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PCL-CNT nano-composites for bone tissue engineering application Feng Luo 1,2 , Tong Wang 1,2 , Junyu Chen 1,2 , Lai Suo 1,2 , Lanlan Pan 1,3 Xibo Pei 1,2 , Qianbing Wan 1,2 1 State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China 2 Department of Prosthodontics, Sichuan University, Chengdu 61004, China 3 Department of Periodontics, the Affiliated Stomatology Hospital of Chongqing Medical University, Chongqing 400015, China Bone injuries and defects, caused by trauma, malformation, osteoporosis and tumours, are therapeutically challenging. Autografts and allografts are ideal treatment approaches for bone regeneration. However, they may be associated with a number of complications, and outcomes are variable. Therefore, bone substitute biomaterials, which not only effectively solved the problem of limited supply, but also provided possibility for reducing immune-complications and enhancing bone formation properties, have shown great potential for bone tissue engineering application. Currently, polycaprolactone (PCL) are widely used for the production of scaffolds for tissue engineering thanks to its superior rheological and viscoelastic properties. Nevertheless, some intrinsic drawbacks, hydrophobic chemical nature, interaction with biological fluids prevent cells adhesion and proliferation, for instance, limited PCL’s application. Carbon nanotubes (CNTs), with excellent osteoinductivity and osteoconductivity, offer a natural platform for obtaining composite micro-fabricated scaffolds thanks to their outstanding mechanical properties and good biocompatibility, even they are not biodegradable. Thus, PCL-CNT nanocomposites, combined PCL and CNTs to maintain their advantages as well as minimise their drawbacks, could be used as effective bone substitute biomaterials for bone regeneration. Keywords: Bone regeneration; Bone tissue engineering; polycaprolactone; Carbon nanotubes; PCL-CNT nano- composites 1. Bone tissue engineering Bone is a nano-composite made of the extracellular matrix (ECM), including organic (mainly collagen) and inorganic (nano-crystalline hydroxyapatite) substances [1]. As a a hard tissue, bone’s main functions are to provide mechanical support for our body, protect internal organs, produce and store blood cells in bone marrow. However, bone injuries and defects, caused by trauma, malformation, osteoporosis and tumours, are therapeutically challenging [2]. Autografts and allografts are ideal treatment approaches for bone regeneration [3]. Unfortunately, they may be associated with a number of complications, and outcomes are variable. Tissue engineering emerged in the early 1990s to address limitations of tissue grafting and alloplastic tissue repair [4]. Bone tissue engineering offers a promising new approach for bone repair. As a scaffold applied in bone regeneration, the scaffold supposed to meet a number of requirements to fullfill the function. Firstly, the scaffold should have porous architecture design to provide sufficient space for cells growth-into and proliferation [5]. Furthermore, a successful scaffold should balance mechanical function with biofactor delivery in the process of new bone formation [4]. Bone scaffold materials have been improved by remarkable advances in the biomedical engineering in the past decades, but still need better biocompatibility and biofunctionality. 2. PCL in bone tissue engineering Recently, artificial materials, particularly polymers, have been of growing importance in various biomedical technologies, including tissue engineering and transplantation medicine. Polycaprolactone (PCL) [Fig. 1], a hydrophobic, semi-crystalline polymer, is one of the earliest polymers synthesised by the Carothers group in the early 1930s [6]. Currently, PCL is widely applied in the production of scaffolds for tissue engineering thanks to its superior rheological and viscoelastic properties [7 - 9]. Shor and his co-workers fabricate PCL scaffolds with a controlled pore size of 350 μm with designed structural orientations using a novel precision extruding deposition (PED) technique [10]. Their studies demonstrated the viability of the PED process to fabricate PCL scaffolds having the necessary mechanical properties, structural integrity, and controlled pore size and interconnectivity desired for bone tissue engineering. Similarly, Porter et al. used PCL nano-fibers as a replacement for common bone graft material, autogenous cancellous bone, and found that the PCL nanofibers provided a biomimetic environment which are beneficial for the mesenchymal stem cells (MSCs) performance [11]. Zanetti et al. also hold the opinion that PCL composites may have appropriate mechanical and biocompatibility properties for use as bone tissue scaffolds [12]. In addition, PCL’s rate of biodegradation is favourable for bone regeneration. The biodegradable nature of PCL may be attributed to the hydrolytic degradation of its ester bond [13]. Degradation behaviours of porous scaffold play a significant role in tissue regeneration via affecting cell vitality, cell growth, and even host response [14]. As living bone constantly undergoes a couple resorptive-formative process known as bone remodelling [15]. The chosen polymer suppose to degrade at a controlled rate in concert with tissue regeneration. However, the degradation behaviours are Polymer science: research advances, practical applications and educational aspects (A. Méndez-Vilas; A. Solano, Eds.) _______________________________________________________________________________________________ 369

