Bone structure

Bone is a hard tissue that contains different kinds of cells including osteoblasts, osteocytes, and osteoclasts (Figure 1) [26]. The inorganic hydroxyapatite and organic type I collagen components are vital to bone tissue. The bone biomineralisation activities are formed by nanosized hydroxyapatite crystals with connective collagen fibrils [27]. The bone possesses a unique combination of strength and stiffness, and it has excellent compressive strength and tensile strength due to the attribution of deep nanostructures of inorganic and organic components.

Figure 1: The bone structure. The magnified image shows the physiological arrangement of the bone matrix. The internal structure of the bone has the 100–500 µm osteon which contains 300 nm collagen fibrils, hydroxyapatite crystals, and 1.5 nm tropocollagen.

Human bones are complex and their asymmetric matrix is constituted of basic components hierarchically organized into distinct structural layers at macro- and nanoscale levels. Cortical (compact) and cancellous (trabecular) bones are two kinds of bone classification based on their macrostructure. A femur is a long bone with a thick cortical covering which is porous and has a cancellous interior. The calvaria is a flat bone with cortical layers on the outside and a cancellous structure on the inside [28,29]. The physical behaviour of the cortical bone is mainly controlled by porosity, mineralization rate, and solid matrix structure (cancellous interior) [30]. Also, the mechanical properties of cancellous bones are controlled by the structural organization of the matrix [31]. The bone microstructure mainly comprises collagen threads of lamellae coiled around layers to form a 200–250 µm diameter osteon which can vary between cortical and cancellous bones [31]. At the scale of 1 µm, collagen fibrils are surrounded by minerals [32] (Figure 1). Crystals, collagens, and non-collagen organic proteins are found at sub-nanoscale levels ranging from 1 to 10 nm [33]. It has been reported that 90% of the proteins identified inside the bone extracellular matrix is produced by bone-forming osteoblasts with a repeating amino acid sequence of [Gly(glycine)–X–Y]n, where X and Y may be proline and hydroxyproline. Collagen fibrils, composed of specific proteins, are usually responsible for mechanical strength. Furthermore, osteoblasts generate a membrane that includes alkaline phosphatase, which cleaves phosphatase groups and causes calcium and phosphate precipitation, resulting in the formation of natural bone minerals with a ratio of 1.67 [27].

Osteoblasts have been predominantly derived from mesenchymal stem cells, which express particular genes for the production of bone morphogenic proteins and wingless (Wnt) pathway elements. It has been revealed that runt-related transcription factors-2 (Runx2), osterix (Osx), and the distal-less homeobox 5 (Dlx5) are primarily responsible for osteoblast differentiation. Specifically, the gene RUNX2 upregulates the genes for collagen type I alpha 1 (ColIA1), alkaline phosphatase (ALP), bone sialoprotein (BSP), bone gamma-carboxyglutamate protein (BGLAP), and osteocalcin (OCN), which are important for the regeneration process [34]. To mimic the natural bone function, the composite materials should be in the form of inorganic and organic composites. To mimic the inorganic portion, researchers have tried to utilize calcium phosphate materials due to their similarity to the native tissue. To mimic the organic portion of the bone, several materials including polymers, proteins, and carbon-based materials have been tested.

