| BackgroundOsteoporosis(OP)is a systemic bone disease.The bone resorption mediated by osteoclasts is significantly stronger than the bone formation mediated by osteoblasts,resulting in the thinning,reduction and fracture of the cancellous trabecula and cortical bone,leading to bone fragility and greatly increased risk of fracture.For patients with severe OP,due to bone loss at the site where the fragility fracture occurs,the original structure is easy to be severely damaged.General treatment methods are usually difficult to restore the original bone anatomy.Also,the prognosis is extremely prone to bone defects.Compared with general bone defects,OP bone defects are characterized with weak bone regeneration,instability of the new bone,and high rate of fracture recurrence.Given all these facts,patients suffering from OP are confronted with huge challenges in clinical treatment.Porous titanium alloy scaffold has become one of the most widely used metal scaffolds for repairing bone defects because of their superior biocompatibility,good corrosion resistance,and suitable elastic modulus.However,due to the lack of osteoinduction and local anti-osteoporosis effects of simple scaffold,there are still high risks of delayed healing,fracture recurrence,and internal plant collapse after repairing OP bone defects.Therefore,it remains important to further enhance the local bone formation capacity of the porous titanium alloy scaffold in order to improve the treatment of OP bone defects.Anti-osteoporosis drugs are the basis for treatment of OP bone defects.However,related drugs are currently used systemically that topical administration cannot be used for OP bone defects to improve drug distribution and utilization.In addition,the antiosteoporosis drugs are expensive and not widely affordable.Poor compliance leads to poor outcomes for many patients.Thereafter,if scaffolds can be combined with anti-osteoporotic drugs(e.g.,Zoledronic Acid,ZOL)to affect the activity of osteoclasts and inhibit bone resorption via local administration,the efficiency of OP bone defect repairment procedure would be greatly promoted.However,if ZOL is directly loaded on the surface of the scaffold,the rapid release of the drug after implantation(i.e.,“burst release effect”)would reduce the repair efficacy of OP bone defects.Thus,local slow-released drug delivery at the defect plays a critical role for the therapy.On the other hand,Gelatin Nanoparticles(GNPs)drug delivery system has plenty of advantages such as satisfactory sealing,high stability,controllable and sustainable release,etc.If GNPs loaded with ZOL can be used to construct a ZOL-GNPs drug delivery system,a local sustainable release of the drug would be expected,leading to a long-term anti-osteoporosis effect.Polydopamine(PDA),one type of surface modifier,can improve the distribution and adhesion of biomolecules.thus,a layer of PDA coated on the surface of the porous titanium alloy scaffold would increase the loading efficiency of ZOL-GNPs.The main objective of this research is to combine “PDA-coated porous titanium alloy scaffold” with ZOL-GNPs to construct a new type of biocompatible and reliable porous titanium alloy scaffold with local controllable slow-released anti-osteoporosis effects.Our research group has mastered the construction method of the scaffold system in the early stage,and has preliminarily studied its influence on osteoblasts after implantation.There is no relevant research reported on how the new type of porous titanium alloy scaffold affects osteoclasts on the repairment of OP bone defects.ObjectiveThe objective of this research is to construct a new type of PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs.In addition,this scaffold exhibits superior match with human bone mechanical characteristics,as well as a sustainable local slow-release of anti-osteoporosis drugs.Experiments are designed and conducted to investigate the mechanism of drug release,explore its biological effects on osteoclasts,and evaluate its influence on bone reconstruction in vivo,thus providing an innovative option for its clinical application.Methods1.The construction of PDA-coated porous titanium alloy scaffold loaded with ZOLGNPs and the investigation on the mechanism of drug release(1)Prepare GNPs based on appropriate specifications,conduct apparent analysis by electron microscope and nanoparticle size analyzer,and load different concentrations of ZOL to prepare ZOL-GNPs.Prepare the porous titanium alloy scaffold with Magics software and electron beam melting technology,and process with PDA coating.(2)Load ZOL-GNPs on the PDA-coated porous titanium alloy scaffold by freezedrying method,and perform tests to study its biomechanical properties.(3)Analyze the mechanism of drug release of PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs by high performance liquid chromatography.2.In vitro study on the effect of PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs on osteoclasts(1)Induction and identification of rat osteoclasts in vitro using bone marrow immature monocyte induction culture technology.(2)Inoculate osteoclasts into new porous titanium alloy scaffolds with different ZOL concentrations,and observe the morphology through electron microscope.(3)Use RT-PCR and Western-Blot technology to detect the expression of osteoclastrelated genes and proteins in different ZOL concentration groups.