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PCL-CNT nano-composites for bone tissue engineering application

Feng Luo1,2, Tong Wang1,2, Junyu Chen1,2, Lai Suo1,2, Lanlan Pan1,3 Xibo Pei1,2, Qianbing Wan1,2 1State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610041, China 2Department of Prosthodontics, Sichuan University, Chengdu 61004, China 3Department of Periodontics, the Affiliated Stomatology Hospital of Chongqing Medical University, Chongqing 400015,

China

Bone injuries and defects, caused by trauma, malformation, osteoporosis and tumours, are therapeutically challenging. Autografts and allografts are ideal treatment approaches for bone regeneration. However, they may be associated with a number of complications, and outcomes are variable. Therefore, bone substitute biomaterials, which not only effectively solved the problem of limited supply, but also provided possibility for reducing immune-complications and enhancing bone formation properties, have shown great potential for bone tissue engineering application. Currently, polycaprolactone (PCL) are widely used for the production of scaffolds for tissue engineering thanks to its superior rheological and viscoelastic properties. Nevertheless, some intrinsic drawbacks, hydrophobic chemical nature, interaction with biological fluids prevent cells adhesion and proliferation, for instance, limited PCL’s application. Carbon nanotubes (CNTs), with excellent osteoinductivity and osteoconductivity, offer a natural platform for obtaining composite micro-fabricated scaffolds thanks to their outstanding mechanical properties and good biocompatibility, even they are not biodegradable. Thus, PCL-CNT nanocomposites, combined PCL and CNTs to maintain their advantages as well as minimise their drawbacks, could be used as effective bone substitute biomaterials for bone regeneration.

Keywords: Bone regeneration; Bone tissue engineering; polycaprolactone; Carbon nanotubes; PCL-CNT nano-composites

1. Bone tissue engineering

Bone is a nano-composite made of the extracellular matrix (ECM), including organic (mainly collagen) and inorganic (nano-crystalline hydroxyapatite) substances [1]. As a a hard tissue, bone’s main functions are to provide mechanical support for our body, protect internal organs, produce and store blood cells in bone marrow. However, bone injuries and defects, caused by trauma, malformation, osteoporosis and tumours, are therapeutically challenging [2]. Autografts and allografts are ideal treatment approaches for bone regeneration [3]. Unfortunately, they may be associated with a number of complications, and outcomes are variable. Tissue engineering emerged in the early 1990s to address limitations of tissue grafting and alloplastic tissue repair [4]. Bone tissue engineering offers a promising new approach for bone repair. As a scaffold applied in bone regeneration, the scaffold supposed to meet a number of requirements to fullfill the function. Firstly, the scaffold should have porous architecture design to provide sufficient space for cells growth-into and proliferation [5]. Furthermore, a successful scaffold should balance mechanical function with biofactor delivery in the process of new bone formation [4]. Bone scaffold materials have been improved by remarkable advances in the biomedical engineering in the past decades, but still need better biocompatibility and biofunctionality.

