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Composite scaffolds loaded with bone mesenchymal stem cells promote the repair of radial bone defects in rabbit model
Biomedicine & Pharmacotherapy 97 (2018) 600–606
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Composite scaffolds loaded with bone mesenchymal stem cells promote the repair of radial bone defects in rabbit model
MARK
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Shi-qiang Ruan, Jiang Deng , Ling Yan, Wen-liang Huang Department of Orthopaedics Surgery, the First People's Hospital of Zunyi City, Zunyi, 563003, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Silk fibroin Chitosan Nano-hydroxyapatite Bone marrow derived mesenchymal stem cells Osteogenesis Bone defect
This study aimed to investigate the efficacy of three-dimensional scaffolds of silk fibroin/chitosan/nano-hydroxyapatite (SF/CS/nHA) and bone marrow derived mesenchymal stem cells (BMSCs) on the repair of long segmental bone defects in rabbits. BMSCs were cultured with SF/CS/nHA in vitro, and cell proliferation, alkaline phosphatase activity and Ca2+ content were examined. A 15 mm segmental defect in the radius was generated in 12 New Zealand White rabbits, which were divided randomly into three groups (n = 4): experimental group with SF/CS/nHA scaffold of induced BMSCs; control group with SF/CS/nHA scaffold; and blank group without any materials. Postoperatively at 12 weeks, osteogenesis effect and the degradation and absorption of SF/CS/ nHA were evaluated by X-ray, hematoxylin eosin staining, and scanning electron microscopy. In vitro, SF/CS/ nHA scaffolds exhibited good biocompatibility and no toxicity. SF/CS/nHA promoted adhesion, growth, and calcium nodule formation of BMSCs compared to control (P < 0.05). In vivo, we observed gradual new bone formation and bone defect gradually recovered at 12 weeks in experimental and control group, but more new bone was formed in experimental group (P < 0.05). In blank group, limited bone formation was observed and bone defect was obvious. In conclusion, SF/CS/nHA scaffolds loaded with BMSCs achieve high efficacy to repair segmental defect in the radius.
1. Introduction Large segmental bone defect remains a big problem for orthopedic surgeons, and the best choice of treatment is autologous bone graft at present [1]. However, this procedure has many disadvantages such as trauma surgery and pain. In addition, limited sources for the graft and complications associated with obtaining the bone prevent the wide application [2]. Although allograft bone grafts and xenograft bone grafts overcome the disadvantages of autogenous bone grafts, they pose the strong possibility of a rejection reaction and the potential spreading of disease [1,3]. Recently, considerable efforts have been focused on materials with physicochemical properties similar to natural bone that have good biological compatibility and activity and induce osteogenesis. These bone graft materials must contain two basic components: (1) bone mineral, namely calcium phosphate hydroxyapatite; and (2) organic matter such as the main collagen fiber and osteogenesis growth factor [1], and include hydroxyapatite (HA), nano-hydroxyapatite (n-HA), calcium phosphate, chitosan and artificial polymers. HA is a good alternative material for bone tissue repair due to good biocompatibility and osteogenic activity [4,5]. However, there are both advantages and
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disadvantages associated with all kinds of single scaffolds used for bone tissue repair. Therefore, it is important to adjust the proportion of material and the corresponding combination to create ideal scaffold materials [6]. BudiRaharjo et al. used chitosan scaffold material coated with nano-hydroxyapatite cultured with stem cells and demonstrated good biocompatibility and osteogenesis induction ability [7]. Da Silva et al. described composite scaffolds composed of chitosan and silk fibroin cultured with bone marrow derived stem cells (BMSCs) in vitro, and they found that these scaffolds could induce cartilage cell differentiation [8]. Wang et al. reported that biomimetic bone substitutes of collagensilk fibroin/hydroxyapatite exhibited good biocompatibility and strong ability to stimulate new bone formation [9]. Yang et al. prepared nanocomposites using silk sericin and hydroxyapatite crystals, and showed that the nucleation of hydroxyapatite crystals mediated by silk sericin promoted osteogenic differentiation [10,11]. Therefore, nanocomposites composed of silk fibroin and hydroxyapatite show promise in tissue engineering. In this study, three-dimensional scaffolds of silk fibroin/chitosan/ nano-hydroxyapatite (SF/CS/nHA) alone or in combination with BMSCs were used to repair radial bone defects in rabbit model. SF/CS/
Corresponding author. E-mail address: [email protected] (J. Deng).
http://dx.doi.org/10.1016/j.biopha.2017.10.110 Received 8 September 2017; Received in revised form 17 October 2017; Accepted 21 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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(Abcam, Cambridge, MA, USA) following the manufacturer’s protocols.
nHA was evaluated in vitro and in vivo for the ability to repair bone defect.
2.6. Construction of animal model of radial bone defect 2. Materials and methods Animal experiments were approved by the Animal Care Committee of the First People's Hospital of Zunyi City (Zunyi, China). New Zealand White rabbits (4 months old, weighed 2.5–3.0 kg) received intravenous injection of pentobarbital (30 mg/kg). In the middle radius, 3 cm long longitudinal incision was generated to expose the radial diaphyseal, and 15 mm long radial cut was produced with a micro-oscillating saw to cause radial defect. The rabbits were then divided randomly into three groups (n = 4): experimental group (treated with 15 × 3 × 3 mm3 scaffold consisting of BMSCs), control group (treated with 15 × 3 × 3 mm [3] scaffold) and blank group (no scaffold). After the closing of the wounds with sutures, the rabbits received intramuscular injection of penicillin at 50 U/kg for 3 days to prevent possible infection.
2.1. Fabrication of three-dimensional composite scaffold materials Silk fibroin, chitosan, and nano-hydroxyapatite (Sigma, St. Louis, MO, USA) were dissolved in 2% solution and then mixed at a volume ratio of 1:1:1 with constant stirring at 55 °C. The solution was then quickly injected into an orifice plate of 9 holes using a 20-ml syringe and frozen at −80 °C for 24 h. The frozen scaffolds were sealed quickly with sealing membrane, which was suctioned using a vacuum dryer for 36 h after creating scaffolds with pores in the corresponding area. After the dried scaffolds were stored in a 75% volume fraction of methanol and 1 mol/L sodium hydroxide, they were again dried in a vacuum after washing with ultrapure water. The scaffolds were soaked for 10 h in EDC and NHS crosslinking agent (Sigma, St. Louis, MO, USA) after the wet composite scaffolds were frozen at −80 °C, dried for 36 h under a vacuum. The prepared three-dimensional composite scaffolds were sealed for storage and then used in the applications [12]. The wafershaped SF/CS/nHA materials were prepared and physical and chemical properties were evaluated at Sichuan University. All materials were sterilized during the preparation.
2.7. Radiographic examination On day 1 and weeks 4, 8, and 12 postoperatively, all models were given X-ray on the surgical area, with uniform exposure conditions to evaluate osteotylus growth and bone defect healing at 42 kV, 100 mA/ s, 1.2 m. The images were evaluated for SF/CS/nHA material absorption and degradation, bone formation and bone defect recovery based on Lane and Sandhu scoring system [13].
