Enhanced Repairing of Critical-sized Calvarial Bone Defects by

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Tissue Engineering and Regenerative Medicine

Enhanced Repairing of Critical-sized Calvarial Bone Defects by Mussel-inspired Calcium Phosphate Cement Zongguang Liu, Jianmei Chen, Guowei Zhang, Junsheng Zhao, Rong Fu, Kuangyun Tang, Wei Zhi, Ke Duan, Jie Weng, Wei Li, and Shuxin Qu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00243 • Publication Date (Web): 21 Apr 2018 Downloaded from http://pubs.acs.org on April 21, 2018

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ACS Biomaterials Science & Engineering

Enhanced Repairing of Critical-sized Calvarial Bone Defects by Mussel-inspired Calcium Phosphate Cement Zongguang Liua, Jianmei Chena, Guowei Zhanga, Junsheng Zhaoa, Rong Fub ,Kuangyun Tangb, Wei Zhia, Ke Duana, Jie Wenga, Wei Li c, *, Shuxin Qua,*

a

Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

b

Department of Plastic Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072, China

c

Department of Burns Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072, China

*Corresponding author: Shuxin Qu Tel.: +86-28-87601897; Fax: +86-28-87601371 E-mail address: [email protected]

Wei Li E-mail address: [email protected] 30 pages, 9 Figures, 1 Table

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Abstract The goal of this study is to investigate the biological response of mussel-inspired calcium phosphate cement (CPC) in vivo. Polydopamine (PDA), which is analogous to that of mussel adhesive proteins, was added in CPC. PDA-CPC was implanted into the femur, muscle and critical size calvarial bone defect of rabbits. Histomorphometry of the sequential fluorescence sections showed that PDA-CPC were capable to form more newborn bone than the control-CPC. More new bone, bone marrow cavity and blood vessel were observed in PDA-CPC than in the control-CPC in decalcified and un-decalcified histological sections. Necrosis bone was not observed in PDA-CPC, whereas it appeared in the control-CPC after 2 weeks. The histological sections in muscle witnessed that more ingrowth of collagen in PDA-CPC than that in the control-CPC. There were no significantly difference in the number of leukocyte between PDA-CPC and the control-CPC in blood. It was confirmed that the addition of PDA enhanced the bone repairing ability and biocompatibility of PDA-CPC. Push-out testing evidenced that PDA increased the bonding strength between PDA-CPC and host bone in the early stage. These present results indicated that PDA-CPC might be one potential bone graft with gratifying biocompatibility and enhanced bone repairing.

Key words: Calcium phosphate cement; Polydopamine; Critical size; Bone defects; Biocompatibility; Enhanced repairing

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1. Introduction Marine mussels can attach onto diverse solid surfaces in the sea by its robust adhesive proteins in byssal plaque. The further studies find that this adhesive versatility of mussels is associated with mytilus foot protein-3 (mfp-3) and mfp-5, which are squirted firstly onto the substrate to from this strong adhesion [1]. Mfp-5 contains 30% of 3, 4-dihydroxy-L-phenylalanine (DOPA), a catechol-containing compound, and 15% of lysine, an amino acid with a side-chain ending in a primary amine group [2]. Analogously, dopamine (DA), a typical catecholamine, also contains catechol moieties of DOPA and amino groups of lysine [3-5], which can form polydopamine (PDA) via self-polymerization in alkaline solution [6]. Intriguingly, PDA has the extraordinary adhesive property due to the strong covalent or non-covalent interactions, e.g. hydrogen bonds or stacking interactions, between catechol moieties and substrates similar to that of mussel adhesive protein [7]. To date, PDA has been broadly explored for surface-modification on biomaterials to improve the hydrophily [8, 9] and biocompatibility. In addition, PDA has been used as intermediate layers to anchor nanoparticles [10-14], drugs and proteins [15-21]. Compared with non-modified surface, PDA modified surface displays the capacity to form a layer of apatite readily after soaking in simulated body fluid (SBF) [10, 22, 23]. It may be attributed to the strong affinity between catechol moieties of PDA and Ca2+, which results in the local supersaturation of Ca2+ and the nucleation site of hydroxyapatite (HA) [24, 25]. Inspired by the adhesive proteins in mussels, our previous studies confirmed that PDA significantly increased the compressive strength 3



of calcium phosphate cement (PDA-CPC) by integrating PDA-CPC into dense structure, and promoting the hydration of CPC [26]. Furthermore, PDA increased the early mineralization capacity of PDA-CPC in vitro to form a layer of micro and nano scale Ca/P based composite, which might be potential for the attachment and proliferation of cells and the growth of tissue subsequently [27]. To date, PDA-based biomaterials showed the excellent biocompatibility to various kinds of cells in vitro, e.g. osteoblasts [8], chondrocytes [28], human mesenchymal stem cells [5], myoblasts [29], and endothelial cells [30]. However, the biological response of cells cultured in vitro is difficult to extrapolate to animal studies, since nearly all cell culture studies only offer a simple and simulated environment similar as that in vivo, and employ relatively short periods of exposure to PDA-based biomaterials. It could not reveal that the response of various cells and tissue for PDA-based biomaterials only via cell culture experiment. It is necessary to study the bone repairing ability of PDA-based biomaterials in vivo for their further application in clinic. In addition, bone tissue generally has some self-repairing ability, which means bone defect can be repaired without any bone filler in case of the limited dimension. Thus, bone defect with critical size is significantly introduced to evaluate the bone repairing ability of bone graft. In the current study, CPC loaded with PDA was implanted into calvaria, femur and muscle of rabbits to study its biological response and bone repairing ability for the critical size bone defects. Moreover, the physicochemical property of PDA-CPC, bonding strength between PDA-CPC and host bone in vivo were also assessed. 4


