Experimental Evidences

There have no researches directly investigating the relationship between the mechanical properties of the nanofibers/nanotubes-reinforced scaffolds and cell/tissue responses. However, many studies examined both the mechanical properties and the cell/tissue responses of these scaffolds. A careful classification of these studies may find some relationship between the mechanical properties and the cell/tissue responses.

Carbon nanotubes (CNTs) are featured with high mechanical properties so that they have been widely employed as reinforcement fillers in scaffolds fabrication, including both ceramics and polymers scaffolds. Several researches consistently found that CNTs reinforced scaffolds can improve both the mechanical properties and proliferation or differentiation capabilities of cells.

Ogihara et al. [88] used CNTs to reinforce an alumina composite for bone tissue replacement. The CNTs/alumina composite demonstrated a 120% increase in fracture toughness. Meanwhile, the CNTs/alumina composite showed superior prolif?erative ability for osteoblasts, chondrocytes, smooth muscle cells and fibroblasts when compared to noncomposite alumina. Further in vivo implantation into 15 week-old Japanese white rabbits found that the CNTs/alumina composite demonstrated direct integration into the bone and a comparable or lessened inflammatory response to that of the alumina control. Chen et al. [86] found that the elastic modulus and compressive strength of chitosan-multiwalled carbon nanotubes/ hydroxyapatite (CS-MWNTs/HA) nanocomposites increased sharply from 509.9 to

1089.1 MPa and from 33.2 to 105.5 MPa with an increase of multiwalled carbon/ chitosan weight ratios from 0 to 5%. As a result, preosteoblast MC3T3-E1 cells cultured on the CS-MWNTs/HA composites spread out well and the cell proliferation on the composites was observed to be higher than that on the CS-HA composites. Similar positive correlation between the mechanical properties and the cell/ tissue responses were observed for CNTs reinforced polymeric scaffolds. Jell et al. [89] added CNTs into polyurethane (PU) scaffolds and found that both the compressive strength and the osteoblasts proliferative ability of the resulting CNT/PU scaffolds were increased. Lin et al. [90] produced MWCNTs-reinforced poly(lactic acid-co-glycolic acid) (PLGA) scaffolds and found that the inclusion of MWCNTs into PLGA scaffolds yielded a 500% increase in the stiffness and a 270% increase in the tensile strength. The proliferative potential was superior to that of pure PLGA scaffolds as well. Similarly, MWCNTs reinforced PLLA scaffolds (PLLA/ MWCNTs) were observed to be more beneficial to the viability of osteoblasts than pure PLLA scaffolds [91]. In vivo test by Sitharaman et al. [92] demonstrated that the inclusion of short (20-80 nm) SWNTs into poly(propylene fumarate) (PPF) scaffolds resulted in 300% increase in bone area as observed in micro-CT analysis and histological analysis. The histological scoring at 12 weeks was also significantly higher than pure PPF scaffolds, indicating higher tissue quality, greater tissue ingrowth, reduced inflammatory cell density, increased connective tissue organization and a greater bone volume. CNTs are also widely employed to reinforce natural polymer scaffolds. Collagen has excellent biocompatibility due to its natural origin. Hirata et al. [93] found that, when coated collagen scaffolds with MWCNT, the resulting composite scaffolds demonstrated earlier osteoblastic differentiation than pure collagen scaffolds based on data of alkaline phosphatase activity and calcium and osteopontin contents. Moreover, the CNT/collagen scaffolds showed a significant increase in bone formation surrounding the scaffolds at both 4 and 8 weeks. Chitosan, as another widely employed natural polymer in biomedical fields, was reinforced with MWCNT by Venkatesan et al. via freezing and lyophilization method [94]. The obtained MWCNT/ chitosan scaffolds showed higher osteoblasts proliferation, alkaline phosphatase and mineralization than pure chitosan scaffolds.

In addition to carbon-based nanotubes, boron nitride nanotubes (BNNTs), as an important noncarbonic nanotubes, can be employed as reinforcement fillers of biomedical scaffolds as well due to their nontoxicity to various cells including osteoblasts, macrophages, human embryonic kidney cells and human neuroblastoma cells [45]. Lahiri et al. [95] prepared BNNTs reinforced polylactide-polycaprolactone (PLC) copolymer composite and investigated the cytocompatibility with osteoblasts and macrophages in vitro. The results show that the BNNTs addition to PLC enhanced the tensile strength and also resulted in an increase in osteoblasts viability and differentiation. BNNTs were employed to reinforce hydroxyapatite (HA) as well. Addition of 4 wt% BNNTs resulted in 120% increment in elastic modulus, 129% higher hardness, and 86% more fracture toughness as compared to HA [96]. Correspondingly, the proliferation and viability of osteoblast cells of BNNTs/HA scaffolds are better than pure HA ones.

Scaffolds reinforced by fibers were observed with enhanced mechanical properties and improved biocompatibility as well. Li et al. [97] reinforced poly(L-lactic acid) (PLLA) porous scaffolds by chitin fibers of 12 pm in diameter, and found that the compressive strength of the resulting composite scaffolds was 4 times higher than the pure PLLA scaffolds. Correspondingly, the reinforced scaffold demonstrated significantly better attachment, proliferation, differentiation, and mineralization of osteoblasts than pure PLLA scaffolds [98]. Being similar to CNTs, carbon nanofibers (CNFs) possess unique mechanical properties as well. Compared with the CNTs, some special characteristics are featured by CNFs such as larger diameter (60-150 nm) and longer length (30-100 pm), and different surface morphology [99], which render them more suitable as a reinforcement agent. Rajzer et al. [100] produced CNFs-reinforced HA fibrous scaffolds for bone tissue engineering. It was demonstrated that the composite scaffolds could establish direct chemical bonds with bone tissue after implantation and the mineralization activity was drastically increased.

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