actively tune the delivery rate to match bone tissue repair. Hence, a new strategy was 11



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developed to mimic the microenvironment of bone tissue at multiple scales to optimize bone defect healing. Our recent study showed that SF could be assembled into nanofibers after lyophilization through adding SF nanofibers with high beta-sheet content as inducers.47 The lyophilized scaffolds were water-insoluble without further treatment. BMP-2 was loaded on SF nanofibers and water dispersible SF-coated HA nanoparticles, respectively (Scheme 1). The BMP-2-loaded SF and HA particles were blended directly to prepare water-insoluble SF/HA composite scaffolds via a freeze-drying process. High bioactivity of BMP-2 was maintained in the scaffolds due to the mild, aqueous process that avoids the use of organic solvents.47 Based on our previous study,31 the HA content in the scaffolds was fixed to 40% to achieve uniform distribution in SF matrices at the nanometer scale. Through adjusting the ratios of BMP-2 loaded on SF and HA, respectively, the delivery behavior was tuned to optimize the osteoinductive capacity of the scaffolds.

Scheme 1. BMP-2 loading and release processes of SF/HA composite scaffolds. 3.2 Characterization of SF/HA scaffolds SF/HA composite scaffolds with and without BMP-2 were prepared via a lyophilization process. Surface and cross-section images of the scaffolds were analyzed to assess the 12


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pore structure and HA distribution. Due to the existence of SF nanofibers, all the SF/HA scaffolds showed good porous structures and nanoscale structures on the pore surface. Cross-section images of pore walls further indicated that the HA particles were homogeneously distributed in SF scaffolds at the nanometer scale, providing a favorable microenvironment for bone regeneration (Figure 1A). Unlike previous methanol-treated SF scaffolds with high beta-sheet content,42 the present SF/HA scaffolds exhibited two main peaks located at 1654 cm-1 and 1627 cm-1, which are attributed to silk I and silk II, respectively. Compared to the peak at 1627 cm-1, the peak at 1654 cm-1 showed higher intensity for all the SF/HA scaffolds, suggesting that these scaffolds are mainly composed of silk I/random structures rather than silk II.27 Another typical peak appeared at 1035 cm-1, which is attributed to P-O bond vibration of HA nanoparticles (Figure 1B). The XRD patterns of the SF/HA scaffolds also displayed specific HA peaks, which corroborated the FTIR results (Figure 1C). Similar mechanical properties appeared among the BMP-2 loaded scaffolds with the compressive strength of 40-46 kPa (Fig S2) without significant difference, which was lower than the silk-HA scaffolds reported previously due to the amorphous state of silk.31 The mechanical properties could be further improved through tuning secondary conformations of silk and would be realized in our following study.32 Therefore, the BMP-2 loading in SF matrices and HA particles had no negative influence on the formation of porous structure or secondary conformation of the SF/HA scaffolds, suggesting the feasibility of the scaffolds as effective drug delivery platform while maintaining suitable morphology and structural features related to material strength. 13



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Figure 1. SEM images (A), FTIR spectra (B) and XRD curves (C) of the silk/HA composite scaffolds. For the images in (A): (a-g) showed the microporous structures of the S-0, S-100, S-50 and H-100 samples while (b-h) indicated the distribution of the HA nanoparticles on the porous walls of the S-0, S-100, S-50 and H-100 samples, respectively.

In (B) and (C): a, b, c and d were S-0, S-100, S-50 and H-100 samples,

respectively. 3.3 Tunable BMP-2 delivery in vitro BMP-2 delivery is an efficient method to enhance the repair of bone defects.49-51 However, the local delivery of BMP-2 is usually limited due to the loss of bioactivity over a short time and undesirable side effects at high doses.52 Although different carriers including scaffolds, hydrogels and nanoparticles have been developed to achieve sustained release of BMP-2,53,54 challenges remain to restrain the initial burst release and effectively tune the released behavior to optimize the therapeutic efficiency of BMP-2. SF-coated HA nanoparticles prepared here exhibited better loading efficiency and sustained delivery than pure SF and HA nanoparticles reported previously,8,17 and achieved improved osteogenesis of BMSCs. The BMP-2 release behavior from the

14


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SF-coated HA nanoparticles was different from that from silk scaffolds, which suggested that more complex and tunable release behaviors could be designed when the two carriers were integrated into a single system. Figure 2 shows the in vitro release curves of BMP-2 loaded in the different components of the SF/HA composite scaffolds. When all the BMP-2 was loaded in SF scaffolds, a typical initial burst release appeared due to the BMP-2 located primarily on the external surface of the SF scaffold. The burst release was partly restrained when 50% of BMP-2 was loaded in SF scaffolds, and then disappeared after all the BMP-2 was loaded on SF-coated HA nanoparticles. The sustained release behavior of BMP-2 appeared for all the SF/HA scaffolds for over 21 days. Noticeably, the release kinetics of BMP-2 was strongly dependent on the ratio of drugs loaded on SF and HA, respectively. The BMP-2 was released within the first 10 days, and thereafter delivered slowly for the S-100 scaffolds, while nearly zero-order release kinetics were observed for the H-100 scaffolds. An intermediate release behavior could be achieved for the S-50 scaffolds. All of these results indicated that the delivery of BMP-2 could be tuned by changing the drug distribution on the SF and HA components, suggesting the possibility of creating a better microenvironment for bone healing.

