The Development Of Bioactive Bone Cement And Its Osteogenic Mechanism

BackgroundBone cement is widely used in total knee/hip arthroplasty,percutaneous vertebroplasty,and bone defects caused by bone tumors,osteomyelitis,and trauma.In 1946,Robert Judet first applied polymethylmethacrylate(PMMA)bone cement to orthopedics.At present,the mainstream bone cement is also PMMA bone cement.However,PMMA bone cement has the disadvantages of biological inertia,nonbiodegradability,potential cytotoxicity,high polymerization temperature and excessive mechanical strength.Therefore,the interface of bone cement and bone cannot form bone integration,and the fixation of the interface of bone cement and bone completely depends on the mechanical interlocking effect between bone cement and bone,Aseptic loosening of the interface of bone cement and bone inevitably occurs sooner or later.According to statistics,75%of joint prosthesis failure is due to aseptic loosening of the interface of bone cement and bone.There have been many studies aimed at improving the above shortcomings of PMMA bone cement,such as adding hydroxyapatite,strontium doped hydroxyapatite,chitosan,and hyaluronic acid into bone cement.However,it was found that the mechanical strength of modified PMMA bone cement weakened,and the biological activity was not improved satisfactorily.Therefore,it is urgent to develop a bioactive material modifying PMMA bone cement to increase the osteogenic capacity of bone cement within the range of mechanical strength requirements of bone cement and without reducing the biocompatibility of bone cement.MethodsDesign of bioactive bone cement system:Firstly,layered magnesium aluminum hydroxide was synthesized,and micron magnesium aluminum hydroxide was generated by self-growth technology.Purchase Commercial PMMA bone cement was purchased and then layered magnesium aluminum hydroxide and PMMA bone cement powder were fully mixed.Subsequently,PMMA liquid initiator was added into the power mixture,and the system solidified to form bioactive bone cement.1.Synthesis of MgAl-LDH(MgAl-layered double hydroxide)microsheetsMg(NO3)2·6H2O,Al(NO3)3·9H2O,and urea were weighed and dissolve in 70 mL of deionized water.After dissolution,transfer the mixed solution to 100 mL Teflon lining,and then put it into an autoclave and heat it at 100℃ for 24 hours.The collected colloidal precipitates were centrifuged and washed three times with deionized water at 4000 rpm.The final white colloid was redispersed in deionized water,which was magnesium aluminum hydrotalcite(MgAl-LDH).The composition of MgAl-LDH was analyzed by X-ray diffraction(XRD).Subsequently,the surface morphology and grain size of MgAlLDH was detected by scanning electron microscope(SEM).2.Preparation of test samples of bone cement and modified bone cementPMMA bone cement and modified PMMA bone cement are divided into the following four types(Table 0-1).After fully mixing the powder of each group,add liquid initiator,fully stir for 1 minute and then leave to set.After the bone cement enters the dough stage,the mold was used to prepare disc-shaped test samples with diameter of 6mm and thickness of 2.5mm for cell and animal experiments;cylindrical mechanical test samples with diameter of 6mm and height of 12mm were prepared with another mold;sheet mechanical test samples with length of 75mm and width of 10mm and thickness of 3.3mm were prepared with other molds.Table 0-1 Composition of bone cement and modified bone cement(wt%)#123.The physical and chemical properties of bone cement and modified bone cementThe temperature changes of pure bone cement and modified bone cement during polymerization were measured by infrared imager.The surface morphology of pure bone cement and modified bone cement was detected by scanning electron microscope(SEM).Energy dispersive spectrometer(EDS)was used to detect the types and distribution of elements in the pure bone cement and modified bone cement.The surface roughness of pure bone cement and modified bone cement was measured by atomic force microscope(AFM).The magnesium/calcium ion release curves of PMMA&COL-I,PMMA&LDH,and PMMA&COL-I&LDH in vitro were detected by ion release test.The bending strength,compressive strength,bending modulus,and compressive modulus of pure bone cement and modified bone cement were tested by universal material testing machine.4.Osteogenesis and biocompatibility of bone cement and modified bone cement in vitroHuman bone marrow mesenchymal stem