2.2. Culture and identificationof BMSCs Rabbit BMSCs were provided by Cyagen Biosciences Inc. (Guangzhou, China). Flow cytometry showed molecular markers as follows：CD90: 90.64%, CD45: 1.24%, CD14: 0.36%. The frozen BMSCs were thawed by rapid thawing method, followed by incubation at a density of 1 × 104 cells/ml at 37 °C in 5% CO2 and 95% air. Third generation cells in logarithmic phase were selected as the test cells, and osteoblasts were identified by direct morphological observation under an inverted phase contrast microscope and Alizarin red staining of mineralized nodules. SF/CS/nHA scaffolds were washed with phosphate-buffered saline (PBS), and then placed in 24-well flat bottom tissue culture plates. BMSCs were seeded at 3 × 104 cells/well and cultured for 10 days.
2.8. Histological examination Postoperative 12 weeks, four rabbits were sacrificed in each group. Then samples were collected, including 1 cm of the bone surrounding the scaffolds, to be fixed in 10% formalin, decalcified, embedded in paraffin, and cut into 5 μm sections. The sections were then stained with collagen type I antibody (Abcam, Cambridge, MA, USA) and counterstained with hematoxylin and eosin (H & E). New bone growth and collagen expression in the interface between the scaffold and host bone were analyzed under microscopy. In addition, the heart, liver, lung, and kidney tissue samples were collected, fixed in 10% formalin, embedded in paraffin, and cut into sections for H & E staining. All sections were evaluated independently by two pathologists in a blind manner.
2.3. Scanning electron microscopy
2.9. Statistical analysis
SF/CS/nHA scaffolds were rinsed and placed into culture plates, BMSCs were then inoculated and cultured. On days 5 and 10 the scaffolds seeded with BMSCs were harvested, fixed by 2.5% glutardialdehyde for 1 h and then dehydrated by graded ethanol (50, 70, 90, 95, 100%). The samples were coated with gold and the morphologies of BMSCs on the surface of variable scaffolds were observed under scanning electron microscope at 20 kV (JEOL JSM-5910, Japan).
All data were expressed as the mean ± standard deviation and analyzed using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). The differences among the groups were analyzed by two-way ANOVA followed by post-hoc test. P < 0.05 was considered significant. 3. Results
2.4. MTT assay 3.1. Morphology of BMSCs cultured with SF/CS/nHA BMSCs were seeded in 96-well plates at 4 × 103 cells/well and allowed to attach for 4 h. The cells were cultured for 14 days. During the period, the cells were labeled with MTT (Sigma) and cell proliferation was detected by MTT assay. Microplate reader (Perkin Elmer HTS7000plus, USA) was used to determine the absorbance value of cultured cells under the wavelength of 570 nm.
BMSCs were cultured with SF/CS/nHA materials for different periods and observed under inverted microscope. BMSCs grew significantly better in experimental group compared with blank group (Fig. 1A and B). In addition, we observed increased number of cells and nuclei, and two to three nuclei in some cells. Thus SF/CS/nHA had good biocompatibility and could induce osteogenesis. Scanning electron microscopy showed that three-dimensional composite scaffolds were porous. The pore connectivity of the internal scaffolds was good, with an aperture of 100–300 μm and a porosity of 90.18 ± 0.96% (Fig. 1C and D). BMSCs in three-dimensional composite scaffolds were fusiform or polygon shaped. In addition, we observed a network structure, indicating the adhesion and growth of BMSCs on the material surface. We also observed the formation of
2.5. Assay of alkaline phosphatase (ALP) activity and Ca2+ content BMSCs were digested with 0.25% trypsin for 5 min, and then collected by centrifugation followed by treatment with Triton-X. By freezing and thawing the cells three times, the lysates were collected and centrifuged at 15,000 rpm at 4 °C for 15 min. ALP activity and Ca2+content in the supernatants were determined by colorimetric kits 601
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Fig. 1. Characterization of BMSCs cultured in vitro. Inverted microscopy observation (×100) showing a large amount of cell growth around SF/CS/nHA in Experimental group (A) but significantly fewer cells in Blank group (B), after culture for 10 days in vitro. Red arrow: BMSCs, black arrow: SF/CS/ nHA. Scanning electron microscopy observation showing that most cells grew on SF/CS/nHA (C × 500, D × 1000) after culture for 5 days. C indicated BMSCs. Scanning electron microscopy observation showing that most cells grew on SF/ CS/nHA and osteoblasts were surrounded by numerous calcium nodules. (E × 500, F × 1000) after culture for 10 days. C indicated BMSCs. Ca indicated calcium nodules. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Fig. 2B, P < 0.05). In addition, we examined Ca2+ content in BMSCs and found that Ca2+content in BMSCs was significantly higher in experimental group than in blank group at all time points (Fig. 2C, P < 0.05).
calcium nodules on the material surface in experimental group (Fig. 1E and F). 3.2. SF/CS/nHA increased BMSCs proliferation, ALP activity and Ca2+ content
3.3. Bone morphology of the rabbits in each group BMSCs grew rapidly within the first 3 to 5 days of culture and reached a peak on day 10. The proliferation of BMSCs was significantly higher in experimental group than in blank group at all time points (Fig. 2A, P < 0.05). Furthermore, we detected the activity of ALP which is a specific marker of osteoblast differentiation. ALP activity was significantly higher in experimental group than in blank group at all time points
No rabbits died postoperatively. At postoperative 1 week, the rabbits had limb hanging, lameness, poor spirit, reduced food intake, and coarse hair. One week later, we observed gradual recovery but no significant differences between experimental group and control or blank group. At postoperative 12 week, we sacrificed the rabbits and collected 602
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Fig. 2. A. MTT assay of the proliferation of BMSCs culture in the presence or absence of SF/CS/nHA. B. ALP assay of BMSCs culture in the presence or absence of SF/CS/nHA. C. Calcium concentration in BMSCs culture in the presence or absence of SF/CS/nHA. d: days. Error bars indicated SDs (n = 3). *P < 0.05, Experimental group vs. Blank group. Fig. 3. A. Perioperative photograph of the bone in Experimental group. B. In Experimental group after surgery for 12 weeks, SF/CS/nHA material was not visible and new bone formed extensively. C. In Control group, SF/CS/nHA material was not visible and new bone formed, but bone defect still existed. D. In Blank group, bone defects were obvious, marginal sclerosis was observed, and the broken ends were connected through the fiber. Black arrow: bone defect edge, blue arrow: fibrous tissue, red arrow: new bone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
bone specimens for gross observation. The implanted SF/CS/nHA fused completely with the host bone in experimental group and control group, and was covered with new bone, indicating bone defect repair. These effects were better presented in experimental group than in control group (Fig. 3A–C). In contrast, in blank group only few new bone formed at the broken bone ends, and sclerosis and bone defects were observed (Fig. 3D). On day 1 and week 12 postoperatively, radiological examination revealed that SF/CS/nHA material gradually degraded and was absorbed in experimental and control group. Moreover, we observed trabecular bone and new bone formation, and the recovery of the radius. In blank group, we observed few new bone formation at the broken bone ends, but obvious marginal sclerosis and bone defects (Fig. 4A–F). According to Lane and Sandhu scoring system with a total score of 11, the scores ranged from 6 to 8 in experimental group and from 1 to 3 in blank group (Table 1).
experimental group. Moreover, we observed trabecular bone and new bone formation, and the recovery of the radius (Fig. 5G). In blank group, we observed only few new bone at the broken bone ends and bone defects were obvious (Fig. 5H).
