CN117180509A - Artificial joint prosthesis material LP01-PS and preparation method thereof - Google Patents
Artificial joint prosthesis material LP01-PS and preparation method thereof Download PDFInfo
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Abstract
The invention provides an artificial joint prosthesis material LP01-PS and a preparation method thereof, belonging to the technical field of artificial joint prosthesis materials. The artificial joint prosthesis material LP01-PS provided by the invention is synthesized by polyether-ether-ketone, LP01 and polydopamine. The LP01-PS rich in LP01 has good elastic modulus, cell and animal biocompatibility, has direct inhibition effects on proliferation, metabolism, diffusion and the like of escherichia coli and staphylococcus aureus, and enhances the inhibition effects of macrophages on proliferation, metabolism, diffusion and the like of S.aureus and E.coli. In addition, the LP01-PS has the effect of promoting bone marrow mesenchymal stem cell osteogenic differentiation, and can improve the mineralization capacity of rat bone marrow mesenchymal stem cells and the osteogenic related gene expression.
Description
Technical Field
The invention belongs to the technical field of artificial joint prosthesis materials, and particularly relates to an artificial joint prosthesis material LP01-PS and a preparation method thereof.
Background
The use of endophytes has been the most basic method for treating orthopedic diseases for centuries, but endophyte loosening and bacterial infection are disastrous complications of orthopedic surgery, bringing great pain and economic burden to patients. Bacterial infection causes local inflammatory reaction, and generated cytotoxin inhibits local osteogenesis reaction, which is an important cause of loosening of the orthopaedics prosthesis; meanwhile, the abrasion particles generated by the loosening of the prosthesis can cause biological immune response, a large amount of macrophages are gathered on the interface to phagocytize various prosthesis material particles, the generation of osteoclasts is induced, so that malignant cycles such as osteolysis and the like occur, and bacterial infection is easily caused by the local immune environment deterioration. Therefore, the development of the orthopaedics prosthesis material with both antibacterial and osteogenic functions has important clinical conversion value.
Polyether ether ketone (PEEK) is one of the common endophyte materials closest to the elastic modulus of bone tissue, is one of the common available materials closest to the bone tissue of human body in terms of mechanical properties, however, the biological inertia limits the clinical wide application of the PEEK, is only widely used in the aspect of interbody fusion cage of spinal cord surgery in clinic at present, and has few reports on the aspects of artificial joint prosthesis, bone fracture plate and the like. Therefore, how to modify the surface of the material can not only keep the inherent mechanical advantage of the PEEK material, but also improve the surface activity of the PEEK material, and the PEEK material is a key of the current PEEK material in the aspects of application of artificial joint prostheses and the like, which needs to be solved and going from basic research to clinic.
The skin acts as a first line of defense, being exposed to the environment and numerous microorganisms. Among these microorganisms, staphylococcus epidermidis is the most abundant bacteria residing on the skin and generally has a benign relationship with its host, playing an important role in host defense by producing lipopeptides that regulate the host's immune response to invasion by harmful pathogens. In the prior art, there is no research on whether the lipopolypeptide LP01 from Staphylococcus epidermidis can be used in plants in artificial joints.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an artificial joint prosthesis material LP01-PS having both antibacterial and osteogenic properties.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an artificial joint prosthesis material LP01-PS, wherein the raw materials of the LP01-PS comprise polyether ether ketone, LP01 and polydopamine.
The invention also provides a preparation method of the artificial joint prosthesis material LP01-PS, which comprises the following steps: sulfonating and hydrothermally treating polyether-ether-ketone to obtain sulfonated polyether-ether-ketone with porous surface; mixing sulfonated polyether-ether-ketone, polydopamine powder, LP01 powder and Tris solution to obtain a mixture, and shaking to synthesize the LP01-PS.
Preferably, the polyether-ether-ketone is a polyether-ether-ketone rod or a polyether-ether-ketone disc.
Preferably, the diameter of the polyether-ether-ketone rod is 2mm, and the length of the polyether-ether-ketone rod is 10mm.