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2. Materials and methods 2.1 CPC preparation The powders of CPC, consisted of 58 wt% of α-tricalcium phosphate (α-TCP), 25 wt% of dicalcium phosphate dihydrate (DCPD), 8.5 wt% of HA, and 8.5 wt% of CaCO3 [26, 27], were blended well and dried overnight. The cement solution was mixed with 10 mM Tris-HCl buffer (pH 8.5) and 40 mg/mL DA (Sigma-Aldrich, Germany), and then exposed to air for 2 d (allowing DA oxidized and cross-linked to form PDA) prior to using. CPC powders and the cement solution were mixed in the ratio of 1 g: 0.3 mL and following placed into cylindrical molds to set with two types of dimensions, Φ 8 × 3 mm and Φ 5 × 4 mm. Then all specimens were hydrated in 100% humidity, 37 °C for 24 h. CPC with and without PDA were named as PDA-CPC and the control-CPC, respectively. 2.2 Surgical procedure Eighteen adult male New Zealand rabbits (Experimental Animal Center of West China Hospital of Sichuan University, Chengdu, China) with an average weight of 2.5 kg were adopted in this experiment. All studies on animals were conducted in accordance with the guidelines for the care and use of laboratory animals of local ethical committee for animal experiments. Rabbits were randomized into two study series, twelve rabbits were created critical size defects (8 mm in diameter, full thickness) [31, 32] in the calvaria and six rabbits were created defects with 5 mm in diameter and 4 mm in height at femur and dorsal muscle. Rabbits in each study series were further divided into two groups 5



equally, PDA-CPC and the control-CPC. The details of the characterization with implantation time were presented in table 1. Before implantation, all specimens were washed ultrasonically for 30 min and dried without heating, and then sterilized by gamma-ray irradiation at a dose of 25 kGy in a vacuum. All rabbits were anesthetized by an intravenous injection of 3 % pentobarbital sodium (1.5 mL/kg), and subcutaneous injection of antibiotic after post-operation. 2.2.1 Calvaria implanted experiment A 25 mm long incision was cut along the sagittal suture on the scalp after shaved, washed with alcohol and disinfected with iodine. Skin, subcutaneous tissue and periosteum were stepwise separated to expose the calvarial bone. Firstly, dental drill was used to create a small cavity (1 mm diameter). Then it was enlarged gradually carefully. Saline was sprayed to prevent hyperthermia at the operative site. Two full-thickness bone defects of 8 mm diameter were created beside the sagittal suture of the calvarial bone. The bone defects were carefully washed to eliminate bone debris and dried with gauze. The calvarial bone defects were implanted by sterilized CPC (Fig. S 1 a), and then the periosteum, subcutaneous tissue and skin were stepwise repositioned and sutured. 2.2.2 Femur implanted experiment A 20 mm long skin incision was cut on the femoral lateral of the distal condyle after shaved, washed with alcohol and disinfected with iodine. Then muscles were separated bluntly to expose the condyle of femur. Bone defects with 5 mm in diameter 6


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and 4 mm in depth were gradually generated in each hind condyle of femur using a dental drill companied with continuous saline irrigation (Fig. S1 b). Sterilized PDA-CPC and the control-CPC were implanted bilaterally in the bone defects of femur. Subsequently, the muscle, subcutaneous tissue and skin were stepwise repositioned and sutured. 2.2.3 Muscle implanted experiment A 40 mm skin incision was created along the vertebral column in dorsal after the rabbits were shaved, washed with alcohol and disinfected with iodine. Subsequently, sterilized CPCs were implanted bilaterally in dorsal pouches created by blunt dissection under aseptic conditions (Fig. S1 c). Finally, the muscular pouches, subcutaneous tissue and skin were stepwise repositioned and sutured. 2.3 Histological preparation 2.3.1 Histological procedure Rabbits were sacrificed by injecting the overdose pentobarbital after post-operation for 2, 4, and 8 weeks. Specimens harvested from calvarial bone and dorsal muscle was fixed in 10 % formaldehyde with a phosphate-buffered solution (PBS). All specimens were divided equally into two groups to prepare un-decalcified and decalcified sections, respectively. All specimens were dehydrated in gradient ethanol from 75 % to 100 %. Then, specimens for the un-decalcified histological section were embedded in polymethymethacrylate. Longitudinal sections were cut into 150 µm thick using a microtome (SP1600, Leica, Germany), and subsequently ground and polished to a final thickness of ~40 µm. The sections were stained with 7



