BMP-2 encapsulated polysaccharide nanoparticle modified biphasic calcium phosphate scaffolds for bone tissue regeneration Zhenming Wang,1 Kefeng Wang,2 Xiong Lu,1 Minqi Li,3 Hongrui Liu,3 Chaoming Xie,1 Fanzhi Meng,4 Ou Jiang,4 Chen Li,1 Wei Zhi1 1

Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, China 2 National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan, China 3 Department of Bone Metabolism, School of Stomatology Shandong University, Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan, China 4 The Second People’s Hospital of Neijiang, Sichuan, China Received 5 February 2014; revised 26 June 2014; accepted 18 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35282 Abstract: Bone morphology protein-2 (BMP-2) encapsulated chitosan/chondrotin sulfate nanoparticles (CHI/CS NPs) are developed to enhance ectopic bone formation on biphasic calcium phosphate (BCP) scaffolds. BMP-2 contained CHI/CS NPs were prepared by a simple and mild polyelectrolyte complexation process. It does not involve harsh organic solvents and high temperature, and therefore retain growth factors activity. These NPs were immobilized on BCP scaffolds, and realize the sustained release of growth factors from the scaffolds. The bare BCP scaffolds, NP loaded scaffolds (BCPNP), and NP loaded and polydopamine coated scaffolds (BCP-Dop-NP) were seeded with bone marrow stroma cells (BMSC) to evaluate the osteoinductivity of the scaffolds. BMSC culture results indicate that all scaffolds favor cell adhesion, proliferation, differentiation. Afterwards, the bare BCP, BCP-NP, and BCP-Dop-NP scaffolds were implanted into rabbits intramuscularly to evaluate the ectopic bone forma-

tion of scaffolds. In vivo results indicate that the BCP-NP and BCP-Dop-NP scaffolds enhance more ectopic bone formation than the bare BCP scaffolds. Both the in vitro and in vivo results demonstrate that BMP-2 encapsulated polysaccharide NPs are effective to improve the osteoinductivity of the scaffolds. In addition, BCP-NP scaffolds induce more bone formation than BCP-Dop-NP scaffolds. This is because BCP-NP scaffolds harness the intrinsic osteoinductivity BCP and BMP2, whereas BCP-Dop-NP scaffolds have polydopamine coatings that inhibit the surfaces biological features of BCP scaffolds, and therefore weaken the bone formation ability of C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: scaffolds. V 00A:000–000, 2014.

Key Words: chitosan, chondroitin sulfate, nanoparticles, BMP-2, biphasic calcium phosphate scaffolds, bone tissue regeneration

How to cite this article: Wang Z, Wang K, Lu X, Li M, Liu H, Xie C, Meng F, Jiang O, Li C, Zhi W. 2014. BMP-2 encapsulated polysaccharide nanoparticle modified biphasic calcium phosphate scaffolds for bone tissue regeneration. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

Biphasic calcium phosphate (BCP) is the mixture of hydroxlapatite (HA) and b-tricalcium phosphate (b-TCP). It has been widely used as bone repairing materials because of its intrinsic bioactivity and osteoconductivity.1 Porous BCP scaffolds have highly interconective macroporous structures that are suitable for vascularization, nutrient delivery, and bone ingrowth.2–4 However, the osteoinductivity of bare BCP scaffolds is not good enough to induce bone regeneration, which limits their applications to repair large bone defects

in orthopedic surgery. One of the efficient approaches to enhance the osteoinductivity of BCP scaffolds is to load bone morphology proteins (BMPs).5–7 BMP-2 is one of the most effective members of BMP family for enhancing bone formation.8–10 However, BMP-2 has short half-life in vivo.11,12 Direct application of BMP-2 to the fracture sites might result in rapid dilution by body liquid and enzymatic digestion, and therefore decrease the activity of BMP-2. Direct application might also lead to burst release of BMP-2 and cause unfavorable side effects such as

Correspondence to: X. Lu; e-mail: [email protected] Contract grant sponsor: 863 Program Contract grant sponsor: NSFC; contract grant numbers: 31070851, 51202152, 81271965 Contract grant sponsor: Program for New Century Excellent Talents in University; contract grant number: NCET-10-0704 Contract grant sponsor: Sichuan Youth Science-Technology Foundation; contract grant number: 2011JQ0010 Contract grant sponsor: Construction Program for Innovative Research Team of University in Sichuan Province; contract grant number: 14TD0050

C 2014 WILEY PERIODICALS, INC. V

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FIGURE 1. Scheme of the preparation process of BMP-2 encapsulated CHI/CS nanoparticles and immobilization of the nanoparticles on porous BCP scaffolds for the study of ectopic bone formation. CHI: chitosan; CS: chondroitin sulfate; BCP-NP: nanoparticles immobilized BCP scaffolds; BCP-Dop-NP: nanoparticles immobilized and polydopamine coated BCP scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