cells(HBMSCs)were cultured with the extract of bone cement and modified bone cement.At the same time,HBMSCs were directly inoculated on the surface of bone cement and modified bone cement.CCK-8(Cell Counting Kit-8)and flow cytometry assays were employed to test the cytocompatibility of bone cement and modified bone cement.The osteogenesis of HBMSCs was semi-quantified by alizarin red S staining and alkaline phosphatase staining.The activated pathways of PMMA&LDH promoting osteogenic differentiation were uncovered by transcriptome sequencing and subsequent ingenuity pathway analysis(IPA),and the above signal pathways were verified by qPCR(Quantitative real-time polymerase chain reaction)and WB(Western blot)assays.5.Osteogenesis and biocompatibility of bone cement and modified bone cement in vivoThe New Zealand white rabbit bone defect model of skull was established to test the osteogenic performance of modified PMMA bone cement in vivo.Alizarin red S and calcein solutions were injected intraperitoneally immediately and 1 month postoperatively,respectively.The intact skull,heart,liver,spleen,kidney and spinal cord were taken at 1 month and 2 months postoperatively.Micro CT quantitatively analyzed the volume of regenerated inorganic mineral in the interface of bone cement and bone.After hard tissue sections,the regenerated bone tissue was observed by laser confocal microscope and methylene blue acid fuchsin staining.The changes of organ microstructure were observed by hematoxylin eosin staining.Results1.Synthesis of MgAl-LDH(MgAl-layered double hydroxide)microsheetsMgAl-LDH microsheets were synthesized by a mild one-step chemical synthesis method(Invention patent No.ZL 2019 1 0162303.6,Authorized patent).2.Preparation of test samples of bone cement and modified bone cementMgAl LDH powder and or CoL-I powder with a certain mass ratio were introduced into PMMA bone cement powder.According to the PMMA bone cement manual,the bone cement powder and liquid initiator were mixed,and the samples to be tested of bone cement and modified bone cement with corresponding sizes are prepared by prefabricated molds.3.The physical and chemical properties of bone cement and modified bone cementThe surface roughness of PMMA,PMMA&COL-I,PMMA&LDH,and PMMA&COL-I&LDH gradually increased,and the maximum polymerization temperatures were 95.2℃,100℃,88.2℃,and 99.7℃ respectively.The working time of modified bone cement was not significantly shortened compared with pure PMMA bone cement.Except that the bending strength of PMMA&COL-I&LDH does not meet the national standard(ISO 5833-2002),the other mechanical strength of modified bone cements meets the requirements of ISO 5833-2002.The magnesium ion sustained-release concentrations of PMMA&LDH and PMMA&COL-I&LDH were maintained at about 1.6μmol/mL and 1.3 μmol/mL respectively.Additionally,the calcium ion sustained-release concentrations of PMMA&COL-I and PMMA&COL-I&LDH were maintained at about 0.30μmol/mL and 0.20μmol/mL.4.Osteogenesis and biocompatibility of bone cement and modified bone cement in vitroThe biocompatibility of modified bone cement is better than that of pure PMMA bone cement.PMMA&LDH and PMMA&COL-I&LDH bone cement can activate p38 MAPK,ERK/MAPK,FGF18,and TGF-β signal pathways in vitro which significantly accelerate the osteogenesis of HBMSCs.5.Osteogenesis and biocompatibility of bone cement and modified bone cement in vivoMicro CT showed that the amount of regenerated bone in the interface of bone and bone cement 2 months postoperatively in PMMA&LDH,PMMA&COL-I&LDH,and PMMA&COL-I groups was 9.26 times,9.12 times,and 5.02 times higher than that in PMMA bone cement group,respectively.The results of laser confocal microscope and acid fuchsin staining were consistent with the results of Micro CT.Additionally,no significant changes of the microstructure of heart,liver,kidney,spleen,and spinal cord were observed.In conclusionCompared with PMMA bone cement,the mechanical properties of PMMA&LDH bone cement declined mildly and the maximum polymerization temperature decreased by 7℃,both of which contribute to relieve stress shielding osteolysis,reduce peripheral cell necrosis and subsequent tissue fibrosis caused by polymerization high temperature,and indirectly promote bone integration.PMMA&LDH bone cement has superior osteogenic ability and biocompatibility in vivo and in vitro.Therefore,LDH modified PMMA is one of promising candidates for promoting bone regeneration in related orthopedic surgery.