Biomaterials for bone tissue engineering scaffolds: a review

Bone tissue engineering has been continuously developing since the concept of “tissue engineering” has been proposed. Biomaterials that are used as the basic material for the fabrication of scaffolds play a vital role in bone tissue engineering. This paper first introduces a strategy for literature search. Then, it describes the structure, mechanical properties and materials of natural bone and the strategies of bone tissue engineering. Particularly, it focuses on the current knowledge about biomaterials used in the fabrication of bone tissue engineering scaffolds, which includes the history, types, properties and applications of biomaterials. The effects of additives such as signaling molecules, stem cells, and functional materials on the performance of the scaffolds are also discussed.


Introduction
Bone and its associated diseases, accounting for half of chronic diseases in people over 50 years old, still remain an important clinical challenge. 1,2 Although bones have a certain healing and/or regeneration capacity, it cannot be accomplished by itself for large segmental bone defects. Large bone defects or injuries, caused by old age, traffic accident, fracture nonunion, bone tumor resection, etc., are serious problems in orthopaedics, and they bring great harms to health and the quality of life. 3,4 Autologous bone graing is still regarded as the "gold standard" for repairing bone defects. However, the drawbacks of autologous bone graing include secondary damages, high donor site morbidity, limitation of special shape, insufficiency of autogenous bone and so on. These weaknesses limit its widespread use in clinical settings.
The term "tissue engineering" was rst used in 1987. 5 It is the utilization of a combination of multidisciplinary approaches to improve or replace biological tissues. In recent years, with the rapid development of tissue engineering technology, bone tissue engineering has become a hopeful approach for repairing bone defects. Scaffolds play a crucial role in bone tissue engineering. Their purpose is to mimic the structure and function of the natural bone extracellular matrix (ECM), which can provide a three-dimensional (3D) environment to promote the adhesion, proliferation, and differentiation and to have adequate physical properties for bone repair. An ideal scaffold should be biodegradable, biocompatible, bioactive, osteoconductive and osteoinductive. Articial bone scaffolds with biomaterials and additives, such as drugs, growth factors (GFs) and stem cells, have been useful for bone repair. The biomaterials (biomedical materials), which are basic components of scaffolds, play an important role in bone tissue engineering. Archaeological ndings showed that materials such as human or animal bones and teeth, corals, shells, wood, and several metals (gold, silver and amalgam) were used for the replacement of missing human bones and teeth. 6 For example, in the ancient times, the Etruscans learnt to replace damaged teeth with articial gra obtained from the bones of oxen. In the early 1960s, the limitations of biological bone substitute materials resulted in the emergence of a multidisciplinary eld called "Biomaterials". 7 Biomaterials are used for the evaluation, treatment, augmentation, repair or replacement of tissues or organs of the body. Ancient alternative materials are mostly bioinert (biologically inert), and these materials interact less with the surrounding tissues and are even toxic to humans. An ideal biomaterial should be non-cytotoxic, printable, biodegradable, bioactive, and osteoconductive in vivo. Due to the various needs of scaffolds, composite materials composed of two or more materials with excellent properties are widely used in bone tissue engineering.
Numerous natural and synthetic polymers such as calcium phosphates, calcium carbonate, and bioactive glasses have been used to fabricate scaffolds. Recent outstanding approaches include the addition of conductive polymers (CPs), inducerons (signaling molecules, unlike bone morphogenetic protein 2 (BMP-2)) and mechanical signals (elastic polymer networks such as hydrogels) to bone tissue engineering scaffolds. With the integration, intercrossing and development of the elds of medicine, biology, materials and other disciplines, biomaterials have been extensively used in the fabrication of bone tissue engineering scaffolds. 8 This article gives a brief introduction to the descriptions of the hierarchical structure, chemical composition of natural bone and strategies for bone tissue engineering. It aims to outline the history, types, properties and development methods of common biomaterials used to fabricate scaffolds. Further, the review also highlights the biomaterial scaffolds with additives. Finally, it examines the combination of advanced technology and biomaterials, and emphasizes the challenges and opportunities of biomaterials in bone tissue engineering scaffolds.

Materials and methods
All studies (in vitro and in vivo) concerning the application of biomaterials to manufacture scaffolds for bone tissue engineering were researched in duplicate in the Medline (PubMed) online database. The PubMed search was performed to look for articles published in English between January 1, 2010 and January 1, 2019. The Medical Subject Heading (abbreviated as MeSH) terms "bone and bones", "biocompatible materials" and "tissue scaffolds" were used together with the keywords "bone tissue engineering", "biomaterials" and "scaffolds" to apply the following search strategy: ( The follow-up period or sample size is not limited. Meta analyses and systematic reviews were not included. Scientic research regarding the following topics was not considered: scaffolds for assisted positioning of transplants and help with surgical planning before the surgery.

Study selection
Two of the authors individually selected the titles and abstracts of the articles obtained by the above-mentioned search. Then, the selected studies were independently carefully sied by both of the reviewers. Any disagreement was determined through discussions between them.