Van Gieson’s solution. For the decalcified histological section, fixed specimens were decalcified in 10 % ethylenediaminetetraacetic acid (EDTA) with 0.1 M Tris-HCl buffer (pH = 7.4; 4 ℃), then embedded in paraffin wax, and cut into sections of 6 mm (RM2125, Lecia, Germany). These sections were mounted on slides coated with gelatin and dried overnight at 50 ℃, stained by hematoxylin and eosin (HE), Toluidine blue and masson's trichrome. All sections were histologically observed (BX63, Olympus, Japan). 2.3.2 Fluorescent labeling and observing A polychrome sequential fluorescent labeling method was carried out to label the bone tissue dynamically. Three kinds of fluorescent were used, e.g. calcein disodium salt (Sigma-Aldrich, USA, 10 mg/kg), tetracycline hydrochloride (Sigma-Aldrich, USA, 25 mg/kg) and xylenol orange tetrasodium (Sigma-Aldrich, USA, 90 mg/kg). The former was dissolved in 2 % NaHCO3 solution, while the latter two reagents were dissolved in physiological saline, respectively. Then they were filter sterilized, and administered intravenously at 2, 4, and 6 weeks after the post-operation [33], respectively. Fluorescent labeling in undecalcified sections were observed by a laser confocal microscope (CLSM, Eclipse Ti, Nikon, Japan). The number of pixels labeled with calcein, tetracycline and xylenol orange staining were represented the bone regeneration and mineralization after 2, 4, and 6 weeks of implantation post-operation. The levels of fluorescence intensity in those images were converted to integrated option density (IOD SUM) using Image-Pro Plus software, which indicated the 8


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amount of bone formation. 2.4 Characterization 2.4.1 In vivo toxicity testing Venous blood samples (~5 mL) were collected from the marginal ear vessels of each rabbit in the calvaria implanted group for blood test after implanted for 3 days. All samples (n=6) were processed within 1 h. Blood cell populations of leukocytes, e.g. granulocytes, lymphocytes and intermediate cells, were determined by hematology analyzer (TEK- II mini, Tecome, China). 2.4.2 X-ray detection Animals were anesthetized by an intravenous injection of 3 % pentobarbital sodium (1.5 mL/kg) before X-ray detection (HF100HA, Mikasa, Japan) after 7 d of implantation. Radiographs with orthotopic and lateral position were collected. 2.4.3 Push-out testing Three samples from each femoral group were obtained for push-out testing after implanted for 2 and 8 weeks. Specimens with circumambient bone were fixed in the center of the clamp. An indenter probe with 4 mm diameter was push down with universal testing machines (5567, Instron, USA) at 0.5 mm/min. The normal load was recorded until implant was pushed out of the bone defect. 2.4.4 XRD analysis X-ray diffraction analysis (XRD, X′Pert Pro, Philips, The Netherlands) was carried out to identify the crystalline phases of samples implanted into femurs after push-out testing. The diffraction patterns were collected with a scanning angle 2θ 9



ranging from 4 ° to 50 ° in step-scan intervals of 0.02 ° with Cu Kα radiation at 40 kV and 40 mA. 2.4.5 SEM observation Scanning electron microscopy (SEM, Quanta 200, FEI, The Netherlands) was employed to observe the morphologies of the interface between femoral implants and host bone after 2 weeks of implantation. Specimens were fixed in glutaraldehyde solution for 7 d, dehydrated in gradient alcohols from 75 % to 100 %, and then dried by critical point drying. In addition, the cross-sections of femoral implants after 2, 4 and 8 weeks of implantation were also observed. All specimens were sputter-coated with a layer of gold prior to examination. 2.5 Statistical analysis Each group had three or more independent experimental specimens. All quantitative data were expressed as mean ± standard deviation (S.D.), and the statistically significant differences were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. A value of p0.05), which were 73.36 ± 2.88 %, 17.42 ± 2.83 % and 9.22 ± 0.75 % for PDA-CPC and 74.62 ± 3.19 %, 17.44 ± 2.76 % and 7.94 ± 1.69 % for the control CPC, respectively. These results indicated that the addition of PDA did not induce the significant inflammatory response and blood toxicity after implanted PDA-CPC.

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3.4 Bonding strength The result of the push-out testing (Fig. 2) showed that the push-out values of PDA-CPC groups were 3.07 ± 0.06 MPa and 5.19 ± 1.08 MPa after 2 and 8 weeks of implantation, respectively, while the control-CPC groups were 2.04± 0.24 MPa and 5.62 ± 0.72 MPa. The present results suggested that the bonding strength between implants and host bone increased with time. Compared with the control-CPC, PDA incorporated into CPC enhanced its early bonding strength (increased from 2.04 MPa to 3.07 MPa) significantly (p

Enhanced Repairing of Critical-sized Calvarial Bone Defects by

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