The Study On Regulating Bone Regeneration And Mechanism Of Hydrogel-based Bioprinting Scaffolds

In clinical cases of bone defects,the bone defect area is usually irregular to preserve the original bone tissue of patients as much as possible.In conventional bone tissue engineering(BTE),cells are inoculated on scaffolds.After being cultured in vitro for a period of time,scaffolds are implanted into the bone defect area in vivo.However,due to the uncontrollable distribution and limited number of cells,this method can’t meet the needs of BTE.Therefore,in order to match bone grafts with the bone defect area,3D bioprinting scaffolds encapsulating cells have become an important development direction of BTE.Gel MA hydrogel is considered as a substitute for extracellular matrix(ECM)and provide biomimetic microenvironment for BTE due to the great biosafety and biocompatibility.However,pure Gel MA hydrogel remains some limitations,such as low mechanical strength and weak bone induction ability.Therefore,the modification of Gel MA hydrogel to obtain composite hydrogels which are suitable for encapsulating cells,bone induced and printable may have great potential in the field of BTE.Based on Gel MA hydrogel,this study synthesized mechanically reinforced bioactive Gel MA/Sr-CSH nanocomposite hydrogel by adding strontium-doped xonotlite(Sr-CSH),and explored its application and related mechanism in BTE.Meanwhile,scaffolds were customized with 3D bioprinting technology,and studied the application in bone and osteochondral repair,providing a new strategy for BTE.Part I Effects of Gel MA/Sr-CSH nanocomposite hydrogel on bone regeneration【Objective】Sr-CSH nanowires was prepared to synthesize organic-inorganic Gel MA/Sr-CSH nanocomposite hydrogel.The effects of Gel MA/Sr-CSH nanocomposite hydrogel on bone marrow-derived mesenchymal stem cells(BMSCs)osteogenic differentiation and the ability to repair calvarial defects were explored.The potential mechanism was studied to lay the material foundation for the subsequent 3D bioprinting scaffolds.【Methods】(1)Sr-CSH and CSH nanowires were synthesized by hydrothermal method.Field emission transmission electron microscopy(FETEM),energy dispersive spectroscopy(EDS),elemental mapping,X-ray diffraction(XRD)and fourier transform infrared spectroscopy(FTIR)were performed to characterize Sr-CSH and CSH.(2)Nanocomposite hydrogels with different contents(0.5%,1%,2%and 4%(w/v))of Sr-CSH or CSH were prepared.Morphological observation was conducted by scanning electron microscopy(SEM).Meanwhile,degradation behavior,swelling properties,mechanical properties and rheological properties were characterized.(3)The optimal CSH and Sr-CSH content was screened by cell counting kit-8(CCK-8)and alkaline phosphatase(ALP)activity assay.(4)The effects of Gel MA,Gel MA/CSH and Gel MA/Sr-CSH on osteogenic and angiogenic differentiation of BMSCs were detected by real-time PCR.(5)The effects of Gel MA,Gel MA/CSH and Gel MA/Sr-CSH on Mitogen-activated protein kinase(MAPKs)signaling pathway in BMSCs were detected by western blot.(6)The effects of Gel MA/Sr-CSH on BMSCs osteogenic differentiation after the addition of MAPKs signaling pathway inhibitors was observed;(7)Gel MA,Gel MA/CSH and Gel MA/Sr-CSH were implanted into the rat calvarial defect model,and the formation and mineralization of new bone were detected by micro computed tomography(Micro-CT),sequential fluorescence labeling and histological staining.【Results】(1)The image of FETEM showed that both CSH and SR-CSH are ultra-long nanofibers with smooth surfaces,a diameter of about 20nm and a length of several microns,and the addition of Sr didn’t affect the morphology and dispersion of nanowires.EDS results showed that Sr element existed in Sr-CSH,but not in CSH,indicating successful incorporation of Sr.The distribution of Ca,Sr,Si and O elements was further detected,and the results showed that Ca,Sr,Si and O were uniformly distributed.The XRD patterns of CSH and Sr-CSH are consistent with Xonotlite,and the addition of Sr didn’t change the XRD patterns.FTIR spectra showed that CSH and SR-CSH had similar waveforms.