| Mineralized collagen fibrils are the basic structural units of natural bone,which are composed of collagen and hydroxyapatite.They are formed under the regulation of non-collagenous proteins(NCPs).Exploring the interaction among collagen,hydroxyapatite and NCPs through the construction of biomimetic collagen mineralization system in vitro is of great research value for understanding the real mineralization mechanism in living organisms and developing highly biomimetic bone repair materials in composition and structure.Based on the feature that NCPs are rich in glutamic and aspartic acid,many studies usually use negatively charged amino acids/peptides as NCPs analogs,which are mixed with hydroxyapatite precursor solution to soak collagen solid matrix(such as sponge and gel).However,it is difficult to regulate the self-assembly behavior of collagen by NCPs analogs in this way,which is not conducive to the study of the interaction mechanism between above three.In view of the necessity of synchronous occurrence of collagen selfassembly and hydroxyapatite formation,this paper firstly investigates the relationship between the aggregation state of collagen at various stages of self-assembly and the structural properties of mineralized collagen composites.Then,introducing amino acids/peptides to regulate collagen self-assembly behavior and hydroxyapatite formation process.The mineralized collagen composites with controllable structure and properties are prepared,and the interaction between NCPs analogs and collagen and hydroxyapatite/precursor is analyzed at the molecular level,providing theoretical and practical basis for understanding of biomineralization mechanism and biomimetic construction of collagen mineralized composites.Under physiological conditions,the self-assembly process of collagen includes nucleation,growth and plateau phase.And,the aggregation state of collagen molecules is different at different stages of collagen self-assembly.Therefore,in this paper,the interaction between collagen and hydroxyapatite at different stages of selfassembly was investigated by starting mineralization at the beginning of the lag period,at the beginning of the growth period,at t1/2(the time to reach half of the total turbidity change),and during the plateau period,respectively.When calcium ions were mixed with PBS,the transparent collagen solution immediately turned milky white,and the initial absorbance of the kinetic curve suddenly increased.This was due to the formation of amorphous calcium phosphate.After a period of incubation at 37 °C,a very sharp wave trough appeared in the mineralization kinetic curves due to volume shrinkage,dehydration,and the amorphous/crystalline conversion.Hydroxyapatite formation was confirmed by Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD)and energy-dispersive spectroscopy.Moreover,the Ca/P ratio was around 1.67.Through scanning electron microscopy(SEM),we observed that the hydroxyapatite had a regular shape and was a rectangular flake.After calcination at800 ℃,the hydroxyapatite dimensions were about nanometer scale.Specifically,separate crystals or only small clusters formed on or in the collagen fibrils when we added aqueous Ca Cl2 at the beginning of the lag and growth period.However,hydroxyapatite formed large clusters attached to the collagen fibrils at t1/2 and during the plateau period.This showed that the aggregation state of collagen molecules could affect the distribution uniformity of hydroxyapatite on collagen fibrils.Compared with the gel appearance of pure collagen samples,the mineralized collagen fibrils formed at the beginning of the lag period were white precipitates,indicating that mineralization could inhibit the self-assembly behavior of collagen;paradoxically,there was more collagen in the latter composites.It is speculated that hydroxyapatite chelated the carbonyl groups of soluble collagen,forming chelate bonds and resulting in coprecipitation along with collagen self-assembly.In addition,the later the initiation of collagen mineralization,the more collagen amount in the mineralized composites,indicating that the inhibition effect of mineralization on collagen selfassembly was weakened.Glutamic acid or arginine(NCPs analogs)were introduced into the synchronous mineralization system.The aim is to elucidate the relationship between glutamic acid/arginine and collagen self-assembly behavior and hydroxyapatite formation by analyzing their effects on collagen mineralization process,as well as the structures and properties of mineralized collagen composites.Kinetic curves showed that,with the increase of glutamic acid concentration,the time for wave trough to appear was prolonged obviously and the intensity of wave trough gradually decreased.It suggested that glutamic acid could stable amorphous calcium phosphate and inhibit the formation of hydroxyapatite,causing a decrease in crystal size.Under neutral conditions,glutamic acid was prone to be negatively charged since it contained carboxyl groups.Once calcium ions were introduced into solution,they were adsorbed to glutamic acid molecules rapidly due to electrostatic interaction,which made the combination of calcium ions and phosphate groups harder and led to the delay of the amorphous-to-crystalline transformation.XRD patterns confirmed that the wave trough was caused by the transformation of amorphous calcium phosphate to crystalline hydroxyapatite.SEM images showed that hydroxyapatite was spherical and deposited on the surface of collagen fibrils.After the introduction of glutamic acid,the average size of hydroxyapatite aggregates decreased with a strong tendency to