Osteoinductive Agents-Incorporated Three-Dimensional Biphasic

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Tissue Engineering and Regenerative Medicine

Osteoinductive Agents-Incorporated Three-Dimensional Biphasic Polymer Scaffold for Synergistic Bone Regeneration Bitao Zhu, Weiguo Xu, Jianguo Liu, Jianxun Ding, and Xuesi Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01371 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Biomaterials Science & Engineering

Osteoinductive Agents-Incorporated Three-Dimensional Biphasic Polymer Scaffold for Synergistic Bone Regeneration Bitao Zhu,†,‡ Weiguo Xu,‡,§ Jianguo Liu,*,† Jianxun Ding,*,‡,§ and Xuesi Chen‡,§

†Department

of Bone and Joint Surgery, The First Hospital of Jilin University, 71 Xinmin

Street, Changchun 130041, P. R. China ‡Key

Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, P. R. China §Jilin

Biomedical Polymers Engineering Laboratory, 5625 Renmin Street, Changchun

130022, P. R. China

Corresponding Authors E-mail: [email protected] (J. Ding). E-mail: [email protected] (J. Liu).

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ABSTRACT: Large-scale bone defects are difficult to be regenerated entirely in clinical practice. Bone tissue engineering has drawn more attention as an alternative to bone grafting owing to its convenience and flexibility. However, the low bioactivity of scaffolds and adverse effects of growth factors have hindered its practical application. Herein, the properties of poly(lactic-co-glycolic acid) (PLGA) scaffold, including mechanical strength and hydrophilicity, were improved by a gelatin coating incorporated with two small molecules, alendronate (ALD) and naringin (NG). Interestingly, these two drugs demonstrated a synergistic effect for repair of rat calvarial defect, as ALD had an inhibitory impact on osteoclast activity and NG had an osteogenic effect on mesenchymal stem cells. From the results of histopathological staining and micro-computed tomography, the PLGA scaffold incorporated with gelatin, ALD, and NG (PLGA+Gelatin/ALD/NG) almost completely repaired the rat calvarial defect with physiological integrity at 16 weeks. In all, this biphasic scaffold can be a promising alternative to the conventional scaffold for clinical application.

KEYWORDS: porous polymer scaffold, osteoinductive drug, controlled release, synergistic effect, bone repair


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INTRODUCTION A large bone defect is particularly challenging to be cured in the clinical practice, and it must meet functional, physiological, and aesthetic requirements.1-2 Although an autograft has been considered the gold standard for bone repair, some problems, including limited availability, donor morbidity, and potential for infection, have restricted its widespread application.3 Fortunately, bone tissue engineering (BTE) has been developed rapidly and can overcome these challenges in bone grafting. BTE, as an emerging interdisciplinary field in materials technology and life science, has drawn increasing attention in recent years. As a convenient and feasible technique, BTE has a much broader scope for clinical applications. Generally, scaffolds, cells, and growth factors constitute the three integral elements of BTE.4 However, some drawbacks require further improvement. The poor interactions with cells and proteins on the surface of the scaffold can lead to severe consequences, such as implant loosening, coagulation, or secondary infections.5-6 Moreover, although growth factors can effectively stimulate cell proliferation and differentiation via various signal pathways during bone healing, the immunogenicity, instability, and high cost preclude their utility in clinical practice.7 Therefore, a new strategy for BTE to overcome the above drawbacks is in demand. Gelatin, a natural polymer material, has already been approved in the food and cosmetics industries for a long time.8 Several advantages, including its high hydrophilicity, biocompatibility, bioactivity, and low immunogenicity and cost, make gelatin a desirable candidate for application in BTE.9-10 Regardless of how it is formulated, whether it is a



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nanoparticle, surface coating, or scaffold, gelatin has shown potentials for improving the properties of materials including mechanical strength and bioactivity.11-12 Owing to the abundance of Arg–Gly–Asp (RGD)-like sequences in gelatin fiber polymer chains, gelatin can also modulate cell adhesion, induce some biological responses, and promote faster healing via the integrin α2β1 signaling pathway.13-14 The process of bone regeneration is associated with bone formation and bone absorption, which mediated by osteoblasts and osteoclasts, respectively. The bioactive molecules (e.g., small molecules, peptides, and growth factors) can guide cell behaviors and prompt bone repair. Due to the short half-life, instability, high cost, and unpredictable therapeutic effect, the growth factors cannot be widely used. However, small molecules such as alendronate (ALD) and naringin (NG) can be incorporated into gelatin coating as osteoinductive factors that overcome the adverse effects associated with protein-based growth factors.15 ALD, one of the conventional anti-osteoporosis drugs,16-17 can potently suppress the production of farnesyl diphosphate synthase (FDPS), a key enzyme in the mevalonate signaling pathway. Therefore, it will inhibit the production of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), so that some small GTPases cannot be synthesized efficiently, leading to osteoclasts' apoptosis.18 NG, a polymethoxylated flavonoid, has been reported to promote bone formation by stimulating the proliferation of mesenchymal stem cells and the differentiation

of

osteoblasts,19

depending

on

the

high

expression

of

some

osteogenesis-related genes, such as bone morphogenetic protein 2 (BMP2).20-21 For the small molecules, they have some advantages toward the growth factors, including low toxicity, low