significant tissue inflammation and cyst-like bone.13 Therefore, appropriate BMP delivery systems that insure controlled release over a prolonged period is necessary for the success of BMP in clinics. Hitherto, several delivery systems have been developed to control the release of BMP, such as b-TCP scaffolds,14 poly-L-lactic acid (PLLA) scaffolds,15 chitosan (CHI) and hyaluronan hydrogels,16 heparin-functionalized poly lactic-co-glycolic acid (PLGA) nanoparticles (NP)-hydrogel complex,17 PLGA nanospheres contained fibrin gel,18 and polyelectrolyte multilayer films.19 Recently, it is reported that NP or microsphere loaded scaffolds could realize sustained release of growth factors and drugs. Hasirci and coworkers20 designed PLGA nanoparticle-grafted porous CHI scaffolds by a soaking method for BMP-2 release in controlled rates. Niu et al.21 developed CHI microsphere contained porous HA/collagen/PLLA scaffolds by a post-seeding technique, which release BMP-2-derived synthetic peptide in a sustained manner for few months. Son et al.22 produced PLGA microsphere loaded porous HA scaffolds via electrostatic interactions to control the long-term release of dexamethasone. To satisfy the diverse requirements of various clinical applications, new systems are necessary to be developed so as to control the release and protect the activity of BMP in a long-term period. Polysaccharide-based NPs by polyelectrolyte complexation are able to encapsulate growth factors at a high loading rate, realize sustained release of growth factors, and protect growth factors from enzymatic degradation.23 CHI/heparin and heparinized CHI/poly(g-glutamic acid) nanoparticles have been employed to delivery fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) for enhancing ischemic tissue regeneration and vascularization.24,25 CHI and CS are commonly used polycation and polyanion to form polysaccharide-based NPs, respectively. CHI is a cationic polysaccharide, which has been used as delivery carriers of therapeutic proteins due to its good biocompatibility and biodegradability.26 CS is an anionic polysaccharide that is structurally similar to the other glycosaminoglycan such as heparin. CS and heparin are capable of electrostatically interacting with positively charged growth factors, including BMP, VEGF, and FGF.25,27–30 Recent studies have shown that growth factors have long-term and desirable

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release characteristics if they are encapsulated into CS-based self-assembled films or nanocomplexes.31–33 In this study, CH/chondrotin sulfate nanoparticles (CS NPs) were developed to delivery BMP-2 on porous BCP scaffolds, and consequently confer strong osteoinductivity on the scaffolds. The CHI/CS NPs were prepared by a simple and mild polyelectrolyte complexation process. It does not involve harsh organic solvents and high temperature and therefore retain growth factors activity. The CHI/CS NPs protect BMP-2 from directly exposing to body fluid, shielding them from deactivation until release, and realizing the sustained release of BMP-2. BMP-2 encapsulation efficiency, size distribution and degradation behavior of NPs were characterized. The BMP-2 encapsulated CH/CS NPs were loaded on porous BCP scaffolds to enhance the osteoinductivity of the scaffolds. The NPs were also immobilized on BCP scaffolds that were coated with polydopamine films. The polydopamine films act as barrier layers so as to inhibit the surfaces characteristics of BCP. Thus, the bone formation of polydopamine-modified BCP scaffolds could be mainly induced by BMP-2 encapsulated NPs. The bare BCP scaffolds, NPs loaded BCP scaffolds and NPs loaded BCP scaffolds with polydopamine films were implanted into rabbits intramuscularly to evaluate the osteoinductivity of the scaffolds in vivo. The experiment process is illustrated in Figure 1. MATERIALS AND METHODS

Materials Low molecular weight CHI (low molecular weight, deacetylation degree 98%), chondroitin 4-sulfate sodium salt from bovine trachea, lysozyme, dopamine hydrochloride, bovine serum albumin (BSA), fluorescein-5-isothiocyanate (FITC), calcein green, and diaminobenzidine (DAB) were purchased from Sigma-Aldrich (USA). Recombinant human bone morphogenetic protein-2 (BMP-2) was purchased from Yu Bo biological technology Ltd, Shanghai, China. Enzyme-linked immunosorption assay (ELISA) kits were purchased from R&D). The BCA assay kits and ALP Assay Kits were purchased from (Nan Jing Jian Cheng, China). Fetal bovine serum (FBS), a-MEM, and 1% penicillin2streptomycin solution were purchased from HyClone. Medium 199 from

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GIBCO. Anti-collagen I antibody was acquired from Abcam. Horseradish peroxidase-conjugated secondary antibody came from DaKo (Glostrup, Denmark). All other regents and solvents were of reagent grade. Preparation and characterization of porous BCP scaffolds The porous BCP scaffolds were made by a foaming method using hydrogen peroxide (H2O2) as the forming agent. The detail of processing was described in our previous publication.34 The weight ratio of HA to b-TCP is 7:3. All specimens were cut into discs with the diameter of 9.6 mm and with the thickness of 3–4 mm. The morphology of scaffolds was observed using a scanning electron microscope (SEM; JSM 6390, JEOL, Japan). The specific surface areas of the BCP scaffolds were measured by applying the Brunauer-EmmettTeller equation to multipoint isotherms of N2 at 77 K using a Quadrasorb SI (Quantachrome Instruments) instrument. Compression tests were performed to measure the mechanical properties of the BCP scaffolds with a universal mechanical testing machine with a 2 kN load cell (Instron 5567). The samples were cylinders with the diameter of 9.6 mm and a height of 18 mm. The crosshead speed was set at 0.5 mm/min, and the load was applied until the sample was compressed to 70% of its original height. The compressive strength was determined by the maximum stress divided by the cross-sectional area. The reported data were the average of six samples. Preparation of NPs BMP-2-loaded CHI/CS NPs were prepared using a polyelectrolyte complexation method. First, CHI solution (2.5 mg/mL) was formed by dissolving CHI in acetic acid (1.0%, v/v) and adjusting pH to 5.0 with NaOH. Then, BMP-2 solution (1.0 lg/lL in PBS, 60 lL) was blended into the CS solution (2.5 mg/mL, 5 mL) with stirring to form BMP-2-CS complexes. Finally, the complexes were added dropwise into the CHI solution (10 mL) under magnetic stirring for 15 min to form CHI/CS NPs. The NPs were collected by centrifugation at 15,000 rpm for 15 min. After centrifugation, supernatant was discarded and NPs were resuspended in DI water for further studies. The BMP-2 concentration in the supernatant was assayed by the ELISA kits. The BMP-2 loading efficiency was determined by the ratio of the encapsulated amount to the initial amount of BMP-2. BSA-loaded CHI/CS NPs were also prepared by a similar process because BSA was used as the model protein for the release study. CS/BSA mixtures (CS 2.5 mg/mL, BSA 0.5 mg/mL, 10 mL) were added into the CHI solution (2.5 mg/mL, 20 mL) to form BSA-loaded CHI/CS NPs. Blank CHI/CS NPs without protein encapsulation were prepared in the same way, which was used as control group. To prepare FITC-CHI/CS NPs, CHI was labeled with FITC according to the method described in our previous publications.35 Characterization of NPs The mean particle size, polydispersity index (PDI), and zeta potential of the NPs were determined by dynamic light scat-

