| Intrafibrillar collagen mineralization is an important structural basis for the excellent mechanical properties and biological functions of hard tissues such as tooth and bone.Collagen fibrils,amorphous calcium phosphate precursors(ACP)and non-collagenous proteins(NCP)have been considered as the three core elements to complete the intrafibrillar collagen mineralization.ACP provides the most important material basis for intrafibrillar collagen mineralization during hard tissue formation.It has been reported that ACP is secreted during the physiological development of mouse skull and long bones,and that ACP is also involved in the process of pathological vascular mineralization.However,ACP is generally unstable and prone to phase transition,forming hydroxyapatite crystals and failing to complete intrafibrillar collagen mineralization.Hence,NCP with polyanion nature has been considered as the key to regulate the whole process.On the one hand,NCP stabilizes calcium-phosphate mineralization precursors to provide minerals,on the other hand,it modifies collagen to regulate the formation of collagen fibrils with intrafibrillar mineralization.However,in recent years,more and more studies have shown that when some key NCP-related genes are knocked out,the process of bone formation and mineralization in mice is not affected;others have found that NCP-induced collagen intrafibrillar mineralization is not the only way for osteoblasts to induce biomineralization.These results all question NCPs as central factors in the regulation of biomineralization.Our previous studies found that the extracellular vesicles secreted in the mineralization area contain not only NCP,but also biological macromolecules-nucleic acids.In addition,extracellular nucleic acids are also present in pathological calcifications such as vascular calcification and breast cancer microcalcification.Nucleic acid molecules,as anionic multifunctional biomacromolecules,possess special molecular structure,which determines that they have a variety of biological properties.The characteristics of nucleic acids,such as the simple and ordered molecular structure,the interaction between non-covalent bonds such as hydrogen bonds and ionic bonds,and the existence of phosphate groups with high charge density in the backbone,endow nucleic acids with excellent flexibility and biocompatibility.However,rather than directly participating in biomineralization,the function of nucleic acid molecules in osteogenesis has been limited to the storage,replication and transmission of genetic information and the synthesis and processing of auxiliary proteins.Has the role of nucleic acids in biomineralization been neglected?Do nucleic acids have the function of regulating mineral formation and matrix mineralization?How about their biological significance?Based on the above issues,in this study,we explore the stabilization mechanism of nucleic acids on calcium phosphate by biomimetic mineralization methods in vitro and characterized by multiple novel techniques.Then,we further explore the mechanism of nucleic acid-ACP precursors inducing collagen mineralization.Meanwhile,based on the above novel findings,we further explore two important biological issues of nucleic acid in biomineralization:the new role of nucleic acid-ACP in pathological mineralization and the new application in hard tissue regeneration.These studies have opened a brand new field of biomineralization,which not only provides a new mechanism for the involvement of nucleic acids in biomineralization,but also provides a new idea for the treatment of pathological mineralization related diseases and the development of synthesizing biomimetic inorganic-organic hybrid materials for bone regeneration.In the first part of the study,we first extracted nucleic acids(DNA and RNA)from cells,and then mixed them with high concentration of calcium and phosphorus solution by biomimetic mineralization methods,and finally obtained a clear nucleic acid stable supersaturated calcium and phosphorus solution.Dynamic light scattering(DLS)and Zeta potential were used to characterize the hydrodynamic diameter and Zeta potential of nucleic acid in calcium phosphate solution.Scanning electron microscopy(SEM)was used to observe its morphology.Fourier transform-infrared spectroscopy(FTIR)was used to detect the vibration of the characteristic functional groups of the nucleic acid-stabilized calcium phosphate solution and further analyze its composition.Subsequently,Transmission electron microscopy(TEM),element mapping and selected area electron diffraction(SAED)were used to analyze the element distribution,morphology and mineral crystallization of the nucleic acid-ACP.Cryo-electron microscopy and selected area electron diffraction were used to capture the different formation states and morphology characteristics of the nucleic acid-ACP.The formation process and mechanism of nucleic acid-ACP were further reproduced by molecular dynamic simulation.In the second part of the study,we mixed the nucleic acid-ACP prepared in the first part with two-dimensional collagen mineralization model(rat tail collagen type I)or threedimensional collagen mineralization model(bovine Achilles tendon-derived type I collagen scaffold and demineralized dentin derived type I collagen scaffold)respectively to evaluate