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cost, and low risk of immune response.22-24 Meanwhile, many studies have demonstrated that sustained release of these small molecules in situ can be more effective than long-term systemic treatment, and will not lead to some side effects, such as osteoclast dysfunction and osteonecrosis.25-26

Scheme 1. Schematic illustration for implantation of PLGA+Gelatin/ALD/NG scaffold into calvarial defects in rats. Although the local use of each molecule has been shown to a positive effect on osteogenesis, there have been no reports of combining them for promoting bone regeneration. The imbalance between the anabolic activities of osteoblasts and the catabolic activities of osteoclasts during bone healing can result in abnormal physiological conditions, such as osteoporosis, hyperostosis, Paget’s disease, and rheumatoid arthritis.27-28 In this study, the hydrophilic gelatin was used to modify poly(lactic-co-glycolic acid) (PLGA) scaffold by physical absorption. As shown in Scheme 1, the performances of PLGA scaffold with the incorporation of ALD and NG into the gelatin coating (PLGA+Gelatin/ALD/NG) was



evaluated in repairing the rat calvarial defect. The potential of the gelatin coating to act as an aggregator for a variety of cells and proteins, as well as the potential synergistic effect of ALD and NG on bone regeneration were investigated. In all, this simple method represents a new alternative strategy for improving the properties of scaffold in BTE. EXPERIMENTAL SECTION Materials. The information for materials is listed in Supporting Information. Preparation of PLGA+Gelatin/ALD/NG Scaffold. The scaffolds were prepared in the form of slices of 2-mm thickness and 5-mm in diameter. They were placed in a vacuum bottle where the air was exhausted. Meanwhile, the gelatin powders were completely dissolved into phosphate-buffered saline (PBS; pH 7.4, 1.0 mg mL−1), and two small molecule drugs (ALD, 0.5 wt.%; NG, 0.5 wt.%) were added. The gelatin solution was then injected into the vacuum bottle, for allowing the gelatin to invade into the internal structure of the scaffolds under negative pressure. The gelatin-coated scaffolds without or with osteoinductive agents were successfully prepared after freeze-drying. Characteristics of Gelatin Coating. After the above surface modification, the internal structures of PLGA scaffolds without or with gelatin coating were examined using a field-emission scanning electron microscope (SEM; Philips XL30, Eindhoven; Netherlands) after fragmented and sputtered with gold spray. The internal structure of the scaffold was detected by fluorescent staining and micro-computed tomography (micro-CT; Bruker, Germany). First, gelatin was labeled with FITC in a thiocarbamide reaction, and then it was coated onto the surface of a PLGA scaffold as described previously. The gelatin-coated


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scaffolds labeled with FITC were sliced into 15-μm sections for fluorescence imaging by confocal laser scanning microscopy (CLSM; Carl Zeiss AG, Oberkochen, Germany). The 10 × 10 × 10 mm scaffold cubes were placed onto a platform with a resolution of 1000 K, and the architectures of the scaffolds were reconstructed using micro-CT. Surgery for Animal Model. A bone defect animal model was established using the six-week-old Sprague Dawley rats (Vital River Laboratory Animal Technology Co., Ltd; Beijing, P. R. China). The protocol for animal surgery was approved by the Institutional Animal Care and Use Committee of Jilin University. The animals were divided into six groups: (1) control group (Control), (2) PLGA scaffold group (PLGA), (3) scaffold incorporated with gelatin coating group (PLGA+Gelatin), (4) PLGA scaffold incorporated with gelatin coating and ALD group (PLGA+Gelatin/ALD), (5) PLGA scaffold incorporated with gelatin coating and NG group (PLGA+Gelatin/NG), and (6) PLGA scaffold incorporated with gelatin coating, ALD, and NG group (PLGA+Gelatin/ALD/NG). First, the rats were anesthetized with 2.0% pentobarbital sodium (30.0 mg kg−1) by intraperitoneal injection. The rat heads were scraped and sterilized with povidone-iodine, and the skin, soft tissue, and periosteum were slit layer by layer for a clear exposure. A round defect with a diameter of 5 mm was created by using a trephine below the junction of the coronal and sagittal seams. The implants were placed into the defect region according to group assignment after ultraviolet irradiation for 30 min. Every tissue layer was carefully closed afterward. All animals were injected with antibiotics for three days after implantation and sacrificed after 8 or 16 weeks.



