| Background Bone defects are caused by trauma,nonunion,bone tumor resection,spinal deformity and infection.Small bone injuries can be healed by patients themselves and do not need surgical intervention,but the bone defects beyond the critical size need surgical intervention.Currently,the "gold standard" for the treatment of bone defects is autologous bone transplantation,However,there are some problems such as limited source and easy complications in the donor site.In addition,allogeneic bone transplantation is also a commonly used method for the treatment of bone defects,but it is easy to infect diseases and cause immune rejection.In view of the above reasons,the use of various materials in the treatment of bone defects is a hot and difficult problem in the field of bone repair and reconstruction in China and abroad.Nano materials have been used in the research of bone defect repair because of their unique physical and chemical properties.Nano carbon materials are the hot materials in bone repair research because of their good mechanical strength,unique physical and chemical properties.At present,the research on nano carbon materials is mainly focused on zero dimensional(nano carbon point,fullerene),one-dimensional(carbon nanotube),two-dimensional(graphene and its oxide),etc.,while the research on three-dimensional carbon nano materials is fewer.The newly developed nano watermelon porous carbon is a new type of three-dimensional carbon nano materials,At present,there are no studies on the biocompatibility,interaction with stem cells and repair ability of bone defects.Therefore,the preparation,biocompatibility,interaction with stem cells and repair of bone defects are studied,which provides a new type of nano carbon materials for the treatment of bone defects.Methods Watermelon was cut into appropriate size and put into stainless steel autoclave.The black carbonaceous hydrogel was obtained by one-step hydrothermal method in an oven at 180℃ for 12 hours.It was immersed in water and ethanol for several days,and then freeze-dried to obtain carbonaceous aerogel.The physical properties of watermelon were studied by scanning electron microscopy,transmission electron microscopy and X-ray photoelectron spectroscopy.Results Field emission scanning electron microscopy confirmed that the carbon carbon aerogels were three-dimensional(3D)porous carbon structure.Transmission electron microscopy(TEM)confirmed that the porous carbon of watermelon is a kind of carbon material with banded or branched structure formed by nanospheres or nanoparticles。X-ray diffraction patterns confirmed that the crystallinity of the nano porous carbon materials was good.Fourier transform infrared spectroscopy(FTIR)confirmed that the nano watermelon porous carbon has many kinds of chemical bonds,such as O-H,C-H,C=O,c-o-c.it is speculated that there are a large number of hydroxyl,carbonyl,carboxyl,aromatic and other functional groups on the surface.The results of XPS were attributed to C Is,O 1s and N 1s respectively.Conclusion(1)in this study,a green,low-cost,convenient and easy to obtain biomaterial watermelon was selected as the source of nano carbon materials.(2)Through X-ray photoelectron spectroscopy,Fourier transform infrared spectroscopy,X-ray diffraction and other means of detection,it was confirmed that the nano porous carbon of watermelon was successfully prepared.(3)Nano watermelon porous carbon has a large number of pore structure,hydrophilic properties,strong mechanical properties,rich surface functional groups,which has the potential to become a new bioengineering material.Background The application of nanomaterials in medicine is a hot issue,and its biosafety analysis is a necessary research before nano materials are applied to animals or human bodies.Nanomaterials from different exposure routes enter the organism in different ways.Some studies have found that nanomaterials implanted in animals can be phagocytized by macrophages,So they enter the circulatory system and disperse to different organs.Therefore,it is necessary to study the function changes of organs in the whole body whether nano materials are used in vein or local application.There are many kinds of nano carbon materials.Although there is no study on the biological safety of three-dimensional nano carbon materials,nano watermelon porous carbon has not been carried out in China and abroad,However,many studies on zero dimensional,one-dimensional and two-dimensional carbon nanomaterials have found that carbon nanomaterials can cause inflammation,fibrosis,cancer,immunosuppression,cardiovascular and nervous system adverse reactions,and can affect the oxidative stress reaction in animals through a variety of ways.In addition,At present,researches in China and abroad focus on the changes of blood routine,liver and kidney function,and the staining changes of liver and kidney pathological sections after the application of nano carbon materials in bone defects,However,there is no evaluation of the oxidative stress response in brain,liver,kidney,thymus,spleen,blood and other organs or systems after the application of nano carbon in local bone defects.For the above reasons,The purpose of this study is to perform a new biocompatible test for porous nano carbon used in bone defects.Methods The rats were divided into three groups:group A(bone defect was not filled),group B(bone