dispersion,resulting in a decrease in the surface roughness of composites.Moreover,the amounts of hydroxyapatite decreased dramatically,further suggesting that glutamic acid could inhibit the precipitation of hydroxyapatite,as previously observed in the turbidity measurement.Glutamic acid could not only affect the crystalline size and distribution of hydroxyapatite but also influence the self-assembly of collagen molecules.When the concentration of glutamic acid was lower than 200 mmol/L,the reduction of hydroxyapatite formation rate was beneficial to collagen self-assembly,which led to the formation of thick fibrils.Therefore,the gel hardness and thermal stability of mineralized collagen fibrils were significantly improved.When the concentration of glutamic acid were 400 and 550 mmol/L,the self-assembly behavior of collagen molecules was inhibited due to the strong electrostatic interaction,resulting in the reduction of the gel strength and thermal stability.Similar to glutamic acid,arginine(0 ~ 40 mmol/L)could also inhibit the formation of hydroxyapatite.It had a negative impact on the crystal size and the amount of hydroxyapatite.The reason might be that the positively charged guanidine group of arginine could be combined with phosphate through hydrogen bonding and electrostatic interaction.At the same time,hydroxyapatite crystals were uniformly dispersed on collagen fibrils,because there was electrostatic repulsion between hydroxyapatite surfaces owing to the adsorption of arginine onto hydroxyapatite.However,the obvious difference between the effects of arginine and glutamic acid were observed for collagen self-assembly.The results showed that when the concentration of arginine was lower than 40 mmol/L,the self-assembly behavior of collagen molecules was continuously retarded with the increase of arginine concentration,resulting in the destruction of the threedimensional network structure of collagen fibrils and a slight decrease in fibril diameter.Therefore,the thermal stability and gel hardness of composites decreased.Apparently,glutamic acid is more suitable to be used as NCPs analogue than arginine.In order to provide a theoretical basis for collagen mineralization,the effect of poly(glutamic acid)chain length on collagen aggregation and self-assembly behavior was studied.After the introduction of glutamic acid and poly(glutamic acid)(100 &1000 k D)into the collagen solution,the particle size distribution,pyrene fluorescence spectrum and AFM image of collagen solutions were firstly analyzed.The results showed that glutamic acid and poly(glutamic acid)(100 k D)could promote the disaggregation of collagen aggregates,leading to the increase of collagen selfassembly rate and degree.However,poly(glutamic acid)(1000 k D)significantly reduced the rate and degree of collagen self-assembly.As the strong electronegativity caused by plentiful acidic carboxyl groups on the long chain,poly(glutamic acid)(100KD)could easily form stable electrostatic interaction and hydrogen bond with the amino group of collagen.These interactions would replace hydrogen bonds involved in the formation of collagen aggregates,promoting the cleavage of collagen aggregates and self-assembly of collagen.However,poly(glutamic acid)(1000 k D)could be tightly entangled with some collagen aggregates,and at the same time neutralize the charge on the collagen in large quantities.Therefore,part of collagen precipitated owing to the salting-out effect,which resulted in that fewer collagen molecules were involved in self-assembly.Furthermore,the steric hindrance was too large to form collagen fibrils quickly.SEM images showed that after the introduction of glutamic acid and poly(glutamic acid)(100 k D),the self-assembly behavior of collagen was improved,so the fibril diameter increased and the porous structure of sponge was compact.However,after the introduction of poly(glutamic acid)(1000k D),the diameter of collagen fibrils continued to increase,but the entanglement between fibrils was loose and the porous structure was destroyed,which was related to the inhibition of collagen self-assembly behavior.In addition,the increase in the molecular weight of poly(glutamic acid)facilitated the lateral aggregation of collagen fibrils,which fused with each other to form large twisted bundles.The results of liquid replacement method showed that glutamic acid and poly(glutamic acid)(100 k D)had little effect on the porosity of collagen sponge,and the porosity was above 70%.However,increasing the molecular weight of poly(glutamic acid)would lead to a slight decrease in the porosity of the collagen sponge,which might be related to the destruction of the porous structure.The poly(glutamic acid)molecule had strong water absorption ability because of hydrophilic group,so the swelling rate of collagen sponge increased with the increase of additive molecular weight.After self-assembly,the gel hardness,thermal stability and enzymatic hydrolysis resistance of collagen fibrils were closely related to the change of collagen self-assembly,which increased first and then decreased.When the molecular weight of poly(glutamic acid)was 100 k D,the maximum value was reached.The confocal laser scanning microscopy images showed that the collagen fibril mixed with glutamic acid and poly(glutamic acid)(100k D)still maintained good biocompatibility.It has been reported that the regulation of NCPs analogs on the formation of hydroxyapatite is closely related to its molecular weight.Therefore,it is of great significance to explore the influence of the molecular weight of poly(glutamic acid)on collagen self-assembly and hydroxyapatite formation