Preferably, the diameter of the polyether-ether-ketone disc is 10mm, and the thickness of the polyether-ether-ketone disc is 1mm.
Preferably, the step of sulphonation and hydrothermal treatment comprises: and (3) sulfonating the polyether-ether-ketone with 98% concentrated sulfuric acid at room temperature for 8min, and washing, and then, carrying out water bath at 120 ℃ for 4h to obtain the sulfonated polyether-ether-ketone with porous surface.
Preferably, the concentration of polydopamine in the mixture is between 1 and 25mg/ml and the concentration of LP01 in the mixture is between 1 and 25mg/ml.
Preferably, the molar concentration of the Tris solution is 0.01M and the pH of the Tris solution is 8.5.
Preferably, the rotational speed of the shaking synthesis is 5rpm and the time is 1 hour.
Preferably, unbound and loosely bound particles are removed after shaking synthesis.
The invention has the beneficial effects that:
the LP01-PS with rich LP01 successfully prepared by the invention has good elastic modulus, cell and animal biocompatibility, has direct inhibition effect on proliferation, metabolism, diffusion and the like of escherichia coli (E.coli) and staphylococcus aureus (S.aureus), promotes expression of Cathelicidin LL-37, mBD-2, mBD-3 and mBD-4 in macrophages, and enhances inhibition effect of the macrophages on proliferation, metabolism, diffusion and the like of the E.aureus and the E.coli. In addition, the LP01-PS prepared by the invention has the effect of promoting bone marrow mesenchymal stem cell osteogenic differentiation, can improve the mineralization capacity of rat bone marrow mesenchymal stem cells (rBMSCs), and the expression of osteogenic related genes ALP, OCN, COL-1, BMP-2, OPN, RUNX2, BSP and OC, and can promote the growth of new bone around the SD rat femoral intercondylar implant material.
Drawings
FIG. 1 shows the preparation, characterization and biocompatibility results of LP01-PS, wherein A is the structure of the LP01 protein and the synthesis of LP 01-PS; b is the contact angle of the surface liquid; c is the drug release profile of the discs and rods; d is a scanning electron microscope result; e and F are HE staining of major organs and weight change of mice after implantation of 25LP01-PS into the backs of mice, respectively; g was 25LP01-PS and release levels of LDH after 3 days co-cultivation with macrophage RAW246.7 and rBMSCs; h is CCK8 count of cell number; scanning electron microscope with I as cell and Actin cell immunofluorescence with J;
FIG. 2 shows the results of biochemical changes in peripheral venous blood and changes in peripheral blood cells, A is the biochemical index of peripheral venous blood at different time points after 0/1/2/4 weeks after 25LP01-PS is implanted into the back of a mouse; b is peripheral venous blood cell change at different time points;
FIG. 3 shows the results of in vitro studies of the direct antibacterial effect of LP01-PS, A is the scanning electron microscope result of the adhesion bacteria on the surface of the materials after the materials of each group are added with equal amounts of S.aureus and E.coli for co-cultivation for 24 hours; b is the living and dying dyeing result of the material surface adhesion bacteria after adding the same amount of S.aureus and E.coli for co-culture for 24 hours; c is the metabolic gene expression change trend of the S.aureus adhered to the surface of the material after the same amount of gold S.aureus is added to each group of material for co-culture for 24 hours; d, adding an equivalent amount of E.coli into each group of materials, and co-culturing for 24 hours, wherein the metabolic gene expression trend of E.coli is adhered to the surfaces of the materials; e is a quantitative analysis result obtained by adding equal amounts of S.aureus and E.coli into each group of materials for co-culture for 24 hours, wherein plankton bacteria and materials in a culture medium show adhesion bacteria, and the quantitative analysis result is measured by a plating dilution method;
FIG. 4 shows the results of in vitro antibacterial property study of LP01-PS, wherein A is a scratch experiment of macrophages after LP01-PS is co-cultured with the macrophages for 3 days; b is a Transwell experiment; c is the extracellular secretion of macrophage antibacterial peptides LL-37, mBD, mBD3, mBD; d is mRNA level expression of macrophage antibacterial peptide; e is quantitative analysis measured by S.aureus and E.coli coating dilution method; f is the metabolic change of S.aureus adhering to the LP01-PS surface; g is the metabolic change of E.coli adhering to the LP01-PS surface; h is a scanning electron microscope with bacteria adhered to the surface; i is live dying;
