Collagen/Polyelectrolyte Complex Induced Intrafibrillar Mineralization

In the field of biomimetic mineralization,how to simulate the exquisite hierarchical structure of biological mineralized bone tissue by artificial synthesis,and then form a hard tissue defect repair material comparable to the performance of natural hard tissue is the current research highlight.Inspired by the characteristics of non-collagenous proteins(NCP)that precisely control the orderly deposition of inorganic mineral in collagen molecules,several international research groups have used polycarboxylate electrolytes to simulate the functional regions of NCP.Thus biomimetic intrafibrillar mineralization has been achieved in vitro.However,in these biomimetic models,the organic template used is bare and unmodified collagen fibril and the non-collagenous protein analogs are dissolved in the mineralized environment.These factors greatly reduce the utilization efficiency of NCP analogs,causing longer mineralization period and limited mineralization depth.In addition,polyelectrolytes in a free state may be toxic to host cells,resulting in poor biocompatibility and other problems.In order to solve the problems existing in the traditional biomimetic mineralization model,this study draws on the characteristics of non-collagenous proteins modification on the surface of collagen matrix in living body,and the non-collagenous proteins' analog,high molecular weight polyacrylic acid(HPAA)is covalently bonded on collagen molecules,constructing a novel collagen/polyelectrolyte complex material.A large number of HPAA carboxyl groups immobilized on collagen molecules are used to rapid enrich the mineral precursors in mineralization solution,and to establish a continuous osmotic pressure difference and charge difference inside and outside the collagen.As a result,heavy and extensive biomimetic intrafibrillar mineralization could be achieved with the minerals enter the collagen fibers continuously and unidirectionally.From the perspective of biomimetic mineralization mechanism,this model highly mimics the positional relationship and interaction relationship between collagen matrix and non-collagenous proteins in vivo,which provides a more persuasive model for the verification of biomineralization concept in the biological mineralization phenomenon.From the field of tissue engineering scaffold materials,we constructed a collagen scaffold material that can spontaneously induc of intrafibrillar mineralization.This property helps us solve the difficulty in in-situ delivery of NCP analogs,which provide a new idea to develop in situ mieralization directed tissue engineering scaffold.1.Scheme 1)High molecular weight polyacrylic acid(HPAA,450 k Da)and low molecular weight polyacrylic acid(LPAA,2 k Da)were chemically-crosslinked to reconstituted type I collagen fibrils or sponges using carbodiimide(EDC)and N-hydroxysulfosuccinimide(NHS).With the help of transmission electron microscopy(TEM)and ruthenium red/ uranyl acetate(UA)double staining,we determined the binding sites of collagen and polyelectrolyte.To comprehensively characterize the physicochemical properties of collagen/polyelectrolyte complex,attenuated total reflection-fourier transform infrared spectroscopy(ATR-FTIR)was used to observe the functional group change of collagen molecules before and after covalent bonding of polyelectrolytes.Change of surface potential of collagen was determined with Zeta potential detection.The methylene blue adsorption experiment and the TNBS experiment quantitatively analyzed the changes of the amine group and the carboxyl group content of the collagen molecule.At last,the high-performance liquid chromatography(HPLC)identified the chemical bond between the collagen and the polyacrylic acid as well as detected the stability.2)In the second part of this study,different mineralization environments were constructed and characterized by dynamic light scattering(DLS),zeta potential analysis and cryo-electron microscopy(cryo-EM).Then,the collagen/polyelectrolyte complex model which could spontaneously induce intrafibrillar mineralization without any NCP and its analogs was screened by TEM.The validity of the collagen/polyelectrolyte model capable of inducing intrafibrillar mineralization was repeatedly verified with different collagen models.Finally,by using cryo-EM and molecular dynamics simulation to simulate the space and