Data extraction
Two of the authors separately summarized the search and sought consensus among other authors in the process. The undermentioned information was recorded: the publication information including the author's name and publication data, the biomaterials applied to manufacture scaffolds and their important characteristics.
3. Structure, mechanical properties and materials of natural bone

Hierarchical structure of bone
As the main part of the human skeletal system, bone plays a crucial role in providing structure, supporting mechanical movement, protecting organs, and producing and hosting blood cells. It has a complex hierarchical structure based on the length and width scale, which consists of the macro scale (trabecular bone, also known as cancellous or spongy bone, and compact bone, also named cortical bone), microscale and submicroscale (haversian canals, osteons and lamellae), nanoscale (brillar collagen) and sub-nanoscale (such as minerals, collagen and so on), as shown in Fig. 1. 11 The structure of natural bone has been presented in various articles. [11][12][13][14][15][16][17][18][19][20][21] Compact bone is nearly solid, except for $3-5% of rooms for canaliculi, osteocytes and so on. 18 However, trabecular bone is an interconnected porous network and has a higher bone surface-to-bone volume (BS/BV) ratio than compact bone.

Mechanical properties of bone
The mechanical properties of natural bone vary greatly with respect to age and the body part. Young's modulus and yield stress of natural bone are anisotropic. A complete understanding of the mechanics of living bones remains an important scientic challenge. Table 1 shows the mechanical properties of natural bone obtained from the reported data. 18 The longitudinal direction of the compact bone is robuster and stiffer than its transverse direction. The trabecular bone has a porous structure, and the porosity and arrangement of the individual trabeculae determine its mechanical properties.

Natural composition of bone
The understanding of the material components of natural bone plays a crucial role in the selection of scaffold materials. Natural bone consists of cells, ECM assembled from collagen brils and hydroxyapatite (HA), and bound minerals. Collagen and HA together account for $95% of natural bone under dry conditions. 21 The composition of natural bone is presented in Table  2. 19 Biological apatites deviate from the stoichiometric composition of HA and contain certain amounts of ion substitution impurities such as Na + , Mg 2+ , Cl À , K + , F À , and Zn 2+ . HA is the major inorganic component of human skeleton.

Bone tissue engineering
Although human bones have a certain self-healing ability, they are powerless for large bone defects. To overcome the problems, bone tissue engineering is proposed on the basis of tissue engineering. Bone tissue engineering aims to induce new tissue repairing and regeneration by the synergy of cells, signals and scaffolds. 8 A scaffold composed of biomaterials is a carrier of cells and signals. It plays a key role in bone tissue engineering. Strategies for bone tissue engineering are shown in Fig. 2. 22 For the large-sized tissues and origins with different shapes, it is necessary to design a temporary support to provide spaces for cell proliferation, differentiation and growth. The support is called scaffold, transplant, template or articial ECM. As noticed before, an ideal scaffold should have biocompatibility, suitable mechanical properties, high porosity and gradient pore structure. As the new tissue grows, the implanted scaffold gradually degrades until the new tissue completely replaces it. The design and fabrication of scaffolds with customization can be obtained by computer-aided design and computer-aided manufacturing (CAD/CAM) technology. Biomaterials are an important part of the scaffolds, and an ideal biomaterial should possess the following characteristics: (1) biocompatibility; (2) biodegradability; (3) easy printing and processing. During the last decades, researchers have shown increasing interest towards biomaterials for their application in bone tissue engineering scaffolds.
Generally, the obtained scaffolds should be biologically investigated. The main approaches of biological research in vitro as forecasting test before pre-clinical can be divided into two main categories: (1) in vitro culture experiments such as scaffold toxicity tests, animal or human cells (such as BMSCs, 23 hMSCs, 24 etc.) and (2) in vivo animal experiments (such as repairing of femur defects in rats). 25 Scaffolds with non-toxic, good biocompatibility are the basis of bone repair and regeneration, in which biomaterials play an important role in the excellent performance of the scaffolds.

History of biomaterials
In the long history of human development, tissues and organs have evolved with respect to function aer millions of years, but humans have been using articial substitutes to repair damaged tissues only for decades. In the year 659 AD, the Chinese rst used dental amalgam to repair defects in teeth. 26 The limitations of bone replacement materials have resulted in the utilization of synthetic alternative materials for bone repair, replacement and enhancement. "Biomaterials" appeared in the early 1960s. 7 The history of using biomaterials for scaffolds based on three different generations is briey introduced below. 8 The rst generation of biomaterials appeared in the 1960s. 27 It aimed to achieve the performance of the biomaterial to match the replaced tissue with the least toxic reaction to the host. They are generally bioinert, and interact minimally with the surrounding tissues. The rst generation of biomaterials mainly includes: metals (such as titanium or titanium alloys), synthetic polymers (such as PMMA and PEEK) and ceramics (such as alumina and zirconia).
The most important feature of the second-generation biomaterials is their bioactive nature, and some could be biodegradable in vivo. They consist of synthetic and natural polymers (e.g. collagen), calcium phosphates, calcium carbonate, calcium sulfates, and bioactive glasses.  The third generation of biomaterials are designed to induce specic benecial biological responses by the addition of instructive substances based on the second-generation biomaterials with excellent properties and/or new biomaterials with outstanding performance. Some of the instructive substances include, but are not limited to, biological factors or external stimuli.