FIG. 5 shows the result of LP01-PS activating the TLR2/P65 signaling pathway of macrophages, A is the cellular immunofluorescence of the TLR2 and P65 signaling pathways of macrophages; b is mRNA detection; c is the protein level detection result and quantitative analysis; d is the bacterial number on the surface of the detection material of a scanning electron microscope after S.aureus and E.coli are co-cultured for 24 hours after macrophages are treated by TLR2 signal path blocker OXPAPC and P65 signal path blocker BAY11-7082, and the survival condition of E.coli on the surface of the detection material is detected by dying and dyeing; e is the elisa secretion and mRNA expression of macrophage antibacterial peptides LL-37, mBD2, mBD3 and mBD; g is the Transwell experimental result of macrophages after treatment of TLR2 signal path blocker OXPAPC and P65 signal path blocker BAY 11-7082; e is E.coli gene metabolism; h is the gene metabolism of S.aureus; i is S.aureus and E.coli quantitative analysis measured by a plate-coating dilution method;
FIG. 6 is an in vitro change in antibacterial capacity of macrophages after lentiviral silencing or high expression of the TLR2/p65 signaling pathway of the macrophages; the method comprises the steps that A is extracellular secretion of four antibacterial peptides of macrophages, B is mRNA expression of the four antibacterial peptides of the macrophages, C is variation trend of quantity of plankton bacteria of E.coli and S.aureus and material surface adhesion bacteria, D and E are variation trend of quantity of S.aureus and E.coli on the material surface displayed by a scanning electron microscope during silencing and high expression, F is mRNA expression quantity results of TCA/PTS energy metabolism pathway related genes lacD and lacB, movement related genes csgA, fliA and fimA, virulence related genes stx2 and ehaA, biofilm synthesis related genes ompA, pgaA and luxS, zinc steady state related genes ykgM, zinT, zunA and Zun B on the material surface; g is mRNA expression quantity results of biomembrane formation specific genes fnbA and fnbB of S.aureus, genes sucA and sucB related to TCA/PTS energy metabolism pathway, main genes agrA and agrC of an agr population stress sensing system of S.aureus, S.aureus sak and HtrA genes for promoting biomembrane degradation;
FIG. 7 shows the results of LP01-PS in vivo macrophage antibacterial action, wherein A and B are quantitative analyses of the number and fluorescence of S.aureus infected in vivo fluorescence systems for detection of bacteria in the back of mice, respectively; c is the expression change of the metabolic gene of S.aureus attached to the surface of LP 01-PS; d is a quantitative analysis of bacteria on the LP01-PS surface and surrounding skin tissue; e and F are the elisa secretion and mRNA expression of macrophage antibacterial peptides LL-37, mBD2, mBD, mBD, respectively; g is a scanning electron microscope result; h is HE staining, immunohistochemical of MPO, giemsa staining;
FIG. 8 shows the results of LP01-PS in promoting osteogenic differentiation and in vivo osteogenesis of rBMSCs, wherein A is the expression of osteogenic genes, B is a transwell experiment of rBMSCs, and C is ALP of rBMSCs and ARS staining by extracellular calcium salt deposition. D is a mechanical experiment of extraction of the new bone after the LP01-PS is implanted into the femur of the rat for 8 weeks, E is micro-CT scanning and three-dimensional reconstruction of the new bone, F is sequence fluorescent staining of the new bone, G is Van Gieson staining (representing the position of SPEEK), H is BMD of the new bone, bone material contact area percentage, BV/TV, tb.N, tb.Th, tb.Sp; i is immunofluorescence of beta-catenin protein of rBMSCs after coculture of LP01-PS and rat rBMSCs, J is WB and quantitative analysis, K is mRNA change of main subunits LRP5, LRP6, axin2 and beta-catenin of WNT/beta-catenin signal pathway of the rBMSCs, L is ARS staining deposited by ALP and extracellular calcium salt of rBMSCs after treatment of rBMSCs by using a chemical blocker of the WNT/beta-catenin signal pathway, and M is expression of osteogenic genes.