4. Discussion Autologous bone grafts are regarded as the best treatment for long segmental bone defects, but the application is restricted due to several disadvantages [1,2]. With the development of tissue engineering, artificial bone materials have demonstrated good biocompatibility, absorbability, degradability, mechanical strength, osteoinduction activity, and processing properties, thus overcoming the disadvantages of autogenous bone grafts. The internal structure of scaffolds is beneficial for the regeneration of normal tissue and appropriate degradation [14,15]. Synthetic polymer materials include poly lactic acid, phosphorus and nitrogen, and beta hydroxybutyric acid. Synthetic inorganic materials include hydroxyl apatite, bioactive glass, and beta tricalcium phosphate. Although natural polymeric materials exhibit good biocompatibility, they have insufficient mechanical strength. Synthetic inorganic materials have good plasticity and mechanical strength but poor hydrophilicity,
3.4. Histological analysis of the bones in each group HE staining of bone tissue lesions showed that most implanted SF/ CS/nHA were absorbed or degraded, while new cartilage and bone formed in experimental and control groups on week 12 postoperatively (Fig. 5A and B). In blank group no new bone and cartilage formed, and massive fibrous tissues formed at the bone defect site (Fig. 5C). Moreover, immunohistochemistry staining showed that most Collagen I fibers formed a weave-like structure in experimental and control groups (Fig. 5D and E), but only very small amount of collagen I fibers formed in blank group (Fig. 5F). At 12 weeks after surgery, scanning electron microscopy showed gradual degradation and absorbance of SF/CS/nHA material in 603
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Fig. 4. Imaging analysis of the bones on Day 1 and 12 Weeks after surgery. A. Blank group on Day 1. B. Control group on Day 1. C. Experimental group on Day 1. D. Blank group after 12 weeks, no new bone formed and bone defect was not restored. E. Control group after 12 weeks, SF/CS/nHA material gradually degraded and was absorbed, new bone formed and bone defect was restored mostly but not completely. F. Experimental group after 12 weeks, SF/CS/nHA material gradually degraded and was absorbed, new bone formed and bone defect was restored completely.
sodium alginate which was then cultured with rabbit bone marrowderived stromal stem cells, and the composite material showed good cell compatibility. Ye et al. prepared three-dimensional scaffolds of carboxyl butyric acid/hydroxyvaleric acid copolymer and single beta tricalcium phosphate which showed better repairing ability and the degradation rate of the composite copolymer was more suitable for bone tissue repair of rabbit tibial bone defect. Yao et al. used composite scaffolds of silk fibroin/hydroxyapatite to repair rabbit articular cartilage defects, and demonstrated that the composite materials had a good scaffold effect and good biocompatibility. Based on these data, in this study we used SF/CS/nHA composite as bone graft material to repair radial bone defects in rabbit model. We performed both in vitro and in vivo experiments to evaluated the biocompatibility, safety and osteoinductive activity of SF/CS/nHA. In addition, we cultured BMSCs with SF/CS/nHA and found that cell adhesion and growth were significantly better compared to BMSCs cultured in the absence of SF/CS/nHA. SF/CS/nHA demonstrated good biocompatibility because −OH, −NH2, and −COOH polar groups in the material promote the adhesion of organic components and the colonization of bone cells on the material surface [21]. ALP is a good marker of osteoblast differentiation. High expression levels of ALP promote the synthesis of collagen fibers, the establishment
Table 1 Modified Lane and Sandhu Radiological Scoring of Bone Formation, Union, and Remodeling. Time
0d 4 wk 8 wk 12 wk
Experimental group
Blank group
formation
union
reconstruction
formation
union
reconstruction
0 1 2–3a 3–4a
0 1 2a 2a
0 0 1a 1–2a
0 1 1–2 1–2
0 0 0 0
0 0 0 0–1
a Significant difference (P < 0.05) was observed between experimental group and blank group at 8 or 12 weeks. Abbreviations: d, days; wk, weeks.
and it is difficult to control the degradation [16]. It is difficult for a single material to meet all requirements of scaffolds, and thus composites of many types of materials are designed to achieve the requirements for ideal bone tissue engineering [17–20]. Zhu et al. prepared scaffolds with nano hydroxyapatite and polylactic acid that were casted with solvent particle leaching technology combined with gas foaming, which demonstrated a good elasticity modulus and better mechanical strength than a single composite material. Xiao et al. prepared a composite bone cement of nano hydroxyapatite/carboxymethyl chitosan/ 604
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Fig. 5. Histological analysis of bone tissue on 12 Weeks after surgery. A–C: HE staining ( ×100). (A) In Experimental group, most SF/CS/nHA material was degraded and absorbed, and extensive new bone formation was observed. (B) In Control group, most SF/CS/nHA material was degraded and absorbed, and new bone formation was observed. (C) In Blank group, there was a large amount of fibrous tissue in the bone defect site. Red arrow: new trabecular bone, black arrow: chondrocytes and cartilage matrix, pink arrow: materials, green arrow: bone marrow cavity, yellow arrow: fibrous tissue, and blue arrow: broken bone ends. D-F: Immunohistochemical staining of Collagen I at the lesion ( ×200). (D) In Experimental group, a large amount of collagen fibers formed around SF/CS/nHA. (E) In Control group, less amount of collagen fibers formed around SF/CS/nHA compared to Experimental group. (F) In Blank group, collagen fiber formation was not apparent. F, collagen I fiber; M, SF/CS/nHA material. G–H: Scanning electron microscopy (×500). (G) In Experimental group, bone defect was barely visible. (H) In Blank group, bone defects were visible. Blue arrow: bone tissue, red arrow: bone defect. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
treatment, SF/CS/nHA was vaguely visible, indicating that it is absorbed and degraded. The radial defect almost recovered and bone defects were repaired in experimental and control groups. In contrast, bone defects were obvious, the broken end was hardened, and the medullary cavity was closed in blank group. Collagen I fibers are important index for bone formation due to the role in the adhesion, differentiation and maturation of osteoblast. Postoperative 12 weeks, immunohistochemical staining of collagen I revealed a large amount of collagen fibers in experimental and control groups, which exhibited a weave-like structure around SF/CS/nHA. However, only few amount of collagen I fibers formed in blank group. These data confirm that SF/CS/nHA composite loaded with BMSCs promote bone formation in vivo. In conclusion, in this study we showed that SF/CS/nHA composite had good physicochemical properties and biological compatibility, was safe, degradable and resorbable, and capable of osteogenic induction. Furthermore, SF/CS/nHA composite loaded with BMSCs demonstrated high efficacy to repair radial defect in rabbit model. To our knowledge,
of calcium nodules, and bone mineralization [22]. In this study we found that ALP activity increased with the prolongation of culture and was significantly higher in BMSCs cultured with SF/CS/nHA than in BMSCs cultured in the absence of SF/CS/nHA. Ca2+ plays important role in the differentiation, maturation, and bone calcification in osteoblasts [23]. Therefore, we detected Ca2+ content and found that it increased significantly in BMSCs cultured with SF/CS/nHA than in BMSCs cultured in the absence of SF/CS/nHA. Taken together, these in vitro experiments confirmed that SF/CS/nHA composite enhanced the differentiation of BMSCs to osteoblasts and stimulated the establishment of calcium nodules to promote new bone formation. Furthermore, we employed rabbit model to evaluate the absorption and degradation, and the osteogenic capability of SF/CS/nHA composite in vivo. The radial defect was established in the rabbits which were then treated with SF/CS/nHA and BMSCs (experimental group), with implanted SF/CS/nHA alone (control group) and with saline (blank group). Gross bone specimen examination, imaging, histochemical and scanning electron microscopy analysis showed that 12 weeks after 605
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this is the first study to combine SF/CS/nHA composite with BMSCs to evaluate the efficacy to repair segmental bone defects. Although our study has several shortcomings such as the lack of the quantitation of the repair outcomes in rabbit model, our results provide the foundation for further development of SF/CS/nHA composite loaded with BMSCs as a promising approach for bone repair and reconstruction.