tering with a laser particle analyzer (ZETA-AIZER, Malvern, UK). All samples were tested in triplicate, and the mean values and standard deviations were calculated. The morphology of NPs was examined with the SEM (JSM 6390). The samples for SEM observation were prepared by dropping 10 lL of NP suspension on a silicon wafer and dried at an ambient atmosphere. FITC-CHI/CS NPs were observed by a fluorescence microscope (DMIL, Leica, Germany). The degradation of CHI/CS NPs was evaluated in PBS with or without lysozyme. The lysozyme concentration of 0.1 mg/mL was chosen to mimic the in vivo physiological conditions.36,37 Immobilization of NPs on BCP scaffolds Two types of BCP scaffolds were used: bare BCP scaffolds and polydopamine coated BCP scaffolds. For bare BCP scaffolds, NPs was firstly dispersed in DI water to form suspension with the concentration of 1.1 mg/mL. Then BCP scaffolds were incubated in 1 mL of NP suspension under mild agitation at 37 C, until the solution dried out. Each scaffold contained 1.1 mg of NPs, which incorporate 3.6 lg of BMP-2. These samples were named as BCP-NP scaffolds. Distribution of NPs in the scaffolds was visualized with the SEM (JSM 6390) and a confocal laser-scanning microscope (CLSM, TCSSP5, Lecia, Germany). For polydopamine coated BCP scaffolds, polydopamine films were coated on the surfaces of BCP scaffolds before NP loading. Dopamine hydrochloride was first dissolved in Tris-HCl (10 mM, pH 8.5) to form Tris-dopamine solution (2.0 mg/mL). Then the bare BCP scaffolds were incubated in the Tris-dopamine solution for 24 h and protected from light. The color of the Tris-dopamine solution changed to dark due to pH-induced oxidation of dopamine. After soaking, the scaffold were washed three times and then dried under vacuum. The resulting products were further treated with glutaraldehyde solution (1.0%, w/v) at room temperature for 12 h to crosslink the amino groups of polydopamine, washed with water, and dried in vacuum. NPs were loaded on the polydopamine coated BCP scaffolds in same route as described above. The samples were denoted as BCP-Dop-NP scaffolds in the subsequent discussion. All samples for in vivo implantation were prepared under sterile conditions. Protein release The BSA release from CHI/CS NPs was determined to study of the kinetics of protein release from NPs. The BSA encapsulated NPs (2.0 mg/mL) were incubated in 10 mL of PBS (pH 7.4) at 37 C under continuous shaking. At predetermined intervals, 500 lL of the release medium was collected and equal amount of fresh PBS was added to the release medium. The amount of released BSA in the collect medium was measured using the BCA assay kit. The absorbance at 562 nm was measured on a microplate reader (MQX200, BioTEK). For protein release kinetics of the scaffolds, BSA-loaded CHI/CS NPs were immobilized on the BCP and BCP-Dop scaffolds as described above. Then, the scaffolds were soaked in 2 mL of PBS. At specific intervals, 1 mL of NP the release medium was collected and a similar