in the synchronous mineralization system.Compared with the samples without additives,poly(glutamic acid)had little effect on the nucleation and growth process of hydroxyapatite,and the kinetic curve remained unchanged.Poly(glutamic acid)(100 KD)and poly(glutamic acid)(1000 KD)have a significant effect on inhibiting the transformation of amorphous calcium phosphate to crystalline hydroxyapatite.It could be speculated that:(1)the chains of poly(glutamic acid)molecules have a large number of carboxyl groups which could chelate calcium ions,at the same time,the long chains of poly(glutamic acid)curled and folded in solution,and the calcium ions were wrapped in curly internal structure,which caused that the collision probability between calcium ions and phosphate groups reduced;(2)The steric hindrance caused by poly(glutamic acid)macromolecules also hindered the binding of calcium ions to phosphate groups and the growth of crystal nuclei.It was worth noting that no significant difference in the inhibition effect of poly(glutamic acid)(100 k D)and poly(glutamic acid)(1000k D)on hydroxyapatite formation,because the whole system became unstable after the addition of the poly(glutamic acid)(1000 k D),which led to the precipitation of partial collagen and poly(glutamic acid),thus the inhibition effect on hydroxyapatite formation weakened.Combined with SEM and ICP-AES,it was found that the higher the molecular weight of the additive,the better the dispersion and the less the amount of the spherical hydroxyapatite on the surface of collagen fibrils.It could also be seen by SEM that with the increase of the additive molecular weight,the mineralized collagen fibrils gradually became thicker,but thinner than the corresponding unmineralized group in the previous section.The reason might be that calcium ions and phosphate groups shielded the surface charges of glutamic acid and poly(glutamic acid)(100 & 1000 k D).After the introduction of glutamic acid and poly(glutamic acid)(100 k D),the inter-fibril density increased,resulting in a decrease in the porosity of the mineralized collagen composite,and an increase in gel hardness,thermal stability and resistance to enzymatic hydrolysis.When the molecular weight of poly(glutamic acid)continued to increase to 1000 k D,the gel hardness,thermal stability and resistance to enzymatic hydrolysis decreased due to the loose of the inter-fibril entanglement and the destruction of the porous structure.CCK-8 and invert fluorescence microscopy experiments showed that the surface of mineralized collagen composites was still suitable for cell growth and showed good biocompatibility after the introduction of glutamic acid and poly(glutamic acid)(100 k D).Cell adhesion experiments showed that all mineralized composites had high cell adhesion ability and had potential application value in the field of bone tissue engineering.In order to further improve the physicochemical and cell adhesion properties of mineralized collagen composites,the composite was modified with the introduction of dopamine.First,poly(glutamic acid)-dopamine(PGA-DA)macromolecule was synthesized by the way of EDS/NHS activation of carboxyl group,and then was used to prepare mineralized collagen composites.The UV,FTIR and NMR results showed that dopamine was successfully grafted onto poly(glutamic acid)(100 k D)molecule with the degree of substitution(grafting rate)of 6.8% and 24.2%,respectively.XRD and EDS mapping experiments showed that the introduction of dopamine did not change the crystal phase structure of hydroxyapatite.Unmodified poly(glutamic acid)could stabilize amorphous calcium phosphate and inhibit the formation of hydroxyapatite,resulting in the reduction of hydroxyapatite amount.However,the formation rate of hydroxyapatite was accelerated and the amount of hydroxyapatite was significantly raised after the grafting of dopamine on the poly(glutamic acid),because the amination reaction consumed part of the carboxyl group,which weakened the ability of the poly(glutamic acid)to capture calcium ions and favored the rapid synthesis of calcium ions to phosphate groups.The SEM results showed that after the introduction of unmodified poly(glutamic acid),the lamellar aggregation clusters of hydroxyapatite decreased,and the distribution of hydroxyapatite on the surface of collagen fibrils was more uniform.However,along the increase of dopamine substitution degree,the hydroxyapatite tended to aggregate again.In addition,after the introduction of dopamine on the poly(glutamic acid)molecule,the collagen fibril winding in the mineralized composites became densier,leading to the decrease of porosity,which was mainly due to the partial oxidation of the catechol structure in PGA-DA into quinone structure,thus cross-linking with the collagen fibrils.Because the oxidative cross-linking was generated by the catechol group,the thermal stability,gel hardness and storage modulus/loss modulus of the mineralized collagen composite improved with the increase of the degree of dopamine substitution,accompanied by a significant decrease in the enzyme degradation.CCK-8 and inverted fluorescence microscopy experiments showed that the mineralized collagen composites displayed good biocompatibility,and high survival rate of BMSCs cells on the surface of composite.Cell adhesion experiments showed that the relative cell adhesion rate increased significantly after the introduction of dopamine,indicating that dopamine could promote the expansion,adhesion and growth of BMSCs cells,which had a broad prospect in the application of bone tissue engineering. |
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