Micro-CT and Histologic Evaluation. The regenerated level of skulls was assessed via radiographic analyses using micro-CT. The specimens were scanned at a resolution of 1000 K (100 kV and 100 mA) and reconstructed by CTVol software (Skyscan1172, Bruker; Germany). The bone morphological parameters of newly formed tissue in the defect region were calculated using CTAn software (Skyscan1172, Bruker; Germany). After micro-CT analyses, the 10% ethylenediaminetetraacetic acid buffer was used for decalcification treatment, which was changed twice per week for four weeks. The decalcified specimens were then embedded in paraffin blocks and cut into 5-mm-thickness sections for hematoxylin and eosin (H&E) staining. In the H&E-stained images, the extent of bone regeneration was assessed according to three features: (1) bone regeneration response at the bone-scaffold interface; (2) bone regeneration within the pores of the scaffold; and (3) extent of bone formation (Tables S1, S2, and S3, Supporting Information). Two blinded observers determined the gross scores. Nanoindentation. The biomechanical analyses of the regenerated bone involved a nanoindentation test. All skull specimens were isolated from the defect region and immersed into PBS for maintaining hydration. The microscopic morphology and force-displacement data of the specimens were determined by using a TriboIndenter nanoindenter (Hysitron Inc., Minneapolis, MN, USA) with a conospherical diamond probe tip with 400-mm radius curvature in five directions (anterior, posterior, central, medial, and lateral). The surface topography could be acquired by the calculation and comparison for each point. A trapezoidal load function was detected in each direction, and the maximum indentation depth was 500


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nm. The reduced modulus and hardness of newly formed tissues were recorded in this manner. Pharmacodynamic Effect. The tartrate-resistant acid phosphatase (TRAP) is a specific enzyme marker for osteoclasts' activity. The rat skull sections were incubated for 1 h with naphthol diphosphate solution after dewaxing treatment. The TRAP in the osteoclasts was dyed red using azo-fuchsin after the hydrolysis of naphthol phosphate by seignette salt. The osteocalcin (OCN) and alkaline phosphatase (ALP) are important proteins in the proliferation and differentiation of osteoblasts. The sections were subjected to antigen unmasking treatment with 0.01 M citric acid−sodium citrate solution after the dewaxing treatment and then incubated with goat serum for 20 min. They were exposed to the OCN antibody at 4 °C overnight, and then rewarmed and incubated with a secondary antibody, Cy5.5, at 37 °C for 40 min. Finally, they were stained with DAPI and mounted with glycerol. The prepared specimens were observed using CLSM. The area and intensity of fluorescence were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). The ALP level of the new bone tissue was determined by using an ALP enzyme-linked immunosorbent assay (ELISA) kit. The tissue was extracted after nitrogen freezing and grinding, and the specimens and reagents were added into 96-well plates by the instructions of manufacturer. Absorbance at 520 nm was detected by a spectrophotometer (Bio Photometer; Hamburg, Germany). Water Contact Angle, Mechanical Strength, Degradation In Vitro, Calcium and Phosphorus

Content

Analyses,

Immunohistochemistry,


Expression

of


Osteogenesis-Related Genes, and Western Blot Analyses. The test methods for water contact angle, mechanical strength, biodegradation in vitro, calcium and phosphorus content analyses, immunohistochemistry, expression of osteogenesis-related genes, and Western blot analyses are presented in the Supporting Information. Statistical Analyses. All the data are represented as the mean ± standard deviation (SD). Every experiment was repeated at least three times unless otherwise specified. Statistical significance was assessed using a Student's t-test and Holm-Sidak method after one-way analysis of variance (ANOVA) test, and the detailed protocols were provided in Supporting Information. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered to reflect generally, highly and greatly significant differences, respectively. RESULTS AND DISCUSSION Improved Properties of PLGA+Gelatin/ALD/NG Scaffolds. The preparation route of PLGA+Gelatin/ALD/NG scaffold is shown in Scheme 1, and the gelatin coating could be covered into the inner surface of PLGA scaffold under negative pressure. However, whether the gelatin was properly coated had an impact on the properties of PLGA+Gelatin scaffold so that SEM, CLSM, and micro-CT determined the internal appearance of scaffolds. In the SEM images, both of scaffolds showed the interconnected porous structures with uniform pore sizes to some extent, and some small pores were distributed onto the internal wall as well. The inner pore wall of PLGA+Gelatin scaffold was found to be rougher and more uneven with a fiber coating compared to that of PLGA scaffold, suggesting the gelatin had been coated onto the inner surface of PLGA scaffold (Figure 1A). As shown in the 3-dimensional