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amount of fresh PBS was added to each sample. In addition, bare BCP scaffolds with directly adsorbed BSA were prepared as the control group. The BSA release from the scaffolds is also determined by the BCA assay kit. All of the experiments were repeated three times. In vitro study Bone marrow stromal cells (BMSC) were cultured on the scaffolds to evaluate their cytocompatibility. BMSC were extracted from one-week-old Sprague–Dawley rats as described in our previous study.38 BMSC (passage 4) were seeded on the scaffolds at a density of 1 3 105 cells/scaffold and cultured in a-MEM supplemented with 10% FBS and 1% penicillin2streptomycin solution at 37 C in a 5% CO2 incubator. For cell attachment study, the cells on the scaffolds were washed twice with PBS, and then fixed with 2.5% glutaraldehyde for 4 h at room temperature after 2 days of incubation. The cells were then subjected to step dehydration with a graded series of ethanol/water solutions (30, 70, 90, 100, and 100%) for 10 min each step. Finally, the cells were dried by critical point drying and goldsputtered prior to SEM observation. Cell proliferation was evaluated by the Alamar Blue assay after 3 and 7 days of culture. First, the culture medium in the 24-well plate was replaced with Alamar blue regents (0.8 mL of Medium 199 without phenol red supplemented with 10% FBS and 10% Alamar Blue). After 4 h incubation, reagents were carefully transferred to 96-well plates. The optical density was measured at 570 and 600 nm against a mediumblank Alamar Blue using the microplate reader (MQX200). Cell differentiation was evaluated by the alkaline phosphatase activity (ALP) assay. After 7 and 14 days of culture, the medium was removed from the wells. Subsequently, the cells on the scaffolds were washed twice with PBS and lysed with 0.8 mL of Triton X-100 (1.0%, v/v). The lysate was centrifugated and the supernatant was employed for ALP activity determination. The final ALP activity was normalized with respect to the total protein content obtained from the same cell lysate. The total protein concentration of the cell lysate of each sample was measured using the BCA Kit. ALP activity was measured using the ALP Assay Kit. In each case, four specimens were tested and the assay was repeated two times. In vivo study Three types of scaffolds, including bare BCP, BCP-NP, and BCP-Dop-NP, were implanted into rabbits intramuscularly to evaluate the osteoinductivity of the scaffolds in vivo. Each group contains six parallel samples. The experiments were performed in accordance with protocols approved by the local ethical committee and laboratory animal administration rules of China. Six Japanese big-ear white rabbits, 2month old, weighing 3 kg, were purchased from a local breeder. The surgical procedure was performed under general sterile conditions. For intramuscular implantation, the rabbits were anesthetized by an intravenous injection of 2.5 wt % pentobarbital sodium with a dose of 1 mL/kg body weight. After the skin was shaved and sterilized with iodine,

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the longitudinal skin incisions were made using blunt dissection near the spine. The length and depth of the incision into the muscle was about 10 and 5 mm, respectively. Each sample was placed in each pouch, three scaffolds per animal. Subsequently, the fascia and skin of the rabbits were sutured. At the end of operation, penicillin with a dose of 400,000 U per rabbits was administered by intramuscular injection once a day during the first postoperative 3 days. After 3 month implantation, all rabbits were sacrificed with air embolism, and the scaffolds with their surrounding tissue were retrieved. All the retrieved samples were fixed in formalin (10%, v/v) for 7 days and then demineralized in EDTA-2Na solution (10%, v/v) for 4 weeks at 4 C. After dehydration through a graded ascending ethanol series, the tissues were embedded in paraffin. Then serial 5-mm-thick sections were prepared for subsequent histological analysis. Hematoxylin and eosin (H&E) and Masson’s trichrome stainings were performed to investigate the ectopic osteogenesis ability of different scaffolds. The stained sections were observed and then digital images were taken with a light microscope (Olympus 603, Japan). With the aid of Image Pro Plus software (Media Cybernetics), newly formed bone volume ratio, expressed as a percentage (area of newly formed bone/area of scaffold pores 3 100%) was measured. Specifically, three tissue sections were selected from each sample and three images of each section were analyzed to obtain the mean value. Ultimately, the newly formed bone volume of each group was expressed as the average level of the three samples implanted at the same time. Immunohistochemistry staining of collagen type I was performed referring to a published study.39 Briefly, the dewaxed paraffin sections were pretreated with 0.3% hydrogen peroxidase for 30 min to inhibit the endogenous peroxidase activity and then treated with 1% BSA in PBS for 20 min. Following that, they were incubated for 2 h at room temperature with anticollagen I antibody (1:200 dilution). After being rinsed with PBS, the sections were incubated with the horseradish peroxidase-conjugated secondary antibody (1:100 dilution) for 1 h at room temperature. The immunoreaction was visualized with DAB. Staining was assessed by a light microscope (Olympus 603) after faintly counterstained with methyl green. Image Pro Plus (Media Cybernetics) was used to measure the integrated optical density (IOD) of collagen type I. As supplementary, the bone formation rate at the middle time-point (6 weeks) was determined using calcein green as the fluorochrome mark. The rabbits received the injection of calcein green (10 mg/kg) intramuscularly before sacrifice. The fluorochrome marks were observed using the CLSM (TCSSP5). RESULTS

Characterization of BCP scaffolds SEM micrograph reveals that porous BCP scaffolds have uniform and highly interconnected macropores [Fig. 2(a)]. Figure 2(b,c) show the scaffolds during surgical operation and after implantation, respectively. The pore size ranges from