formation, Acta Biomater. 6 (3) (2010) 1149–1157. [9] J. Wang, Q. Yang, C. Mao, S. Zhang, Osteogenic differentiation of bone marrow mesenchymal stem cells on the collagen/silk fibroin bi-template-induced biomimetic bone substitutes, J. Biomed. Mater. Res. A 100 (11) (2012) 2929–2938. [10] M. Yang, G. Zhou, Y. Shuai, J. Wang, L. Zhu, C. Mao, Ca2+-induced self-assembly of Bombyx mori silk sericin into a nanofibrous network-like protein matrix for directing controlled nucleation of hydroxylapatite nano-needles, J. Mater. Chem. B Mater. Biol. Med. 3 (12) (2015) 2455–2462. [11] M. Yang, Y. Shuai, C. Zhang, Y. Chen, L. Zhu, C. Mao, H. OuYang, Biomimetic nucleation of hydroxyapatite crystals mediated by Antheraea pernyi silk sericin promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells, Biomacromolecules 15 (4) (2014) 1185–1193. [12] C.M. Murphy, M.G. Haugh, F.J. O'Brien, The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering, Biomaterials 31 (3) (2010) 461–466. [13] J.M. Lane, H.S. Sandhu, Current approaches to experimental bone grafting, Orthop. Clin. North Am. 18 (2) (1987) 213–225. [14] M. Salerno, E. Cenni, C. Fotia, et al., Bone-targeted doxorubicin-loaded nanoparticles as a tool for the treatment of skeletal metastases, Curr. Cancer Drug Targets 10 (7) (2010) 649–659. [15] J.N. Yewle, D.A. Puleo, L.G. Bachas, Enhanced affinity bifunctional bisphosphonates for targeted delivery of therapeutic agents to bone, Bioconjug. Chem. 22 (12) (2011) 2496–2506. [16] J.S. Lee, W.Y. Park, J.K. Cha, et al., Periodontal tissue reaction to customized nanohydroxyapatite block scaffold in one-wall intrabony defect: a histologic study in dogs, J. Periodontal Implant Sci. 42 (2) (2012) 50–58. [17] T. Crouzier, F. Sailhan, P. Becquart, R. Guillot, D. Logeart-Avramoglou, C. Picart, The performance of BMP-2 loaded TCP/HAP porous ceramics with a polyelectrolyte multilayer film coating, Biomaterials 32 (30) (2011) 7543–7554. [18] F. Liao, Y. Chen, Z. Li, et al., A novel bioactive three-dimensional beta-tricalcium phosphate/chitosan scaffold for periodontal tissue engineering, J. Mater. Sci. Mater. Med. 21 (2) (2010) 489–496. [19] F.J. Martínez-Vázquez, F.H. Perera, P. Miranda, A. Pajares, F. Guiberteau, Improving the compressive strength of bioceramic robocast scaffolds by polymer infiltration, Acta Biomater. 6 (11) (2010) 4361–4368. [20] U. van der Pol, L. Mathieu, S. Zeiter, et al., Augmentation of bone defect healing using a new biocomposite scaffold: an in vivo study in sheep, Acta Biomater. 6 (9) (2010) 3755–3762. [21] R. Bos, H.C. van der Mei, H.J. Busscher, Physico-chemistry of initial microbial adhesive interactions–its mechanisms and methods for study, FEMS Microbiol. Rev. 23 (2) (1999) 179–230. [22] A.G. Dias, M.A. Lopes, A.T. Trigo Cabral, J.D. Santos, M.H. Fernandes, In vitro studies of calcium phosphate glass ceramics with different solubility with the use of human bone marrow cells, J. Biomed. Mater. Res. A 74 (3) (2005) 347–355. [23] N.J. Hallab, K.J. Bundy, K. O’Connor, R.L. Moses, J.J. Jacobs, Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion, Tissue Eng. 7 (1) (2001) 55–71.
