Chapter 8 Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 Construction of Bio-Inspired Composites for Bone Tissue Repair Junchao Wei,*,1 Lina Wang,1,2 Lan Liao,3 Jiaolong Wang,3 Yu Han,4 and Jianxun Ding*,4 1College of Chemistry, Nanchang University, Nanchang 330031, P. R. China of Science, Nanchang Institute of Technology, Nanchang 330029, P. R. China 3Department of Prosthodontics, Affiliated Stomatological Hospital of Nanchang University, Nanchang 330006, P. R. China 4Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China *E-mail: firstname.lastname@example.org (J. Wei); email@example.com (J. Ding) 2College Materials with excellent mechanical properties and biofunctions are key points of bone tissue repair. With the increase of knowledge about bone tissue structure and the development of nanotechnology, tough materials have been designed to mimic the structure of bone. Based on the structure of bone and nacre, we briefly introduced the factors affecting the mechanical properties of composites and also introduced the most widely used techniques, such as, electrospinning, phase separation, and three-dimensional (3D) printing method, to acquire porous scaffolds. In addition, the biofunctionalization of scaffolds was also introduced in this chapter. 1. Introduction Due to various diseases, accidents and aging of population, bone defect has been a common problem. Autologous bone graft is the gold standard for treating bone defect, however, it also brings a lot of side effects to patients. Although Allograft and Xenograft has brought some promise, sometimes donors’ shortage and immune response limit their application, and thus much work has been carried out to design alternative bone graft materials, which may be composed of © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 polymers, metals, and ceramics. Tissue engineering, which applies the principles of engineering and life sciences towards the development of biological substitutes to restore, maintain or improve the tissue functions (1, 2), has been an exciting and promising method to repair the bone defect. During the process of tissue engineering, the tissue engineering scaffold is a key factor to affect the functional reconstruction of tissues. The general requirement for a tissue engineering scaffold is that the scaffold materials should not only fill the defect area, but also supply both structure and mechanical support (3), even bio-functions to realize the regeneration of bone (4). And thus, much attention has been paid to the materials used in bone tissue engineering (5, 6). The materials of bone tissue engineering scaffolds or bone tissue regenerative composites should have proper mechanical properties to support the growth of bone tissue, furthermore, as for the load-bearing place, the mechanical properties are the key factors, and this point is very important for bone substitutes or bone fixation devices. Secondly, the porous structure should also be realized for the supplement of the ample space to support the growth of new tissues. Thirdly, enough biofunctions such as biocompatibility, bioactivity, bone conductivity and bone inductivity are also critical factors for materials used in bone tissue engineering. So far, various methods have been developed to fabricate polymer composites used in bone tissue repair. Bio-inspired idea has been widely used to design new materials or scaffolds that can mimic the functions of native bone tissue. However, before designing ideal bio-inspired composites, it is vital to understand the structure of native tissue and also it is much helpful to understand the interactions between polymers, nanofillers, and cells. In this chapter, we firstly give a brief introduction of bone structure and the reason why it is tough and strong, and then materials used in bone tissue repair was introduced, finally, methods about how to construct bone-inspired composites were introduced. This short review may give an idea about how to construct strong and tough materials used in bone tissue engineering, and also give some suggestion on how to design biofunctionalized scaffolds. 2. Materials for Bone Tissue Repair 2.1. Natural Strong and Tough Materials—Bone & Nacre Bone is mainly composed of collagen (mostly type I) and hydroxyapatite (Ca10(PO4)6(OH)2, HA). Both the HA and collagen consists about 95% weight of the total bone and formed a tough, strong and low weight materials. Collagen is a kind of natural polymer, its mechanical strength is very low, while HA is a kind of inorganic bioceramic, its mechanical properties is poor, and much brittle. However, an interesting thing is that the natural bone is typically strong and tough, due to the hierarchical structure of HA and collagen, and especially that the HA and collagen arranged in an order way. Briefly, the HA nanoplates are mostly arranged along its c-axis and arranged parallel to the collagen fibrils, the arrangement repeat periodically (Figure 1). 