Mechanism Of Chondroitin Sulfate Inducing Collagen Mineralization And Its Role In Heterotopic Ossification

Objective:Chondroitin sulfate（CS）is an important extracellular matrix component in mineralized tissues.It participates in dentinogenesis and bone formation,and can also be used in bone tissue engineering,with the ability to promote bone defect repair.However,the mechanism in which CS functions is unclear.This study explored the regulatory effect of CS on mineralization through in vitro biomimetic mineralization and multiple characterization techniques,and utilized molecular simulation to explain the mechanism.On this basis,the correlation between CS deposition and the occurrence of pathological calcification in the Achilles tendon was found.This discovery will further broaden the application prospects of CS in tissue engineering and provide new ideas for the treatment of ectopic calcification related diseases.Methods:In the first part of the experiment,H&E staining,Alcian blue staining,1,9-dimethylmethylene blue（DMMB）quantitative spectrophotometry,and confocal laser scanning microscopy（CLSM）were used to characterize the relationship between CS and heterotopic ossification（HO）.In the second part of the experiment,first of all,using in vitro biomimetic mineralization methods,different concentrations of CS were mixed and proportioned with high concentration of calcium phosphate solution,and finally the optimal concentration of CS that can form amorphous calcium phosphate（ACP）was determined.Dynamic light scattering（DLS）and zeta potential were used to detect the hydration diameter and potential of CS-ACP.Transmission electron microscopy（TEM）,energy dispersive spectroscopy（EDS）,and selected area electron diffraction（SAED）were used to analyze the particle morphology,elemental distribution,and mineral crystal form of the sample.In the third part of the experiment,a two-dimensional collagen mineralization model（type I rat tail collagen）and a three-dimensional collagen mineralization model（type I collagen scaffold derived from bovine Achilles tendon）were co-incubated with the CS-ACP mineralization solution prepared in Experiment 2,and then the efficiency of collagen mineralization mediated by different concentrations of CS were evaluated.Transmission electron microscopy was used to characterize the deposition position of minerals and explore the mineralization form of collagen fibers.Scanning electron microscopy（SEM）and energy dispersive X-ray spectroscopy（EDX）were used to observe the morphological changes and element distribution of collagen fibers before and after mineralization.X-ray diffraction（XRD）was used to identify the crystal form of minerals in the collagen.Thermogravimetric analysis（TGA）was used to detect the degree of mineralization of collagen fibers,and Fourier-transform infrared spectroscopy（FTIR）was used to further clarify the minerals composition.The formation process of CS-ACP was reproduced using molecular simulation.In the fourth part of the experiment,the bound CS model was constructed by cross-linking CS to the surface of ammoniated glass.The combination of CS and glass carrier was verified using CLSM.High performance liquid chromatography（HPLC）was used to detect whether the glass carrier and CS had formed stable chemical cross-linking.On the basis,SEM and TEM were used to study the effect of bound CS on collagen mineralization.In the fifth part of the experiment,CLSM was used to characterize the correlation between hyaluronidase-1（HYAL1）and mineral in calcified regions.Chondroitinase ABC（Ch ABC）was used to simulate the specific hydrolysis of CS by HYAL1 in vitro,and the amount of CS in the hydrolysate was estimated by DMMB assay at different digestion times.Scanning electron microscope was used to further test in vitro whether free CS could induce intrafibrillar mineralization of Achilles tendon.Results:PartⅠ:Correlation between Achilles tendon HO and CS depositionH&E staining of rat Achilles tendon tissue sections at 6 weeks after surgery showed that the HO model was successfully constructed.Alcian blue staining and DMMB analysis showed an increase in acid GAG content in the ectopic ossification region compared to the control group.Confocal laser scanning microscopy results showed the regions of calcified collagen were coincident with the distribution of CS.Part II:Formation and Characterization of CS-ACPThe supersaturated calcium phosphate solution（Ca P）with 2 mg/m L of free CS was in a turbid state after three days of standing.Transmission electron microscopy results showed that the particles agglomerated into needle shaped particles with a complex multimodal structure in hydrodynamic diameter.The Ca P solutions added with 3 mg/m L and 5 mg/m L of free CS remained clear after three days of standing.With the increase of CS concentration,TEM results showed