the role of nucleic acid-ACP in mediating collagen mineralization.TEM,element mapping and SAED were used to observe the morphology of collagen and the mineral crystals in collagen;SEM was used to observe the mineral deposition on collagen fibrils;Cryo-electron microscopy combined with SAED and three-dimensional reconstruction were used to characterize the mineralization process of collagen.Subsequently,immunofluorescence staining was used to analyze the distribution of minerals and DNA on collagen fibrils;Uranyl acetate staining was used to determine the specific sites where DNA molecules bound to collagen fibrils;A polyanion modified collagen fibril model were used to probe the adsorption of collagen fibrils to DNA;FTIR was used to explore the binding mode between DNA and collagen fibrils;Molecular dynamic simulation was used to further verify the interaction between DNA and collagen fibrils;Different molecular weight of DNA were used to clarify the mechanism of nucleic acids-ACP inducing collagen mineralization.In the third part of the study,considering the existence of extracellular DNA in pathological calcification in vivo,we first constructed a variety of pathological mineralization models in vivo(heterotopic ossification model of heart valve,muscle and Achilles tendon)and in vitro(heterotopic ossification model of vascular smooth muscle cells).Subsequently,TEM was used to characterize the form of collagen mineralization;Immunofluorescence staining was applied to characterize the correlation between calcification distribution and nucleic acid deposition in the above heterotopic ossification model;Subsequently,we further investigated whether DNA-ACP can induce heterotopic ossification in vivo by implanting DNA-ACP collagen scaffolds into the muscles of mice legs.After 3 weeks of implantation,Micro-computed tomography(Micro CT)and threedimensional image reconstruction technology were used to observe the heterotopic ossification in the tissue,and immunofluorescence staining was applied to identify whether nucleic acid-ACP was involved in the formation of heterotopic ossification;TEM and SEM were used to further explore collagen fibril mineralization in heterotopic ossification induced by DNA-ACP.Subsequently,in vitro experiments were conducted to evaluate the effect of DNase addition on collagen mineralization and the regulation of cell-mediated matrix calcification.Immunofluorescence and alizarin red staining were applied to evaluate the ability of DNase to inhibit matrix calcification.This is the first biological significance of the present study,that is,the mechanism of DNA-ACP-mediated pathological mineralization.In addition,we added RNase to the RNA-ACP system to explore whether specific nuclease has a general inhibitory effect on the nucleate-mediated collagen mineralization,providing a new direction for the application of RNA-ACP in bone tissue engineering.In the fourth part of the study,because RNA degradation is easier to control and RNA can quickly induce the mineralization of collagen fibrils,we have prepared biomaterials with controllable mineralization and rapid bone regeneration.First,we constructed a mouse skull defect model,and implanted RNA-ACP-collagen scaffold and RNase-CaP-collagen scaffold into the skull defect.Samples were collected at 3 and 6 weeks after implantation.Micro CT,three-dimensional reconstruction and quantitative analysis were used to evaluate new bone regeneration;Histochemical staining(HE staining,von Kossa staining and Masson staining)and semi-quantitative analysis were conducted on the serial sections of undecalcified calvarial hard tissues to evaluate the formation of new bone tissue.Subsequently,the biocompatibility and the osteogenic effect of RNA-ACP-collagen scaffolds and RNase-CaP-collagen scaffolds were further investigated.CCK8 was used to evaluate the initial adhesion and proliferation of bone marrow derived mesenchymal stem cell(BMSC)treated with RNA-ACP,RNase-CaP and BMSC in control group.Immunofluorescence staining was used to characterize the morphological changes and mineralization of collagen in the above groups;SEM and TEM were used to characterize the spreading and adhesion of cells and the mineralization of collagen fibrils;Quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the expression of osteoblast-related genes and autophagy-related genes under the above conditions;The expression of osteoblast-related protein Osteocalcin(OCN)and LC3 was further characterized by immunofluorescence staining,to further clarify the mechanism of RNAACP or RNase-CaP in regulating bone defect repair.This is the second biological significance of the present study,that is,the biological application of nucleic acid-ACP in hard tissue regeneration.2.ResultsPart Ⅰ Nucleic acids stabilizing amorphous calcium phosphate(ACP)and the mechanism of ACP formation(1)As measured by dynamic light scattering,the mean hydrodynamic diameter of DNAstabilized CaP was 52.4 nm and RNA-stabilized CaP was 29.6 nm.The zeta potential of pristine DNA was-37.7 mV and RNA was-28.4 mV.After adding calcium and phosphate ions,the zeta potential changed to-19.7 mV for DNA-stabilized CaP and-10.3 mV for RNA-stabilized CaP.SEM showed that stabilization of the mineralization medium in the form of nanoparticles after adding nucleic acids to the mineralization medium.FTIR showed that stretching vibrations assigned to PO43-(1000-1245 cm-1)were indicative of the presence of mineralization medium-derived phosphate and the amorphous state of the mineral.