Different natural or synthetic materials have been used for scaffold fabrication in bone tissue engineering field 9,26−29. Natural materials have shown more appropriate regeneration properties, mostly due to their similarity to mammalian cell ECM. In this regard, Col is the most prevalent protein in human tissues and extensively used for biomaterial research 9. Col exists in bone ECM and forms bone cell and tissue structural support, combined with mineral materials like calcium phosphate crystals. Col exclusive properties, including availability, biocompatibility, biodegradability, high porosity, and low immunogenicity, have resulted in extensive bone tissue engineering application 9. Col provides easy processing and combination with other materials. Col has been used as injecting hydrogels, films and membranes, sponges and micro/nanospheres for bone regeneration studies 9.
The macroscopic structure of Col and Col-rGO are shown in Fig. 1A and B, respectively. Graphene coating has darkened the scaffolds color. The structure and stability of the scaffold were not changed after the coating process. Figure 2 depicts morphological and structural characteristics of scaffolds during the synthesis process, resulted from FESEM. The results showed 3D porous structures in both scaffolds. The 3D structure of the Col scaffold did not change after GO coating and reduction process and 3D porous framework could be seen (Figs. 1 and 2). Bone scaffolds should provide 3D porous structure mimicking in vivo like bone-forming environment. Col's structure and composition flexibility have resulted in different structure scaffolds, including 3D porous Col sponges and hydrogels. An optimized protocol based on chemical crosslinking and freeze-drying resulted in a 3D porous scaffold (Fig. 2) which is in accordance with our previous studies 14,24 and Liu et al.'s study 30.
Raman spectroscopy was used to detect the typical graphene peaks on Col-rGO scaffolds. Fig. 3 shows three main peaks, D (Range of ~1300-1400 cm-1), G (Range of ~1500-1600 cm-1) and 2D (Range of ~2700-2900 cm-1) in Col-rGO scaffolds confirming successful coating of graphene on Col scaffolds. The G/2D ratio was used for determining graphene layers number. The ratio showed the coating of multi-layer graphene-coated structures. FTIR spectra of Col and Col-rGO scaffolds are shown in Fig. 4. Col scaffold spectra resulted amide I, II, III, and amide A peaks at 1628, 1535, 1233 and 3274 cm−1, respectively 24,31. Col-rGO scaffolds spectra showed the same amide groups in addition to 1615 and 2929 cm−1 peaks related to CO-NH stretching peak 14,32. Chemical crosslink bonds Col amine or hydroxyl groups and GO sheets oxygen-containing functional groups (Carboxyl group (COOH) and alkoxy) (Fig. 3 and 4).
The liquid displacement method showed 96.4% porosity for Col and 94% for Col-rGO scaffolds, respectively. The porosity difference was not significant between scaffolds before and after rGO coating showing fabrication of highly porous scaffolds for cellular studies. Compressive testing was used to determine the effects of graphene coating on the Col scaffold's mechanical properties. Stress-strain curves of compression test on Col and Col-rGO scaffolds was used to determine elastic modulus and mechanical strength. The obtained results showed 325 ± 18 kPa of Elastic modulus for Col-rGO scaffold compared to that of 115 ± 16 kPa for Col samples. Bone is a load-bearing tissue with high strength properties and in result bone tissue scaffolds need to have sufficient mechanical properties. It is shown that Col has weak mechanical properties for bone structural support and bone differentiation 9,33, requiring an additional part to enhance scaffold strength. It reported that Col's mechanical properties could be enhanced by crosslink to graphene-based nanoparticles 14–16. Liu et al. 30 significantly increased the elastic modulus of Col from 0.2 MPa to 0.34 MPa adding 0.1% GO. In this study, Col and Col-rGO scaffolds represented 115 ± 16 kPa and 325 ± 18 kPa elastic modulus, respectively, similar to our previous study 14. Graphene nanosheets coating on Col structure remarkably enhanced mechanical strength of the scaffold by 2.8 folds. Using this scaffold, cells use mechanosensing and differentiate on a substrate similar to native bone tissue. In addition, implantation of a high-strength scaffold prevents additional scaffold and bone remodeling and damage to the injured area. Besides, the limited electrical conductivity of natural biomaterials challenges their practical applications. Its reported that osteoinductive properties of graphene incorporated PLGA films enhance bone differentiation of stem cells and guiding bone tissue regeneration 20. Therefore, exceptional mechanical and electrical conductivity of rGO which is significantly more electrical conductive than GO on Col scaffolds seems to be helpful in bone repair.
Cytotoxicity of the samples was evaluated using MTT assay. After cell culture, MTT results (Fig. 5) showed more biocompatibility in Col-rGO scaffolds than alone Col, which was significant after 96 h (p > 0.05). These results confirmed that addition of graphene not only did not induce any cytotoxic effects but also enhanced cell viability and proliferation. The adhesion of BMSCs on scaffolds was evaluated by FESEM microscopy. After 48 h of hBMSCs seeding on scaffolds, images were taken that are shown in Fig. 6. Stem cells were adhered and grown on both scaffolds. In both groups, the stem cells were attached and expanded on 3D scaffolds and penetrated into pores. In Col-rGO scaffolds, 3D adherence and expansion with natural morphology are apparent. Cells used the 3D porous structure of graphene-coated scaffolds for enhanced adhesion, proliferation and cell-cell contact.