(4)Osteoclasts were cultured under different ZOL concentration of the new porous titanium alloy scaffold extracts,and the morphology observation,bone resorption capacity and apoptosis detection of osteoclasts were carried out.3.In vivo study on OP bone defect animal model of PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs(1)The osteoporosis model of New Zealand white rabbits was established by castration method,and then the osteoporotic femoral condyle defect model was constructed on this basis.New porous titanium alloy scaffolds with different ZOL concentrations were implanted into OP bone defect animal models.(2)Using Micro-CT scanning and histological staining methods to evaluate the effect of the new porous titanium alloy in promoting new bone formation and bone-scaffold integration under different ZOL concentrations.(3)Perform biomechanical tests on the new porous titanium alloy scaffold implanted in the OP bone defect model.Results1.The construction of PDA-coated porous titanium alloy scaffolds loaded with ZOLGNPs and the study of its drug release rate(1)Preparation of ZOL-GNPs: GNPs were successfully prepared and loaded with different concentrations of ZOL.The average particle size of GNPs was 243.6±63.4 nm.The scanning results of electron microscopy showed that they had good spheroidization,no adhesion between spheres,and uniform dispersion.(2)Construction of PDA-coated porous titanium alloy scaffolds loaded with ZOLGNPs: a porous titanium alloy scaffold with PDA coating was successfully prepared,with a pore size of 520±35 μm and a porosity of 57±4.2%,and loaded with different ZOL concentrations.The elastic modulus of the scaffold is about 1.81 GPa,close to the elastic modulus of normal human cancellous bone.(3)The drug release rate of PDA-coated porous titanium alloy scaffolds loaded with ZOL-GNPs: the percentage of drug released on the first day increased significantly(10 μmol/L: 11.1%,50 μmol/L: 13.2%,100 μmol/ L: 9.6%),which manifested as the burst release of ZOL;in the following 27 days,the drug release gradually increased slowly(10μmol/L: 35.4%,50 μmol/L: 37.9%,100 μmol/L: 34.7%).The ZOL loaded with the new scaffold maintains a stable release rate for at least several weeks in vitro after the initial burst release.2.In vitro study of the effect of PDA-coated porous titanium alloy scaffold with ZOLGNPs on rabbit osteoclasts(1)Successful induction of osteoclasts in vitro: after TRAP staining,the osteoclasts are large,multinucleated,with pseudopodia and protrusions under the microscope,indicating that mature osteoclasts have been induced,forming the foundation for subsequent experiments.(2)Electron microscopic observation of osteoclasts and the new porous titanium alloy scaffold: osteoclasts adhered to the surface of the scaffold to varying degrees;a relatively large number of cells adhered and proliferated in the Control group and 1μmol/L group,the cells spread well,and the pseudopodia stretched sufficiently.Compared to the control group,the cell morphology of the 10μmol/L group was not fully extended.With the increase of the ZOL concentration,the osteoclast morphology of the osteoclasts gradually became insignificant,and spheroid cells appeared.Apoptotic cells appeared in each phase at 500 μmol/L.(3)RT-PCR and Western-Blot detection results: The relationship between the expression level of osteoclast-related genes and the concentration of ZOL was detected by RT-PCR.The expression of Ctsk gene increased with the increase of ZOL concentration at the beginning.Among them,the expression is highest in the 10μmol/L group,and then drops rapidly when it reaches 50μmol/L,the lowest at 100μmol/L,and the expression of related genes cannot yet be detected at 500 μmol/L;the trend of TRAP gene expression is similar to that of Ctsk,but the highest level appears in the 50 μmol/L group.Western-Blot analysis further verified the concentration-dependent effect of ZOL on osteoclast differentiation.The expression levels of osteoclast markers Ctsk and TRAP showed similar trends with the expression levels of their related genes at different ZOL concentrations(0 μmol/L-100 μmol/L)(4)Osteoclasts were cultured under different ZOL concentration of the new porous titanium alloy scaffold extracts.1)TRAP staining and fusion index analysis of osteoclasts under the condition of extract:In control group(0μmol/L),the osteoclast cell body is larger,and obvious cell fusion can be observed,forming multinucleated,mature broken cells with pseudopods.With the increase of ZOL concentration,cell fusion is gradually not obvious,and there are fewer multinucleated and mature osteoclasts;there are basically no TRAP-positive cells in the 100μmol/L group and 500μmol/L group.The analysis of the fusion index results indicated that both the 1 μmol/L group and the 10 μmol/L group(R: 32.34±3.09;33.67±1.25)could enhance the differentiation of osteoclasts,but compared with the control group(0μmol/L)(R: 31.18±2.08)there was no significant statistical difference.In the 50 μmol/L group(R:8.54±0.39),the number of TRAP-positive multinucleated osteoclasts was significantly lower than that in the control group(0 μmol/L)(P <0.01),while in the 100 μmol/L group and 500 μmol/L group,almost all osteoclasts were apoptotic.2)Detection of the bone resorption capacity of osteoclasts under the condition of the extract: after culturing the mouse BMMs under the condition of the new porous