2. PCL in bone tissue engineering

Recently, artificial materials, particularly polymers, have been of growing importance in various biomedical technologies, including tissue engineering and transplantation medicine. Polycaprolactone (PCL) [Fig. 1], a hydrophobic, semi-crystalline polymer, is one of the earliest polymers synthesised by the Carothers group in the early 1930s [6]. Currently, PCL is widely applied in the production of scaffolds for tissue engineering thanks to its superior rheological and viscoelastic properties [7 - 9]. Shor and his co-workers fabricate PCL scaffolds with a controlled pore size of 350 μm with designed structural orientations using a novel precision extruding deposition (PED) technique [10]. Their studies demonstrated the viability of the PED process to fabricate PCL scaffolds having the necessary mechanical properties, structural integrity, and controlled pore size and interconnectivity desired for bone tissue engineering. Similarly, Porter et al. used PCL nano-fibers as a replacement for common bone graft material, autogenous cancellous bone, and found that the PCL nanofibers provided a biomimetic environment which are beneficial for the mesenchymal stem cells (MSCs) performance [11]. Zanetti et al. also hold the opinion that PCL composites may have appropriate mechanical and biocompatibility properties for use as bone tissue scaffolds [12]. In addition, PCL’s rate of biodegradation is favourable for bone regeneration. The biodegradable nature of PCL may be attributed to the hydrolytic degradation of its ester bond [13]. Degradation behaviours of porous scaffold play a significant role in tissue regeneration via affecting cell vitality, cell growth, and even host response [14]. As living bone constantly undergoes a couple resorptive-formative process known as bone remodelling [15]. The chosen polymer suppose to degrade at a controlled rate in concert with tissue regeneration. However, the degradation behaviours are

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usually influenced by polymer structures and properties and environmental conditions (medium, temperature, and pH) [16]. Wu et al. investigated the in vitro degradation behaviours of three-dimensional tissue engineering porous scaffolds made from polymers systematically up to 26 weeks in phosphate buffer saline solution at 37 C [17]. It is believed that the ideal in vivo degradation rate may be similar or slightly less than the rate of tissue formation [18]. Eventually, the three-dimensional space occupied by porous scaffolds is replaced by newly formed tissue [16]. Furthermore, the degradation products should not be toxic and must be easily excreted by metabolic pathways. For PCL, the nontoxic degradation metabolites that are formed ultimately and either secreted directly or metabolised in the Krebs cycle [19].

Fig. 1 Structures made from PCL: Nanospheres (a,b). Nanofibres (c,d). Foams (e,f). Knitted textiles (g,h,i). Selective laser sintered scaffold (j-o). Fused deposition modeled scaffolds (p–u) [5]. Nevertheless, some intrinsic drawbacks, hydrophobic chemical nature, interaction with biological fluids prevent cells adhesion and proliferation, for instance, limited PCL’s application [20]. In addition, PCL does not have adequate mechanical properties to be applied in high load bearing situation, particularly which has limited its use in bone tissue engineering. As aforementioned, bone is a kind of hard tissue, normal trabecular bone’s mechanical strength stress is 5 MPa, while the compression modulus is 50 MPa [21]. To overcome these disadvantages, tissue engineering science has focused efforts on developing PCL-based bone scaffolds, which must fulfil certain specific requirements such as biocompatibility, osteointegration ability, biodegradability, and capacity of bioresorption [22]. Moreover, they should have compatible mechanical properties (similar to those of bone) and simple processing [23].

3. CNTs in bone tissue engineering

As Lahiri et al. put forwarded, one of the most effective methods of increasing the mechanical properties (elastic modulus and tensile strength) of a polymer is by reinforcing with a second-phase material [24]. In terms of reinforcement, carbon nanotubes (CNTs) [Fig. 2] supposed to be the candidate with most potential, due to their high mechanical properties (Young’s modulus 0.2-1 TPa, tensile strength 11-63 GPa) and fiberlike structure [25 - 27]. The addition of CNTs to PCL results in an increase in the crystallisation temperature and a decrease in the percent crystallinity confirming the heterogeneous nucleating effect of the nanotubes [28]. The CNT reinforced polymer composites can be used as a new generation high load bearing applications [29 - 31 ].There are some reports on preparation of MWCNT/PLLA composite and MWCNT/PCL composite to increase the mechanical proper- ties of polymers [31, 32]. Lahiri et al. studied the use of CNTs as reinforcement to enhance the mechanical properties of a polylactide-caprolactone copolymer (PLC) matrix. They attributed the increase in the viability of human osteoblast cells compared to the PLC matrix to the combined effect of CNT content and surface roughness of the composite films [24]. Our previous studies also demonstrated that the MWNTs/PCL composite, fabricated via solution evaporation technique, improved its mechanical properties with the addition of MWNTs (0.25-2 wt%) and enhanced the proliferation and

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differentiation of rat bone-marrow-derived stroma cells (BMSCs) [33]. Accordingly, we concluded that MWNTs/PCL composite scaffolds have the potential for bone tissue engineering and the relatively low concentration of MWNTs (0.5 wt%) is preferred [Fig. 3].