Go and rGO cytotoxicity depends on the number of layers, concentration, lateral dimensions, shapes, and cell types 12,24. For example, our previous study on different concentration ranges of rGO from 200–800 µg/mL on Col scaffold showed that 400 µg/ml concentration allows highest cell viability for HUVECs cells 14. Another study reported that 0.1% GO–Col aerogels group shows better cytocompatibility than 0.05% and 0.2% groups on rat BMSCs 30. The addition of GO to alginate-chitosan-Col based composite scaffolds did not change the metabolic activity of MC3T3 osteoblast cells. It provided better support for cell proliferation compared to the GO nonincorporated group 17. GO addition induced increased osteogenesis and in result, calcium mineral deposition. Cytotoxicity results of this study on Col-rGO showed more biocompatibility than alone Col on hBMSCs after 96 h seeding (Fig. 5). The same results were achieved about cell adhesion (Fig. 6). Despite adhesion and expansion on both scaffolds, Col-rGO scaffolds showed enhanced adhesion, proliferation, expansion, called cells developing spreading cytoplasmic projections, penetration into pores and cell-cell contact.
Different structural and physicochemical characteristics of Col-rGO scaffolds could be involved in these cellular results. The 3D structure of the Col-rGO scaffold provides high surface area for hBMSCs stem cells to attach and grow in an in vivo like structure and in different directions. Tissue scaffold should have 3D structure and interconnected pores for appropriate cell adhesion and interconnection, in addition to nutrients and oxygen transport. Significant porosity of 94% for Col-rGO scaffold (higher than 83% for the GO–Col scaffold synthesized by mixing of Col and GO solutions 30) is sufficient for cell migration and vascularization 34,35. High porosity facilitates stem cells interconnection and medium transport. Creating of cellular framework could be seen in Col-rGO scaffolds which is necessary for constructing larger tissue. Besides, the scaffolds' topological properties provide additional support for better cell adhesion, proliferation, and viability. It is confirmed that ripples and wrinkles on graphene nanosheets result better cell adhesion 25,36,37. Stiffer scaffolds activate molecules involved in cell adhesion and proliferation 30,38. It’s been reported that high strength, 3D structure and porous graphene foam induced spontaneous osteogenic differentiation for hMSCs 39. Other surface properties like existing oxygen-containing functional groups such as hydroxyl, carboxyl and epoxy and free surface π electrons allow hydrogen bonding, π–π interactions, and other surface reactions, providing additional surface reaction sites 24,40. Kolanthai et al. 17 reported negatively charged surface of GO is favored by osteoblast cell adhesion, growth and proliferation. Surface sites can adsorb serum proteins, such as fibronectin, in culture media and provide a hydrophilic surface, enhancing stem cell adherence, viability, and proliferation. These interactions are confirmed in other studies using different graphene materials 24,37,41,42. The abovementioned properties encounter some reported graphene cytotoxicity damages like ROS production 43,44, physical damage to the cell membrane by graphene sharp edges 45,46.
Histological analysis was used to examine the presence of new bone formation in the defect area. H & E (Fig. 7) and alizarin red (Fig. 8) staining were performed on rabbit cranial defect samples. H&E results showed no necrosis or inflammation in both groups. Above mentioned advantages of Col-rGO scaffolds could be involved in animal study results. Testing this theory, an 8 mm critical-sized rabbit cranial defect model was used. As shown in Fig. 7, H&E results showed that new osteogenesis and cell migration in different regions of the implanted Col-rGO scaffolds are more than Col counterparts. Alizarin red staining was used to determine mineralization and bone formation. Alizarin red results (Fig. 8) showed that compared to the Col scaffolds, mineralization in central regions of the defect could be seen for implanted Col-rGO scaffolds. Similarly, the surrounding area of the defects shows more mineralization and new bone formation.
Enhanced in vivo results follows the biological, physicochemical and topological properties of the Col-rGO scaffolds like 3D, porous structure, biocompatibility, enhanced cell adhesion, high mechanical strength and specific surface factors, which are discussed above. Obtained results are in accordance with similar in vivo studies. For example, 0.1% GO–Col aerogel implant in rat cranial defect models showed better bone repair than Col aerogel 30. In vivo results of graphene-contained scaffolds indicate the scaffold's osteogenesis properties without any external ingredients like growth factors or cells. Different studies have been used external osteogenesis factors along with scaffold. For example, 47 used ECM components, cells and growth factors along with Col scaffold for osteogenesis. Besides the discussed reasons, another two advantages of Col–rGO scaffolds for better in vivo bone-forming results could be angiogenic and antibacterial properties of graphene. Addition of rGO flakes within MSC spheroids upregulated the expression of VEGF growth factors, cell–ECM interaction and allowed cell signaling cascades 42. Low levels of ROS production by GO incorporation and activation of phospho-eNOS and phospho-Akt by rGO could be pro-angiogenic signaling factors inducing vascularization 42,48,49. Govindarajan et al. investigated that 3D porous Col aerogel shows cytocompatibility and wound healing and angiogenesis effects 50. Our scaffold fabricated by Col–rGO400 µg/ml confirmed VEGF induced angiogenesis after subcutaneous implant for 4 weeks 24. In addition, graphene materials present antibacterial and antifungal properties 51,52, promising for bone tissue engineering. Our previous study confirmed that the Col-rGO scaffold induces antibacterial effects against E. coli, S. aureus and S. pathogens through the loss of bacterial membrane integrity and generation of oxidative stress 14. Col–rGO scaffold's antibacterial properties could be advantageous in preventing biofilm and implant infection and subsequent successful practical applications.