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(3D) images by micro-CT, one piece of the scaffold was cut out to observe the internal structure better and prevent any impact from the surrounding. The internal structure of PLGA+Gelatin scaffold was much denser and more complex than that of PLGA scaffold as a whole (Figure 1B). Apart from the compact structure, the FITC-labeled gelatin layer could show the porous structures with uniform density under CLSM in both of 2-dimensional (2D) and 3D model constructs (Figure 1C). Meanwhile, for PLGA scaffold, the mean pore size was 273.2 ± 63.5 μm. By contrast, the pore size of the PLGA+Gelatin scaffold was smaller, at 155.9 ± 14.3 μm (Figure 1D). Owing to the open structure and internal negative pressure of scaffold, the gelatin could be invaded into and uniformly covered onto the inner surface of scaffold, instead of forming a thick gelatin coating at the surface (Figure S1). Although the pore size was slightly decreased, the pore size of our scaffolds was sufficient to allow for cell invasion and migration. Additionally, because of the abundant integrin in gelatin fibers, PLGA+Gelatin scaffold could be in favor of cell adhesion and proliferation via the integrin α2β1 signaling pathway. One of our purposes is to improve the properties of PLGA scaffold. In the water contact angle test, the water drop hung onto the surface of PLGA scaffold and challenging to invade. However, the water drop could slightly penetrate the internal structure of PLGA+Gelatin scaffolds (Figure S2A). The mean water contact angle of PLGA scaffold was 113.0°, larger than 81.1°of PLGA+Gelatin scaffold (Figure S2B). Because of the lack of polar function, the aliphatic polyesters (e.g., PLGA) exhibit high hydrophobicity that can result in reduced cell



interactions and poor protein absorption. The water contact angle of PLGA scaffold was higher than 90° so that the surface of the scaffold was considered to be hydrophobic. Conversely, for water contact angles less than 90°, it was hydrophilic. As bone regeneration starts with material degradation and cell adhesion, the cell–material interaction is significant for bone formation. The increased hydrophilicity of PLGA+Gelatin scaffold, therefore, appears to promote the cell adhesion to the surface of scaffold.29

Figure 1. Properties of scaffolds. (A) SEM and (B) micro-CT imaging of the PLGA and PLGA+Gelatin scaffolds. (C) CLSM of a PLGA+Gelatin scaffold labeled with FITC in 2D and 3D models. (D) Mean pore size of the PLGA and PLGA+Gelatin scaffolds. Student's t-test determines significance (n = 6; **P < 0.01). The degradation process of a polymer scaffold is driven by bulk erosion due to hydrolysis, and the new bone tissue cannot be formed until the scaffold has been completely degraded.30


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Therefore, the scaffolds were soaked in PBS buffer to observe the degradation process by measuring weight loss. During the first two weeks, the weight of scaffolds did not significantly decrease. However, after two weeks, the remaining mass of PLGA+Gelatin scaffolds declined rapidly. On day 40, 75.5% of PLGA+Gelatin scaffold remained compared to 85.2% of PLGA scaffold (Figure S2C). It appeared that PLGA+Gelatin scaffolds would continue to undergo degradation at a fast speed. However, this process was difficult to be confirmed, because the scaffolds became too brittle to be weighed.

As shown in Figure S3,

the scaffolds had been fallen apart in the 28th day, and small fragments could be found according to the process of bulk erosion. Because of the high hydrophilicity of gelatin, the presence of gelatin improved the hydrophilicity of PLGA scaffold, and PLGA+Gelatin scaffold exhibited a rapid degradation rate. The stiffness and mechanical strength of the scaffolds also play an essential role in the acceleration of bone formation. As shown in Supplementary Figure S2D and S2E, the compression modulus (Modulus) of PLGA scaffold reached up to 1.2 MPa, and the maximum compression load (Load) reached up to 140.5 N. However, as for PLGA+Gelatin scaffold, Modulus was 1.9 MPa, and Load was 216.2 N, suggesting that PLGA+Gelatin scaffolds exhibited a better performance with regard to the mechanical strength. In addition, the stress−strain curve of scaffolds was tested with displacement from 0 mm to 4.0 mm. As shown in Figure S2F, the stress of the same displacement in PLGA+Gelatin scaffold required more force, and the slope was much sharper. When the displacement reached 1.5 mm, the load of PLGA scaffold achieved a plateau of 55.3 N, suggesting that the maximum