titanium alloy scaffold with different ZOL concentrations for 10 days,the bone resorption capacity was quantified by the bone plate resorption area assessment.The bone plate resorption area formed by the 1 μmol/L-ZOL group(28.22±4.17)and the 10 μmol/L-ZOL group(33.41±3.23)was significantly higher than that of the control group(0 μmol/L)(22.52±2.78),while at 50 μmol/L the bone plate resorption area was significantly lower than that of the control group(0μmol/L).With the increase of the concentration,the bone plate resorption area continued to decrease(50 μmol/L-ZOL group: 8.41±2.80;100 μmol/L-ZOL group: 1.32±0.45;500 μmol/L-ZOL group: 0.11±0.03).3)Detection of osteoclast apoptosis under the condition of extract: after 7 days of culture of mouse BMMs in the extract of new porous titanium alloy scaffold with different ZOL concentration,the osteoclast apoptosis was tested by flow cytometry.The results indicated that the apoptosis rate of osteoclasts increased linearly with the concentration of ZOL.When the concentration of ZOL was increased from 1 μmol/L to 500 μmol/L,the apoptosis rates were(15.77%±0.99%,18.57%±4.08%,54.77%±0.62%,82.57%±3.01%and 91.23%±5.80%).All the experimental groups showed significantly higher apoptosis rate than the control group(0 μmol/L)(5.67%±0.53%,P <0.01).3.The in vivo study of the PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs on the animal model of osteoporotic bone defect(1)Construction of OP bone defect model and implantation of the scaffold: After the New Zealand white rabbit osteoporosis model was established and verified successfully,the OP bone defect model was constructed.In this study,the OP femoral condyle defect model was constructed by surgery(bilateral hind limbs),and then implanted with PDA-coated porous titanium alloy scaffolds loaded with ZOL-GNPs with different ZOL concentrations(0 μ mol/L,10 μ mol/L,50 μ mol/L,100 μ mol/L,500 μ mol/L),postoperative X-ray photograph showed that the scaffolds were all in good position.(2)Micro-CT scan reconstruction and data analysis: no matter in 8 weeks or 12 weeks,the comparative analysis of the bone volume fraction(BV/TV),bone trabecular thickness(Tb.Th),and bone trabecular number(Tb.N)indicates that there is a concentrationdependent positive correlation in low concentration ZOL;these values of these parameters climb up with the increase of ZOL concentration;when the ZOL concentration is 50μmol/L,those parameters reach their maximum value.At 100umo/L and 500 μmol/L groups,the value of those parameters are all decreased.There is even no statistical difference between the 500 μmol/L group and the 1 μmol/L group(P>0.05).(3)Histological examination(modified Von-Gieson staining)results: 8 weeks and 12 weeks after the scaffold implantation,the Von-Gieson staining was used to compare the bone ingrowth around the implanted scaffold;there were new bone formation at the bonetitanium interface.There was less bone ingrowth inside the scaffold at 8W,but obvious bone ingrowth was visible inside the scaffold at 12 W.With the increase of ZOL concentration,the amount of new bone gradually increased.The new bone ingrowth was maximum at ZOL concentration of 50 μmol/L.Then the amount of new bone gradually decreased with the increase of the ZOL concentration.At the ZOL concentration of 500 μmol/L,there was only a small amount of bone ingrowth.(4)Biomechanical testing and analysis of implanted porous titanium alloy scaffolds:the implanted scaffolds with different ZOL concentrations were subjected to vertical mechanical compression experiments at 8 weeks and 12 weeks after scaffold implantation.The results showed that there was no statistical difference between the elastic modulus of the scaffold before and after the implantation in the 0 μmol/L,100 μmol/L,and 500 μmol/L groups.However,the scaffold elastic modulus was significantly higher after the implantation(P<0.05)for 1 μmol/L,10 μmol/L and 50 μmol/L of ZOL.The scaffold elastic modulus of the 50 μmol/L group is the highest(8W: 2.78±0.09 Gpa;12W: 2.91±0.10 Gpa),close to the elastic modulus of human cancellous bone,exhibiting a satisfactory compression resistance.Conclusions1.This project successfully constructed a PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs.Its elastic modulus is close to that of human cancellous bone.Also,it can reduce the effect of stress shielding and maintain a stable ZOL release rate for several weeks.2.The new porous titanium alloy scaffold has anti-osteoporosis effect by local slowrelease of drugs,effectively inhibiting the bone resorption of osteoclasts,and promotes good osseointegration between new bone tissue and the scaffold.However,this property is related to the concentration of ZOL.3.When the ZOL concentration is too high or too low,it will affect the local antiosteoporosis effect of the scaffold.At a ZOL concentration of 50 μmol/L,it can effectively inhibit the fusion formation and bone resorption of osteoclasts,thereby promoting generation of new bone.In summary,this PDA-coated porous titanium alloy scaffold loaded with ZOL-GNPs not only has good mechanical properties and biocompatibility,but also has an antiosteoporosis effect via local slow release of drug.These properties indicate a promising alternative to traditional implants,providing a new method for repair and reconstruction treatment of OP bone defect. |
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