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Figure 2. BMP-2 release profiles from the different silk/HA composite scaffolds: (A) the release behavior of BMP-2 within 24 hours, (B) the sustained release of BMP-2 for 3 weeks.

Figure 3. The cytocompatibility of the scaffolds in vitro: (A) Confocal microscopy images and SEM images of BMSCs cultivated on day 1 and day 12 on different silk/HA composite scaffolds. The arrows point to the cells; (B) BMSCs proliferation behavior on different silk/HA composite scaffolds on day 1, 3, 6, 9, 12. 3.4 In vitro cytocompatibility of the scaffolds BMSC attachment and proliferation behavior were used to evaluate cell responses on the BMP-2 loaded SF/HA scaffolds. As control, the BMP-2 free SF/HA scaffolds (S-0) were used to assess the influence of BMP-2 loading on cytocompatibility. Figure 3 shows typical cell growth on the different scaffolds. The BMSCs grew well on the surfaces of the scaffolds from day 1 to day 12. At day 1, BMSCs attached and distributed evenly on all the scaffolds with a spread morphology. Then cells increased and formed continuous monolayers on the scaffolds by day 12 (Figure 3A), indicating good cell proliferation. DNA results indicated that cell numbers increased up to 12 days 16


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without reaching a plateau, confirming cytocompatibility of the SF/HA scaffolds (Figure 3B). Compared to the BMP-2 free scaffolds (S-0), all the BMP-2 loaded scaffolds showed similar cell attachment and proliferation behavior. Therefore, the in vitro cell culture results indicated that the drug loading processes had no negative influence on the cytocompatibility of the scaffold system. 3.5 Osteogenic differentiation of BMSC in vitro The different SF/HA scaffolds showed good cytocompatibility in vitro and were subsequently used in cellular differentiation. The expression of collagen type I, a marker for osteogenic differentiation, was measured through immunofluorescence staining. The highest expression of collagen I appeared in the H-100 scaffolds possibly due to suitable BMP-2 release behavior inside the scaffolds, confirming the osteoinductivity in our present SF-HA scaffold systems (Figure 4A). The level of ALP activity and Runx2 protein expression, two key events during the early time points of osteogenesis,35-37,55,56 were examined to assess the osteogenic differentiation of BMSC on these scaffolds. Significantly higher ALP activity of cells was observed on the BMP-loaded scaffolds than those on the BMP-free scaffolds, suggesting the osteogenic activity of the loaded BMP-2. The ALP activity was further enhanced following the adjustment of BMP-2 release rate, achieving the highest expression level in the MSCs cultured on the H-100 scaffolds (Figure 4B). Similar Runx2 expression results were observed, which also showed the highest expression in the MSCs cultured on the H-100 scaffolds (Figure 4C). These results suggested that the osteogenic differentiation of the MSCs could be improved through tuning the release behavior of the BMP-2. Both the ALP activity and 17



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Runx2 expression in MSCs for all the scaffolds exhibited a similar trend that increased and peaked on day 7 and then decreased on days 14 and 21, similar to some studies reported previously.55-57 The results implied that subsequent stages of bone regeneration such as osteogenic maturation and bone formation happened after 7 days,57 which was supported by significant and sustained increase in levels of osteocalcin and calcium content in MSCs from 1 to 21 days for all the scaffolds. Similar to the ALP activity and Runx2 expression results, significantly higher calcium mineralization and osteocalcin expression were observed in the BMP-2 loaded scaffolds than in the BMP-2 free scaffolds (Figure 4D, E). The calcium content and osteocalcin expression also increased following the adjustment of BMP-2 release rate, with the highest results in the H-100 scaffolds. It is not surprising that with the loading and sustained release of BMP-2 from the SF-HA scaffolds an enhancement of BMSC differentiation toward bone was observed. A critical role of BMP-2 in inducing osteogenic differentiation of BMSCs has been explored and reported in many previous studies.58 As BMP-2 carriers, HA and SF materials have shown superior capability to maintain the activity and sustained release of BMP-2.8 Although the influence of release behaviors of BMP-2 on the osteogenic differentiation of BMSCs has been revealed,59 few studies achieved active control of the BMP-2 release for optimizing osteogenic capability of the systems. Unlike previous studies,26,31 BMP-2 was loaded on SF matrices and HA nanoparticles, respectively, in our present system. The release behavior could be easily tuned through changing the BMP-2 ratios loaded on the SF and HA. It is worth noting that more studies are still 18


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needed to further tune the BMP-2 release behavior based on the system for optimized outcomes since we can’t make sure that the H-100 scaffolds are the best candidate for the bone regeneration. An advantage of the present SF-HA scaffold system is that it provides a promising system for actively designing BMP-2 sustained release behaviors and then achieving optimal matrices for bone regeneration, which remains a challenge for other BMP-2 loading systems. Although it seems difficult to create similar BMP-2 loaded system with other biocompatible materials such as gelatin and collagen, the mechanical and hierarchical tunability of silk materials suggests a possibility of further improvement of silk-based bone regeneration system,34 which might also be superior to other biomaterials.