(2)TEM combined with elemental mapping and selected area electron diffraction confirmed the presence of amorphous nucleic acids-stabilized CaP(DNA-ACP and RNA-ACP)phase.The distribution of nucleic acids derived nitrogen overlapped with the distributions of elemental calcium and phosphorus within those particulates.Cryo-electron microscopy showed the morphology of nucleic acids-ACP in their hydrated status.The nucleic acidsACP existed in solution as electron-dense nanoparticulates.These amorphous nanoparticles consisted of prenucleation clusters(PNC)with sizes 1 nm in diameter.These results confirmed that poly anionic nucleic acids could stabilize saturated CaP to form prenucleation nanoclusters and ACP.Accordingly,MD simulation with rigorous theoretical modeling approaches was used to predict,reproduce and analyze the nucleic acids-ACP formation process.Addition of calcium ions to nucleic acids(DNA and RNA)caused nucleic acids aggregation due to binding of Ca2+ to the polyanionic domains of the nucleic acids.This binding resulted in reduction of electrostatic repulsion among nucleic acids strands.Subsequent addition of phosphate ions produced prenucleation clusters externally around the nucleic acids aggregates.Liquid-like nucleic acids-ACP was formed eventually via coalescence of the prenucleation clusters.In addition,in RNA-ACP system,moderate calcium concentration promotes self-assembly of RNA to form RNA-ACP with specific structure.These results reveal the mechanism of nucleic acid stabilizing amorphous minerals and the mechanism of ACP formationPart Ⅱ Nucleic acids stabilizing ACP mediate collagen mineralization(3)SEM and TEM showed that three-dimensional collagen scaffolds were heavily mineralized,achieving intrafibrillar and extrafibrillar mineralization.The collagen fibrils were further examined with cryo-electron microscopy to monitor the evolution of mineralization mediated by nucleic acids-ACP.Intrafibrillar minerals were seen along the C-axis of the collagen fibrils as early as 3 h after the assembled fibrils were immersed into a freshly-prepared mineralization medium.More extensive intrafibrillar mineralization was identified after 5 h and mineralization of the entire fibril was accomplished as early as 24 h.S AED showed that hydroxyapatite(HAP)orderly deposited along the long axis of collagen fibrils.This is the first time complete in vitro mineralization of pristine collagen fibrils mediated by DNA-ACP and RNA-ACP is reported within such a short time.(4)The previous experiments confirmed that DNA-ACP and RNA-ACP have similar physical and chemical characteristics,and both can efficiently induce the mineralization of collagen fibrils.In order to further explore the mechanism of nucleic acids-ACP-mediated collagen mineralization,we selected DNA,which is easier to characterize in experimental methods to explore the mechanism of collagen fibril mineralization.With the use of confocal laser scanning microscopy(CLSM),we observed that nucleic acids were adsorbed on the collagen fibrils even after they were completely mineralized.TEM showed that ruthenium red-stained,electron-dense filamentous aggregates were attached randomly along the surface of the collagen fibril.To further investigate the interactions involved in binding of DNA with collagen,we resorted to the use of anionic collagen models for analysis.Images taken with CLSM indicated that the fluorescence intensity of DNA decreased only slightly when these molecules interacted with anionic polyacrylic acidbound collagen.This suggests that ion association is not the only driving force in DNAcollagen interaction.FTIR showed the strength of the hydrogen bonds.The MD simulation confirmed that DNA binds with collagen fibrils.Through calculation of the binding energy between DNA and collagen,we found that energy attributed to van der Waals attraction and nonpolar solvation energy,including hydrogen bonds are favorable for binding.TEM showed DNA with molecular weight higher than 40 kDa stabilized a supersaturated mineralization medium and induced collagen mineralization.The above results confirm the mechanism that nucleic acids with high molecular weight can bind with collagen fibrils through hydrogen bonds,thus inducing the rapid mineralization.Part Ⅲ DNA-ACP inducing ectopic mineralization and strategy to prevent(5)TEM of specimens retrieved from the pathological calcification models showed that collagen fibrils within the calcified regions were heavily-mineralized via intrafibrillar mineralization.CLSM of the sections prepared from the pathological calcification models and mouse vascular smooth muscle cells(VSMCs)demonstrated regions of calcified collagen that coincided with extracellular DNA deposition.Based on these findings,we surmised that introduction of DNA-ACP to soft connective tissues such as skin and muscle induces ectopic calcification in vivo.To clarify this notion,a murine intramuscular implantation model was used to validate the proposition.Micro-CT and 3D reconstruction further confirmed that ectopic bone was formed in DNA-ACP group only.The CLSM images of specimens harvested from after 3 weeks of implantation showed that the DNAACP collagen scaffolds were intensely calcified with extracellular DNA deposition.Both SEM and TEM confirmed intra/extrafibrillar mineralization of the collagen fibrils in the DNA-ACP group.Taken together,these data indicate that ectopic mineralization may be induced in vivo in the presence of DNA-ACP.