Fig. 2 CNTs with controlled structures synthesised in CVD: (a) random SWCNT network; (b) vertically aligned MWCNTs; (c, d) patterned growth of vertically aligned MWCNTs; (e) pyramid like structure of vertically aligned MWCNTs; (f, g) soybean peroxidase (SBP) immobilised on (f) vertically aligned and (g) random CNTs [27]. Besides, it was well documented that carbon nanotubes (CNTs), including single-walled carbon nanotube (SWNT) and multi-walled carbon nanotube (MWNT), offer a natural platform for obtaining micro-fabricated scaffolds for cellular growth attributed to their outstanding mechanical properties, biocompatibility [34, 35] As bone-tissue compatibility of CNTs and CNT influence on bone formation are important issues, the effects of CNTs on bone also attract a lot of attention. In terms of bone regeneration, CNTs can be considered as osteoproductive material which can elicit both intracellular and extracellular cell response at the material surface, which is desirable in bone regeneration since they promote rapid bone integration performance [34]. It is reported that MWNTs adjoining bone show high bone-tissue compatibility and accelerate bone formation stimulated by recombinant human bone morphogenetic protein-2 (rhBMP-2) [36]. Li et al. evaluated the attachment, proliferation, osteogenic gene expression, ALP/DNA, protein/DNA and mineralization of human adipose-derived stem cells cultured in vitro on MWNTs. The results indicated that MWNTs might stimulate inducible cells in soft tissues to form inductive bone by concentrating more proteins, including bone-inducing proteins [37]. Usui et al. demonstrated that MWCNT have very good bone-tissue compatibility and help in the bone repair by accelerating its growth [36]. Furthermore, CNTs get closely integrated in the grown bone without toxic effect [38]. CNTs also shows minimised local inflammatory reactions and is well integrated into the newly formed bone after implantation for bone regeneration [36, 39]. These findings should encourage development of clinical treatment modalities involving CNTs.

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Fig. 3 Experimental and computational effective moduli for (a) tensile and (b) compressive specimens. Data represents mean±standard deviation for n=6, *P<0.05 (compared to pure PCL specimens) [33].

4. PCL-CNT nano-composites in bone tissue engineering

Thus, PCL-CNT nano-composites, combined PCL and CNTs together to maintain their advantages as well as minimise their drawbacks, could be used as an effective bone substitute biomaterial for bone regeneration [20]. With this materials combination, the achieved scaffolds are supposed to obtain bioactivity, biocompatibility, porosity, and size pore, and mechanical properties compatible with the bone tissue, as well as electrical conductivity for inducing bone healing. For instance, Sanchez-Garcia et al. present the properties of nano-bio-composites of PCL containing carbon nanotubes as a function of filler content. It is found that carbon nanotubes can be used to enhance the conductivity, thermal, mechanical and to enhance gas barrier properties of thermoplastic biopolyesters [40]. Otherwise, polymer-based nano-composites containing biocompatible and bioactive nano-components seem to be excellent materials that could be used in many biomedical applications. The carbon nanotubes can modified the bone cell growth and proliferation rate. Our previous work also reported the rat bone-marrow-derived stroma cells (BMSCs) on the composite scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP [Fig. 4. 5] [33]. Wiecheć et al. seed the human osteoblast-like MG 63 cells with PCL/CNT nano-composites for biological evaluation. Results of this study confirm that the PCL/CNT nano-composites are appropriate to the growing and proliferation of human osteoblast-like cells [41]. Besides, by “grafting from” approach based on in-situ ring-opening polymerisation, biodegradable PCL has been covalently grafted onto the surfaces of multi-walled carbon nanotubes (MWNTs). It was convenient for us to control the grafted PCL content via adjusting the feed ratio of monomer to MWNT-supported macro-initiators (MWNT-OH). When fabricating MWNT/PCL composite fibers by in situ polymerisation, Saeed et al. found that the carbon nanotubes were well dispersed and oriented along the finer axes [42]. More importantly, the carbon nanotubes retain their inherently tubelike characteristic and the PCL also hold its own biodegradability in the biodegradation experiments. These results indicate possible application for PCL/CNT nano-composites in biomaterials, biomedicine and artificial bones [43].