BMP-2 ENCAPSULATED POLYSACCHARIDE NP MODIFIED BCP SCAFFOLDS

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Characterization of CHI/CS NP NPs were produced by polyelectrolyte complexation of positively charged CHI with negatively charged CS at certain weight ratios. Table I shows the effect of CHI/CS weight ratios on the particles size, zeta potential values, PDI, and yield of the NPs. The mean particle size of NP ranges from 482 to 698 nm. The PDI ranges from 0.193 to 0.175. All the NPs have positive zeta potential (130 to 144 mV). The increase of weight ratio of CHI to CS leads to an increase of particle surface charges. This is because the amount of positively charged CHI significantly exceeds that of negatively charged CS, and therefore some of the excessive CHI molecules are entangled on the surfaces of the NPs. The NPs that were prepared with the weight ratio of 2:1 has the highest yield, and good dispersity, which were used for the rest of the study. The BMP-2 loading efficiency of the CHI/CS NPs is 91%. SEM micrograph reveals that the CHI/CS NPs are spherical, and the average size is about 400 nm [Fig. 3(a)]. The particle size from SEM is smaller than that from the particle analyzer. This is because dried polysaccharide NPs are observed under SEM, whereas NPs in aqueous solution are analyzed by the particle analyzer.40,41 In water, CHI/CS NPs are hydrophilic and swell, and therefore present a large hydrodynamic size. The fluorescence microscope image reveals the good dispersion of FITC-grafted NPs [Fig. 3(b)]. Degradation of NPs and protein release from NPs The degradation of CHI/CS NPs in PBS proceeded slowly during the whole degradation period. As shown in Figure 4(a), about 85% of its initial mass remained after 6 weeks of incubation in PBS. With the introduction of lysozyme, the weight loss of CHI/CS NPs increased. Only 75% of particle weight remained. The fast degradation rate with the existence of lysozyme is due to the accelerated degradation of the CHI, which ultimately destroys the compact structure of NPs. The release tests reveal that the protein in the CHI/CS NPs exhibits sustained release manner in PBS [Fig. 4(b)]. In the initial 6 days of incubation, the cumulative released BSA from CHI/CS nanoparticles is 44.0 6 5.0% and 47.1 6 3.8% with and without lysozyme, respectively. In the subsequent 15 days, about 1% of BSA is released per day. In the later period, the release rate slows down. The total cumulative release is 64.8 6 0.9% and 70.1 6 1.4%. FIGURE 2. (a) SEM micrograph of porous BCP scaffolds; (b) BCP scaffolds in the intramuscular pouch during surgery operation; (c) The implanted scaffolds retrieved after 12 week implantation. More tissue is observed on BCP-NP and BCP-Dop-NP scaffolds than on bare BCP scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

50 to 500 lm. The porosity of the scaffolds is about 81% as determined by a water replacement method. The compressive strength of the scaffolds was 2.22 6 0.34 MPa. The specific surface area of the BCP scaffold is 0.59 m2/g and the mass of each scaffold is 0.2 g; 3.6 mg of BMP is loaded on each scaffold, which is calculated by the loading efficiency of NPs and described in the subsequent section. Thus, the BMP concentration on the scaffold surface is 3.05 ng/cm2.

NP loaded BCP scaffolds CLSM image reveals that FITC-NPs distribute uniformly throughout the whole scaffolds [Fig. 5(a)]. SEM micrograph TABLE I. Particle Sizes, Zeta Potentials, and Yield of Nanoparticles Prepared with Different CHI/CS Weight Ratios in Deionized Water (n 5 3) Chi:CS Weight Ratio (w/w) 2.0:1.0 4.0:1.0 8.0:1.0

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Mean Particle Size (nm)

Zeta Potential (mv)

PDI

Yield

698.7 6 24.3 30.6 6 0.4 0.193 6 0.012 44.6 6 2.1% 606.7 6 30.6 35.1 6 1.3 0.185 6 0.026 30.6 6 1.8% 482.8 6 9.0 44.1 6 1.3 0.175 6 0.036 12.3 6 2.3%

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results indicate that the number of BMSC increases on all three types of scaffolds during the culture period, which reveals that all scaffolds have no cytotoxicity and favor cell proliferation [Fig. 7(d)]. BMSC on BCP-NP and BCP-Dop-NP scaffolds have significantly higher proliferation rate than those on bare BCP scaffolds. ALP activity test results indicate that ALP activity increase on all three types of scaffolds during the culture period, which reveals that all scaffolds favor cell differentiation [Fig. 7(e)]. The ALP activity of BMSCs on NP-immobilized BCP scaffolds is higher than that on bare BCP scaffolds. In summary, BMSC culture results indicate that all scaffolds favor cell adhesion, proliferation, and differentiation. The BMP encapsulated NPs loaded BCP scaffolds provide a favorable environment for proliferation and differentiation of BMSC, and the released BMP-2 plays a key role in the early stage of osteogenetic differentiation. Ectopic bone formation Fluorochrome analysis indicates that bone mineralization happens as early as 6 weeks after implantation. The calcein green that were injected at 6 weeks is visible in all implants (Fig. 8). Bare BCP scaffolds are only weak stained and abundant cancellated fibrous tissue fills in the ceramic pores [Fig. 8(a)]. In contrast, BCP-NP and BCP-Dop-NP scaffolds both are heavily stained and present dense lamellar bone

FIGURE 3. (a) SEM micrograph of BMP-2 encapsulated CHI/CS NPs; (b) Fluorescence microscope image of FITC labeled CHI/CS NPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

reveals that certain amounts of NPs adhere on the edge of pore walls of the scaffold [Fig. 5(b)], whereas others locate in the center of pores [Fig. 5(c)]. The BSA in BCP-NP and BCP-Dop-NP scaffolds release in a sustain manner (Fig. 6). The release kinetics of BSA from BCP-NP scaffolds shows linear profiles (36.9 6 3.0%) in the initial 6 days, and a sustained release of 0.5% per day in the subsequent period. A similar release pattern was observed in BCP-Dop-NP scaffolds. In contrast, BSA that is directly adsorbed on the BCP scaffolds release relative fast. About 71.6 6 5.1% of BSA is release in the initial 6 days. These results indicate that BSA encapsulated CHI/CS NPs on BCP scaffolds can achieve sustained release of BSA, compared with direct adsorption. In vitro assay BMSC were cultured on the scaffolds to evaluate the cytocompatibility of the scaffolds. SEM observation shows that BMSC could attach and spread well on all three types of scaffolds after 2 days of incubation [Fig. 7(a–c)]. BMSC on the scaffolds present flat and spindle shape, and attach tightly on the scaffold surfaces, which suggests that all scaffolds support cell adhesion and spreading. Alamar blue test

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FIGURE 4. (a) The degradation of CHI/CS NPs in PBS with and without lysozyme (0.1 mg/mL); (b) The release of BSA from CHI/CS NPs.