Detailed Description
The invention provides an artificial joint prosthesis material LP01-PS, wherein the raw materials of the LP01-PS comprise polyether ether ketone, LP01 and polydopamine.
In the invention, the LP01 is a lipopeptide LP01 derived from staphylococcus epidermidis, and the molecular formula is shown as follows:
The protein structure is shown in FIG. 1A, and the specific source and specific preparation method of the lipopeptide LP01 are not particularly limited, so long as the synthetic molecular formula is as described above, and in the specific embodiment of the present invention, the lipopeptide LP01 derived from human staphylococcus epidermidis is synthesized by Shanghai Jier Biochemical Co. The specific sources of the polyether-ether-ketone and the polydopamine are not particularly limited, and the products which are conventionally and commercially available in the field can be adopted.
The invention also provides a preparation method of the artificial joint prosthesis material LP01-PS, which comprises the following steps: sulfonating and hydrothermally treating polyether-ether-ketone to obtain sulfonated polyether-ether-ketone with porous surface; mixing sulfonated polyether-ether-ketone, polydopamine powder, LP01 powder and Tris solution to obtain a mixture, and shaking to synthesize the LP01-PS.
In the invention, the polyether-ether-ketone is preferably a polyether-ether-ketone rod or a polyether-ether-ketone disc, the diameter of the polyether-ether-ketone rod is preferably 2mm, and the length of the polyether-ether-ketone rod is preferably 10mm; the diameter of the polyether-ether-ketone disc is preferably 10mm, and the thickness is preferably 1mm. In the present invention, the step of sulfonation and hydrothermal treatment preferably comprises: sulfonating polyether-ether-ketone with 98% concentrated sulfuric acid at room temperature for 8min, cleaning, and then carrying out water bath at 120 ℃ for 4h to remove residual acidic substances on the surface of the material, thereby obtaining sulfonated polyether-ether-ketone with three-dimensional porous surface; the washing is preferably repeated with deionized water, the number of times of washing is preferably 3, and the water bath is preferably performed in a hydrothermal kettle. In the present invention, after the sulfonated polyether ether ketone, the polydopamine powder, the LP01 powder and the Tris solution are mixed to obtain a mixture, the polydopamine concentration in the mixture is preferably 1 to 25mg/ml, more preferably 5 to 25mg/ml, and the LP01 concentration in the mixture is preferably 1 to 25mg/ml, more preferably 5 to 25mg/ml. In the present invention, the molar concentration of the Tris solution is preferably 0.01M, and the pH of the Tris solution is preferably 8.5; the rotation speed of the shaking synthesis is preferably 5rpm, and the time of the shaking synthesis is preferably 1 hour; after shaking synthesis, unbound and loosely bound particles are preferably removed by gentle flushing with double distilled water, preferably 5 times.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Data in the examples below are expressed as mean.+ -. Standard deviation (mean.+ -. SD), and statistical analysis is performed by graphpad prism 9.0 (Graphpad software, la Jolla, calif.) software, and statistical differences are considered to be present when p < 0.05 by comparison of group differences by one-way analysis of variance (ANOVA) and unpaired t-test (Student's t test).
Example 1
The raw material of PEEK was purchased from Geiger Corp, germany, and then cut into rods (rod) of diameter 2mm and length 10mm or discs (disc) of diameter 10mm and thickness 1 mm. And sulfonating PEEK with 98% concentrated sulfuric acid at room temperature for 8 minutes, repeatedly flushing with deionized water for 3 times, and then carrying out water bath for 4 hours at 120 ℃ in a hydrothermal kettle to remove residual acidic substances on the surface of the material, thereby obtaining the surface porous Sulfonated PEEK (SPEEK). The SPEEK rod was placed in a beaker and 0.01M Tris pH 8.5 and 25mg/ml polydopamine powder (PDA, shanghai aladine biochemistry limited, china) was added. Then, artificially synthesized LP01 powder (99% by Shanghai Jier Biochemical Co., ltd.) was added to make the content of the LP01 powder in the beaker 25mg/mL. These mixtures were shaken at 5rpm for 1 hour, and then gently rinsed with double distilled water to remove any loosely bound particles to give LP01-PS, designated 25LP01-PS. The synthesis process is shown in the right side of FIG. 1A.