deformation was achieved and the scaffolds began to break down. In contrast, the load of PLGA+Gelatin scaffold was 80.0 N when displacement reached 1.5 mm and still increased continuously with constant stress, which indicated Young’s moduli of PLGA+Gelatin scaffold was significantly improved compared to that of PLGA scaffold (Figure S2F). Due to a large amount of gelatin distributed throughout the internal structure of PLGA scaffold, it forms a more complex structure so that PLGA+Gelatin scaffold can gain a significant improvement in mechanical properties and bear a higher load, corresponding to the 3D reconstruction by micro-CT. Enhanced Skull Bone Regeneration by PLGA+Gelatin/ALD/NG Scaffolds. The detailed assessment of newly formed tissues was carried out by micro-CT. As shown in Figure 2A, the PLGA+Gelatin/ALD/NG group showed clear evidence of the best performance for bone repair after eight weeks, and the defect was nearly filled. Meanwhile, the sagittal images from micro-CT were photographed to observe the mineralization status of the new tissues. It found that the control group retained a mostly open area with minimal mineralization, and showed minimal bone mineralization mostly confined to the defect edges. However, in the PLGA+Gelatin/ALD/NG group, the size of the defect area was significantly decreased, and a dotted line could be observed, indicating the formation of the newly formed mineralized tissue (Figure 2A). Bone morphological parameters, including bone volume (BV), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were selected for the quantitative evaluation for bone regeneration. At eight weeks, in the PLGA+Gelatin group,


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BV was increased to 0.47 mm3 compared to 0.18 mm3 in the control group. In the PLGA+Gelatin/ALD/NG group, BV was 1.84 mm3, BV/TV was 23.1%, Tb.N was 1.56 mm−1 and Tb.Th was 0.17 mm. All of them were significantly higher than the corresponding measurements in the PLGA+Gelatin/ALD group and PLGA+Gelatin/NG group. At 16 weeks, all groups exhibited significant improvement for bone repair, corresponding to the 3D reconstructed and sagittal images. Particularly for the PLGA+Gelatin/ALD/NG group, the defect appeared to be almost wholly repaired with an integral structure. BV and BV/TV were 2.3 mm3 and 32.7%, respectively, which were much higher than that in the PLGA+Gelatin/ALD group as well as PLGA+Gelatin/NG group. Meanwhile, both Tb.N and Tb.Th were higher in the PLGA+Gelatin/ALD/NG group than that in other groups. Therefore, PLGA+Gelatin/ALD/NG scaffold was demonstrated to have the highest potential for osteogenesis, suggesting the combination of ALD and NG could effectively facilitate bone formation rather than a single molecule. Furthermore, NG appeared to have a better osteogenic effect compared to ALD. The BV in the PLGA+Gelatin/NG group was 1.2 mm3, which was 1.5 times higher than that in the PLGA+Gelatin/ALD group. Additionally, both BV/TV and Tb.N in the PLGA+Gelatin/NG group were also significantly higher than that in the PLGA+Gelatin/ALD group, suggesting PLGA+Gelatin/NG scaffold could exert a more powerful effect on accelerating bone regeneration than PLGA+Gelatin/ALD scaffold (Figure 2B). As mentioned before, NG can inhibit HMG-CoA reductase and promote the expression of BMP2.21 However, ALD mainly has an inhibitory action to osteoclasts presented at a relatively small number. Because of the



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positive effect on osteoblasts by NG and inhibitory activity on osteoclasts by ALD, a synergistic effect was shown in the PLGA+Gelatin/ALD/NG scaffold, which exhibited the best osteoinductive potential rather than PLGA+Gelatin/ALD scaffold or PLGA+Gelatin/NG scaffold.

Figure 2. Micro-CT assessments. Three-dimensional reconstructions, sagittal images, and analyses of bone morphological parameters, i.e., BV, BV/TV, Tb.N, and Tb.Th, in control, PLGA,

PLGA+Gelatin,

PLGA+Gelatin/ALD,


PLGA+Gelatin/NG,

and

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PLGA+Gelatin/ALD/NG groups after (A) 8 weeks or (B) 16 weeks. Holm-Sidak methods analyze significance (n = 5; *P < 0.05, **P < 0.01). The surface morphology and mechanical strength were determined by nanoindentation technology. As shown in Figure 3A, the surface appearance of newly formed tissue in the PLGA+Gelatin/ALD/NG group was smooth and homogeneous, suggesting the formation of cortical bone. Different from the connective fibrous tissue, the surface of cortical bone should be much smoother (Figure 3A). In addition, the hardness and reduced modulus of newly formed tissue in the defect region were measured at 16 weeks. The hardness of newly formed tissue was significantly increased by adding the gelatin coating, ALD, and NG. In the PLGA+Gelatin/ALD/NG group, it could be reached at 44.0 KPa, and highest among each group. The reduced modulus was 1.4 GPa, significantly higher than 1.1 GPa in the PLGA+Gelatin/NG group and 0.8 GPa in the PLGA+Gelatin/ALD group, suggesting that the newly

formed

tissue

had

been

gradually

transformed

into

bone

PLGA+Gelatin/ALD/NG scaffold could promote bone formation (Figure 3B).