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H&E staining results reveal that different amount of new bone forms on three groups of scaffolds that are retrieved after 12 week implantation. The bare BCP scaffolds have a small amount of newly formed bone that is spread in a few scaffolds pores [Fig. 9(a)]. The newly formed bone islands have woven structures, and are distributed in separate pores [Fig. 9(b,c)]. These results indicate that the BCP scaffold itself has a limited degree of osteoinductivity, which is not enough to induce abundant bone formation in a short term. In contrast, the BCP-NP scaffolds demonstrate extensive newly formed bone that is filled in the most of scaffolds pores [Fig. 9(d,e)]. The nearby new bone islands integrate to form a whole bone area. The higher magnified image reveals that primary lamellar bone is mineralized [Fig. 9(f)]. These results reveal that the BCP-NP scaffolds induce a large amount of new bone formation, suggesting that the bone regenerative capacity of NP modified BCP scaffolds are much higher than that of bare BCP scaffolds. The BCP-NP scaffolds having superior osteoinductivity than the bare BCP scaffolds are attributed to the sustained release of BMP-2 from CHI/CS NPs. In the BCP-Dop-NP scaffolds, moderate amount of newly formed bone together with a certain number of empty scaffold pores are observed [Fig. 9(g–i)]. Statistical analysis suggests that the bone volume ratio of the BCP-NP scaffolds is the highest, and that of the bare BCP scaffolds is the lowest among three groups (Fig. 10). Masson’s trichrome staining was performed to further explore bone maturity among various groups. Generally, primary bone mainly made up of collagen type I is be stained into blue while highly mineralized bone is stained into red. As shown in Figure 11, the bare BCP scaffolds present many empty pores, and only a small amount of blue stained bone tissue appears [Fig. 11(a)]. In the BCP-NP group, primary bone (blue) and highly mineralized bone (red) both are frequently observed [Fig. 11(b)]. In the BCP-Dop-NP scaffolds, abundant primary bone (blue) presents while little mature bone (red) is detected [Fig. 11(c)]. In summary,

FIGURE 5. (a) FITC labeled CHI/CS NPs uniformly distribute on the BCP scaffold as revealed by CLSM; (b) SEM micrograph of CHI/CS NPs on the edge of the BCP scaffold; (c) CHI/CS NPs in the central of the BCP scaffold. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

[Fig. 8(b,c)]. These results indicate that the osteoinductivity of BCP scaffolds is further enhanced by the loading of BMP2 encapsulated CHI/CS NPs.

FIGURE 6. BSA as the model protein to study protein release from various BCP scaffolds in PBS at pH 7.4. In BCP scaffolds, BSA was directly absorbed on the surface. In BCP-NP and BCP-Dop-NP scaffolds, BSA was encapsulated in CHI/CS NPs. BSA release was quantified using BCA Kits and normalized to the total protein on each scaffold.

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FIGURE 7. Attachment and spreading of BMSC on bare BCP (a), BCP-NP (b), and BCP-Dop-NP (c) scaffolds after 2 days of incubation. (d) Cell proliferation of BMSC on the scaffolds after 3 and 7 days of incubation. (e) ALP activity of BMSC on the scaffolds after 7 and 14 days of incubation. * Indicates the significant difference (p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

Masson’s trichrome staining demonstrates that BMP-2 functionalized BCP scaffolds induce higher and more advanced mineralization. This could be attributed to a sustained release of BMP-2 from CHI/CS NPs, which results in an early bone formation and maturation. Note that the bone formation in the BCP-Dop-NP scaffolds is less than that in the BCP-NP scaffolds, which suggests that polydopamine coatings weaken the osteoinductivity of the BCP scaffolds. The collagen type I activity was detected by immunohistochemistry at 12 week post implantation to evaluate of new bone quality in different groups. In the bare BCP group, the collagen type I immunoreactivity is faintly detected only in certain individual areas [Fig. 12(a)]. In the BCP-NP scaffolds, it is highly expressed and the positive areas are wide spread in almost all pores [Fig. 12(b)]. In the BCP-Dop- NPs groups, the expression level of collagen type I is moderate. It is strongly expressed in several pores but hardly detected in the other ones [Fig. 12(c)]. Statistical analysis reveals that IOD value of collagen type I of the BCP-NP scaffolds is the highest, and that of the bare BCP scaffolds is the lowest among the three groups (Fig. 13), which corroborates the histological findings.