154 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 Figure 1. Hierarchical structure of Bone. Reproduced with permission from ref. (7). Copyright 2014, Nature publishing group. The scheme of bone’s resistance to external force is based on its multiple deformation scales, ranging from nanoscale protein to microscale physiological structure (7, 8). As for the cortical bone, the origins of its fracture resistance ability rise from both intrinsic mechanisms that promote ductility and extrinsic mechanisms that act to shield the growing cracks (8). The intrinsic ductility origins from the smallest length scales, which is mainly from the molecular uncoiling of mineralized collagen. Most importantly, when the load is added to the bone, the stress may transfer between the HA plates and the collagen fibrils, when the stress is too high, fibrillar sliding may happens, which will make the materials tough to resist to the tension. Besides, many other factors contribute to the bones, such as the collagen fibers structure, the phase interaction between HA and collagen, the intermolecular crosslinking, these factors realize the increased strength of bone. These factors make it possible to dissipate energy. So, in order to design strong composites, basic requirements are: realizing the ordered arrangement of fillers, and enhancing the phase compatibility between fillers and polymer matrix. Nacre is another kind of strong materials. It is a brick-and-mortar structure consisting of 95% vol. layered aragonite (CaCO3) plates and a thin layer of protein molecules (Figure 2). Generally, the mechanical properties of both CaCO3 and protein molecules are very poor. However, the mechanical toughness of nacre is three orders of magnitude higher than that of CaCO3. The fracture toughness of CaCO3 is 0.25 Mp·m1/2, while that of nacre is about 10 Mpa·M1/2, nearly 40 times that of CaCO3 (9). The mineral aragonite is brittle, and it provides the strength of nacre. Due to the existence of protein chains (Figure 2a), it is possible for nacre to realize elastic deformation when external load added. The organic molecules work as glue to connect with the plate aragonites and realize the stress distribution, so the nacre shows toughness (Figure 2b). 155 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 Figure 2. Hierarchical structure of nacre. Structure illusion of nanoparticle glued by protein chains (a), the schemes of its toughness (b) and the brick-and-mortar structure (c). Reproduced with permission from ref. (7). Copyright 2014, Nature publishing group. During recent years, much work has been carried out to prepare nacre or bonelike bioinspired materials. Great progresses have been carried out, most of which are focused on the structure-properties characteristic. The unique structure of bone is not only the arrangement of compositions, but also its complex formation process, in which cells function and involvement place an important role. The exact understanding of the biomineralization process and structure has contributed a lot to the design of polymer composites, although much more things need to be deciphered clearly. Nowadays, although it is difficult to prepare materials that can completely mimic the structure of bone or nacre, bioninspired idea has been used to prepare materials from different points, such mechanical properties, porous structure and biofunctions, which may realize their further application in bone regeneration. 2.2. Materials Used for Bone Tissue Repair There are three kinds of materials used in bone tissue repair: metals, ceramics and polymers (3). They can be used in different parts due to their properties. Metals, such as titanium alloy and stainless alloy which are biocompatible and have good mechanical properties, have been used as bone plates or bone screws, however, this kind of materials always need second operation. Ceramics are most inorganic materials exist in bioactive glass, HA, tricalcium phosphate (TCP) and so on, these materials with bone conductivity or bone inductivity are the most widely investigated inorganic materials, however, these materials are brittle and can not be used in load bearing parts. Polymers with good mechanical properties and excellent biocompatibilities may satisfy the requirements of tissue regeneration, and have been widely used in tissue engineering. According to the source of polymers, they can be classified as natural and synthetic polymers. Generally, both degradable and nondegradable polymers can be used in bone tissue repair. For example, ultra-high molecular 156 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 weight polyethylene and polyether ether ketone (PEEK) have been used as bone substitute or artificial kneel, however, when tissue engineering is considered, biodegradable polymers show much more potential, such as collagen, gelatin, poly(L-lactide) (PLLA), poly(lactide-co-glycolide) (PLGA) and so on, have been widely used. However, pure polymers can only mimic part functions of native tissues, they still lack of enough biofunctions to induce bone formation, or biomechanical properties to satisfy bone loading requirements, so polymer composites, especially polymers complexed with inorganic bioceramics have been widely designed. An ideal polymer composite may have hierarchical structure and possess the advantages of different composites, realizing a synergistic effect to put forward the applications in bone repair. Up to now, various polymer composites have been used in bone tissue repair field, such as polymer-polymer blends and polymer-inorganic nanocomposites. The most widely investigated bioceramic/polymer composites are hydroxyapatite/polymer composites. Collage, gelatin, PLLA, PLGA and their HA composites have been widely investigated or reviewed (10). Besides, multicomponent composites contains more than two kinds of polymers or two kinds of inorganic component are also well investigated due to their combination of multi-advantages of different composites and show much better synergistic effects. Although much progress has been achieved, there still need a long way to prepare ideal composites which can mimic the properties of natural tissue and realize the rapid bone substitute or rebuilt of bone tissue. 