Recent trends in the application of widely used natural and synthetic polymer nanocomposites in bone tissue regeneration

Abstract

The goal of a biomaterial is to support the bone tissue regeneration process at the defect site and eventually degrade in situ and get replaced with the newly generated bone tissue. Nanocomposite biomaterials are a relatively new class of materials that incorporate a biopolymeric and biodegradable matrix structure with bioactive and easily resorbable fillers which are nano-sized. This article is a review of a few polymeric nanocomposite biomaterials which are potential candidates for bone tissue regeneration. These nanocomposites have been broadly classified into two groups viz. natural and synthetic polymer based. Natural polymer-based nanocomposites include materials fabricated through reinforcement of nanoparticles and/or nanofibers in a natural polymer matrix. Several widely used natural biopolymers, such as chitosan (CS), collagen (Col), cellulose, silk fibroin (SF), alginate, and fucoidan, have been reviewed regarding their present investigation on the incorporation of nanomaterial, biocompatibility, and tissue regeneration. Synthetic polymer-based nanocomposites that have been covered in this review include polycaprolactone (PCL), poly (lactic-co-glycolic) acid (PLGA), polyethylene glycol (PEG), poly (lactic acid) (PLA), and polyurethane (PU) based nanocomposites. An array of nanofillers, such as nano hydroxyapatite (nHA), nano zirconia (nZr), nano silica (nSi), silver nano particles (AgNPs), nano titanium dioxide (nTiO₂), graphene oxide (GO), that is used widely across the bone tissue regeneration research platform are included in this review with respect to their incorporation into a natural and/or synthetic polymer matrix. The influence of nanofillers on cell viability, both in vitro and in vivo, along with cytocompatibility and new tissue generation has been encompassed in this review. Moreover, nanocomposite material characterization using some commonly used analytical techniques, such as electron microscopy, spectroscopy, diffraction patterns etc., has been highlighted in this review. Biomaterial physical properties, such as pore size, porosity, particle size, and mechanical strength which strongly influences cell attachment, proliferation, and subsequent tissue growth has been covered in this review. This review has been sculptured around a case by case basis of current research that is being undertaken in the field of bone regeneration engineering. The nanofillers induced into the polymeric matrix render important properties, such as large surface area, improved mechanical strength as well as stability, improved cell adhesion, proliferation, and cell differentiation. The selection of nanocomposites is thus crucial in the analysis of viable treatment strategies for bone tissue regeneration for specific bone defects such as craniofacial defects. The effects of growth factor incorporation on the nanocomposite for controlling new bone generation are also important during the biomaterial design phase.

Keywords: Biocompatibility; Biomaterial; Bone tissue regeneration; Nanocomposite; Nanofillers.

Fig. 1.

Proliferation of OB-6 pre-osteoblasts on CS and CS composite scaffolds at day 10 and 20. The proliferation was better on CS-nHA and CS-nCZ groups; scale = 250 μm [52].

Fig. 2.

Histological Images of nSI/CS composite scaffold. A (2 weeks), C (4 weeks), and E (8 weeks) with H&E staining. B (2 weeks), D (4 weeks), and F (8 weeks) with Mallory’s trichrome staining [44].

Fig. 3.

Influence of ATP nanoparticles on CS/GP hydrogel crosslinking [56].

Fig. 4.

H&E Staining after 12 weeks of implantation along the decalcified regions; green stars signifies new bone formation whereas yellow arrows identify new blood vessels [73].

Fig. 5.

Fluorochrome-labelling analysis of the hBMSCs for cell proliferation for the three scaffold groups with varying amounts of the paste binder; culture done for (A) 1 day, (B) 7 days; cell nuclei in blue and actin in red [93].

Fig. 6.

Control sample bone growth from only bone defect wall (a,b,c,d) and contact and distance osteogenesis from the center as well as wall of the bone defect in sulfated CNC aerogel implanted samples (e,f,g,h) [101].

Fig. 7.

OB-6 preosteoblasts proliferating along cracks on the microparticles on day 10 prepared (a) with 5% w/v ZrCl₄ and (b) with 10% w/v ZrCl₄ [103].

Fig. 8.

Runx2 and Osteocalcin expression data in Relative Fluorescence Intensity [109].