DISCUSSION

Repair of large osseous defects or critical-sized bone defects is still a challenge in the fields of orthopedic surgery. Present therapeutic approaches include autologous and artificial bone grafting. Although autogenous bone

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grafting is considered as the gold standard in the treatment of bone defects and nonunions, it is associated with donor site complications and the limitations of harvested bone.42,43 Therefore, developing artificial bone substitutes is critical for orthopedic surgery. The success of artificial bone substitutes depends on multiple factors. The scaffolds for mechanical support and the growth factors for regulating vascularization and bone regeneration are two important aspects that need to be paid attention.44 In this study, BCP scaffolds decorated with BMP-2 contained CHI/ CS NPs were developed, which exhibit strong osteoinductivity and could potentially serve as bone substitute materials. These BCP scaffolds have macrosized pores with good interconnectivity that favors bone ingrowth. BMP-2 is encapsulated in CHI/CS NPs, which preserve the activity of BMP-2 and realize the long-term sustained release of BMP-2. Protein release tests reveal that protein encapsulated in CHI/CS NPs exhibit sustained release over 40 days, whereas protein that is directly absorbed scaffolds shows a high release rate. The long term release of protein could be explained by the slow degradation of the CH/CS NPs and the stable immobilization of the CHI/CS NPs on BCP scaffold surfaces. First, the release of protein from the NPs is controlled by degradation of polysaccharides.37,45 The degradation in PBS with and without lysozyme shows that the NPs have slow degradation rate and long-term stability. The slow stability of NPs are originated from the strong interaction between CHI and CS, which leads to high crosslinking

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FIGURE 8. CLSM image of bone formation after 6 week implantation. The new bone is labeled by using calcein green as the fluorochrome mark. Bare BCP scaffolds (a) are only weak stained and abundant cancellated fibrous tissue fills in the pores; BCP-NP scaffolds (b) and BCP-Dop-NP scaffolds (c) both are heavily stained and present dense lamellar bone. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

density of polysaccharide NPs, and therefore precludes penetration and accessibility of body liquid and enzyme to the encapsulated protein. Second, the immobilization of the NPs is based on the electrostatic interaction between BCP scaffold surfaces and NPs. BCP scaffolds are negatively charged at physiologic pH due to the presence of phosphate components.22,46 The NPs have positive zeta potential in the range of 125 to 140 mv because excessive positively charged CHI molecules are entangled onto the surfaces of NPs. The results of rabbit ectopic model indicate that the bare BCP scaffolds can induce bone formation to a certain extent, which could be ascribed to the high porous structures and the intrinsic osteoinductivity of BCP.45 However, BMP-2 encapsulated NPs enhance the osteoinductivity of BCP scaffolds, and induce more mature and highly mineralization bone. The high osteoinductivity of NP loaded scaffolds is mainly from the local release of BMP-2 that diffuses into the interconnected inner pores of porous BCP scaffolds, which increases the concentration of BMP-2 in the micropores. Thus, osteogenetic cells that contact BCP scaffolds are exposed to effective BMP-2 concentration, which leads cells to proliferate, differentiate, and form bone tissue that infiltrate into the micropores.47 It should be noted that the efficacy of BMP is highly dependent on the loading method although incorporation of BMP to scaffolds generally enhances the osteogenetic properties of scaffolds.6 Direct physical adsorption is a simple approach to load BMP on scaffolds. Luvizuto et al.14 direct adsorbed BMP-2 on b-TCP scaffolds and evaluated their bone formation in critical-sized calvaria defects. The results show that BMP-2 fails to enhance bone formation. Lan Levengood et al.48 adsorbed BMP-2 to gelatin microparticles and incorporated these microparticles into the macropores of BCP scaffolds; the results show that BMP does not significantly influence the new bone volume fraction after the scaffolds were implanted in the ramus of hemi-mandible of pigs. Those negative results might be attributed to the burst release of BMP-2 in the early stage and therefore the lack of BMP-2 in the later stage of implantation. However, there are also reports that direct physical adsorption of BMP on porous scaffolds indeed promotes bone formation.49,50 In these reports, a high dose of BMP-2, ranging from tens to hundreds microgram, is used. Such a high dose might result in side effects, such as soft tissue swelling and high cost of surgery.13 Other loading methods have been developed to realize the sustained release of BMP and reduce the dose of BMP-2 in order to lessen the risk of side effects and high cost of surgery. Crouzier et al.19 employed polyelectrolyte multilayer films as rhBMP-2 carrier, which were coated on macroporous TCP/HA granules to induce ectopic bone formation. Jun et al.51 designed silica xerogel-CHI hybrids as a coating material to incorporate BMP-2 on porous HA scaffolds for bone tissue engineering. These study demonstrates that appropriate BMP-2 loading methods lead to significantly more bone volume and bone coverage in the defect site, even with low dose of BMP.18,52 Our results are consistent with those previous reports. BCP scaffolds loaded with

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FIGURE 9. H&E staining images of various scaffolds retrieved after 12 week implantation. The new bone matrix is stained red via H & E. (a–c): Bare BCP scaffolds; (d–f): BCP-NP scaffolds; (g–i): BCP-Dop-NP scaffolds. Original magnification: (a, d, g) 340; (b, e, h) 3100; (c, f, i) 3400; Low magnification images show a small amount of newly formed bone (a), extensive and moderate amount of new bone (d, g) fills in the pores of scaffolds. High magnification images show evidence of new bone with woven structures (b, e, h). Higher magnified images reveal primary lamellar bone in the pores of the scaffolds (c, f, i). m: material; nb: new bone. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

low dose of BMP-2 (3.6 lg/scaffold) induce ectopic bone formation in 3 month implantation, which further confirms that a long-term sustained release of BMP from scaffolds favors bone formation.

FIGURE 10. Quantitative evaluation of newly formed bone on various scaffolds after 12 week implantation. * indicates the significant difference (p < 0.05).