Example 2
The difference from example 1 is that the concentration of polydopamine powder in the beaker is 5mg/ml, the concentration of LP01 powder is 5mg/ml, and the remainder is the same as in example 1, giving LP01-PS, designated 5LP01-PS.
Example 3
The difference from example 1 was that the concentration of polydopamine powder in the beaker was 1mg/ml, the concentration of LP01 powder was 1mg/ml, and the remainder was the same as in example 1, giving LP01-PS, designated as 1LP01-PS.
Comparative example 1
The difference from example 3 is that no LP01 powder was added, the remainder being PS in the same way as in example 3.
Example 4
The pure SPEEK without polydopamine powder and LP01 powder was used as a control group and was designated as Con group.
The contact angles of the materials of each of the groups example 1, example 2, example 3, comparative example 1 and Con were tested using a liquid contact angle meter (LSAMOB-L, LAUDA Scientific, hazi, germany), and the drug release profile of LP01-PS in PBS was examined using a microplate reader (FC, thermo, new York, USA). In addition, the surface of each group of materials was observed using a FEI NovaNano450 scanning electron microscope (JEOL, JSM-6310LV, tokyo, japan). The results are shown in FIG. 1, B, C, D, respectively.
As can be seen from FIG. 1B, the more the LP01 powder was added, the denser the lipoprotein balls enriched on the porous surface, and the larger the liquid contact angle of LP01-PS. drug release profiles of disc and rod in PBS showed that at equal rich concentrations, disc released LP01 more rapidly than rod (fig. 1C). This is related to a larger disc surface area. LP01 had poor solubility in PBS and scanning electron microscopy showed that a large number of LP01 aggregated into grape cluster-like spheres embedded in the surface three-dimensional pores of sulfonated PEEK (fig. 1D). Since LP01 is mostly concentrated inside the surface pores of sulfonated PEEK, the surface of non-sulfonated PEEK is flat. Therefore, the surface of the 25LP01-PS group has the most fine and most dispersed pores, and the optimal physicochemical properties such as liquid contact angle.
Example 5
Assessment of cellular and animal biocompatibility of LP01-PS
RAW246.7 (CL-0190,Wuhan Zishan Biotechnology Co.LTD) and rBMSCs (STCC 5011, wuhan Zishan Biotechnology Co. LTD) were used for cell compatibility testing. RAW246.7 and rBMSCs were cultured in DMEM (MT 15013LX, corning, new York, USA) medium supplemented with 10% fetal bovine serum (SH 3010902HI, gibco, grand Island, USA). A total of 5 groups of discs from examples 1-3, comparative example 1 (Con group) and example 4 (Con group) were placed horizontally in 24-well plates, respectively. Cytoskeleton was detected by cellular immunofluorescence (LSM 510meta, zeiss, oberkochen, germany) and cell morphology, adhesion, cell pseudopodia were observed using a scanning electron microscope (JEOL, JSM-6310LV, tokyo, japan). Cell proliferation was examined at various time points by CCK-8 (C0038, beyotime Bio-Tech) and changes in Lactate Dehydrogenase (LDH) in the medium were examined by ELISA (C0017, beyotime Bio-Tech). Wherein 25LP01-PS was co-cultured with macrophage RAW246.7 and rat rBMSCs for 3 days, the results obtained are shown in FIG. 1, G, H, I, J, respectively. The results showed no significant difference in LDH release (fig. 1G) and cell number (fig. 1H) for each group. Both cell types adhere well to different sets of material surfaces and can produce sufficient pseudopodia (fig. 1I). Cell immunofluorescence showed that the cytoskeleton on the cell surface grew well and was uniformly distributed for each group (fig. 1J). The LP01-PS material has good biological safety at the cellular level