3. How To Mimic the Properties of Bone During the bone repair process, five special targets should be considered, osteogenesis, vascularization, growth factors, mechanical environment and osteoconductive scaffolds (3). To realize successful bone regeneration, at least three of the targets should be involved. Thus, it is vital to design materials with proper mechanical properties, especially for load-bearing parts. The excellent properties are inevitable, besides, the materials should also have a proper structure and bioproperties to support the growth of new tissues. 3.1. Construction of Composites with Excellent Mechanical Properties Bone tissue has excellent mechanical properties, thus many efforts have been focused on preparing bone inspired composites, for one purpose to obtain strong and light weight materials, and another purpose is to prepare bone regenerative materials or related medical devices, especially for load bearing bone repair, the mechanical properties are always the vital factors. Up to now, plenty polymer composites have been designed, however, mostly, the mechanical properties are far from their theoretical value. A critical challenge is to transfer the excellent mechanical properties from nanoscale to macroscale (11). As for filler-reinforced composites, the obtained properties are always far from their ideal results. The key problem is that they could not realize the homogeneous dipsersion of nanofillers and easily control their arrangement. 157 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 Another factor is that the phase interaction between the fillers and polymer matrix are not as strong as the natural bone composition (12). Nicholas Kotov’s research demonstrated that it is possible to produce composites with properties that can compare with the theoretical values by tuning the spatial and orientation of nanofillers (11). They used a bottom up method called layer-by-layer assembly to prepare a kind of poly(vinyl alcohol)/Montmorillonite (PVA/MTM) composites. With the LBL method, an interlayer structure was formed which can mimic the structure of nacre, due to the controlled structure organization, the clay platelets in polymer matrix arranged orderly, due to much hydroxyl groups of PVA chains and SiO4 groups in MTM, the phase interaction between PVA and MTM are strong. Besides, when the film was crosslinked with GA, the interaction will be much stronger, and the mechanical properties can arrive to its theoretical value. The final tensile strength of the crosslinked PVA/MTM was 400±40Mpa, and the modulus was 106±11Gpa. By tuning the arrangement of nanofillers, various strong bio-inspired composites have been designed. Recently, Robert O Ritchie has reported a kind of hydroxyapatite/poly(methyl methacryalate) (HA/PMMA) composites with layered structure (preparation scheme is shown in Figure 3) (13), which can mimic the nacre structure and work as a kind of tissue engineering scaffolds. The strength, elastic stiffness and work of fracture were 100 Mpa, 20 Gpa and 2075 J·m-2, respectively. These results are nearly two orders of magnitude than monolithic HA. Figure 3. Schematic illustration of fabrication of HA/PMMA composite with nacre-mimetic structure. Reproduced with permission from ref (13). Copyright 2015, John wiley and Sons. 158 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 Another important factor to enhance the mechanical properties of polymer composites is to improve the phase compatibility of nanofillers and polymer matrix. Up to now, many works have been reported to tune the surface properties of nanoparticles by grafting polymer chains on the surface of nanofillers (14–18). As for bone tissue regenerative materials, bioceramics such as hydroxyapatite, bioactive glass are most important to endow polymers with bone conductivity or bone inductivity. However, pure bioceramic particles are brittle and their phase interaction with polymers are always too weak, and thus polymer grafted bioceramics have been prepared. For example, Chen’s group have used ring opening polymerization method to graft polymers on the surface of HA (19–21), PLLA was grafted on the surface of HA and the tensile strength of PLLA-g-HA/PLLA (75 Mpa) was much higher than that of pure HA/PLLA composite (less than 60 Mpa). Besides, PLLA-g-HA can also be used to prepare porous scaffold and showed excellent osteogenesis properties (22). Wei used poly(benzly-l-glutamate) to modify the surface of HA not only change its biocompatibility, but also increase its phase compatibility with Polymer matrix, and the results showed that only 0.3% content can make the mechanical properties increased a lot (18). Besides, Wei’s group also used PBLG to modify the surface of SiO2@GO hybrid and then prepared its PLLA composites, the results showed that the tensile strength of PLLA composites can arrive to 88.9 Mpa, much higher than that of pure PLLA, PLLA/GO or PLLA/SiO2 (23). 