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One interesting finding of this study is that the bone formation of the BCP-Dop-NP scaffolds is not as good as that of the BCP-NP scaffolds, which could be ascribed to the polydopamine coating on the BCP-Dop-NP scaffolds. The polydopamine coating is stable and not easy to be degraded. It acts as an impermeable cap, preventing the release of Ca21 and PO32 from 4 BCP scaffolds.53 Thus, the intrinsic osteoinductivity of BCP is precluded and the bone formation of the BCP-Dop-NP scaffolds is only dependent on the BMP-2 release from CHI/CS NPs. Previous study also showed that the polymer coatings on calcium phosphate (CaP) scaffolds inhibit the surfaces biological features of CaP scaffolds and weaken the bone formation ability of scaffolds.19,22 However, the BCP-NP scaffolds directly bind BMP-loaded NPs on surfaces, and therefore harness both the bioactive surface characteristics of BCP scaffolds and the BMP in the NPs. Our results further emphasize the important role of BCP in inducing bone formation, and demonstrate that an optimum delivery system should combine the advantages of both growth factors and scaffolds. CONCLUSIONS

These results indicate that CHI/CS NPs are effective carriers for BMP-2 release. They have high protein loading efficiency

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and strong ability to preserve the activity of growth factors. These NPs can be loaded on BCP scaffolds uniformly throughout the whole scaffolds, and realize the sustained release of growth factors from the scaffolds. The in vivo results indicate that scaffolds with BMP-2 encapsulated NPs induce more ectopic bone formation than bare BCP scaffolds, which indicates that BMP-2 encapsulated NPs are

FIGURE 11. Masson’s trichrome staining of different groups: (a) BCP, (b) BCP-NP, and (c) BCP-Dop-NP scaffolds. Masson’s trichrome stains bone matrix collagen into blue, and mineralized mature bone into red. (a) In bare BCP scaffolds, many pores are empty and only a small amount of blue stained bone tissue presents; (b) in BCP-NP scaffolds, primary bone (blue), and highly mineralized bone (red) both is frequently observed; (c) in BCP-Dop-NP scaffolds, abundant primary bone (blue) presents whereas little mature bone (red) is detected. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 12. Immunohistochemical staining for collagen type I on deparaffinized sections of different groups: (a) BCP scaffolds, (b) BCPNP scaffolds, and (c) BCP-Dop-NP scaffolds. Extensive immunoreactive area (brown) is observed in (b). In (a, c), only little scattered positive areas are detected, as indicated by the blue arrows. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

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FIGURE 13. Quantification of collagen type-I activity after 12 weeks of implantation. * Indicates the significant difference (p < 0.05).

effective to improve the osteoinductivity of the scaffolds. In addition, the BCP-NP scaffolds induce more bone formation than the BCP-Dop-NP scaffolds. This is because the BCP-NP scaffolds combine the osteoinductivity of both BMP-2 and BCP, whereas the BCP-Dop-NP scaffolds have polydopamine coatings that inhibit the surfaces biological features of BCP scaffolds, and therefore weaken the bone formation ability of scaffolds. These results suggest that the scaffolds that harness both the intrinsic osteoinductivity of the BCP and growth factor could better induce good bone formation. The BCP scaffolds with BMP-encapsulated polysacharide NPs is a promising artificial bone substitutes at clinical applications. REFERENCES 1. Roohani-Esfahani S, Lu Z, Li J, Ellis-Behnke R, Kaplan D, Zreiqat H. Effect of self-assembled nanofibrous silk/polycaprolactone layer on the osteoconductivity and mechanical properties of biphasic calcium phosphate scaffolds. Acta Biomater 2012;8: 302–312. 2. Song G, Habibovic P, Bao C, Hu J, van Blitterswijk CA, Yuan H, Chen W, Xu H. The homing of bone marrow MSCs to nonosseous sites for ectopic bone formation induced by osteoinductive calcium phosphate. Biomaterials 2013;34:2167–2176. 3. Abbah SA, Liu J, Goh JCH, Wong H-K. Enhanced control of in vivo bone formation with surface functionalized alginate microbeads incorporating heparin and human bone morphogenetic protein-2. Tissue Eng Part A 2012;19:350–359. 4. Sun X, Kang Y, Bao J, Zhang Y, Yang Y, Zhou X. Modeling vascularized bone regeneration within a porous biodegradable CaP scaffold loaded with growth factors. Biomaterials 2013;34: 4971–4981. 5. Polak SJ, Levengood SKL, Wheeler MB, Maki AJ, Clark SG, Johnson AJW. Analysis of the roles of microporosity and BMP-2 on multiple measures of bone regeneration and healing in calcium phosphate scaffolds. Acta Biomater 2011;7:1760–1771. 6. Bose S, Tarafder S. Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: A review. Acta Biomater 2012;8:1401–1421. -Guasch E, Helder MN, ten Bruggenkate CM, 7. Overman JR, Farre Schulten EA, Klein-Nulend J. Short (15 minutes) bone morphogenetic protein-2 treatment stimulates osteogenic differentiation of human adipose stem cells seeded on calcium phosphate scaffolds in vitro. Tissue Eng Part A 2012;19:571–581. 8. Kleinschmidt K, Ploeger F, Nickel J, Glockenmeier J, Kunz P, Richter W. Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2. Biomaterials 2013;34:5926–5936.

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