The mice were implanted subcutaneously with 25LP01-PS on the backs of the mice BABL/c mice (Shanghai SLAC Laboratory Animal co., ltd) and the weight changes of the mice were recorded at different time points. Peripheral venous blood biochemical changes were detected using a full-automatic blood biochemical analyzer (ADVIA 2400, zeiss, oberkochen, germany), and peripheral blood cell changes were detected using a full-automatic blood cell analyzer (BC 6800, mindray, shenzhen). 1. After 4 weeks, the mice were removed and HE stained for heart, liver, spleen, lung, kidney, and observed for changes such as denaturation of organ tissues using a light microscope (LSM 510meta, zeiss, oberkochen, germany). The results are shown in fig. 1 and E, F and fig. 2. The LP01-PS was subcutaneously implanted on the back of mice for 0, 7, 28 days, and the mice were sacrificed to collect their main viscera for HE staining observation. The results of HE staining showed good status of each group of organs (fig. 1E). At different time points, there was no statistical difference in the change in body weight of mice (fig. 1F). As can be seen from fig. 2, the differences in the biochemical indexes of the peripheral blood cells and the peripheral blood of each group of mice at different time points have no statistical significance. The LP01-PS material has good biological safety at animal level.
Example 6
In vitro antibacterial experiments
S.aureus (SF 8300, sangon Biotech, shanggai) and E.coli (ATCC 35218, sangon Biotech, shanggai) were resuspended in PBS buffer and the bacterial concentration was adjusted to 10 7 CFU/ml. Bacterial suspensions were divided into 5 groups, respectively control (Con, example 4), PDA-containing medium (PS, comparative example 1), LP01-PS containing 1mg/ml drug (1 LP01-PS example 3), LP01-PS containing 5mg/ml drug (5 LP01-PS, example 2) and LP01-PS containing 25mg/ml drug (25 LP01-PS, example 1).
(1) In vitro studies of direct antibacterial activity of LP01-PS
First, 10mm×10mm×1mm discs were placed in 24-well plates, respectively, and 1 disc was placed per well. Then inoculating 500ul of the culture medium with a concentration of 10 to each well 6 CFU/ml bacterial liquid and co-culturing at 37 ℃ for 24 hours. Planktonic bacteria in the original TSB medium were quantified using SPM method (110704011, BKMAN, changde, china). The cultured specimens were removed with sterile forceps and gently rinsed with fresh PBS to remove non-adherent bacteria. The discs were placed in 1ml fresh PBS, shaken for 10 minutes using an ultrasonic cleaner (150W, B3500S-MT, branson Ultrasonics Co, shanghai) and mixed for 2 minutes using a mixing Vortex (Vortex Genie 2,Scientific Industries,Bohemia,USA) to remove adherent bacteria. Then calculating the number of viable bacteria in the bacterial suspension by using SPM method, wherein the number of each group of planktonic bacteria and material surface adhesion bacteria is log 10 CFU/ml.
Subsequently, the discs were placed in a new 24-well plate, washed with PBS, stained with LIVE/DEAD BacLight mixed dye (L7007, invitrogen, carlsbad, USA) for 30 minutes, and observed and photographed under a fluorescence microscope (LSM 510meta, zeiss, oberkochen, germany). Dead bacteria are stained red, while living bacteria are stained green. Next, 2.5% glutaraldehyde was used for 48 hours at 4℃and then dried with gradient alcoholic solutions (50, 60, 70, 80, 90, 95 and 100% v/v). After drying, the discs were sprayed with gold and the bacterial distribution on the surface of each group of material was observed using a scanning electron microscope (JEOL, JSM-6310LV, toky, japan).
For the study of the direct antibacterial properties of LP01-PS on S.aureus, the expression levels of the following genes were detected using the rt-PCR method (T100, bio-Rad, shanghai, china): the expression levels of the biofilm formation-specific genes fnbA and fnbB mRNA are known for s.aureus biofilm formation. Expression levels of TCA/PTS energy metabolic pathway related genes sucA and sucB, and changes in metabolic levels of s.aureus were evaluated. Expression levels of the major genes agrA and agrC in the agr population stress induction system were evaluated for stress response capacity of s.aureus. The expression levels of genes sak and HtrA promoting s.aureus biofilm degradation are known for s.aureus biofilm degradation.
