| Background:3D bioprinting is a new emerging biomanufacturing technology in recent years.It can be used to produce biomimetic tissue-engineered bone structures,and it is also used in other regenerative medicine fields,such as skin bioprinting and heart bioprinting.It is an additive manufacturing technology based on traditional 3D printing,using biological materials loaded with living cells(ie,bioink),stacked layer by layer according to a preset path.In 3D bioprinting,in order to improve the survival rate of laden cells,hydrogel with high water content and good cell compatibility is usually used as the matrix material of the bioink.However,at present,most of the common bioinks in the field of bone bioprinting exist the following problems: 1)Low mechanical strength,which is not enough to print the construct whose height in the Z-axis direction exceeds 1cm;2)Poor osteoinductive ability,additional osteoinductive factors,such as BMP-2,are needed;3)It is difficult to provide a simulated natural suitable microenvironment for extracellular matrix(ECM).As a kind of biomaterial with great clinical application potential,nanosilicate(n Si)has attracted more and more interest in recent years.It has excellent biological properties,for example,good cell compatibility,improving the mechanical strength of nanocomposites,and the degradation products are not only non-toxic,but also have strong osteogenic induction activity.At present,many studies have focused on in vitro experiments,and rare research has been done on the osteogenic effect and its biocompatibility of n Si in vivo.In addition,the microenvironment of cells is of great significance for the maintenance of their biological activities and functions.Natural ECM mainly consists two major categories:fibrous structural proteins(such as collagen)and polysaccharides(such as hyaluronic acid).Simulating the natural ECM as much as possible in structure and composition is of great significance for the functionalization of 3D bioprinted structures.Objective:1.Prepare a functional and biomimetic nanocomposite hydrogel that can be used in 3D bioprinting.The hydrogel is based on gelatin-sodium alginate,which can not only mimic the structure and composition of natural ECM,but also increase the strength of the nano-composite hydrogel by adding different concentrations of n Si,and then increase the Z-axis printable height.2.Prepare a bioprinted tissue engineering bone scaffold that can induce osteogenic differentiation of encapsulated bone marrow mesenchymal stem cells(BMSCs)without adding any osteogenic factor,which greatly decreases biological manufacturing cost.Cell viability,proliferation and differentiation of BMSCs encapsulated in 3D bioprinted tissue engineering bone scaffolds were evaluated in vitro in normal medium(NM)without any osteogenic factor and osteoinductive medium(OIM)3.The 3D bioprinted BMSCs-loaded tissue-engineering bone scaffolds were directly implanted into the 8mm critical bone defect of the rat skull.To investigate the effect of bone healing with or without cells and with or without n Si,and investigate whether n Si is biologically toxic to rat heart,liver,spleen,lung,and kidney in vivo,providing a reference for further clinical transformation.Methods:1.Four kinds of nanocomposite hydrogels(0%n Si/1%n Si/2%n Si/3%n Si)with10%gelatin-1%alginate as basic material and consisting of different concentrations of n Si.The morphology of n Si was characterized by a cryo-transmission electron microscope(Cryo-TEM);the presence of gelatin,sodium alginate,and n Si components in nanocomposite hydrogels was characterized by Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD);element content in 0%n Si and 2%n Si hydrogels and the internal pore structure of hydrogels with different proportions were characterized by energy dispersive X-ray(EDX)and scanning electron microscope(SEM).Finally,the swelling rates and water content of the four hydrogels were characterized.2.The shear rate scan and shear stress scan were used to characterize the rheological characteristics of the four hydrogels at body temperature of 37℃ and room temperature of 25℃.Through uniaxial compression experiments,the stress-strain curves were drawn.Then,the compression moduli of the four hydrogels were calculated.By analyzing the printability of the four hydrogels,an optimal formulation was finally selected for the following 3D bioprinting,and the top view and side view of the 3D printed scaffold was observed by SEM.Finally,a child tibial plateau model was printed using the optimal formulation.3.The SD rats BMSCs were extracted and purified by adherent culture,and the BMSCs obtained were identified by three aspects: cell morphology,flow cytometry,and multipotential differentiation potential.4.Optimizing the entire 3D bioprinting process,prepare tissue engineering bone scaffolds loaded with BMSCs,and evaluate the printed cell viability and its 3D distribution within bioink by live-dead cell staining;CCK-8 test was used to detect the cell proliferation ability and to evaluate the cytotoxicity of nanosilicate;the