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Fig.4 SEM images of the BMSCs cultured on composite scaffolds (CNT-0.5 and CNT-2) and pure PCL scaffold under the same culture condition. Magnification: (a) 600×, (b) 1200× [33]. Furthermore, besides the superior mechanical capacity as well as biocompatibility, another important feature for bone substitute scaffolds, is that, after the implantation in the bone-defected site, the formation of layers of bio-active bone-like hydroxyapatite (Ca10(PO4)6(OH)2) (HA) between the surface of biomaterials and the bone tissue [44].

Hydroxyapatite, the main inorganic component of bone tissue, is the ideal candidate for enhancing bioactivity of composites [45, 46]. The formation of hydroxyapatite in vitro of bone scaffolds can be implemented in simulated body fluid (SBF), which has substantially identical ion composition and ion concentration as human plasma [47]. Investigators have fabricated a bone-like apatite layer on various types of organic polymer substrates and titanium metal by soaking in SBF solution [48 - 50]. Costa et al. further studied the differential regulation of osteoblast and osteoclast activity of hydroxyapatite coatings by incubation of polycaprolactone with SBF, which is a straight-forward method to directly modify surface complexity and roughness [51]. As the chemical composition of molecular structures of minerals formed are similar to the minerals of natural bone, the bioactive layer is supposed to promote the integration between biomaterials and bone tissue as well as bone healing [52 - 54]. Therefore, once the synthetic biomaterial is able to exchange via ion and remineralisation in SBF, and induce the formation of bone-like apatite layer on its surface, the material can be considered to own a relatively superior bioactivity [55]. It is a convenient way to evaluate whether the material is bioactivity or not. Fortunately, it was reported that the surface of PCL, both two-dimensional (2 D) and three-dimensional (3 D), formed a dense and uniform bone-like apatite layer, which is strongly adhered to the PCL surface and remained intact after a tape-detachment test, by immersing into SBF for 24 h [17, 48, 51]. In this regard, it is concluded that the scaffold also shows great potential for bone tissue engineering. Currently, some papers also try to using PCL/CNT composites in various fields regarding tissue engineering. The diversity of applications for which PCL/CNTs composites can be used for is greatly increased by the addition of surface functionalisations; addition of functional groups to the surface can alter the solubility, dispersion and cellular uptake of composites [56 - 58]. For example, as reported, the myocardium is unable to regenerate itself after infarct, resulting in scarring and thinning of the heart wall. Wickham et al. [Fig. 6] devoted to develop a patch to buttress and bypass the scarred area, while allowing regeneration by incorporated cardiac stem/progenitor cells (CPCs) [59]. With further development, PCL/T-CNT meshes or similar patches may become a viable strategy to aid restoration of the postmyocardial infarction myocardium. The CNT/PCL conductive polymer composite, developed by layer by layer spray, also showed promising results as a possible sensor for the identification of organic vapours [60]. Rana et al. prepared films consisted of chitosan grafted (CNT-CS) and chitosan-co-polycaprolactone grafted (CNT-CS-PCL) multiwalled carbon nanotubes using a spray layer-by-layer technique for Vapor Sensing [61]. Kim et al. described a one-pot method for the mass production of polymeric microspheres containing water-soluble carbon-nanotube (w-CNT)-taxol complexes as a sustained drug delivery system [62].

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Fig. 5 A. Live/dead staining of BMSCs cultured for 1 day. (a) Live cells on pure PCL. (b) Live cells on MWNTs/PCL composite (CNT-0.25). (c) Dead cells on MWNTs/PCL composite. The live BMSCs were stained green and appeared to have adhered and attained a normal polygonal morphology on all materials. Dead cells were stained red and were very few on all materials. B. BMSCs proliferation on pure PCL and MWNTs/PCL composite scaffolds. (a) Cells on pure PCL; and (b) cells on MWNTs/PCL composite (CNT-0.25); (c) confluent monolayer of BMSCs on MWNTs/PCL composite (CNT-0.5). C. Percentage of live BMSCs, PLive. Each value is mean ± deviation, n = 5. [33] Another promising field, nano-materials refer to the size of those material which are less than 100 nanometers in diameter. Due to their size, nanoparticles exhibit properties like high surface area, functionalizable surface chemistry and controllable structure, which are unlike that of the same material in bulk size. Nanoparticles themselves have the potential to have therapeutic benefit. Through manipulation of their elemental composition, size, shape, charge and surface modification or functionalisation it may be possible to target particles to specific organs where they may elicit their therapeutic effect [63]. With truly nanometric features, CNTs show exceptional mechanical, thermal, optical and electrical properties which may facilitate better structural and interfacial integrity in biomedical devices. In addition, CNTs could be potentially assembled into complex, multifunctional and three-dimensional (3D) structure. These advantages suggest carbon nanostructures with well defined shapes and functions as a next-generation material to repair and regenerate hard tissues both in vitro and in vivo [27].