Figure 4. Osteogenic differentiation of BMSCs on the different scaffolds: (A) Immunofluorescence staining for collagen type I of BMSCs cultivated on day 3, 7, 14, and 21. Alkaline phosphatase activity (B), mRNA levels of RUNX2-related transcription factor (C), Calcium quantification (D) and osteocalcin (E) quantified by real-time polymerase chain reaction (*≤0.05,**≤0.01,***≤0.001). 3.6 In vivo response In order to distinguish the osteoconductive and osteoinductive capacities, ectopic bone 19



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formation model rather than bone defect model was used to further confirm the improved osteoinductive potential of the BMP-2-loaded SF-HA scaffolds. The H-100 scaffolds with best osteogenic capability in vitro were implanted ectopically beneath the skin of rats. The S-100 scaffolds, with a similar composition and BMP-2 release behavior with the SF-HA scaffolds reported previously,25 were also implanted beneath the skin of rats as control. All animals used in this experiment survived and were healthy after 4 and 8 weeks, without evident inflammatory or immune responses. The H-100 scaffolds showed significantly enhanced ectopic bone formation compared to the S-100 scaffolds. HE stained images of both types of scaffolds are shown in Figure 5. At the early implanted stage (4 weeks), both of the S-100 and H-100 scaffolds induced bone formation with viable osteocytes. Some new blood vessels also appeared inside S-100 and H-100 scaffolds, which would further facilitate the mineralization and maturation of the new bone. After 8 weeks of implantation, bone formation was clear with the signs of active cubic osteoblasts inside S-100 scaffolds, suggesting their osteoinductive potential due to the existence of HA and sustained released BMP-2 in the scaffold systems. Compared to the S-100 scaffolds, the H-100 scaffolds demonstrated more newly formed bone (Figure 5B). Significant positive staining of OC was observed on H-100 scaffolds after 4 and 8 weeks post-implantation, indicating better osteogenesis capacity and bone repair ability than the S-100 scaffolds. The superior osteoinducivity of the H-100 scaffolds should be attributed to optimizing BMP-2 release behavior from the scaffolds since both of the scaffolds have the same silk/HA composition, nanostructure and BMP-2 load. 20


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Both HA and SF have been studied as BMP-2 delivery carriers for more than 10 years.8,25,60 The sustained release of BMP-2 was achieved for these carriers, endowing improved osteogenesis capacity. However, there is still lack of methods for designing BMP-2 delivery behavior actively for optimization of bone repair. Here, different ratios of BMP-2 were loaded in the SF scaffolds and HA nanoparticles, respectively, to tune BMP-2 delivery and also improve bone repair ability of the scaffolds. Although the amount of ectopic bone formation looks minor, the present study provides a promising strategy for creating preferred microenvironments for bone repair. Further studies including bone defect model (microCT and histology) will be continued to assess clinical possibility of our present silk-HA composite scaffolds system.

Figure 5. The osteogenic capacity of the different scaffolds when they were implanted ectopically beneath the skin in vivo. (A) Haematoxylin and eosin staining of the S-100 and H-100 scaffolds at 4, 8 weeks post-implantation in SD rats and (B) immunohistochemistry staining of osteocalcin. Red arrows point to osteoblast, while black arrows point to blood vessels. NB indicated new bone.

4. CONCLUSIONS A new HA nanoparticle-embedded SF scaffold system for tunable BMP-2 delivery was

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developed. BMP-2 retained bioactivity and achieved sustained release behavior through adjusting the ratio of loaded BMP-2 on SF and HA. In vitro and in vivo osteogenesis studies revealed the critical influence of BMP-2 release behavior on osteogenesis of the scaffolds. Compared to the BMP-2-loaded SF-HA scaffold system reported previously, the present scaffolds provide improved bone repair ability, suggesting promising application in bone tissue engineering. ACKNOWLEDGMENTS The National Basic Research Program of China (973 Program 2013CB934400), NSFC (21174097) and the NIH (R01 DE017207) supported this work. We also thank the Excellent Youth Foundation of Jiangsu Province (BK2012009), the Natural Science Foundation of Jiangsu Province (Grants No BK20140397) and the second affiliated hospital of Soochow university preponderant clinic discipline group project funding (NO.XKQ2015010) for support of this work.

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