tissue

and


Figure 3. Surface topography and mechanical strength. (A) Surface morphology. (B) Hardness and reduced modulus of newly formed tissue at 16 weeks. Holm-Sidak test determines significance (n = 5; *P < 0.05, **P < 0.01). Histologic staining of the tissues in the defect region was performed to identify the tissue type. As shown in Figure 4A, some fibrous connective tissues were observed at the defect region and the implanted scaffold was found to be partially degraded after eight weeks. For the control group and PLGA group, no distinct biological interaction for bone formation occurred despite the presence of monocytes and macrophages. These mononuclear phagocytes infiltrated into the internal pores of scaffolds and lead to an inflammatory response. Although a great deal of fibrous tissue had grown into the gelatin-modified scaffolds, the calcified extracellular matrix was deposited and gradually became new bone tissue. Especially for the PLGA+Gelatin/ALD/NG group, some new bone tissue was found to be formed across the defect area, and the integral bone structure started to be reconstructed. At 16 weeks, because of the degradation of the scaffolds and the biological responses between the small molecules and the cells, a significantly different level of bone regeneration could be observed. In the control group and PLGA group, there was little bone formation, and some fibrous tissue remained. Meanwhile, the fibrous tissue could also be found in the PLGA+Gelatin group, but relatively less than that in the PLGA group, and it was gradually mineralized to form bone tissue. In both the PLGA+Gelatin/ALD group and PLGA+Gelatin/NG group, the defect region has been steadily replaced by mature bone tissue. In the PLGA+Gelatin/ALD/NG group, the mature bone tissue had been formed and


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well-connected with the original tissue, showing an integral structure of rat skull. The scores of the gross evaluations in each group are shown in Figure 4B, suggesting that the gelatin coating and small molecules had a positive effect on promoting bone formation as well as the highest potential of PLGA+Gelatin/ALD/NG scaffold for bone repair. Due to the lack of osteoconductive property of the scaffold, the defect was hardly repaired in the control group. As previously proved, the gelatin-modified scaffold had a fast degradation rate so that the bone tissue could be formed earlier. With the help of RGD sequences in gelatin coating, the cells could be recruited into the defect, and fibrous tissue grew into so that PLGA+Gelatin scaffold could play a better role for bone repair rather than PLGA scaffold alone. Furthermore, ALD and NG, as osteoinductive agents, could promote the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and inhibit the osteoclasts' activity, leading to the formation of new bone tissue. Therefore, PLGA+Gelatin/ALD/NG scaffold could affect osteoblasts and osteoclasts, and exhibit the best performance for bone regeneration. The calcium and phosphorus contents can reflect the extent of bone maturation and are determined using inductively coupled plasma mass spectrometry. For the normal mature bone tissue, the calcium content was 87.4 mg g−1, and phosphorus content was 46.5 mg g−1. At eight

weeks,

there

was

little

difference

in

the

calcium

content,

except

the

PLGA+Gelatin/ALD/NG group, in which the calcium content of newly formed tissue was significantly higher, nearly twice that of newly formed tissue in the other groups. The phosphorus content increased gradually with the addition of gelatin, ALD, and NG. At 16



week, the calcium and phosphorus contents of the tissue in the PLGA+Gelatin/ALD/NG group were very similar to those of healthy bone tissue, indicating that mature bone tissue had been formed in the defect region (Figure 4B).

Figure 4. Histological evaluation. (A) Bone regeneration at 8 weeks and 16 weeks. (B) Immunohistochemical staining for collagen type I (Col I) at 16 weeks. (C) Gross evaluation of hematoxylin and eosin (H&E) staining (i.e., pore response, extent of bone formation, and bone-scaffold interface), calcium (Ca), and phosphorous (P) content of newly formed tissue