(2)The image of SEM showed that Gel MA,Gel MA/CSH and Gel MA/Sr-CSH all had similar sponge-like structures with porous morphology,indicating that the incorporation of nanowires didn’t affect the structure of Gel MA,and the nanowires were well dispersed in Gel MA.XRD and FTIR spectra proved the existence of CSH and Sr-CSH in nanocomposite hydrogels.The degradation behavior showed that the doping of nanowires could slow down the degradation of Gel MA hydrogel,and Gel MA/Sr-CSH had the effect of slow release of Sr.The results of swelling property showed that the doping of low content nanowires didn’t affect the swelling ratio and equilibrium water content of Gel MA.The results of mechanical properties showed that the doping of nanowires could improve the mechanical properties of Gel MA.The rheological properties showed that Gel MA,Gel MA/CSH and Gel MA/Sr-CSH were stable cross-linking.(3)The results of cell proliferation and ALP activity showed that Gel MA/2%Sr-CSH had the best osteogenic effect,which was used in subsequent experiments.(4)RT-PCR results showed that Gel MA/2%Sr-CSH could significantly promote the expression of osteogenesis and angiogenesis related genes.(5)Western blot results showed that Gel MA/2%Sr-CSH could promote the phosphorylation of ERK and p38,and the ALP activity was significantly inhibited by the addition of ERK and p38 inhibitors.(6)In the rat calvarial defect model,BV/TV,sequence fluorescence and new bone area were the highest in Gel MA/2%Sr-CSH group.【Conclusion】(1)In this study,Sr-CSH nanowires were synthesized by a simple hydrothermal method,and an organic-inorganic nanocomposite Gel MA/Sr-CSH hydrogel was synthesized for in situ bone regeneration.(2)Gel MA/Sr-CSH hydrogel had good swelling property,high water content,enhanced mechanical properties and stable rheological properties.(3)Gel MA/Sr-CSH hydrogel promoted the osteogenic differentiation of BMSCs through ERK/p38 signaling pathway,showed good osteogenic ability in vitro and in vivo,and provided a potential composite scaffold material and 3D printing bioactive ink for Gel MA hydrogels in BTE.Part II 3D bioprinting scaffold based Gel MA/Sr-CSH nanocomposite hydrogel to regulate bone regeneration【Objective】Gel MA/Sr-CSH nanocomposite hydrogel was used as bioink and BMSCs as seed cells to construct 3D bioprinting scaffolds.The effect of Gel MA/Sr-CSH nanocomposite hydrogel on the osteogenic differentiation of BMSCs in 3D bioprinting scaffolds and the ability of 3D bioprinting scaffolds to repair rat’s calvarial defects were investigated.【Methods】(1)The shear-thinning properties of Gel MA,Gel MA/CSH and Gel MA/Sr-CSH were determined by rheometer.(2)Cell density was screened by Live/Dead staining and ALP activity assay.(3)Live/Dead staining was used to screen the content of nanowires.(4)ALP activity,real-time PCR,immunofluorescence staining and alizarin red staining were used to evaluate the osteogenic differentiation of BMSCs in Gel MA,Gel MA/0.5%CSH and Gel MA/0.5%Sr-CSH 3D bioprinting scaffolds.(5)The pure Gel MA scaffold and Gel MA,Gel MA/0.5%CSH and Gel MA/0.5%Sr-CSH 3D bioprinting scaffolds were implanted into the rat calvarial defect model,and the formation and mineralization of new bone were detected by Micro CT scanning,sequence fluorescence labeling and histological staining.【Results】(1)Rheological results showed that the viscosity of Gel MA,Gel MA/CSH and Gel MA/Sr-CSH hydrogels decreased with the increase of shear rate and exhibited a shear-thinning characteristic,which made them suitable for 3D bioprinting.The 3D bioprinting scaffolds had clear shape and uniform pore size.(2)Live/Dead staining showed that Gel MA hydrogel had good biosafety.ALP activity test showed that the 2×10~6 cells/m L group showed the best ALP activity,and there was no statistical difference among the other three groups(P<0.05),so the density of 2×10~6cells/m L was selected for subsequent experiments.