3.2. Construction of Porous Scaffolds for Bone Tissue Repair The natural bone is porous structure, while tissue engineering scaffolds also need porous structure to support the growth of tissues. Many methods, such as electrospinning, phase separation and 3-D printing have been used to prepare porous structure scaffolds to realize the regeneration of bone tissue. Here, we will give a brief introduction about these methods. 3.2.1. Electrospinning Electrospinning method has been widely used to construct fibrous porous scaffold to mimic the fibrillar architecture of extracellular matrix (ECM). These fibrous biomimetic scaffolds can supply microenvironment for the regenerative of bone tissue. The basic progress of electrospinning contains three parts (Figure 4a). Firstly, polymer solutions were extruded from a conductive spinneret, and then voltage was applied between the spinneret and grounded collector. When the electric potential in the polymer solution overcomes the surface tension of polymer solution droplet, the droplet will eject to the collector, during this period, the solvent will evaporate, and polymer fibers will be collected on the collector. 159 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 Figure 4. Schemes of basic electrospinning process (a) and fibers with different orientation, random fiber (b), parallel fiber (c), crossed fiber (d), patterned fiber net (e), 3D fibrous stack (f), wavy fiber (g), helical fiber (h), and twisted fibers (i). Reproduced with permission from ref. (26). Copyright 2014, Elsevier. The electrospinning fibers have showed potential applications in tissue engineering, the morphologies of electrospun fibers, such as fiber size, porosity, fiber orientation may affect the attached cells behaviors, thus many efforts have been worked to tune the structure or morphologies of fibers (24). Firstly, the polymer concentration is a key factor, it has to exceed a critical concentration so that enough polymer chains entangle within the polymer solution, then polymer fibers can be formed via electrospinning. Otherwise, dilute polymer solutions will spray into beads or uniform polymer fibers with much more beads aggregate. It is also important to choose polymers with proper molecular weight. If the molecular weight is too low, the polymer chains can not entangle well and it is difficult to form fibers. If the molecular weight is too high, the polymer entangles a lot and increases the solution viscosity, which means the surface tension of droplet is much higher, and thus it is also difficult to form fibers, unless high pressure voltage was used. So it is vital importance to tune the polymer solution, some times other additive, such as surfactant or amphiphilic molecules should be added. Besides, the solvent can also be a critical factor to affect the solution viscosity, proper solvent, sometimes mix solution is a prerequisite to obtain designed fibers (25). In addition, with different kinds of jet or needle, and different kinds of collectors can realize different morphologies electrospun fibers, 160 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. fibers with different morphologies or arrangement such as random or orientation aligned fibers can be prepared via different technologies (26). Electrospinning as an important method to prepare new tissue engineering is not only used to prepare polymer materials, up to now, various polymer blend or polymer-inorganic composites have been prepared, Such as PLA/PCL (27), PLLA grafted hydroxyapatite/PLLA (28), gelatine/chitoasan/hydroxyapatite/graphene oxide. With the increasing requirements for tissue engineering, various functional molecules (growth factor, proteins) and drugs can be loaded with fibers and enrich the properties of tissue engineering scaffolds (29, 30) (see part 3.3). Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 3.2.2. Phase Separation Phase separation is a simple method to prepare porous or fibrous structure that can mimic the native structure of ECM and has been widely used in tissue engineering scaffolds. The basic principle of phase separation is based on the thermally unstability of polymer solutions and tend to separate into two or more phases under certain conditions (31–33), briefly, either exposuring the polymer solution to a immiscible solution or cooling down to a certain temperature, phase separation would happen and form a polymer-rich phase and a polymer-poor phase. The basic procedure of phase separation includes polymer dissolution, phase separation and gelation, solvent extraction, freezing and freeze drying (31). By tuning the parameters of phase separation, porous scaffolds with different pore sizes and shapes can be obtained (34), for example, by tuning the temperature below or above the polymer solutions, both closed and open pores can be obtained, respectively; Under lower temperature, the solvent can crystallize quickly and the crystal size will be smaller, when the solvent crystal was removed, the scaffolds, with smaller pores will be obtained, otherwise, scaffolds with large pores can be formed. even the cooling rate and freezing temperature may have also vital affection on the pore structure (35). For example, when frozen at -80 °C and 190 °C, PLLA scaffolds with different pore sizes 47±8 and 22±4μm were prepared (36), many other kinds of scaffolds, such as chitosan, PLLA/chitosan have also been prepared by phase separation method (37). Sometimes it is difficult to realize precise control of the microstructure, some modified methods or combination of phase separation methods and other methods, such as solvent casting, porogen leaching and supercritical method have been used (38), polymer solution can be cast around salt sugar and other porogen. By tunning the size of porogen agents, scaffolds with different porous structures can be obtained, and thus it is vital important in tissue engineering, due to that the pore size has an important effect on the cell behavior and bone formation (39). 