Disclosure The authors report no conflicts of interest in this work. Acknowledgments This study was financially supported by the Science and Technology plan projects in Guizhou province (No. [2016]1420) and the National Natural Science Foundation of China (No. 81660367). References [1] R.Z. LeGeros, Properties of osteoconductive biomaterials: calcium phosphates, Clin. Orthop. Relat. Res. 395 (2002) 81–98. [2] E.S. Jang, J.W. Park, H. Kweon, et al., Restoration of peri-implant defects in immediate implant installations by Choukroun platelet-rich fibrin and silk fibroin powder combination graft, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 109 (6) (2010) 831–836. [3] W.R. Moore, S.E. Graves, G.I. Bain, Synthetic bone graft substitutes, ANZ J. Surg. 71 (6) (2001) 354–361. [4] P. Gentile, V. Chiono, F. Boccafoschi, et al., Composite films of gelatin and hydroxyapatite/bioactive glass for tissue-engineering applications, J. Biomater. Sci. Polym. Ed. 21 (8–9) (2010) 1207–1226. [5] S.I. Roohani-Esfahani, S. Nouri-Khorasani, Z. Lu, R. Appleyard, H. Zreiqat, The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites, Biomaterials 31 (21) (2010) 5498–5509. [6] Z. Wang, M. Li, B. Yu, L. Cao, Q. Yang, J. Su, Nanocalcium-deficient hydroxyapatitepoly (e-caprolactone)-polyethylene glycol-poly (e-caprolactone) composite scaffolds, Int. J. Nanomed. 7 (2012) 3123–3131. [7] R. Budiraharjo, K.G. Neoh, E.T. Kang, Hydroxyapatite-coated carboxymethyl chitosan scaffolds for promoting osteoblast and stem cell differentiation, J. Colloid Interface Sci. 366 (1) (2012) 224–232. [8] M.L. Alves da Silva, A. Crawford, J.M. Mundy, et al., Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Composite scaffolds loaded with bone mesenchymal stem cells promote the repair of radial bone defects in rabbit model
MARK
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Shi-qiang Ruan, Jiang Deng , Ling Yan, Wen-liang Huang Department of Orthopaedics Surgery, the First People's Hospital of Zunyi City, Zunyi, 563003, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Silk fibroin Chitosan Nano-hydroxyapatite Bone marrow derived mesenchymal stem cells Osteogenesis Bone defect
This study aimed to investigate the efficacy of three-dimensional scaffolds of silk fibroin/chitosan/nano-hydroxyapatite (SF/CS/nHA) and bone marrow derived mesenchymal stem cells (BMSCs) on the repair of long segmental bone defects in rabbits. BMSCs were cultured with SF/CS/nHA in vitro, and cell proliferation, alkaline phosphatase activity and Ca2+ content were examined. A 15 mm segmental defect in the radius was generated in 12 New Zealand White rabbits, which were divided randomly into three groups (n = 4): experimental group with SF/CS/nHA scaffold of induced BMSCs; control group with SF/CS/nHA scaffold; and blank group without any materials. Postoperatively at 12 weeks, osteogenesis effect and the degradation and absorption of SF/CS/ nHA were evaluated by X-ray, hematoxylin eosin staining, and scanning electron microscopy. In vitro, SF/CS/ nHA scaffolds exhibited good biocompatibility and no toxicity. SF/CS/nHA promoted adhesion, growth, and calcium nodule formation of BMSCs compared to control (P < 0.05). In vivo, we observed gradual new bone formation and bone defect gradually recovered at 12 weeks in experimental and control group, but more new bone was formed in experimental group (P < 0.05). In blank group, limited bone formation was observed and bone defect was obvious. In conclusion, SF/CS/nHA scaffolds loaded with BMSCs achieve high efficacy to repair segmental defect in the radius.
1. Introduction Large segmental bone defect remains a big problem for orthopedic surgeons, and the best choice of treatment is autologous bone graft at present [1]. However, this procedure has many disadvantages such as trauma surgery and pain. In addition, limited sources for the graft and complications associated with obtaining the bone prevent the wide application [2]. Although allograft bone grafts and xenograft bone grafts overcome the disadvantages of autogenous bone grafts, they pose the strong possibility of a rejection reaction and the potential spreading of disease [1,3]. Recently, considerable efforts have been focused on materials with physicochemical properties similar to natural bone that have good biological compatibility and activity and induce osteogenesis. These bone graft materials must contain two basic components: (1) bone mineral, namely calcium phosphate hydroxyapatite; and (2) organic matter such as the main collagen fiber and osteogenesis growth factor [1], and include hydroxyapatite (HA), nano-hydroxyapatite (n-HA), calcium phosphate, chitosan and artificial polymers. HA is a good alternative material for bone tissue repair due to good biocompatibility and osteogenic activity [4,5]. However, there are both advantages and
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disadvantages associated with all kinds of single scaffolds used for bone tissue repair. Therefore, it is important to adjust the proportion of material and the corresponding combination to create ideal scaffold materials [6]. BudiRaharjo et al. used chitosan scaffold material coated with nano-hydroxyapatite cultured with stem cells and demonstrated good biocompatibility and osteogenesis induction ability [7]. Da Silva et al. described composite scaffolds composed of chitosan and silk fibroin cultured with bone marrow derived stem cells (BMSCs) in vitro, and they found that these scaffolds could induce cartilage cell differentiation [8]. Wang et al. reported that biomimetic bone substitutes of collagensilk fibroin/hydroxyapatite exhibited good biocompatibility and strong ability to stimulate new bone formation [9]. Yang et al. prepared nanocomposites using silk sericin and hydroxyapatite crystals, and showed that the nucleation of hydroxyapatite crystals mediated by silk sericin promoted osteogenic differentiation [10,11]. Therefore, nanocomposites composed of silk fibroin and hydroxyapatite show promise in tissue engineering. In this study, three-dimensional scaffolds of silk fibroin/chitosan/ nano-hydroxyapatite (SF/CS/nHA) alone or in combination with BMSCs were used to repair radial bone defects in rabbit model. SF/CS/
Corresponding author. E-mail address: [email protected] (J. Deng).
http://dx.doi.org/10.1016/j.biopha.2017.10.110 Received 8 September 2017; Received in revised form 17 October 2017; Accepted 21 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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(Abcam, Cambridge, MA, USA) following the manufacturer’s protocols.
nHA was evaluated in vitro and in vivo for the ability to repair bone defect.
2.6. Construction of animal model of radial bone defect 2. Materials and methods Animal experiments were approved by the Animal Care Committee of the First People's Hospital of Zunyi City (Zunyi, China). New Zealand White rabbits (4 months old, weighed 2.5–3.0 kg) received intravenous injection of pentobarbital (30 mg/kg). In the middle radius, 3 cm long longitudinal incision was generated to expose the radial diaphyseal, and 15 mm long radial cut was produced with a micro-oscillating saw to cause radial defect. The rabbits were then divided randomly into three groups (n = 4): experimental group (treated with 15 × 3 × 3 mm3 scaffold consisting of BMSCs), control group (treated with 15 × 3 × 3 mm [3] scaffold) and blank group (no scaffold). After the closing of the wounds with sutures, the rabbits received intramuscular injection of penicillin at 50 U/kg for 3 days to prevent possible infection.
2.1. Fabrication of three-dimensional composite scaffold materials Silk fibroin, chitosan, and nano-hydroxyapatite (Sigma, St. Louis, MO, USA) were dissolved in 2% solution and then mixed at a volume ratio of 1:1:1 with constant stirring at 55 °C. The solution was then quickly injected into an orifice plate of 9 holes using a 20-ml syringe and frozen at −80 °C for 24 h. The frozen scaffolds were sealed quickly with sealing membrane, which was suctioned using a vacuum dryer for 36 h after creating scaffolds with pores in the corresponding area. After the dried scaffolds were stored in a 75% volume fraction of methanol and 1 mol/L sodium hydroxide, they were again dried in a vacuum after washing with ultrapure water. The scaffolds were soaked for 10 h in EDC and NHS crosslinking agent (Sigma, St. Louis, MO, USA) after the wet composite scaffolds were frozen at −80 °C, dried for 36 h under a vacuum. The prepared three-dimensional composite scaffolds were sealed for storage and then used in the applications [12]. The wafershaped SF/CS/nHA materials were prepared and physical and chemical properties were evaluated at Sichuan University. All materials were sterilized during the preparation.
2.7. Radiographic examination On day 1 and weeks 4, 8, and 12 postoperatively, all models were given X-ray on the surgical area, with uniform exposure conditions to evaluate osteotylus growth and bone defect healing at 42 kV, 100 mA/ s, 1.2 m. The images were evaluated for SF/CS/nHA material absorption and degradation, bone formation and bone defect recovery based on Lane and Sandhu scoring system [13].
