The Role Of Genetically Engineered Peptide EHABP In Inducing Intrafibrillar Mineralization

ObjectivesNatural bone is an organic-inorganic nanocomposite material that is characterized by its prominent mechanical properties such as high strength and toughness.These impressive mechanical properties are attributed to the organic matrix and the inorganic mineral components and their unique hierarchical geometrical arrangements.The organic collagen scaffold is used as the "template" for the biomineralization,the mineralized collagen fibrils with embedded HA crystals are preferentially oriented with their c-axes parallel to the longitudinal axis of the fibrils.This field has attracted a number of researchers to conduct the cutting edge researches with a view of biomimetic to imitating the nanostructures of hard tissues by designing ideal biomineralization models in vitro.The ideal bone substitutes should resemble with the natural bone's structure and properties.In recent years,many researchers are attempted to synthetise bone substitutes through the biomimetic mineralization model in vitro with different methods.In the past,the construction of nanostructures via the immersion of collagen scaffolds in simulated body fluid(SBF)has been previously attempted.However,this approach does not seem to truly mimic bone structure,since the minerals were found to deposit on the external regions of the collagen fibrils rather than on the interior.Nanomaterials fabricated using such approaches can be classified as "top-down" materials.Crystals precipitated from an aqueous solution form a critical cluster size which could initiate nucleation and grow by ionic deposition on the surface of the fibrils.Studies show that infiltrated minerals are dominant contributors to the elasticity and rigidity of the hard tissues.The lack of analogue non-collagenous proteins(NCPs)may be ultimately responsible for the limited biomimetic success.Among the major biological building blocks in biology,proteins are vital in the control of precise microstructure formation.Proteins participate actively in important physiological functions such as in the transportation of raw materials,enzymatic reactions for inorganic synthesis,controlled nucleation,growth,and the morphogenesis of inorganic crystals.In the mineralization process,non-collagenous proteins(NCPs),which only account for approximately 3%of the organic component,but play a significant role in manipulating the growth of crystals.For instance,osteopontin(OPN)?dentin phosphophoryn(DPP)and dentin matrix protein-1(DMP-1).However,it is very difficult to extract pure,native NCPs to use in biomimetic approaches.Hence various polypeptides and polymers have been explored to play the role of NCPs in biomimetic biomineralization,such as polyaspartate(pAsp),and poly acrylic acid(PAA).Over the last few years,genetically engineered peptides have been widely applied to regulate the growth of inorganics.A genetically engineered peptide for inorganics(GEPI)that specifically and selectively binds to hydroxyapatite surface as defined by a sequence of amino acids.In our previous research,we selected the enamel hydroxyapatite binding peptide(EHABP)through a phage display biopanning process,which has a high degree of affinity to natural tooth enamel surfaces that contains abundant amounts of HA particles.In this study,the role of EHABP has been explored in biomineralization.The peptide enables the liquid precursor phase minerals to be drawn into the interstices of the collagen fibrils.The intrafibrillar mineralization had been confirmed by various measurements.At meanwhile,compared with PAA-mediated mineralized collagen,the EHABP could act as a stabilizer to induce intrafibrillar mineralization effectively.The possible mechanism for the temporary inhibition of hydroxyapatite nucleation was explored.Materials and methodsPART1We obtained HA-binding peptides through purification to 95%and of molecular weight 954.06 g/mol(Shenggong,Shanghai,P.R.of China).The EHABP was dissolved in deionized water to prepare a stock solution(10 mmol/L)and stored at 4 ?.In this experiment,the mineralization solutions were prepared by mixing equal volumes of CaCl2 2H2O(4.5 mM)(Sigma,St.Louis,MO)and K2HPO4(2.1 mM)(Sigma,St.Louis,MO)according to the method described by Gower et al.The final concentration of EHABP(Mw:954.06 g/mol)was used as directing agent at different concentrations of 0.1 mM?0.2mM?0.4mM ? 