(6)TEM showed that after 5 days of mixing of collagen fibrils with DNA-ACP system,a large amount of minerals deposit inside of the collagen fibrils.However,when DNase was added to the DNA-ACP system,there is no mineral depositing inside the collagen fibrils,indicating the collagen mineralization was inhibited.In cell-mediated matrix calcification,immunofluorescence revealed that there was a positive correlation between DNA deposition and matrix calcification in MC3T3-E1 mineralization model in vitro.Adding DNase could not only reduce DNA deposition,but also reduce the matrix calcification in MC3T3-E1 cells.Alizarin red staining also confirmed that the matrix calcification of MC3T3-E1 was significantly reduced compared with the control group(P<0.001).In addition,introduction of DNase to the pathological calcified cell mineralization model of VSMCs significantly inhibited the matrix calcification compared with the control group(P<0.001).Therefore,DNase can inhibit the mineralization of collagen fibrils and the extracellular matrix calcification.We proposed that the use of nuclease may effectively inhibit pathological calcification.In addition,RNase can also inhibit the mineralization of collagen fibrils in RNA-ACP system.Therefore,the application of specific nucleases generally inhibits the nucleic acids-mediated mineralization of collagen fibrils.Part Ⅳ RNA-ACP-Incorporated Collagen Scaffolds Promoting Rapid Bone Repair(7)Micro-CT showed that regenerated bone was observed in the RNA-ACP group only and not in the control or the RNase-CaP group after 3 and 6 weeks implantation.The results indicated that collagen scaffolds containing RNA-ACP nanomachines possessed better reossification potential.The radiographic findings were supported by histological staining of the regenerated bone with hematoxylin&eosin,Masson’s trichrome and von Kossa staining.In the RNA-ACP group,many of the scaffolds had already been replaced by newly-formed trabecular bone after 3 weeks.Multiple blood vessels and marrow cavities could be seen within the trabecular bone.At 6 weeks,cortical bone as well as Haversian canals were clearly identified.There was more regenerated bone with complete osseous closure along the junction of the defects.After 3 and 6 weeks,collagen scaffolds in the control group were completely degraded.In the RNase-CaP group,new bone was barely visible;the defect was mostly filled with soft tissue.(8)CCK-8 assay showed that RNA-ACP mineralization medium promoted initial BMSC attachment and had excellent biocompatibility.CLSM and SEM were further showed that collagen fibrils in the RNA-ACP group had already been mineralized after 24 h.After culturing for 7 days,collagen fibrils in the RNA-ACP group were highly mineralized intrafibrillarly.The BMSC were well-extended with many filopodia identified on the surface of the mineralized collagen scaffolds.For those collagen fibrils that were cultured in RNase-CaP medium for 7 days,mineralization of collagen fibrils was inhibited.Consequently,qRT-PCR showed that osteogenic differentiation-related genes were all significantly upregulated in the RNA-ACP medium group,compared with the control group or the RNase-CaP group(P<0.001).Immunofluorescence staining showed that the expression of osteocalcin protein was concomitantly observed in collagen scaffolds cultured RNA-ACP medium.Autolysosomes could also be identified by TEM during osteogenic differentiation of the BMSC in vitro in the RNA-ACP group.In addition,qRT-PCR further showed that RNA levels of autophagy-associated genes significantly increased during osteogenesis when the BMSC were cultured in collagen scaffolds supplemented within RNA-ACP for 7 days.These results demonstrated that autophagy is activated during rapid osteogenesis in collagen scaffolds incorporated with RNA-ACP.Thus,the enhanced bone regeneration in the RNA-ACP collagen system may be attributed to the role of autophagy in promoting osteoblast differentiation and function.Addition of RNase to the RNA-ACP system inhibited collagen mineralization and decreased BMSC proliferation and osteogenesis capacity,causing relatively low bone repair.3.ConclusionIn this study,we find that nucleic acids can stabilize supersaturated calcium phosphate to form ACP precursors,regulating mineral formation,and effectively inducing collagen mineralization.Focusing on natural macromolecular nucleic acid on biomineralization,we explored the relationship between collagen,nucleic acids and minerals,and further find the brand-new mechanism of nucleic acids induced collagen mineralization.Inspired by nucleic acids mediated mineralization,we discovered that DNA can induce ectopic mineralization and DNase is probably a powerhouse strategy in solving the issue of unwarranted ectopic mineralization in the human body.In addition,the use of RNA-ACP for rapid and controllable collagen mineralization has clinical translation values in bone tissue engineering and regenerative medicine. |