2.2. Culture and identificationof BMSCs Rabbit BMSCs were provided by Cyagen Biosciences Inc. (Guangzhou, China). Flow cytometry showed molecular markers as follows：CD90: 90.64%, CD45: 1.24%, CD14: 0.36%. The frozen BMSCs were thawed by rapid thawing method, followed by incubation at a density of 1 × 104 cells/ml at 37 °C in 5% CO2 and 95% air. Third generation cells in logarithmic phase were selected as the test cells, and osteoblasts were identified by direct morphological observation under an inverted phase contrast microscope and Alizarin red staining of mineralized nodules. SF/CS/nHA scaffolds were washed with phosphate-buffered saline (PBS), and then placed in 24-well flat bottom tissue culture plates. BMSCs were seeded at 3 × 104 cells/well and cultured for 10 days.
2.8. Histological examination Postoperative 12 weeks, four rabbits were sacrificed in each group. Then samples were collected, including 1 cm of the bone surrounding the scaffolds, to be fixed in 10% formalin, decalcified, embedded in paraffin, and cut into 5 μm sections. The sections were then stained with collagen type I antibody (Abcam, Cambridge, MA, USA) and counterstained with hematoxylin and eosin (H & E). New bone growth and collagen expression in the interface between the scaffold and host bone were analyzed under microscopy. In addition, the heart, liver, lung, and kidney tissue samples were collected, fixed in 10% formalin, embedded in paraffin, and cut into sections for H & E staining. All sections were evaluated independently by two pathologists in a blind manner.
2.3. Scanning electron microscopy
2.9. Statistical analysis
SF/CS/nHA scaffolds were rinsed and placed into culture plates, BMSCs were then inoculated and cultured. On days 5 and 10 the scaffolds seeded with BMSCs were harvested, fixed by 2.5% glutardialdehyde for 1 h and then dehydrated by graded ethanol (50, 70, 90, 95, 100%). The samples were coated with gold and the morphologies of BMSCs on the surface of variable scaffolds were observed under scanning electron microscope at 20 kV (JEOL JSM-5910, Japan).
All data were expressed as the mean ± standard deviation and analyzed using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). The differences among the groups were analyzed by two-way ANOVA followed by post-hoc test. P < 0.05 was considered significant. 3. Results
2.4. MTT assay 3.1. Morphology of BMSCs cultured with SF/CS/nHA BMSCs were seeded in 96-well plates at 4 × 103 cells/well and allowed to attach for 4 h. The cells were cultured for 14 days. During the period, the cells were labeled with MTT (Sigma) and cell proliferation was detected by MTT assay. Microplate reader (Perkin Elmer HTS7000plus, USA) was used to determine the absorbance value of cultured cells under the wavelength of 570 nm.
BMSCs were cultured with SF/CS/nHA materials for different periods and observed under inverted microscope. BMSCs grew significantly better in experimental group compared with blank group (Fig. 1A and B). In addition, we observed increased number of cells and nuclei, and two to three nuclei in some cells. Thus SF/CS/nHA had good biocompatibility and could induce osteogenesis. Scanning electron microscopy showed that three-dimensional composite scaffolds were porous. The pore connectivity of the internal scaffolds was good, with an aperture of 100–300 μm and a porosity of 90.18 ± 0.96% (Fig. 1C and D). BMSCs in three-dimensional composite scaffolds were fusiform or polygon shaped. In addition, we observed a network structure, indicating the adhesion and growth of BMSCs on the material surface. We also observed the formation of
2.5. Assay of alkaline phosphatase (ALP) activity and Ca2+ content BMSCs were digested with 0.25% trypsin for 5 min, and then collected by centrifugation followed by treatment with Triton-X. By freezing and thawing the cells three times, the lysates were collected and centrifuged at 15,000 rpm at 4 °C for 15 min. ALP activity and Ca2+content in the supernatants were determined by colorimetric kits 601
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Fig. 1. Characterization of BMSCs cultured in vitro. Inverted microscopy observation (×100) showing a large amount of cell growth around SF/CS/nHA in Experimental group (A) but significantly fewer cells in Blank group (B), after culture for 10 days in vitro. Red arrow: BMSCs, black arrow: SF/CS/ nHA. Scanning electron microscopy observation showing that most cells grew on SF/CS/nHA (C × 500, D × 1000) after culture for 5 days. C indicated BMSCs. Scanning electron microscopy observation showing that most cells grew on SF/ CS/nHA and osteoblasts were surrounded by numerous calcium nodules. (E × 500, F × 1000) after culture for 10 days. C indicated BMSCs. Ca indicated calcium nodules. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Fig. 2B, P < 0.05). In addition, we examined Ca2+ content in BMSCs and found that Ca2+content in BMSCs was significantly higher in experimental group than in blank group at all time points (Fig. 2C, P < 0.05).
calcium nodules on the material surface in experimental group (Fig. 1E and F). 3.2. SF/CS/nHA increased BMSCs proliferation, ALP activity and Ca2+ content
3.3. Bone morphology of the rabbits in each group BMSCs grew rapidly within the first 3 to 5 days of culture and reached a peak on day 10. The proliferation of BMSCs was significantly higher in experimental group than in blank group at all time points (Fig. 2A, P < 0.05). Furthermore, we detected the activity of ALP which is a specific marker of osteoblast differentiation. ALP activity was significantly higher in experimental group than in blank group at all time points
No rabbits died postoperatively. At postoperative 1 week, the rabbits had limb hanging, lameness, poor spirit, reduced food intake, and coarse hair. One week later, we observed gradual recovery but no significant differences between experimental group and control or blank group. At postoperative 12 week, we sacrificed the rabbits and collected 602
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Fig. 2. A. MTT assay of the proliferation of BMSCs culture in the presence or absence of SF/CS/nHA. B. ALP assay of BMSCs culture in the presence or absence of SF/CS/nHA. C. Calcium concentration in BMSCs culture in the presence or absence of SF/CS/nHA. d: days. Error bars indicated SDs (n = 3). *P < 0.05, Experimental group vs. Blank group. Fig. 3. A. Perioperative photograph of the bone in Experimental group. B. In Experimental group after surgery for 12 weeks, SF/CS/nHA material was not visible and new bone formed extensively. C. In Control group, SF/CS/nHA material was not visible and new bone formed, but bone defect still existed. D. In Blank group, bone defects were obvious, marginal sclerosis was observed, and the broken ends were connected through the fiber. Black arrow: bone defect edge, blue arrow: fibrous tissue, red arrow: new bone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
bone specimens for gross observation. The implanted SF/CS/nHA fused completely with the host bone in experimental group and control group, and was covered with new bone, indicating bone defect repair. These effects were better presented in experimental group than in control group (Fig. 3A–C). In contrast, in blank group only few new bone formed at the broken bone ends, and sclerosis and bone defects were observed (Fig. 3D). On day 1 and week 12 postoperatively, radiological examination revealed that SF/CS/nHA material gradually degraded and was absorbed in experimental and control group. Moreover, we observed trabecular bone and new bone formation, and the recovery of the radius. In blank group, we observed few new bone formation at the broken bone ends, but obvious marginal sclerosis and bone defects (Fig. 4A–F). According to Lane and Sandhu scoring system with a total score of 11, the scores ranged from 6 to 8 in experimental group and from 1 to 3 in blank group (Table 1).
experimental group. Moreover, we observed trabecular bone and new bone formation, and the recovery of the radius (Fig. 5G). In blank group, we observed only few new bone at the broken bone ends and bone defects were obvious (Fig. 5H).