defect was filled with gelatin sponge),and group C(bone defect was filled with 10%porous nano carbon).The incision was sutured and the postoperative treatment was carried out according to the standard.The following indexes were compared among group C,group A and group B after modeling,body weight and visceral index were measured at the fourth week to know the general effect of nano watermelon porous carbon in bone defect on the whole body of rats;The contents of total glutathione(GSH),nitric oxide(NO),superoxide dismutase(SOD)and malondialdehyde(MDA)in brain,liver,kidney,thymus and spleen were determined.The objective is to evaluate the effect of nano watermelon porous carbon bone defect on systemic oxidative stress.The effect of nano watermelon porous carbon bone defect on rat blood cells was evaluated by blood routine test.The pathological sections of brain,liver,kidney,thymus and spleen were observed,and the objective is to evaluate the effect of nano watermelon porous carbon bone defect on the morphology of multiple organs and organs in rats.Results Compared with group A and group B,there was no significant difference in body weight and visceral index between group C and group A and group B(p>0.05);There was no significant difference in the protein levels of total glutathione(GSH),nitric oxide(NO),superoxide dismutase(SOD)and malondialdehyde(MDA)in brain,liver,kidney,thymus and spleen(p>0.05);No pathological changes were found in brain,liver,kidney,thymus and spleen sections by HE staining.Conclusion When the nano watermelon porous carbon is applied locally in the bone defect,it will not affect the overall nutritional status of rats,the oxidative stress level of brain,liver,kidney,thymus,spleen and blood,and the morphology of blood cells,brain,liver,kidney,thymus and spleen,and it has good biological safety ability.Background Bone defect is a difficult problem in clinical treatment.At present,allogeneic bone,autogenous bone,ceramics,hydroxyapatite and other materials are mainly used to repair bone defects,but the treatment of bone defects is still very difficult.Therefore,it is necessary to find some new osteogenic materials to assist the treatment of bone defects.In recent years,with the continuous development of nanomaterials science,in particular with the continuous development of carbon nanomaterials,zero dimensional(fullerene,carbon point),one-dimensional(carbon nanotubes),two-dimensional(graphene)and other carbon nano materials have been introduced into the biomedical field for research.Because of their good mechanical properties and large surface area,they have been used in the field of bone defect research.It is found that these nano carbon materials have a certain ability to promote osteogenesis.In this experiment,a new kind of watermelon derived nano porous carbon was prepared.It was confirmed by scanning electron microscope and transmission electron microscope that it had three-dimensional porous structure.There was no study on the interaction between the material and stem cells and the promotion of osteogenic differentiation in China and abroad,Therefore,this experiment is the first time to study the adhesion,proliferation,apoptosis,osteogenic differentiation and other aspects of nano watermelon porous carbon on bone marrow mesenchymal stem cells at home and abroad.It is also the first time to study the mechanism of nano watermelon porous carbon promoting bone marrow mesenchymal stem cells osteogenic differentiation.Methods The effect of nano watermelon porous carbon on the proliferation of bone marrow mesenchymal stem cells was evaluated by cell viability assay.The morphology of the cells was evaluated by observing the morphology of the cells.By alkaline phosphatase staining and quantification,objective is to evaluate the effect of nano watermelon porous carbon on the early osteogenic ability of bone marrow mesenchymal stem cells(BMSCs)differentiating into osteoblasts;Western blotting and real-time fluorescent quantitative polymerase chain reaction were used to detect the effect of nano watermelon porous carbon on osteogenic signal protein and gene expression of bone marrow mesenchymal stem cells;By alizarin red staining and alizarin red quantitative experiment,objective is to evaluate the effect of nano watermelon porous carbon on the mineralization ability of bone marrow mesenchymal stem cells(BMSCs)transformed into osteoblasts.Results At low concentration(2.5μg/ml、5μg/ml、10μg/ml),the results showed that nano watermelon porous carbon suspension had no effect on the proliferation of bone marrow mesenchymal stem cells(p>0.05).At high concentrations(20μg/ml、50μNano watermelon porous carbon suspension inhibited the proliferation of bone marrow mesenchymal stem cells(p<0.05).At the concentration of 10μg/ml,the activity of alkaline phosphatase(ALP)of bone marrow mesenchymal stem cells was significantly increased(p<0.05),2.5 μg/ml,5μg/ml and 20μg/ml of nano watermelon porous carbon induced osteogenic medium suspension.The activity of alkaline phosphatase(ALP)of bone marrow mesenchymal stem cells(BMSCs)was also increased by the suspension of nano watermelon porous carbon induced osteogenic culture(p>0.05),At 50μg/ml,the activity of alkaline phosphatase(ALP)of bone marrow mesenchymal stem cells was inhibited by the suspension of nano watermelon porous carbon induced osteogenic culture(p>0.05).At