3.2.3. 3D Printing Although various methods such as elecrospinning, phase separation solvent casting, and salt leaching and many other methods have been designed to prepare porous scaffolds, it is still a challenge to precise control the hierarchical structure 161 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 such as pore size, shape and pore interconnectivity. Inspired by the complex structure of biological materials, especially some native tissue structure such as bone or nacre, 3D printing has been used to fabricate various scaffolds with intricate microstructure (40, 41). 3D printing applies additive manufacturing approaches, combine computer assisted design (CAD) software and printing machine together to prepare products by a layer-by-layer method, and the basic procedure has been introduced in many references (42, 43). The basic characteristics of 3D printing can be described as follows (44): A) supply of building blocks or raw materials in a continuous or stepwise method. B) A programme that contains the structure information should be supplied to determine the assembly of materials. C) Mechanisms or equipments that can fulfill the programme and control the assembly of building blocks. D) A consolidation step to fix the deposited materials and make the printed structure as designed. Up to now, various technologies, such as deposition modeling, stereolithography, ink-jet printing have been used to prepare tissue engineering scaffolds and can easily realize on-demand fabrication of customized products with precise structures (44, 45). For bone tissue engineering, 3D printing is very convenient to construct scaffolds with desired structure. Meantime, with the development of 3D technology, various materials, such as ceramic, metallic, polymers and polymer composites can be used for 3D print (46, 47). Cho used sterolithography method and prepared three-dimensional (3D) porous scaffolds of poly(propylene fumarate)/diethyl fumarate (PPF/DEF) (48), which have sufficient mechanical stability and are non-toxic. After post-modification, the scaffold can enhance the adhesion and proliferiation of MC3T3-E1 preosteoblast cells, showing potential application in bone tissue repair. Polyester, such as PLA, PCL, PLGA have been widely used in bone tissue engineering, when combined with 3D technology, various scaffolds have been prepared with these biodegradable polymers (49). Inorganic materials, such as HA, α-TCP, β-TCP and bioactive glass have also been used as 3D printing materials to build bone tissue engineering scaffolds (50). In order to combine the bone inductivity of bioceramics and the biodegradability of polymers, bioceramics, HA, TCP, bioactive glass have been used as fillers to prepare polymer composites used for bone tissue engineering. For examples, PLLA/HA, chitosan/HA, collagen/HA and alginate/bioactive glass have been prepared with 3-D printing (51–53). Via 3-d printing method, scaffolds which can mimic the structure of native bone tissue can be prepared easily; however, the mechanical properties of porous scaffolds are still challenges. The porous structure always results in low mechanical properties, and always used in non-load bearing place. However, by tuning the delicate printing method, it is a good method to realize the requirements of complicated structures. For example, PolyJet 3-D printing method based on ink jet technology can realize deposition of multi-materials, which enables the possibility to prepare both strong and tough materials. This method would be much useful to prepare polymer composites that can mimic the bone (44, 54). Buelher had used multi-material 3D printing to print composites with bone-inspired topologies that exhibit superior fractural mechanical properties, and the computational model predictions of the fracture behaviors and trends in mechanical properties are in accordant with the experimental results, 162 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 demonstrating that it is possible to design composite materials and then use 3-D printing to synthesize the desired materials with expected mechanical performance (54). 3-D printing will have much more promising perspective in tissue engineering. However, many critic requirements, such as specific technical, material and cellar aspects of the printing process will bring more challenges (43). The increasing resolution requirements may make 3-D printed products mimic the tissue structure more exactly. The ideal materials should not only be biocompatible, but also can be easily manipulated by the printing technology to acquire complex 3-D structure and maintain its bioproperties. The cells used should be easily available, and can reproduce all the functions of tissue or organ system (43), so multidiscipline should forged together to meet the challenges and thus further improve the application of 3-D printing in tissue engineering. 