dynamic relationship between collagen,polyelectrolyte and mineral ions,we explore the interaction mechanism between the three factors and the principle of biomimetic mineralization.3)In the third part of this study,to provide experimental basis for tissue engineering applications of the novel collagen/polyelectrolyte composite materials,a series of technical detection were used to characterize the features and changes in mineralized collagen/polyelectrolyte composite materials.X-ray diffraction(XRD)was used to identify the mineral crystal form in the collagen fibrils.Thermogravimetric analysis(TGA)was used to detect the mineral content and thermodynamic degradation mode of the material.Dynamic FTIR was used to track the hydroxyapatite formation process of mineralized collagen.Stress-strain curve test and atomic force microscopy(AFM)to test the mechanical properties of mineralized collagen/polyelectrolyte composite materials.With the help of MTT and flow cytometry,biocompatibility of the novel material was evaluated.2.Results Part I Development and characterization of collagen/polyelectrolyte composite 1)With the help of crosslinking agents EDC and NHS,the carboxyl groups of polyacrylic acid were activated and covalently bonded with the amine groups from the side chain of collagen molecules,forming a collagen/polyelectrolyte complex with a large number of functional carboxyl groups on the surface.2)TEM of reconstituted PAA-bound collagen fibrils stained with ruthenium red,a cationic dye showed that the bound PAA appeared as electron-dense filamentous aggregates on the fibril surface that obscured the banding patterns of bare collagen fibrils.The results of FTIR showed that collagen molecules covalently bound with polyacrylic acid had augmented amide I,II,III and B peaks compared with bare collagen,that were attributed to increases in amide linkages after crosslinking.Collagen matrices with bound PAA demonstrated marked increases in negative surface zeta potential(collagen:-3.04 ± 0.37 m V,collagen/HPAA complex:-17.17 ± 1.98 m V,collagen/LPAA complex:-15.19 ± 1.22 m V,p<0.05 when comparing with control group)and carboxyl groups(collagen: 0.460 ± 0.017 m M/g,collagen/HPAA complex: 1.907 ± 0.084 m M/g,collagen/LPAA complex: 1.807 ± 0.035 m M/g,p<0.05 when comparing with control group).Decreases in amine groups was caused by the formation of O=C...N-H linkages(collagen: 0.132 ± 0.007 m M/g,collagen/HPAA complex: 0.033 ± 0.004 m M/g,collagen/LPAA complex: 0.023 ± 0.002 m M/g,p<0.05 when comparing with control group).3)The binding/release characteristics of PAA-bound collagen sponges were examined by high performance liquid chromatography.Milli-Q water used for storing PAA-bound collagen sponges did not contain traceable amounts of polyelectrolyte after one month of immersion.Likewise,PAA was not detected after extraction with 4M guanidine HCl.By contrast,PAA was detected after the PAA-collagen sponges were treated with 0.1 mg/m L bacterial collagenase.The results indicate that PAA binds tightly to type I collagen via covalent crosslinking,and that the ability of PAA-collagen to stabilize calcium phosphate(Ca P)solution in the vicinity of collagen fibrils is not caused by PAA dissociation.Part II Biomimetic intrafibrillar mineralization of collagen/polyelectrolyte complex and exploration of mineralization mechanism 4)DLS results indicated that pure Ca P is extremely unstable.In the absence of NCP analogs(HPAA)as nucleating inhibitors,calcium ions and hydrogen phosphate ions aggregate into crystal precipitates rapidly with extremely large particle size(955.4 nm).In contrast,the size of HPAA-Ca P is stably distributed in the small particle peak region(18.2 nm and 91.3 nm).In the presence of collagen/polyelectrolyte complex,the Ca P solution exhibits three discrete peak regions(15.7 nm,78 nm,and 1000 nm),indicating that the collagen/polyelectrolyte complex effectively stabilizes the surrounding mineral ions.However,aggregation still occurs in areas away from the complex.5)HPAA-bound collagen model,a collagen/polyelectrolyte complex model capable of inducing intrafibrillar mineralization in an environment in which no additional polyelectrolyte was added,was rapidly screened by TEM.Then the effectiveness of the collagen/polyelectrolyte complex to induce intrafibrillar mineralization was verified by two different collagen models(three-dimensional recombinant collagen sponge