For the study of the direct antibacterial properties of LP01-PS on e.coli, the expression levels of the following genes were detected using the rt-PCR method (T100, bio-Rad, shanghai, china): detecting the expression level of TCA/PTS energy metabolic pathway related genes lacD and lacB mRNA of E.coli on the surface of the material, and knowing the metabolic pathway of E.coli. And simultaneously detecting mRNA expression levels of the vitality related gene csgA, fliA, fimA and the virulence related genes stx2 and ehaA, and evaluating the growth state and potential virulence of E.coli. Meanwhile, the mRNA expression level of the e.coll biofilm synthesis-related gene ompA, pgaA, luxS was detected, the biofilm formation ability of e.coll was evaluated, and the mRNA expression levels of the zinc steady-state-related genes ykgM, zinT, zunA and ZunB were evaluated, and the environmental adaptation ability of e.coll was evaluated.
The results are shown in FIG. 3. In the LP01-PS and bacteria co-culture system, the more LP01 is loaded on the surface of the material, the scanning electron microscope shows that the number of S.aureus and E.coll on the surface of the LP01-PS group material is gradually reduced (figure 3A), and the bacterial live-dead staining shows that the death proportion of E.coll on the surface of the LP01-PS group material is gradually increased (figure 3B). The quantitative analysis result of the mRNA of the material surface adhesion bacteria shows that the expression level of S.aureus biofilm formation specific genes fnbA and fnbB, TCA/PTS energy metabolic pathway related genes sucA and sucB is reduced, the expression level of the agr population stress sensing system main genes agrA and agrC of S.aureus, S.aureus sak and HtreA genes for promoting the degradation of the biofilm is increased (figure 3C), the expression level of the E.coli TCA/PTS energy metabolic pathway related genes lacD and lacB, the motion related genes csgA, fliA and fimA, virulence related genes stx2 and ehaA, the expression level of the biofilm synthesis related genes ompA, pgaA and luxS, and zinc steady state related genes ykgM, zinT, zunA and Zun B mRNA is reduced, and the difference among groups is statistically significant (figure 3D). The smaller the number of planktonic and material surface adherent bacteria measured by the dilution plating method, the more statistically significant the differences between groups (fig. 3E).
(2) In vitro study of LP01-PS to enhance macrophage antibacterial Properties
To investigate the effect of LP01-PS on macrophage antibacterial properties, different groups of materials were placed in 24-well plates, respectively, and were divided into Con group, PS group, 1LP01-PS group, 5LP01-PS group, and 25LP01-PS group. Each group had 3 duplicate wells, and one 10mm 1mm disc was placed in each well. Will contain 2.5X10 5 Cells of RAW264.7 were seeded into each well, and DMEM cell culture medium (MT 15013LX, corning, newYork, USA) containing 100U/ml penicillin, 100U/ml streptomycin, and 10% fetal bovine serum (SH 3010902HI, gibco, grand Island, USA) was then added. The plates were placed at 37℃in 5% CO 2 Is cultured in a cell culture vessel. After 1 day of culture, most cells successfully grew in adherent fashion, and then fresh cell culture medium was replaced and culture continued for 3 days.
Changes in macrophage TLR2/P65 signaling pathway mRNA and protein levels were detected using rt-PCR, westernblot and immunofluorescence methods, and macrophage secretion levels of Cathelicidin LL-37, mouse beta-dephenosin 2, mouse beta-dephenosin 3, and mouse beta-dephenosin 4 were detected by the Elisa method. Changes in the level of LL-37, mBD2, mBD3 and mBD mRNA in RAW246.7 were detected using rt-PCR. After 3 days of cell culture, when the degree of cell fusion reached 90%, new cell culture medium was exchanged, and S.aureus and E.coli (500 ul, 1X 10) were added 6 CFUs/mL), co-culture 24 hours. Finally, bacterial proliferation and metabolic indicators were tested for each group.