the concentrations(2.5μg/ml、5μg/ml、10μg/ml、20μg/ml)of nano watermelon porous carbon suspension,the osteogenic ability of osteogenic cells was significantly increased after osteogenic differentiation(p<0.05),The results showed that nano watermelon porous carbon(CG)suspension with high concentration(50μG/ml)inhibited the osteogenic and calcification ability of bone marrow mesenchymal stem cells(p>0.05).At the concentration of 10μg/ml,the expression of Runx-2 in bone marrow mesenchymal stem cells was significantly increased(p<0.05),and at other concentrations(2.5μg/ml、5μg/ml、20μg/ml、50μg/ml),the expression of Runx-2 in BMSCs was also increased(p>0.05).At 5μg/ml、5μg/ml、10μg/ml、20μg/ml,nano watermelon porous carbon suspension significantly increased the expression of β-catenin and BMP-2 of thebone marrow mesenchymal stem cells significantly higher than control group(p<0.05).10μg/ml is the best concentration to improve the expression of β-catenin and BMP-2.10μg/ml and 20μg/ml nano watermelon porous carbon suspension significantly increased the expression of OCN in bone marrow mesenchymal stem cells(p<0.05).10μg/ml is the best concentration to up regulate the expression of OCN in bone marrow mesenchymal stem cells.Conclusion At low concentration(2.5μg/ml、5μg/ml、10μg/ml),the results showed that nano watermelon porous carbon suspension had no effect on the proliferation of bone marrow mesenchymal stem cells(p>0.05).At high concentrations(20μg/ml、50μNano watermelon porous carbon suspension inhibited the proliferation of bone marrow mesenchymal stem cells(p<0.05).At the concentration of 10μg/ml,the activity of alkaline phosphatase(ALP)of bone marrow mesenchymal stem cells was significantly increased(p<0.05),2.5 μg/ml,5μg/ml and 20μg/ml of nano watermelon porous carbon induced osteogenic medium suspension.The activity of alkaline phosphatase(ALP)of bone marrow mesenchymal stem cells(BMSCs)was also increased by the suspension of nano watermelon porous carbon induced osteogenic culture(p>0.05),At 50μg/ml,the activity of alkaline phosphatase(ALP)of bone marrow mesenchymal stem cells was inhibited by the suspension of nano watermelon porous carbon induced osteogenic culture(p>0.05).At the concentrations(2.5μg/ml、5μg/ml、10μg/ml、20μg/ml)of nano watermelon porous carbon suspension,the osteogenic ability of osteogenic cells was significantly increased after osteogenic differentiation(p<0.05),The results showed that nano watermelon porous carbon(CG)suspension with high concentration(50μG/ml)inhibited the osteogenic and calcification ability of bone marrow mesenchymal stem cells(p>0.05).At the concentration of 10μg/ml,the expression of Runx-2 in bone marrow mesenchymal stem cells was significantly increased(p<0.05),and at other concentrations(2.5μg/ml、5μg/ml、20μg/ml、50μg/ml),the expression of Runx-2 in BMSCs was also increased(p>0.05).At 5μg/ml、5μg/ml、10μg/ml、20μg/ml,nano watermelon porous carbon suspension significantly increased the expression of β-catenin and BMP-2 of thebone marrow mesenchymal stem cells significantly higher than control group(p<0.05).10μg/ml is the best concentration to improve the expression of β-catenin and BMP-2.10μg/ml and 20μg/ml nano watermelon porous carbon suspension significantly increased the expression of OCN in bone marrow mesenchymal stem cells(p<0.05).10μg/ml is the best concentration to up regulate the expression of OCN in bone marrow mesenchymal stem cells.Background Bone defect is a difficult problem in clinical treatment,and it is also a hot and difficult research topic at home and abroad.As one of the bone defect repair materials,nano carbon material is regarded as a promising material for clinical bone defect treatment because of its special mechanical properties and strong surface area.Nano watermelon porous carbon is a three-dimensional nano carbon material,At present,there is no study on the effect of nano watermelon porous carbon on the repair of bone defects in vivo.In view of the good osteogenic effect of the material at the cellular level,the osteogenic properties of nano watermelon porous carbon in the bone defects of animals are further evaluated.Methods The rats were divided into three groups:group A(bone defect was not filled),group B(bone defect was filled with gelatin sponge),and group C(bone defect was filled with 10μg/ml)immersion).The incision was sutured and the postoperative treatment was carried out according to the standard.The following indexes were compared among group C,group A and group B after modeling.The area and volume of new bone and its proportion in the volume and area of bone defect were compared.The skull defect was decalcified after lweeks and 4weeks by microCT.After paraffin fixation,HE staining,Masson staining and Goldner staining were performed to evaluate the morphological changes of the defect.Results The percentage of new bone volume in nano watermelon porous carbon group was significantly higher than that in blank group and negative control group(p<0.05).The area of newly formed bone,collagen area in Masson staining and mineralized bone area in Goldner staining in nano watermelon porous carbon group were more than those in blank group and negative control group.Conclusion Nano watermelon porous carbon can promote the repair of rat skull defect,which is a potential bone defect repair material. |
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