and two-dimensional recombinant single layer rat tail collagen fibril).6)Cryo-EM in-situ vitrification technology was used to restore the true morphology of the liquid phase mineralization precursors and their dynamic relationship with collagen fibers.HPAA-bound collagen was found to rapidly adsorb surrounding pre-nucleation cluster(PNC)singlets to form chain-like PNC aggregates,while discrete low electron density ACPs were also observed around the collagen.According to their size and high-water content,we speculated that all of the three mineralization precursors: PNC singlets,PNC aggregates and ACP have the potential to entering the collagen.But PNC aggregates dominated both in quantity and in the positional relationship with collagen,so it is mainly a large amount of PNC aggregates that enter the interior of collagen to form intrafibrillar mineralization.7)According to molecular dynamics simulation,we found that HPAA-bound collagen could not enter the internal space of collagen due to its excessive molecular weight.At the same time,it caused a difference in charge and osmotic pressure in the extrafibrillar environment due to its significant anionic properties.As a result,negative pressure was formed inside the collagen,which constituted the driving force of amorphous mineralization precursors to penetrate into the fibers,which is the key to intrafibrillar mineralization of collagen.Part III Physicochemical and biocompatibility characterization of the intrafibrillarly mineralized collagen/polyelectrolyte complex 8)XRD results confirmed that the minerals contained in the collagen/polyelectrolyte composite material after biomineralization were hydroxyapatite,and the mineral content was 77.03% according to thermogravimetric analysis,which was significantly higher than the 66.07% of the control group(p<0.05),indicating that the materials were highly close to the natural mineralized bone in terms of organic and inorganic composition.9)The dynamic FTIR tracking mineralization processes showed that compared with the traditional biomimetic mineralization method,the collagen/polyelectrolyte composite material significantly improved in mineralization rate and mineralization degree.This complex can solve the long mineralization period and limited mineralization depth which often occur in traditional mineralization strategy.10)The stress-strain curve results show that the toughness modulus of the collagen/polyelectrolyte complex(352.7±81.4KPa)after biomimetic mineralization is significantly higher than that of the traditional biomineralized collagen(266.7±121.5Kpa,p<0.05).The Young's modulus of bare collagen,extrafibrillarly mineralized collagen,intrafibrillarly mineralized collagen with traditional method,and the biomineralized collagen/polyelectrolyte complex increased in turn,and the differences between the groups were statistical significance(p<0.05 when comparing in pairwise).These results indicats that the collagen/polyelectrolyte composites possessed optimum stiffness,toughness and uniformity after intrafibrillar mineralization.11)MTT assay and flow cytometry demonstrated that polyelectrolyte bound collagen sponge did not adversely affect MSC activity,proliferation and apoptosis compared to bare collagen sponge(for each experiment,p>0.05 when comparing with control group).Indicating that the collagen/polyelectrolyte composites have good biocompatibility,which improves the experimental basis for the subsequent exploration of in vivo application.3.Conclusion In summary,our findings provide an excellent biomimetic model for elucidating biomineralization mechanisms.The novel collagen/polyelectrolyte complex highly simulated the structure characteristics of collagen/ NCP complex found in vivo,which make the derived in vitro biomimetic mineralization mechanism more reference to the biomineralization principles hide in Nature Furthermore,we demonstrated that the collagen/polyelectrolyte complex could effectively induce rapid,extensive and uniform intrafibrillar mineralization spontaneously,which sheds light on potential solutions to problems existing in the current biomimetic mineralization method.Finally,from a materials perspective,enhancement of intrafibrillar mineralization by HPAA-bound collagen produces more consistently infiltrated minerals within bulk collagen matrices,with better mechanical properties and potential flaw reduction to increase fatigue resistance.