4. Discussion Autologous bone grafts are regarded as the best treatment for long segmental bone defects, but the application is restricted due to several disadvantages [1,2]. With the development of tissue engineering, artificial bone materials have demonstrated good biocompatibility, absorbability, degradability, mechanical strength, osteoinduction activity, and processing properties, thus overcoming the disadvantages of autogenous bone grafts. The internal structure of scaffolds is beneficial for the regeneration of normal tissue and appropriate degradation [14,15]. Synthetic polymer materials include poly lactic acid, phosphorus and nitrogen, and beta hydroxybutyric acid. Synthetic inorganic materials include hydroxyl apatite, bioactive glass, and beta tricalcium phosphate. Although natural polymeric materials exhibit good biocompatibility, they have insufficient mechanical strength. Synthetic inorganic materials have good plasticity and mechanical strength but poor hydrophilicity,
3.4. Histological analysis of the bones in each group HE staining of bone tissue lesions showed that most implanted SF/ CS/nHA were absorbed or degraded, while new cartilage and bone formed in experimental and control groups on week 12 postoperatively (Fig. 5A and B). In blank group no new bone and cartilage formed, and massive fibrous tissues formed at the bone defect site (Fig. 5C). Moreover, immunohistochemistry staining showed that most Collagen I fibers formed a weave-like structure in experimental and control groups (Fig. 5D and E), but only very small amount of collagen I fibers formed in blank group (Fig. 5F). At 12 weeks after surgery, scanning electron microscopy showed gradual degradation and absorbance of SF/CS/nHA material in 603
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Fig. 4. Imaging analysis of the bones on Day 1 and 12 Weeks after surgery. A. Blank group on Day 1. B. Control group on Day 1. C. Experimental group on Day 1. D. Blank group after 12 weeks, no new bone formed and bone defect was not restored. E. Control group after 12 weeks, SF/CS/nHA material gradually degraded and was absorbed, new bone formed and bone defect was restored mostly but not completely. F. Experimental group after 12 weeks, SF/CS/nHA material gradually degraded and was absorbed, new bone formed and bone defect was restored completely.
sodium alginate which was then cultured with rabbit bone marrowderived stromal stem cells, and the composite material showed good cell compatibility. Ye et al. prepared three-dimensional scaffolds of carboxyl butyric acid/hydroxyvaleric acid copolymer and single beta tricalcium phosphate which showed better repairing ability and the degradation rate of the composite copolymer was more suitable for bone tissue repair of rabbit tibial bone defect. Yao et al. used composite scaffolds of silk fibroin/hydroxyapatite to repair rabbit articular cartilage defects, and demonstrated that the composite materials had a good scaffold effect and good biocompatibility. Based on these data, in this study we used SF/CS/nHA composite as bone graft material to repair radial bone defects in rabbit model. We performed both in vitro and in vivo experiments to evaluated the biocompatibility, safety and osteoinductive activity of SF/CS/nHA. In addition, we cultured BMSCs with SF/CS/nHA and found that cell adhesion and growth were significantly better compared to BMSCs cultured in the absence of SF/CS/nHA. SF/CS/nHA demonstrated good biocompatibility because −OH, −NH2, and −COOH polar groups in the material promote the adhesion of organic components and the colonization of bone cells on the material surface [21]. ALP is a good marker of osteoblast differentiation. High expression levels of ALP promote the synthesis of collagen fibers, the establishment
Table 1 Modified Lane and Sandhu Radiological Scoring of Bone Formation, Union, and Remodeling. Time
0d 4 wk 8 wk 12 wk
Experimental group
Blank group
formation
union
reconstruction
formation
union
reconstruction
0 1 2–3a 3–4a
0 1 2a 2a
0 0 1a 1–2a
0 1 1–2 1–2
0 0 0 0
0 0 0 0–1
a Significant difference (P < 0.05) was observed between experimental group and blank group at 8 or 12 weeks. Abbreviations: d, days; wk, weeks.
and it is difficult to control the degradation [16]. It is difficult for a single material to meet all requirements of scaffolds, and thus composites of many types of materials are designed to achieve the requirements for ideal bone tissue engineering [17–20]. Zhu et al. prepared scaffolds with nano hydroxyapatite and polylactic acid that were casted with solvent particle leaching technology combined with gas foaming, which demonstrated a good elasticity modulus and better mechanical strength than a single composite material. Xiao et al. prepared a composite bone cement of nano hydroxyapatite/carboxymethyl chitosan/ 604
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Fig. 5. Histological analysis of bone tissue on 12 Weeks after surgery. A–C: HE staining ( ×100). (A) In Experimental group, most SF/CS/nHA material was degraded and absorbed, and extensive new bone formation was observed. (B) In Control group, most SF/CS/nHA material was degraded and absorbed, and new bone formation was observed. (C) In Blank group, there was a large amount of fibrous tissue in the bone defect site. Red arrow: new trabecular bone, black arrow: chondrocytes and cartilage matrix, pink arrow: materials, green arrow: bone marrow cavity, yellow arrow: fibrous tissue, and blue arrow: broken bone ends. D-F: Immunohistochemical staining of Collagen I at the lesion ( ×200). (D) In Experimental group, a large amount of collagen fibers formed around SF/CS/nHA. (E) In Control group, less amount of collagen fibers formed around SF/CS/nHA compared to Experimental group. (F) In Blank group, collagen fiber formation was not apparent. F, collagen I fiber; M, SF/CS/nHA material. G–H: Scanning electron microscopy (×500). (G) In Experimental group, bone defect was barely visible. (H) In Blank group, bone defects were visible. Blue arrow: bone tissue, red arrow: bone defect. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
treatment, SF/CS/nHA was vaguely visible, indicating that it is absorbed and degraded. The radial defect almost recovered and bone defects were repaired in experimental and control groups. In contrast, bone defects were obvious, the broken end was hardened, and the medullary cavity was closed in blank group. Collagen I fibers are important index for bone formation due to the role in the adhesion, differentiation and maturation of osteoblast. Postoperative 12 weeks, immunohistochemical staining of collagen I revealed a large amount of collagen fibers in experimental and control groups, which exhibited a weave-like structure around SF/CS/nHA. However, only few amount of collagen I fibers formed in blank group. These data confirm that SF/CS/nHA composite loaded with BMSCs promote bone formation in vivo. In conclusion, in this study we showed that SF/CS/nHA composite had good physicochemical properties and biological compatibility, was safe, degradable and resorbable, and capable of osteogenic induction. Furthermore, SF/CS/nHA composite loaded with BMSCs demonstrated high efficacy to repair radial defect in rabbit model. To our knowledge,
of calcium nodules, and bone mineralization [22]. In this study we found that ALP activity increased with the prolongation of culture and was significantly higher in BMSCs cultured with SF/CS/nHA than in BMSCs cultured in the absence of SF/CS/nHA. Ca2+ plays important role in the differentiation, maturation, and bone calcification in osteoblasts [23]. Therefore, we detected Ca2+ content and found that it increased significantly in BMSCs cultured with SF/CS/nHA than in BMSCs cultured in the absence of SF/CS/nHA. Taken together, these in vitro experiments confirmed that SF/CS/nHA composite enhanced the differentiation of BMSCs to osteoblasts and stimulated the establishment of calcium nodules to promote new bone formation. Furthermore, we employed rabbit model to evaluate the absorption and degradation, and the osteogenic capability of SF/CS/nHA composite in vivo. The radial defect was established in the rabbits which were then treated with SF/CS/nHA and BMSCs (experimental group), with implanted SF/CS/nHA alone (control group) and with saline (blank group). Gross bone specimen examination, imaging, histochemical and scanning electron microscopy analysis showed that 12 weeks after 605
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this is the first study to combine SF/CS/nHA composite with BMSCs to evaluate the efficacy to repair segmental bone defects. Although our study has several shortcomings such as the lack of the quantitation of the repair outcomes in rabbit model, our results provide the foundation for further development of SF/CS/nHA composite loaded with BMSCs as a promising approach for bone repair and reconstruction.