Biomaterials for bone graft substitutes

Hydroxyapatite and its composites have been widely utilized/studied biomaterials for bone tissue engineering [35,36]. Hydroxyapatite with several polymeric materials has been used to mimic the natural function of the bone. Different kinds of polymeric materials have been utilized including chitosan, alginate, fucoidan, carrageenan, and ulvan from natural polymeric materials. Polycaprolactone (PCL), poly ᴅ,ʟ-lactic-co-glycolic acid (PLGA), and polylactic acid (PLA) have been extensively studied with hydroxyapatite to develop bone mimetic scaffolds [37]. Bone morphogenetic protein 2 (BMP-2) is one of the widely utilized growth factors for the treatment of bone-related diseases and defects [38,39]. Growth factors are responsible for bone formation which happens through the stimulation of different kinds of cells in our body. However, the drawbacks of using growth factors and enzymes are stability, high cost, and low availability. To overcome these issues, bioactive materials are often studied to mimic the natural function of growth factors. Several researchers are studying nanomaterial-based chitosan composites regarding their osteoinductive properties [40,41]. Chitosan is combined with several polymeric materials and nanoparticles to mimic the natural function of the bone (Table 1) [42,43]. Chitosan biomaterials enhance the proliferation of osteoblasts and the formation of bone minerals by promoting gene expression of type I collagen, osteopontin, osteonectin, and osteocalcin. Chitosan mixed with different functionalized materials such as silver, magnesium oxide, and bioactive glass has aided the treatment of infected bone defects with a biodegradable behaviour at the bone defect site [44,45]. The N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HACC) is a chitosan biomaterial used for the treatment of infected bone defects. The arrangements of amino acids in the chitosan (degree of deacetylation) determines the growth and support necessary for osteoblast differentiation. Increasing the degree of deacetylation in the chitosan, the positive charge density increases, which results in high electrostatic interaction which electrically stimulate the osteoblasts to proliferative and differentiate [46-48]. Chitosan biomaterials containing graphene oxides were used as substrates for the generation of hydroxyapatite, which has a high elastic flexibility and tensile strength for bone tissue engineering applications [49]. Carbon nanotubes were also used as fillers in chitosan to increase flexibility, porosity, and mechanical strength of chitosan biomaterials for applications in bone tissue engineering [50]. The porous structure of the chitosan with an absorbable collagen sponge encourages osteoblast stem cells to attach to the surface to proliferate and differentiate promoting bone development. As compared to absorbable collagen sponges, the increase in bone mineral density, defect closure, and new bone formation on rat calvaria defects indicate a strong healing effect and new bone formation on chitosan/absorbable collagen sponges [51].

Table 1: Combinations of chitosan with several polymeric materials and nanoparticles to mimic the natural bone function.