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at 8 and 16 weeks. Holm-Sidak methods analyze significance (n = 5; *P < 0.05, **P < 0.01). To verify the composition of the newly formed tissue, immunohistochemical staining for Col I was performed. Col I has been shown to be present at a much higher level in bone tissue than other forms of collagen.31 After 16 weeks, the bone tissue demonstrated high immunoreactivity to anti-Col I than the fibrous tissue. In the control group, abundant connective tissues invaded the inside of scaffold, which were characterized by light staining. By contrast, deep yellow staining was observed in the healthy bone tissue, especially for the mature bone. In the PLGA+Gelatin/ALD/NG group, the new bone tissue was identical to mature bone tissue with excellent bone integrity, corresponding to the level of calcium and phosphorus contents as mentioned. The PLGA+Gelatin/ALD/NG scaffold with unique structure and composition could accelerate the bone maturation and develop a good connection to the surrounding tissue. Mechanisms of Carnival Bone Regeneration by PLGA+Gelatin/ALD/NG Scaffolds. Because of the pharmacological function of NG and ALD, a synergistic effect was expected. As shown in Figure 5A, the TRAP-dyed area was markedly decreased in the PLGA+Gelatin/ALD group and PLGA+Gelatin/NG/ALD group, suggesting that ALD could effectively inhibit osteoclasts' activity. Furthermore, the structure of newly formed tissue was found to be disordered and irregular in the control group and PLGA group, instead of being well-organized and compact in drug-loading groups. Moreover, OCN fluorescence staining revealed that NG improved the activity of osteoblasts. As shown in Figure 5B, the stained regions for high osteoblast's activity could be observed in the PLGA+Gelatin/NG group and



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the PLGA+Gelatin/ALD/NG group. Interestingly, PLGA+Gelatin/ALD scaffold also exhibited a positive osteogenic effect, suggesting ALD had a positive impact on osteoblasts. The fluorescent area and density in the PLGA+Gelatin/ALD/NG group were highest among each group, indicating the synergistic action of ALD and NG for bone regeneration (Figure 5C

and

5D).

Additionally,

the

ALP

level

of

newly

formed

tissue

in

the

PLGA+Gelatin/ALD/NG group was significantly higher than that in the PLGA+Gelatin/ALD group and PLGA+Gelatin/NG group (Figure 5E), also demonstrating the synergistic action of ALD and NG. Owing to the inhibitory impact of ALD on osteoclasts and the positive effect of NG on osteoblasts, PLGA+Gelatin/ALD/NG scaffold had a high potential for bone repair. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses were carried out to investigate the underlying osteogenic mechanism of the PLGA+Gelatin/ALD/NG scaffold. The genes for osteogenesis-related proteins, including BMP2, osterix (OSX), osteopontin (OPN), and bone sialoprotein (BSP), were selected for RT-PCR. As shown in Figure 6A, at eight weeks, the relative expression of BMP2 in the PLGA+Gelatin/ALD/NG group was significantly higher than that in other groups, more than twice that in the PLGA+Gelatin/ALD group and PLGA+Gelatin/NG group, and 10-times that in the control group. However, the expression levels of OSX, BSP, and OPN in the PLGA+Gelatin/ALD/NG group were slightly higher compared to the other groups. At 16 weeks, BMP2, OSX, BSP, and OPN were all expressed significantly. In particular, the expression of osteogenesis-related genes in the PLGA+Gelatin/ALD/NG group was the


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highest among all groups, suggesting PLGA+Gelatin/ALD/NG scaffold could promote the expression of the osteogenesis-related genes to accelerate bone formation.

Figure 5. Pharmacodynamic effects. (A) Tartrate-resistant acid phosphatase (TRAP)



staining in the central region of the calvarial defect (red arrows represent areas of high osteoclast activity). (B) Osteocalcin (OCN) fluorescence staining at 16 weeks. (C) Fluorescence intensity and (D) fluorescence area analyses for OCN staining. (E) Alkaline phosphatase (ALP) level of newly formed tissue at 16 weeks. Holm-Sidak procedure determines significance (n = 5; **P < 0.01, ***P < 0.001). According to the central law of genetics, the findings for selected proteins in the Western blot test were similar to that of the osteogenesis-related genes in the RT-PCR experiment. BMP2, OSX, OPN, and BSP were used for qualitative and quantitative analyses of the western blot. A quantitative gray-scale analysis was performed. As shown in Figure 6B, the levels of BMP2, OSX, OPN, and BSP were highest in the PLGA+Gelatin/ALD/NG group. In addition, the BMP2 level in the PLGA+Gelatin/NG group was 2.2 times higher than in the PLGA+Gelatin/ALD group, and the level of OSX in the PLGA+Gelatin/NG group was 1.7 times higher than that in the PLGA+Gelatin/ALD group, in agreement with the high osteogenic potential of NG compared to ALD (Figure 6B and 6C). As described previously, NG could promote the differentiation of MSCs into osteoblasts via the BMP2 signaling pathway, which consistent with our observation that the relative expression of BMP2 in the PLGA+Gelatin/NG group and PLGA+Gelatin/ALD/NG group was upregulated. OSX, a zinc finger protein, is highly expressed in bone developing and can interact downstream of the Runx2 signal pathway.32 OPN, a so-called "bone-bridging" protein, can communicate with cell-surface receptors, and one of significant receptors is the RGD sequence, which is abundant in bone tissue matrix and may mediate important