Simple biomaterial scaffolds
Biomaterials such as metals, natural polymers, synthetic polymers, ceramics, and their composites have been widely used in biomedical elds for decades. Fig. 3a indicates the values (normalized by density) of stiffness and the strength of various materials by an Ashby plot. 17 Natural materials, except silk that exhibits excellent toughness, have much lower values of strength and toughness than engineering materials. However, many natural materials have a toughness value that far exceeds their composition and their homogeneous mixture (as shown by the dashed line in Fig. 3b). 17 Selection of matrix material plays a crucial role in the properties of bone scaffolds. Various polymers have been developed to fabricate bone tissue engineering scaffolds. An overview of different biomaterials including their characteristics, advantages, and disadvantages is given in Table 3.

Composite biomaterial scaffolds
Composite biomaterials are designed to combine two or more materials. The purpose of using composite materials is mainly to improve the processability, printing performance, mechanical properties and bioactivity of the scaffolds. Ti6Al4V, HA, b-TCP and BG are widely used as bioactive biomaterials due to specic biological reactions between scaffolds and living tissues. Bioresorbable biomaterials applied in bone tissue engineering are generally natural polymers (such as collagen, gelatin, silk broin, and chitosan), synthetic polymers (such as PLA, PGA, and PCL) and ceramic (such as HA, b-TCP, and BGs). Scaffolds containing additives (such as GFs) have been used in clinical applications because of their excellent bone regeneration capabilities. The general composite biomaterial scaffolds with additives (signaling molecules, stem cells, functional materials, and so on) for bone tissue engineering are summarized in Table 4, which include metal matrix composites, polymer matrix composites, ceramic matrix composites, and functional composites.
Bioactive metal matrix composites are widely used in clinical medical settings because of their outstanding mechanical properties, excellent biocompatibility, thermal stability, and corrosion resistance. Titanium, tantalum and their respective alloys are considered to be the preferred biomaterials for scaffolds. However, the high costs of manufacturing scaffolds limit their widespread development. Ti6Al4V is an outstanding representative of metal matrix composites. Young's modulus of the suitable porous Ti6Al4V scaffolds can be similar to natural bone and improve the mechanical shielding to the living tissue. 114,115 The Ti6Al4V scaffolds can signicantly increase bone ingrowth, osteointegration, and osteogenesis by covering the tantalum coating, 116 adding simvastatin/hydrogel, 117 or polydopamine-assisted hydroxyapatite coating (HA/pDA), 118 as summarized in Table 4. Although metal matrix composites, such as Ti6Al4V, have many outstanding advantages; the nonbiodegradable properties of metal matrix composites fundamentally limit their potential to become ideal materials.  Ta-coated scaffolds  116  Ti6Al4V Simvastatin The combination of hydrogel hardness and BMP-2 has a synergistic effect on cellular osteogenic differentiation 132 In recent years, the application of polymer matrix composites and ceramic matrix composites has made great progress in bone tissue engineering scaffolds. Polymer composites have various excellent properties, such as biodegradability and mechanical properties. [122][123][124][125][126][127][128][129][130][131] Ceramic materials, especially HA, are the main inorganic constituents of natural bone. 19 Composite materials composed of ceramic materials and polymer materials have desirable properties for the manufacturing of scaffolds for bone tissue engineering. 125,126,130,131 The composite scaffolds with additives (signaling molecules, stem cells and functional materials) have superior performance compared to just composite scaffolds ( Table 4). The composite scaffolds with additives could further enhance the performance of the scaffolds. As shown in Fig. 4a, scaffolds with bioactive polydopamine coating have the capacity to automatically t into irregular defects at higher temperatures. Wang et al. fabricated BP-SrCl 2 /PLGA scaffolds for rat femoral defects, and the nearinfrared light-triggered platform signicantly enhanced bone regeneration, as seen in Fig. 4b. 129

Conclusion
In this paper, the summarized literature, which involves biomaterials for bone tissue engineering scaffolds, has been reviewed. The application and properties of various biomaterials used to fabricate scaffolds have also been elaborated. In particular, composite materials such as metal matrix composites, polymer matrix composites, ceramic matrix composites, and functional composites have been discussed. It was found that additives such as signaling molecules, stem cells, and functional materials can enhance the performance of the scaffolds. Although it was impossible forty years ago to nd a material that is not repelled by living tissue, nowadays biomaterials have been used for bone repair. Improved performance of ideal biomaterials is required for their positive interactions with host tissues. The approaches for bone regeneration will make giant steps with the exploitation of novel biomaterials and new strategies, particularly the deep integration of nanotechnology, stem cell science and other elds.

Conflicts of interest
There are no conicts to declare.