5. 3 D structure of PCL-CNT nano-composites

When talking about 3D structure, it is well documented that porosity and pore size of biomaterial scaffolds play a critical role in bone formation in vitro and in vivo [64]. Bone is a structure composed of cancellous and cortical bone. Cancellous is spongy in nature having 50–90 vol% porosity, while cortical bone with less than 10 vol% porosity [65]. Scaffolds for osteogenesis should mimic the ECM properties in order to optimise integration into surrounding tissue. Porous bone scaffolds, or 3D structure scaffolds, can be made by lots of methods, e.g. chemical/gas foaming [66], solvent casting, particle/salt leaching [67], foam-gel [68]. Park et al. devised a new method for biosynthesis of CNT-based 3D scaffold by in situ hy bridising CNTs with bacterial cellulose (BC), which has a structure ideal for tissue-engineering scaffolds [39]. This 3D scaffold was achieved simply by culturing Gluconacetobacter xylinus, Bc-Synthesising bacteria, in medium containing

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CNTs. An amphiphilic comb-like polymer (APCLP) was adsorbed on CNTs to overcome CNT’s aggregation problem. The APCLP-adsorbed CNT-BC hybrid scaffold (CNT-BC-Syn) showed homogeneously distributed CNTs throughout the 3D microporous structure of BC. More importantly, the CNT-BC-Syn scaffolds also showed excellent osteoconductivity and osteoinductivity that led to high bone regeneration efficacy. Fig. 6 SEM images showing topological difference between (A) PCL sheets and (B) PCL fibers. Scale bar, 20 lm. The incorporation of thiophene-conjugated carbon nanotubes (T-CNTs) did not alter the morphology of the meshes, as seen in SEM micrographs of electrospun PCL (C) and PCL/T-CNT (D) fibrous meshes. Scale bar, 10 lm. [59] Among the different technology methods, three dimensional printing (3DP) is becoming more and more popular due to the ability to directly print porous scaffolds with designed shape, controlled chemistry and interconnected porosity. Bose et al. reviewed recent advances in 3D printed bone tissue engineering scaffolds along with current challenges and future directions [66]. Gonçalves et al. produced a three-phase composite scaffold via 3D printing, including nanocrystalline hydroxyapatite (HA), carbon nanotubes (CNT), mixed in a polymeric matrix of polycaprolactone (PCL), aimed at bringing together the properties of all into a unique material to be used in tissue engineering as support for cell growth [23]. The 3D printing technique allows producing composite scaffolds having an interconnected network of square pores in the range of 450-700 μm [Fig. 7]. The three-phase composites composites show typical hydroxyapatite bioactivity, good cell adhesion and spreading at the scaffolds surface, indicating that the scaffolds with CNT and PCL are promising materials in the field of bone regenerative medicine.

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Fig. 7 Technical drawing (A); 3D simulation (B); 10CNT scaffold (C); and final configurations of the 0CNT scaffold (D-side view, E-top view) [23].

6. 3 D structure of PCL-CNT nano-composites

In summary, both CNTs and PCL have superior properties which are favourable for tissue engineering application. The PCL-CNT nano-composites, have excellent mechanical properties, biocompatibility as well as osteogenesis ability, indicating CNT/PCL have great potential in bone regeneration application. In addition, We predicted that the CNT/PCL scaffold with 3D structure which can mimic the ECM properties is very promising. Finally, as an emerging field, we need pay more attention supposed to the long-term degradation and biocompatibility about the CNT/PCL nano-composites.

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