3.3. Composites with Special Biofunctions Although most biocompatible polymers or polymer composites can be made into scaffolds that can mimic the structure of native tissue, but it is difficult to mimic the biofunctions. The cell functions and growth factors play critical roles in the process of tissue regeneration, so various methods have been used to prepare composites with special biofunctions. Firstly, the native biomacromolecues or polymers found in the ECM can be used as ideal scaffolds to mimic the biofucntions. Collagen, the most organic content of bone, has been used for a long time. It is not only used alone, but also blended with various polymers or inorganic particles. Other materials, such as hydroxyapatite, the inorganic component of bone, have also been widely used in tissue engineering or bone repair, due to its bone conductivity or bone inductivity properties. In order to further improve the biofunctions, increasing works have been focused on the combination of scaffolds and biomacromolecules, such as protein or growth factors (BMP, IGF). The growth factors can control osteogenesis, bone tissue regeneration and ECM formation via recruiting and differentiation osteoprogenitor cells to specific lineages (55). So it is critical to incorporate protein or growth factors in the tissue engineering scaffolds. Generally, there are two kinds of method to prepare biomacromolecules contained scaffolds. One method is pre-treat method, which means that biomolecules were added into the scaffolds while the preparation process. The other method can be called post-treatment method, which means the biomacromolecules were adsorbed or anchored on the scaffolds by physical or chemical interactions. For example, biomolecules contained electrospinning scaffolds can be prepared via different blend electrospinning, coaxial electrospinning and covalent immobilization methods (56), and these methods can realize the functional molecules be adsorbed or covalently anchored on the surface of fibers or encapsulated within the fibers. The configurations of biomolecules have key effects on their signal transduction activity, and thus it is critical to keep the structure of growth factors. Mussel-inspired method with 3,4-dihydroxyphenethylamine (DOPA) contained peptide has been widely used, recently Ito’s group designed a method to prepare 163 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 DOPA containing insulin-like growth factor-1(IGF-1) and anchor the IGF-1 on the surface titanium (Figure 5). This method can be used to prepare novel cell-growth enhancing materials and thus have much potential in tissue engineering (57). Zhang’s group also used the mussel-inspired method and immobilized collagen mimetic peptide and osteogenic growth peptide on the surface of L-lactic acid oligomer modified hydroxyapatite/Poly(lactide-co-glycoclide) composite film (58). The results demonstrated that it is a good method to immobilize biomacromolecues on the surface of implants with bioinspired method and realize their enhanced osteointegration of bone implants. Figure 5. Preparation of DOPA contained IGF-1 derivatives and its immobilization on the surface of titanium. Reproduced with permission from ref (57). Copyright 2016, John wiley and Sons. To encapsulate special cells in the scaffolds is also a good method to improve the biofunction of scaffolds, for example bio-printing has been designed to prepare cells contained scaffolds (43), and it can combine the biocompatible materials, cells and other components into a functional living tissue and thus will have much more applications. 4. Perspective In this chapter, we briefly introduce the structure of bone and how to construct the bio-inspired composites used in the field of bone tissue repair. Due to the requirements of bone tissue regenerative, tough materials with excellent mechanical properties and scaffolds with porous structure and special biomolecules are needed in bone tissue repair. Although many progresses 164 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2017. Downloaded by GEORGETOWN UNIV on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1253.ch008 have been achieved, there still need a long way to prepare ideal scaffolds or substitutes that can real mimic the bone structure or properties. However, by further understanding the structure of native bone and the interactions between materials and tissues, new hints maybe brought out to the materials design, and the challenges will be overcame via the combination of materials, engineering, biology, medicine, and others. To our opinion, in the next few years, more interests will be placed on the design of new functional materials with special biofunctions, or prepared new functional polymer composites, which may be used for bone implant medical devices or manufactured into scaffolds with desired structure. As a basic requirement, how to realize enhancements of both toughness and strength, and make the mechanical properties satisfied for bone tissue will still be an interesting point. New preparation method or modification method will be continuously investigated to realize the multi-functions of tissue engineering scaffolds, such as how to keep the long term stability of growth factors or realize its controlled release in the scaffolds, and how to control the scaffold structure exactly, especially in the nanoscale level. 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