that the nanoparticles were more evenly dispersed,and the diameters also increased with the growing of CS concentration.Due to the electronegativity of CS,the zeta potential of Ca P solution also slightly changed.High-angle annular dark-field scanning transmission electron microscopy（HAADF-STEM）combined with elemental patterns of nanoparticles shows that the distribution of calcium and phosphorus is consistent with that of sulfur from CS.The SAED results indicate that the nanoparticles are in an amorphous state.The above results indicate that CS in the free state can stabilize the Ca P solution and form CS-ACP nanoparticles in a concentration dependent manner.Part III:Mineralization of collagen fiber models induced by CS-ACPTransmission electron microscopy results showed that obvious intrafibrillar mineralization was observed after co-incubation with CS-ACP for 24 hours,and on day 3,intrafibrillar mineralization was more widespread.Transmission electron microscopy images of three-dimensional collagen models cultured in mineralization medium for 5 days showed that mineral crystals infiltrated into collagen sponge fibers.Elemental analysis with EDX spectroscopy showed that the locations of calcium,phosphorus,and sulfur were coincident with the locations of mineralized collagen in the SEM images.The X-ray diffraction peak at 25.86（°2θ）is characteristic of the 002 plane（Miller index）of HAp.The stretching and bending vibrations of the O-P-O peak of the phosphate group were observed by FTIR results.Thermogravimetric analysis testing results showed that the mineral content of the collagen sponge reached 40.4%after soaking in CS-ACP for 5 days.The above studies confirm that CS-ACP can induce collagen mineralization and has excellent mineralization induction efficiency.Meanwhile,the results of SEM and TEM indicated that the ability of CS-ACP to induce intrafibrillar mineralization is concentration-dependent.When the concentration of CS is low（1 mg/m L and 2 mg/m L）,intrafibrillar mineralization cannot be achieved.The molecular dynamics results reveal the formation mechanism of CS-ACP.The hydrogen bonds between CS molecules provide an energy source for initiating CS self-assembly,and then CS collects Ca²⁺from the environment through electrostatic interactions,further promoting CS assembly.After adding HPO₄^2-to the system,the amount of Ca²⁺and HPO₄^2-around CS increased with the extension of simulation time.Subsequently,Ca²⁺and HPO₄^2-gather around CS under electrostatic interaction to form CS-ACP.Part IV:Effects of bound CS on collagen mineralizationConfocal imaging verified that the binding of CS with aminated glass matrix.And no elution peak of CS in the aminated glass stock solution was detected by HPLC,which indicated that the glass carrier and CS form stable covalent bonds through chemical crosslinking,and there is no presence of free CS in the environment.After the collagen fibers were soaked in mineralization medium containing bound CS for 3 days,both SEM and TEM results showed that HAp crystals were deposited only on the surface of the collagen fibers.The clearly discerned periodicity of the collagen fibers was an additional indicator that minerals did not precipitate within the collagen.The above results indicated that bound CS cannot induce intrafibrillar mineralization.Part V:Free CS can induce mineralization of collagen fibers in the Achilles tendonImmunofluorescence results indicate that HYAL1 and CS co-deposited in the calcified area,so it is speculated that free CS may originate from the hydrolysis of HYAL1.The Ch ABC was used to hydrolyze Achilles tendon tissue in vitro,and DMMB results showed that the content of CS in the hydrolysate increased significantly with the prolongation of hydrolysis time.Subsequently,the treated Achilles tendon tissue was incubated in a mineralization medium containing free CS.SEM results showed that intrafibrillar mineralization occurred in the Achilles tendon,and the degree of mineralization deepened with the increase of hydrolysis time.The above results indicated that free CS can induce intrafibrillar mineralization of Achilles tendon collagen in vitro.Conclusion:This study proposed an innovative mechanism for CS to regulate collagen mineralization.The presence of CS（free or bound）can significantly affect the results of collagen mineralization.The free CS can stabilize the Ca P mineralization system and achieve extensive intrafibrillar mineralization.When CS is fixed to a glass plate through chemical crosslinking to simulate the bound CS in vivo,it does not have the effect of inducing intrafibrillar mineralization.The ability of free CS to promote collagen mineralization provides a new strategy for the development of novel biomimetic mineralized materials,as well as new orientation for targeted treatment of pathological calcification diseases.