Fig. 9.

(A) Radiographs of nHA only and (B) Fucoidan/nHA nanocomposite scaffold. H&E staining (x100) displaying (C) the filling of bone defect with Fucoidan/nHA nanocomposite biomaterial, and (D) identification of osteoblasts (represented by ) and fat cells (represented by ) [116].

Fig. 10.

PCL/HA (20%) nanocomposite incubation data in human osteosarcoma cell lines for analysis of cell viability; p-value > 0.05; n=5 for statistical significance [125].

Fig. 11.

(A) Fluorescent images denoting live cells (in greens spots) and dead cells (in red spots) for cell viability analysis on RGO/PCL nanofibrous mat and other control samples. (B) Cell spreading analysis (using toluidine blue staining) of 3T3s after 24 hours of culture on RGO/PCL biomaterial as well as other control samples. (C) Cell adhesion analysis (using SEM micrographs) for 3T3s on RGO/PCL nanofibrous mat as well as other control samples after 24 hours of cell culture [134].

Fig. 12.

Scanning electron microscope images showing a) PCL nanofibers only, b) PCL/TNT at 2.5A, c) PCL/TNT at 2A, and d) PCL/TNT at 1.5 A magnetron current [135].

Fig. 13.

Histologic images: (A) PLGA/TNT, (B) pure PLGA, (C) empty defect. Histomorphometric analysis: (D) new bone formation (%), (E) residual scaffold (%). G – Graft, NB – New bone formation, and CT – connective tissue. Asterisk and double asterisk denote the significant difference between time points and scaffolds respectively [147].

Fig. 14.

SEM images for biomineralization by mesenchymal stem cells on PLGA (A), PLGA/SF (C), and PLGA/SF/GO nanofiber scaffolds 14 days post implantation; corresponding EDS images for mineral identification are given along with on the right [148].

Fig. 15.

Alizarin Red staining of MC3T3-E1 cells (on 21st day of cell culture) on (A) PLGA, (B) PLGA/HA, (C) PDA-PLGA, (D) PDAPLGA/HA, (E) PDA-PLGA/BMP-2, and (F) PDA-PLGA/HA/BMP-2. [149]

Fig. 16.

Cell viability analysis of MC3T3-E1 cells on (a, b. c. d) electrospun PCL and (a’, b’, c’, d’) electrospun PCL/GO/PEG (at 0.25 wt.%); cell culture done for 1 (a, a’), 5 (b, b’), 7 (c, c’), and 9 (d, d’) days respectively; scale at 200 μm [153].

Fig. 17.

Energy dispersion spectroscopy for detecting apatite-like formation on the nano calcium membranes after 10 days of SBF incubation [154].

Fig. 18.

Schematic for preparation of 3D printed PLA/HA-CS composite hydrogels through freeze gelation technique [165].

Fig. 19.

MG 63 cells stained with DAPI (blue) and actin filaments with Oregon Green 488 phalloidin (green) after 5^th and 7^th day culture (images captured using confocal laser scanning microscopy). (a) and (b) – S1 (0 wt.% nHA), (c) and (d) – S2 (10 wt.% nHA), (e) and (f) – S3 (20 wt.% nHA), and (g) and (h) – S4 (30 wt.% nHA); scale bars are representing 100 μm [167].

Fig. 20.

Relative gene expressions (alkaline phosphatase (a), osteocalcin (b), osteonectin (c), collagen I (d), and Runx 2 (e)) at 7^th day (with osteogenic differentiation of human mesenchymal stem cell lines) for control sample, PLLA nanofiber only, BG/PLLA nanocomposite scaffold, and BG nanofiber only; between group significant difference at P < 0.05 [168].

Fig. 21.

Cell growth using bone marrow-derived mesenchymal stem cells on various scaffold compositions [175].

Fig. 22.

MC3T3-E1 Cell Viability and Proliferation for a 5-day period with live cells (red) and dead cells (green) for scaffolds – Electrospun scaffolds of (a) PU (pure), (b) PU/ZnO-fMWCNTs (0.1 wt.%), (c) PU/ZnO-fMWCNTs (0.2 wt.%), and (d) PU/ZnO-fMWCNTs (0.4 wt.%) [178].