division,contact,and extension of cells in three dimensions hydrogel was evaluated by H&E staining and cytoskeleton-nuclear staining.5.The p-NPP azo method was used to detect the activity of alkaline phosphatase of the laden cells within the scaffold in NM and OIM;the expression of the osteogenesis-related genes: Runx2,Osterix,ALP,and Col 1a1,OCN and OPN in NM and OIM was detected by q RT-PCR technology;Alizarin red staining and SEM observation were used to evaluate the mineralization capacity of the laden cells within the scaffold,and EDX was used to further analyze the elemental distribution and quantitative elemental analysis of the calcium nodules.6.Building a SD rat calvarial defect model(8mm),implant four kinds of scaffolds with/without cells and with/without n Si,and select 2 weeks,4 weeks,8 weeks and 12 weeks as observation points.Micro-computed tomography(Micro-CT),calcein-alizarin red sequential fluorescent labeling,Van-Gieson staining,H&E staining,and Masson staining were used to evaluate the effect of bone repair and angiogenesis on bone defects;the biocompatibility of nanocomposite hydrogels was evaluated by H&E staining of heart,liver,spleen,lung,and kidney.Results:1.Cryo-TEM shows that the n Si used in this experiment is a nanosheet with a diameter of about 50nm;FTIR and XRD proved the composition of the 2% n Si nanocomposite hydrogel: gelatin,alginate and n Si;in the gelatin-alginate polymer network,the internal pore diameter of the nanocomposite hydrogel increased with the increase of the n Si concentration;EDX results showed that the main elements of the gelatin-alginate hydrogel were C and O.The main elements of the gelatin-alginate-nanosilicate nanocomposite hydrogel include C,O,Mg and Si(Li cannot be detected,due to machine reasons);the water absorption and swelling experiments of the four hydrogels show that with the increasing of the concentration of nanosilicate,its water content and swelling rate decreased.2.Rheological tests show that compared with pure gelatin or/and alginate,the nanocomposite hydrogels with n Si have significantly improved rheological properties,and both show better shear-thinning.2%n Si and 3%n Si have similar G ′ and G ″;the compression modulus of the four hydrogels calculated by uniaxial compression experiments are: 46.98 ± 1.54 k Pa,57.82 ± 1.69 k Pa,101.41 ± 3.11 k Pa,123.59 ±2.10 k Pa.Finally,2%n Si was selected as the best ratio for the following 3D bioprinting.The SEM results showed that the scaffold printed with this formulation was clear and without collapse,and the height(Z-axis direction)of the child tibial plateau model printed with this formulation can reach to 15 mm and consists of 60 layers.3.The morphology of BMSCs used in this experiment is normal,showing a typical spindle or spindle shape.The three lines differentiation of osteogenesis,adipogenesis and cartilage are obvious.The results of flow cytometry showed that the BMSCs were of high purity.4.For 3D bioprinted scaffolds loaded with cells,live-dead cell staining proves that the cells have a high survival rate and uniform spatial distribution;CCK-8 testing shows that the scaffolds with/without nanosilicate can promote cell proliferation,and the addition of n Si had no effect on the biocompatibility of the hydrogel;H&E staining showed that the cells were evenly distributed inside the scaffold;cytoskeleton-nucleus staining showed that the laden cells could maintain normal morphology,and the cells could divide,stretch and contact normally.5.Quantitative analysis of ALP,q RT-PCR detection of osteogenic genes,alizarin red staining,and SEM/EDX observations have shown that in the absence of any osteogenic factor,the scaffolds containing nanosilicate can obviously induce the osteogenic differentiation and mineralization of the encapsulated BMSCs,and produce calcium nodules.6.The results of in vivo experiments show that the nanocomposite hydrogel scaffolds loaded with cells show the best bone repair effect,and there are more new blood vessels inside the scaffold,while the nanocomposite hydrogel scaffolds without cells and the gelatin-alginate scaffolds with cells exhibited similar bone repair effects,but they were inferior to cell-loaded nanocomposite hydrogel scaffolds.Conclusions:This research has developed a functional and biomimetic nanocomposite bioink that can be 3D bioprinted and can be used for the biological manufacture of functional tissue engineering bones.The addition of nanosilicate not only improves its printability,but also increases the print height in the Z-axis direction,which provides a possibility for the printing of tissues and organs of the size of human anatomy in the clinic in the future.In addition,the bioink has obvious osteogenic induction effect,and can induce osteogenic differentiation and mineralization of encapsulated BMSCs in the absence of any osteogenic induction factor.Systematic in vivo experiments have proven that BMSCs-loaded nanocomposite hydrogel tissue engineering bone scaffolds have good bone repair effect and good biocompatibility. |
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