0.8mM.While poly acrylic acid(PAA)(Mw:1.8kDa,Sigma)was used as the directing agent at a concentration of 500 ?g/mL.The diluted peptide and the poly acrylic acid were stirred with equal volumes of the calcium and the phosphate solutions.The pH of the mineralized solution was maintained at 7.4 using Tris-buffered saline(TBS).We measured the OD values of EHABP-mediated,PAA-mediated and TBS solutions without polymer at different time points.Through checking the turbidity degree of the three solutions,we can recognize whether the solutions are in the steady state.Different concentrations of EHABP-mediated solutions were added to separate wells of a 96 well-plate and each well was scanned by the spectrophotometer.The wavelength used for the measurements was 590 nm and the temperature was 25 ?.The samples were tested immediately after mixing the components resulting in t=0 min as the earliest time point in the time-dependent analysis.Furthermore,we had a visual observation on three solutions if they were changed from clear to turbid.Also we did TEM observations to obtain the morphology of the nanodroplets or nanoparticles in three solutions.PART 2 Supply Co.,Inc.,MA,USA.The collagen matrix was mineralized by EHABP-mediated solutions,incubated at 37 ?.The mineralized collagen samples were recovered from the mineralization solution after different periods of time for the all measurements,including TEM(transimission electron microscopy)?SEM(scanning electron microscopy)?STEM(scanning transimission electron microscopy)?XRD(X-Ray diffraction)?TGA(thermo gravimetric and differential thermal analysis)?XPS(X-ray photoelectron spectroscopy.These methods were used to observe if the mineral was in the inside of collagen fibrils,the content of minerals and the components of the mineral.Moreover,in order to detect the combination of EHABP and the collagen scaffold,we labeled FITC-sequence onto one side of the EHABP and visualized using the LCFM(Laser confocal fluorescence microscopy).PART 3The collagen matrix was mineralized by PAA-mediated solutions,incubated at 37 ?.After different periods of time,the mineralized collagen samples were recovered from the mineralization solution for the measurements,including TEM(transimission electron microscopy)?SEM(scanning electron microscopy)?TGA(thermo gravimetric and differential thermal analysis).Then compared with EHABP-mediated mineralized collagen from those tests.Results1.We demonstrated that the EHABP could stabilize the amorphous calcium phosphate(ACP)for at least 14days through OD measurments.The peptide enables the liquid precursor phase minerals to be drawn into the interstices of the collagen fibrils through PILP process.This time-frame is adequate to facilitate calcium phosphate liquid-precursor intrafibrillar mineralization.2.The stabilization of amorphous calcium phosphate is dependent on the formation of EHABP-ACP nanodroplets.These droplets will be attracted onto the the vicinity of the collagen fibrils and infiltrated to the interior of collagen fibrils through PILP process.After phase transition,TEM results show that the mineralized collagen fibrils with embedded HA crystals are preferentially oriented with their c-axes parallel to the longitudinal axis of the fibrils.3.In addition,since the PAA has been widely applied as a polymer in several biomaterials,we compared the use of EHABP and PAA on the control of the mineralization process.TEM images present abundant nanodroplets using EHABP,the diameter of these nanodroplets were smaller that may infiltrate into the inside of the collagen fibers more easily and faster than bigger nanoparticles.While the TGA results suggest that in the same period of time,the EHABP-mediated mineralized collagen contained 55.12%minerals.This demonstrated that the EHABP could act as a stabilizer to induce intrafibril lar mineralization effectively.ConclutionThrough a PILP process a degree of mineral penetration was achieved which resulted in hierarchical nanostructures,the EHABP was employed as a stabilizer at a low concentration to induce the amorphous calcium phosphate to infiltrate into the interior of the collagen fibers.A highly hierarchical assembly of intrafibrillar hydroxyapatite crystallites in type I collagen scaffolds,mineralized in vitro using EHABP was confirmed by SEM,TEM,STEM,TGA,and OD measurements.Thus,we believe that the EHABP could act as biomimetic analogues of non-collagenous proteins to induce intrafibrillar mineralization using stabled amorphous calcium phosphate.The EHABP-mediated mineralized collagen was successfully rebuilt in hierarchical nanostructures that resembled the properties found in natural bone.This ideal model has brought new concepts in the area of biomimetic mineralization,and also has the great potential in the area of bone defects regeneration.