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cell-matrix and cell-cell interactions.33 Because of the RGD motif present in the gelatin coating, the expression of OPN was high in all of the gelatin-coating groups. Notably, the expression of OPN in the PLGA+Gelatin/ALD group was slightly reduced. Some studies have demonstrated that OPN is an activator of the NF-κB ligand (RANKL)/osteoprotegerin signaling pathway, can activate the immune system, and promote the adhesion and proliferation of osteoclasts. However, ALD, an osteoclast inhibitor, could inhibit the RANKL signal pathway and inhibit bone resorption, thereby promoting bone regeneration. BSP has is one of the primary proteins for osteoblasts and expressed in the final stage of cell differentiation. BSP protein and mRNA have been reported to be found in mature osteoblasts but not in precursor cells. Therefore, we selected BSP for the evaluation of bone maturation. The expression of BSP was significantly highest in the PLGA+Gelatin/ALD/NG group among all the groups, corresponding with fact mature bone tissue had been formed.



Figure 6. Osteogenic analyses. (A) Expression of osteogenesis-related genes by reverse transcription-polymerase chain reaction (RT-PCR) (i.e., BMP2, OSX, OPN, and BSP). (B) Photograph of protein electrophoresis. (C) Quantitative analyses of protein levels. Holm-Sidak test determines significance (n = 5; *P < 0.05, **P < 0.01). CONCLUSIONS In this study, two small molecules, ALD and NG, were incorporated into the gelatin coating of PLGA scaffold as osteoinductive agents. Apart from the improved properties by gelatin coating, including mechanical strength and hydrophilicity, NG can positively facilitate bone regeneration by promoting osteoblasts' proliferation and differentiation, and ALD can suppress bone resorption by inhibiting the activity of osteoclast. The achievement of bone regeneration by the two drugs in combination was significantly higher than that of either drug alone. Additionally, the rat calvarial defect can be almost wholly repaired with physiological integrity using PLGA+Gelatin/ALD/NG scaffold within 16 weeks. Taken all together, PLGA scaffold can be optimized using a simple surface coating, and the synergistic actions of ALD and NG have an excellent effect on bone repair. Therefore, a new strategy for bone defect has been developed and expected to be applied to clinical practices in the future. However, some limitations have to be improved, such as the defect size was a little small, which may be repaired by itself and influence the evaluation of bone regeneration. Furthermore, the results from animal models are not sufficient evidence for clinical practice. It is difficult to monitor the level of bone formation in real-time and accurately evaluate the feedbacks of rats in our experiment. Extensive research will be carried out for further clinical translation, including


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critical bone defect models, randomized clinical trials, and precise assessment of pharmacological effects.

ASSOCIATED CONTENT Supporting Information The following files are provided for free. The surface morphological structure of PLGA+Gelatin scaffold is presented in Figure S1. The physical characterizations are presented in Figure S2. The degradation of scaffold is presented in Figure S3. The evaluation standards of bone regeneration are listed in Tables S1, S2, and S3, the primer sequences of osteogenesis-related genes are listed in Table S4. Water contact angle measurements; tests of mechanical strength and biodegradation in vitro; calcium and phosphorus content analyses; immunohistochemistry assessments; animal studies; expression of osteogenesis-related genes; Western blot analyses; statistical analyses.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] (J. Ding). E-mail: [email protected] (J. Liu).

ORCID Bitao Zhu: 0000-0002-7592-5214



Weiguo Xu: 0000-0002-0146-8532 Jianguo Liu: 0000-0003-0051-4987 Jianxun Ding: 0000-0002-5232-8863 Xuesi Chen: 0000-0003-3542-9256

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was financially supported by the National Key Research and Development Program of China (Grant No. 2016YFC1100701), the National Natural Science Foundation of China (Grant Nos. 51873207, 51803006, 51673190, 51603204, 51673187, and 51520105004), and the Science and Technology Development Program of Jilin Province (Grant Nos. 20160204015SF, 20160204018SF, and 20170101102JC).

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For Table of Contents Use Only

Osteoinductive Agents-Incorporated Three-Dimensional Biphasic Polymer Scaffold for Synergistic Bone Regeneration

Bitao Zhu, Weiguo Xu, Jianguo Liu, Jianxun Ding, and Xuesi Chen


Osteoinductive Agents-Incorporated Three-Dimensional Biphasic

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