formation, Acta Biomater. 6 (3) (2010) 1149–1157. [9] J. Wang, Q. Yang, C. Mao, S. Zhang, Osteogenic differentiation of bone marrow mesenchymal stem cells on the collagen/silk fibroin bi-template-induced biomimetic bone substitutes, J. Biomed. Mater. Res. A 100 (11) (2012) 2929–2938. [10] M. Yang, G. Zhou, Y. Shuai, J. Wang, L. Zhu, C. Mao, Ca2+-induced self-assembly of Bombyx mori silk sericin into a nanofibrous network-like protein matrix for directing controlled nucleation of hydroxylapatite nano-needles, J. Mater. Chem. B Mater. Biol. Med. 3 (12) (2015) 2455–2462. [11] M. Yang, Y. Shuai, C. Zhang, Y. Chen, L. Zhu, C. Mao, H. OuYang, Biomimetic nucleation of hydroxyapatite crystals mediated by Antheraea pernyi silk sericin promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells, Biomacromolecules 15 (4) (2014) 1185–1193. [12] C.M. Murphy, M.G. Haugh, F.J. O'Brien, The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering, Biomaterials 31 (3) (2010) 461–466. [13] J.M. Lane, H.S. Sandhu, Current approaches to experimental bone grafting, Orthop. Clin. North Am. 18 (2) (1987) 213–225. [14] M. Salerno, E. Cenni, C. Fotia, et al., Bone-targeted doxorubicin-loaded nanoparticles as a tool for the treatment of skeletal metastases, Curr. Cancer Drug Targets 10 (7) (2010) 649–659. [15] J.N. Yewle, D.A. Puleo, L.G. Bachas, Enhanced affinity bifunctional bisphosphonates for targeted delivery of therapeutic agents to bone, Bioconjug. Chem. 22 (12) (2011) 2496–2506. [16] J.S. Lee, W.Y. Park, J.K. Cha, et al., Periodontal tissue reaction to customized nanohydroxyapatite block scaffold in one-wall intrabony defect: a histologic study in dogs, J. Periodontal Implant Sci. 42 (2) (2012) 50–58. [17] T. Crouzier, F. Sailhan, P. Becquart, R. Guillot, D. Logeart-Avramoglou, C. Picart, The performance of BMP-2 loaded TCP/HAP porous ceramics with a polyelectrolyte multilayer film coating, Biomaterials 32 (30) (2011) 7543–7554. [18] F. Liao, Y. Chen, Z. Li, et al., A novel bioactive three-dimensional beta-tricalcium phosphate/chitosan scaffold for periodontal tissue engineering, J. Mater. Sci. Mater. Med. 21 (2) (2010) 489–496. [19] F.J. Martínez-Vázquez, F.H. Perera, P. Miranda, A. Pajares, F. Guiberteau, Improving the compressive strength of bioceramic robocast scaffolds by polymer infiltration, Acta Biomater. 6 (11) (2010) 4361–4368. [20] U. van der Pol, L. Mathieu, S. Zeiter, et al., Augmentation of bone defect healing using a new biocomposite scaffold: an in vivo study in sheep, Acta Biomater. 6 (9) (2010) 3755–3762. [21] R. Bos, H.C. van der Mei, H.J. Busscher, Physico-chemistry of initial microbial adhesive interactions–its mechanisms and methods for study, FEMS Microbiol. Rev. 23 (2) (1999) 179–230. [22] A.G. Dias, M.A. Lopes, A.T. Trigo Cabral, J.D. Santos, M.H. Fernandes, In vitro studies of calcium phosphate glass ceramics with different solubility with the use of human bone marrow cells, J. Biomed. Mater. Res. A 74 (3) (2005) 347–355. [23] N.J. Hallab, K.J. Bundy, K. O’Connor, R.L. Moses, J.J. Jacobs, Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion, Tissue Eng. 7 (1) (2001) 55–71.
Disclosure The authors report no conflicts of interest in this work. Acknowledgments This study was financially supported by the Science and Technology plan projects in Guizhou province (No. [2016]1420) and the National Natural Science Foundation of China (No. 81660367). References [1] R.Z. LeGeros, Properties of osteoconductive biomaterials: calcium phosphates, Clin. Orthop. Relat. Res. 395 (2002) 81–98. [2] E.S. Jang, J.W. Park, H. Kweon, et al., Restoration of peri-implant defects in immediate implant installations by Choukroun platelet-rich fibrin and silk fibroin powder combination graft, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 109 (6) (2010) 831–836. [3] W.R. Moore, S.E. Graves, G.I. Bain, Synthetic bone graft substitutes, ANZ J. Surg. 71 (6) (2001) 354–361. [4] P. Gentile, V. Chiono, F. Boccafoschi, et al., Composite films of gelatin and hydroxyapatite/bioactive glass for tissue-engineering applications, J. Biomater. Sci. Polym. Ed. 21 (8–9) (2010) 1207–1226. [5] S.I. Roohani-Esfahani, S. Nouri-Khorasani, Z. Lu, R. Appleyard, H. Zreiqat, The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites, Biomaterials 31 (21) (2010) 5498–5509. [6] Z. Wang, M. Li, B. Yu, L. Cao, Q. Yang, J. Su, Nanocalcium-deficient hydroxyapatitepoly (e-caprolactone)-polyethylene glycol-poly (e-caprolactone) composite scaffolds, Int. J. Nanomed. 7 (2012) 3123–3131. [7] R. Budiraharjo, K.G. Neoh, E.T. Kang, Hydroxyapatite-coated carboxymethyl chitosan scaffolds for promoting osteoblast and stem cell differentiation, J. Colloid Interface Sci. 366 (1) (2012) 224–232. [8] M.L. Alves da Silva, A. Crawford, J.M. Mundy, et al., Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix
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