In vitro and in vivo biocompatibility and osteogenesis of graphene-reinforced nanohydroxyapatite polyamide66 ternary biocomposite as orthopedic implant material

Abstract

Graphene and its derivatives have been receiving increasing attention regarding their application in bone tissue engineering because of their excellent characteristics, such as a vast specific surface area and excellent mechanical properties. In this study, graphene-reinforced nanohydroxyapatite/polyamide66 (nHA/PA66) bone screws were prepared. The results of scanning electron microscopy observation and X-ray diffraction data showed that both graphene and nHA had good dispersion in the PA66 matrix. In addition, the tensile strength and elastic modulus of the composites were significantly improved by 49.14% and 21.2%, respectively. The murine bone marrow mesenchymal stem cell line C3H10T1/2 exhibited better adhesion and proliferation in graphene reinforced nHA/PA66 composite material compared to the nHA/PA66 composites. The cells developed more pseudopods, with greater cell density and a more distinguishable cytoskeletal structure. These results were confirmed by fluorescent staining and cell viability assays. After C3H10T1/2 cells were cultured in osteogenic differentiation medium for 7 and 14 days, the bone differentiation-related gene expression, alkaline phosphatase, and osteocalcin were significantly increased in the cells cocultured with graphene reinforced nHA/PA66. This result demonstrated the bone-inducing characteristics of this composite material, a finding that was further supported by alizarin red staining results. In addition, graphene reinforced nHA/PA66 bone screws were implanted in canine femoral condyles, and postoperative histology revealed no obvious damage to the liver, spleen, kidneys, brain, or other major organs. The bone tissue around the implant grew well and was directly connected to the implant. The soft tissues showed no obvious inflammatory reaction, which demonstrated the good biocompatibility of the screws. These observations indicate that graphene-reinforced nHA/PA66 composites have great potential for application in bone tissue engineering.

In vitro effect of graphene structures as an osteoinductive factor in bone tissue engineering: A systematic review

“…The most dominant cell types used in the included articles were mesenchymal stem cells (MSCs), including human adipose-derived stem cells (hADSCs), [17][18][19][20][21][22] Goat ADSCs, 23 human dental pulp stem cells (hDPSCs), 24 human alveolar bone marrow stem cells (hABMSCs), 25 human periodontal ligament stem cells (hPDLSCs), 26,27 and bone MSCs (BMSCs), [28][29][30][31] such as human BMSCs 19, 32-41 (hBMSCs), murine BMSCs (mBMSCs), 4, 42-49 rabbit BMSCs (rBMSCs), 50 murine MSCs cell line C3H10T1/2 51,52 and Caprine BMSCs (cMBSCs). 53 The type of the MSCs in fifteen studies 19,32,36,40,[54][55][56][57][58][59][60][61][62][63][64] was not defined.…”

“…141 In order to overcome these limitations, graphene and its derivatives can be incorporated into polymers through different methods to produce composites exhibiting synergistic effects over the two individual components with improved properties. 34,36,37,46,52,61,63,66,68,70,73,75,78,91 Recently, graphene-polymer composites have been used for orthopedic applications. 142,143 For example, GO in Ct matrix was shown to affect interactions between cells and scaffold leading to improved cell proliferation.…”

“…Cell adhesion and spread within the scaffold are usually the first steps following seeding of cells onto the biomaterials, and therefore, have a significant effect on modulating the forthcoming cell responses, including cell proliferation and differentiation. 47 In most reviewed studies, presence of graphene and its derivatives resulted in accelerated cell adhesion and spreading, 19,21,52 yet, there are few studies showing no difference between cell density on GO-coated substrates and noncoated substrate . 62,79 Besides, in a study by Kanayama et al, 70 GO and RGO films were found to show less DNA content compared to controls.…”

“…This can be attributed to graphene enhancing the adsorption of the growth factors and chemicals containing benzene rings, such as β-glycerophosphate and Dex. 52 Also, the electric characteristics of CNMs may be a significant factor in this improvement. 205 Nayak et al 62 compared the effect of common growth factors (BMP2) and graphene on osteogenesis.…”

“…In addition, MTT assay performed in 21 included studies was discovered to be not successful in predicting the toxicity of graphene and its derivatives due to the spontaneous reduction of MTT by them, leading to a false positive signal. 100 Therefore, to evaluate the toxicity of graphene, it is much better to use alternate assays, such as water-soluble tetrazolium salt (WST-8) assay, 58 ROS assay, 41 CCK-8 assay 52,76,77,79 and DNA counting 25, 36, 37, 71 3. In nine included studies the type of the MSCs, and in one study, type of the preosteoblast cells were not defined.…”