S. No. Materials Methods Cell line/animal Bacteria Ref. 1 chitosan–silver coating rabbit Staphylococcus aureus [52] 2 chitosan–silver electrophoretic deposition MG-63 cells/rat Staphylococcus aureus [53] 3 chitosan–diatomite freeze-drying MG-63 cells/Saos-2 cells/human osteoblasts – [54] 4 chitosan–silica cross-linking – – [55] 5 chitosan–silver self-assembly – Staphylococcus aureus and Escherichia coli [56] 6 chitosan–collagen cross-linking MC3T3-E1 cells – [57] 7 chitosan–carbon nanotubes sonication – – [58] 8 chitosan–carbon nanotubes electrophoretic deposition MC3T3-E1 cells – [59] 9 chitosan–reduced graphene oxide self-assembly MG-63 cells – [60] 10 chitosan–graphene oxide sonication and lyophilisation MC3T3-E1 cells – [61] 11 chitosan–graphene oxide solvent casting MG-63 cells Staphylococcus aureus and Staphylococcus epidermidis [62] 12 chitosan–tetraethoxysilane sol–gel human osteoblasts – [63] 13 chitosan–bioactive glass sol–gel/coprecipitation human osteosarcoma cells – [64] 14 chitosan–mesoporous silica nanoparticles electrospinning MC3T3-E1 cells – [65] 15 chitosan film–graphene oxide–hydroxyapatite–gold hydrothermal and gel casting C3H10T1/2 Escherichia coli, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa [66] 16 chitosan–silver nanoparticle reduction human adipose-derived mesenchymal stem cells – [67] 17 chitosan–carbon nanotubes–gelatin sonication – Bacillus subtilis, Staphylococcus aureus and Listeria monocytogenes, Escherichia coli 0157, Salmonella enteritidis, Salmonella typhi, and Klebsiella pneumoniae [68] 18 chitosan–hydroxyapatite–zinc oxide stirring and self-assembly MG-63 cells Escherichia coli XL1B, Lysinibacillus fusiformis, and Bacillus cereus [69] 19 chitosan–hydroxyapatite–zinc oxide–palladium coating dental pulp stem cells Pseudomonas aeruginosa [70] 20 chitosan–zinc–gelatin electrophoretic deposition rat bone marrow stromal cells/Sprague Dawley rat Escherichia coli and Staphylococcus aureus [29] 21 chitosan–graphene oxide–hydroxyapatite ultrasonication MG-63 cells – [71] 22 chitosan–graphene oxide–polyvinylpyrrolidone electrospinning rat bone marrow mesenchymal stem cells/Sprague Dawley rat – [72] 23 chitosan–graphene oxide–hydroxyapatite layer-by-layer assembly technique mouse mesenchymal stem cells – [73] 24 polysaccharide 1-deoxylactit-1-yl chitosan–silver nanoparticles coating human adipose-derived stem cells/mini-pig Staphylococcus aureus and Pseudomonas aeruginose [74] 25 chitosan–nanohydroxy-
apatite–nanosilver freeze-drying osteoprogenitor cells Staphylococcus aureus and Escherichia coli [75] 26 chitosan–polyurethane–silver nanoparticle electrospinning NIH 3T3 cells Porphyromonas gingivalis [76] 27 carboxylated chitosan–silver–hydroxyapatite facile gas diffusion MG-63 cells Staphylococcus aureus [77] 28 chitosan–nanohydroxy-
apatite–silver cross-linking and lyophilisation MC3T3-E1 cells Escherichia coli [78] 29 chitosan–nanohydroxy-
apatite–silver in situ hybridization human osteoblasts Escherichia coli [79] 30 chitosan–multiwalled carbon nanotubes–hydroxyapatite sonication and cross-linking MC3T3-E1 cells – [80] 31 titanium oxide–naringin–chitosan dropping osteoblasts – [81] 32 chitosan–nanosilicon dioxide–chondroitin sulfate cross-linking MG-63 cells – [82] 33 chitosan–nanosilicon dioxide-gelatin cross-linking MG-63 cells – [83] 34 chitosan–octa(tetramethyl-
ammonium)–polyhedral silsesquioxane Freeze-drying MG-63 cells/ Saos-2 cells/3T3 cells – [84] 35 chitosan–bioactive glass–silver nanoparticle electrophoretic deposition MG-63 cells Staphylococcus aureus [85] 36 carboxymethyl chitosan–copper ion–alginate cross-linking MC3T3-E1 cells Staphylococcus aureus [86] 37 chitosan–glycyl-ʟ-histidyl-ʟ-
lysine–copper ions–mesoporous silica nanoparticles stirring MC3T3-E1 cells Escherichia coli and Staphylococcus aureus [87] 38 chitosan–nanohydroxy-
apatite–silver–magnesium ions microwave-assisted coprecipitation human fibroblast skin cells Escherichia coli and
Staphylococcus aureus [88] 39 chitosan–titanium oxide–selenium electrodeposition osteoblasts Escherichia coli [89] 40 chitosan–silver–ion-loaded calcium phosphate electrospinning bone marrow stromal cells Staphylococcus mutans [90] 41 chitosan–carboxymethyl cellulose–silver nanoparticle modified cellulose nanowhiskers freeze-drying MG-63 cells Escherichia coli and Enterococcus hirae [91] 42 chitosan–silver–strontium–
hydroxyapatite ultrasonication human bone marrow mesenchymal stem cells Staphylococcus aureus [92] 43 chitosan–collagen–functional-
ized multiwalled carbon nanotubes–hydroxyapatite lyophilisation/freeze drying MG-63 cells – [93] 44 chitosan–zein–polyurethane–
functionalized multiwalled carbon nanotubes electrospinning MC3T3-E1 cells Escherichia coli, Staphylococcus aureus, Micrococcus luteus, and Staphylococcus epidermidis [94] 45 chitosan–titanium substrate–titanium oxide nanotubes–alginate self-assembly osteoblasts Escherichia coli and Staphylococcus aureus [95] 46 chitosan–melatonin–titanium oxide–gelatin spin-assisted layer-by-layer mesenchymal stem cells – [96] 47 chitosan–alginate–hydroxy-
apatite–silver nanoparticles freeze-drying MG-63 cells Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa [97] 48 chitosan–copper–bioactive nanoparticles–glycero-
phosphate–silk fibroin stirring MC3T3-E1 cells/rat – [98] 49 chitosan–titanium–silica–
silver–acemannan induction plasma spray coating osteoblasts/rat Staphylococcus epidermidis [99] 50 chitosan–nanohydroxyapatite–
nanocopper–zinc freeze-drying rat progenitor cells Escherichia coli and Staphylococcus aureus [100] 51 chitosan–calcium phosphate–graphene oxide–silver nanoparticles freeze-drying bone marrow stromal cells/Sprague Dawley rat Staphylococcus epidermidis and Escherichia coli [101] 52 chitosan–silver-doped hydroxyapatite–iron oxide ball milling NIH-3T3 cells Staphylococcus aureus and Escherichia coli [102] 53 chitosan–bone morphogenic protein–silver–hydroxyapatite coating bone marrow stromal cells/Japanese big-ear white rabbit Staphylococcus epidermidis and Escherichia coli [103] 54 chitosan–silk cryogel–silver–strontium–
nanohydroxyapatite freeze-drying rat bone marrow stromal cells/Sprague Dawley rat Escherichia coli and Staphylococcus epidermidis [104] 55 chitosan–calcium silicate–gelatin–silver