(3)The results of Live/Dead staining showed that BMSCs still maintained good biosafety of Gel MA when the doping content of CSH and Sr-CSH was 0.5%.However,when the nanowires’doping content was 1%,there were more dead cells,fewer live cells,indicating that the nanowires’doping content of 1%caused cell death and was not suitable for bioink.Therefore,0.5%nanowires’doping content was used in subsequent experiments.(4)The results of ALP activity,RT-PCR,immunofluorescence and alizarin red staining showed that Gel MA/0.5%Sr-CSH 3D bioprinting scaffold exhibited the best osteogenic differentiation.(5)In the rat calvarial defect model,BV,BV/TV,sequence fluorescence and new bone area were the highest in Gel MA/0.5%Sr-CSH group.【Conclusion】(1)The printability and biosafety of the organic-inorganic composite Gel MA/Sr-CSH hydrogel were confirmed as a suitable bioink.(2)Based on Gel MA/Sr-CSH hydrogel bioink,3D bioprinting scaffolds were constructed and the ability of Sr to promote osteogenic differentiation of BMSCs in vitro was verified.(3)The ability of 3D bioprinting scaffolds based on Gel MA/Sr-CSH nanocomposite hydrogel bioink to promote the repair of rat calvarial defects in vivo and the prospect of Gel MA/Sr-CSH+BMSCs bioprinting scaffolds in BTE were confirmed.Part III 3D bioprinting of biphasic multicellular scaffolds to regulate the osteochondral repair【Objective】The 3D bioprinting scaffold of Gel MA/Sr-CSH+BMSCs was used as the osteogenic layer of the biphasic scaffold,and the 3D bioprinting scaffold of Gel MA+ACs/BMSCs was used as the chondrogenic layer of the biphasic scaffold.The effect of this two-phase,multicellular and co-culture 3D bioprinting scaffold in rat’s osteochondral repair in vivo was explored.【Methods】(1)Primary articular chondrocytes(ACs)from rats were cultured by enzymic method and identified by alcian blue and toluidine blue staining;(2)In vitro cell viability of biphasic 3D bioprinting scaffolds was detected by Live/Dead staining;(3)Real-time PCR and immunofluorescence staining were used to evaluate the chondrogenic differentiation of cells in Gel MA/BMSCs and Gel MA/ACs+BMSCs 3D bioprinting scaffolds;(4)ALP activity,real-time PCR and immunofluorescence staining were used to evaluate the osteogenic differentiation of BMSCs in Gel MA and Gel MA/Sr-CSH 3D bioprinting scaffolds;(5)Gel MA,Gel MA+Cells and Biphasic+Cells 3D bioprinting scaffolds were implanted into the osteochondral defect model of the femoral intercondyle in rats,and the formation and mineralization of new bone were detected by Micro CT scanning and histological staining.ICRS score and histological staining were used to detect chondrogenesis.【Results】(1)Under an inverted microscope,the primary ACs presented triangles or polygons attached to the bottom of the petri dish.After subculture,ACs showed polygon or spindle shape,and were dyed blue and purple by alcian blue and toluidine blue,respectively,which proved the successful culture of ACs.(2)Live/Dead staining results showed that biphasic 3D bioprinting scaffolds showed high cell viability in vitro.(3)Gel MA/ACs+BMSCs 3D bioprinting scaffolds exhibited better chondrogenic differentiation.(4)Gel MA/Sr-CSH 3D bioprinting scaffolds exhibited better osteogenic differentiation.(5)The biphasic+Cells group showed the highest BV/TV and new bone area.At the same time,the biphasic+Cells group also displayed the best ICRS score and cartilage repair in the rat femoral intercondylar osteochondral defect model.【Conclusion】(1)Based on the anisotropic biological structure of osteochondral tissue,biphasic multicellular scaffolds were constructed by 3D bioprinting technology to achieve the spatial distribution of multiple cells.(2)Based on the bidirectional regulation of Sr-containing bioink,we demonstrated the ability of 3D bioprinting biphasic multicellular scaffolds to promote the repair of integrated osteochondral defects between the femoral condyles of rats in vivo.