Construction And Evaluation Of Injectable BP/PPF Bone Cement

Objectives:Bone cement is a minimally invasive material used to repair bone defects,offering advantages such as reduced damage,ease of operation,fewer complications,and less pain.However,the commonly used polymethyl methacrylate(PMMA)bone cement has limitations and defects.Polypropylene fumarate(PPF)is an absorbable and degradable polyester that can be thermally crosslinked with NVP to create an injectable and absorbable bone cement material.Black phosphorus(BP)nanosheets are two-dimensional nanomaterials that enhance osteogenic performance and provide a photothermal effect to PPF bone cement.This study aims to prepare a new type of injectable and degradable BP/PPF bone cement by combining PPF with BP nanosheets.The preparation method was investigated and optimized,followed by exploration of the physical and chemical properties of the material.Additionally,degradation performance,photothermal performance,biocompatibility,osteogenic performance,and in vivo toxicity experiments were conducted to provide a theoretical basis for clinical application of this composite material.Methods: Part Ⅰ:PPF with varying molecular weights were prepared using a two-step method,and characterized by Fourier transform infrared spectroscopy,nuclear magnetic resonance hydrogen spectrum,and gel permeation chromatography.BP nanosheets were obtained through liquid phase peeling method,and their size and particle size were analyzed using TEM and DLS.The structure of BP nanosheets was characterized by XPS,XRD,Raman spectrum,and infrared spectrum.Subsequently,BP/PPF bone cement was prepared and observed under scanning electron microscope.Part Ⅱ :The prescription of BP/PPF was optimized,and the molecular weight of PPF and the content of BP nanosheets were analyzed through single factor analysis(n=5).Subsequently,based on the results obtained from single factor analysis,a three-factor three-level orthogonal design experiment(L9(33),n=5)was conducted.The prescription of BPO,DMPT,and NVP was optimized by evaluating compressive strength,elastic modulus,crosslinking time,and crosslinking temperature to obtain the optimal formulation.Part Ⅲ:Under the optimal formulation conditions,different compositions of BP/PPF bone cement were assigned as experimental groups(n=3),while PPF bone cement served as the control group(n=3).The degradation performance of bone cement was investigated through an in vitro degradation experiment using PBS solution.Additionally,phosphate release ability in vitro for BP/PPF bone cement was examined using a phosphate detection kit.Furthermore,infrared thermal imaging instrument recorded photothermal performance under 808 nm near-infrared light irradiation for BP/PPF bone cement.Biocompatibility assessment involved CCK-8 cytotoxicity experiments and live-dead cell staining to evaluate cell viability on the surface of bone cement observed via scanning electron microscope.Osteogenic performance evaluation included alizarin red staining along with alkaline phosphatase and osteocalcin determination for BP/PPF bone cement.Finally,endogenous toxicity experiments were performed in mice(n=40)to verify biological safety of BP/PPF bone cement;this consisted of a control group with 10 mice and an experimental group with 30 mice.Results:(1)The average molecular weight of PPF prepared by two-step method was between 1000-3000 Da,and the PPF had a high proportion of carbon-carbon double bonds as shown by 1H NMR and IR spectra.The average particle size of BP nanosheets was 135.40±53.82 nm.XPS,XRD,Raman spectroscopy and infrared spectroscopy showed that the BP nanosheets were prepared into bone.Scanning electron microscopy showed that BP nanosheets were attached to the surface of bone cement,indicating that BP/PPF bone cement was successfully prepared.(2)Single factor analysis showed that the molecular weight of PPF significantly affected the physical properties of bone cement.The compressive strength,elastic modulus and crosslinking temperature of bone cement increased with the increase of molecular weight of PPF,and the crosslinking time shortened with the increase of molecular weight of PPF.When the average molecular weight of PPF was close to 3000 Da,the elastic modulus increased and the compressive strength decreased.The brittleness of bone cement was increased.The addition of BP nanosheets could increase the compressive strength of bone cement,reduce the elastic modulus of bone cement,prolong the cross-linking time of bone cement,and reduce the cross-linking of bone cement.The difference was statistically significant(p < 0.05).The content of BP nanosheets had no significant effect on the compressive strength,elastic modulus,cross-linking temperature and cross-linking time of bone cement,and there was no statistical difference between the groups.The results of orthogonal analysis showed that the importance of compressive strength was as following:A(BPO)> C(NVP)> B(DMPT),with BPO having a positive effect,DMPT and NVP having a negative effect.The influence importance on elastic modulus was A(BPO)> B(DMPT)> C(NVP),where BPO was positive,DMPT and NVP were negative.The influence importance on crosslinking time was B(DMPT)> A(BPO)> C(NVP),where A,B and C were all negative.The importance of the influence on crosslinking temperature was B(DMPT)> A(BPO)> C(NVP),in which BPO and DMPT were both negative effects.Finally,A1B1C1 was selected as the best cement formula for the next experiment by comprehensive balance method.(3)The addition of BP nanosheets accelerated the degradation rate of PPF bone cement,and the higher the content of BP nanosheets,the faster the degradation rate.The addition of BP nanosheets resulted in a good photothermal effect on PPF bone cement.The cement with BP nanosheets increased by up to 22.6℃under near-infrared light irradiation,and almost no loss of photothermal properties under cyclic light irradiation.In vitro phosphate release showed that 200 BP/PPF cement had a large phosphate release in the first 14 days.Under near-infrared light,the phosphate release was higher in the first 14 days,but tended to be the same around the 28 th day.CCK-8 toxicity test and/or dead cell staining showed that BP/PPF had good biocompatibility,and the cell survival rate of all experimental groups was higher than80%.Scanning electron microscopy showed that MC3T3-E1 cells adhered to the material surface.Alizarin red staining showed a large number of calcium nodules stained red under the microscope in the BP/PPF group.The results of ALP and OCN assays also demonstrated that PPF bone cement with BP nanosheets had a good osteogenic effect.The pathological staining sections of the heart,liver,spleen,lung and kidney in the experimental group and the control group showed no obvious pathological changes,and BP nanosheets had no obvious biological toxicity in mice.Conclusions: BP/PPF cement showed similar mechanical properties to bone,excellent photothermal properties,cytocompatibility,and osteogenic properties.BP/PPF cement is an effective injectable degradable cement with great potential in the field of bone regeneration.