In vitro studies of LP01-PS to enhance macrophage antibacterial action through TLR2/P65 signaling pathway
The 12 disks of 25LP01-PS were randomly divided equally into 4 groups and loaded into 24 well plates, 1 disk per well. When RAW246.7 reached 90% confluence, it was replaced with fresh medium. A total of 4 groups, 25LP01-PS group without any chemical blocker, 25LP01-PS+OxPAPC group with 30ul/ml OxPAPC, 25LP01-PS+Bay11 group with Bay 11-708235uM, 25LP01-PS+OxPAPC+Bay11 group with 30ul/ml OxPAPC and Bay11-70825uM. After 3 days, S.aureus or E.coli (500 ul, 1X 10) 6 CFUs/mL), co-culture at 37℃for 24 hours. Then, bacterial proliferation and metabolism indexes of the four groups are detected.
For macrophage TLR2/P65 signaling pathway gene silencing, mouse TLR2 or NF- κb/P65 specific short hairpin RNAs were cloned into the pll3.7 vector (Addgene, shanghai, china). 4mg of pLL3.7 construct containing specific shRNA, 24g of packaging plasmid psPAX and 2.G2 g of envelope plasmid pMD were prepared and then 293T cells were transfected by calcium phosphate precipitation. After 48 hours, lentiviruses containing mouse TLR2 or NF-. Kappa.B/p 65 specific shRNA were collected and transfected into RAW246.7. 10 was injected around RAW246.7 of a 10cm dish with a microinjector 12 50ul of/L lentivirus for silencing TLR2 or NF- κB/p65 genes is used for detecting the efficiency of shRNA blocking macrophage TLR2 or NF- κB/p65 expression by rt-PCR and Western blot, and each group of RAW246.7 meeting silencing requirements is prepared. The 12 disks of 25-LP01-PS were randomly divided equally into 4 groups and loaded into 24 well plates, 1 disk per well.
The 25LP01-ps+scramble group was added to the lentiviral empty vector transfected RAW246.7 that did not express shRNA, the 25LP01-ps+tlr2 shRNA group was added to the lentiviral transfected RAW246.7 that expressed TLR2 shRNA, the 25LP01-ps+p65 shRNA group was added to the lentiviral transfected RAW246.7 that expressed p65 shRNA, and the 25LP01-ps+tlr2/p65 shRNA group was added simultaneously to the lentiviral transfected RAW246.7 that expressed TLR2 and p65 shRNA. When RAW246.7 reached 90% fusion, S.aureus or E.coli (500 ul, 1X 10) 6 CFUs/mL), co-culture at 37℃for 24 hours. Then, the bacterial increase in the above four groups was examinedAnd (5) the index of reproduction and metabolism.
For high expression of macrophage TLR2/P65 signaling pathway genes, TLR2 or NF-. Kappa.B/P65 over-expression plasmids (zorin, shanghai), psPAX, pmD2G plasmids were co-transfected into 293T cells. After about 48 hours, lentiviral supernatants were collected, and concentrated virus was purified by membrane filtration and ultracentrifugation. Flow cytometry detects the high expression lentivirus titer of TLR2 or NF- κB/p65 genes. 10 was injected around RAW246.7 of a 10cm dish with a microinjector 12 50ul of lentivirus with high expression of TLR2 or NF- κB/p65 gene of/L is used for detecting the high expression efficiency of TLR2 or NF- κB/p65 of macrophage by rt-PCR and Western blot, and each group of RAW246.7 meeting the high expression requirement is prepared. The 12 disks of 1-LP01-PS were randomly divided equally into 4 groups and loaded into 24 well plates, 1 disk per well. The 1LP01-ps+mock group was added with lentiviral empty vector transfected RAW246.7 not expressing mRNA, the 1LP01-ps+tlr2 over group was added with lentiviral transfected RAW246.7 expressing TLR2 mRNA, the 1LP01-ps+p65 over group was added with lentiviral transfected RAW246.7 expressing p65 mRNA, and the 1LP01-ps+tlr2/p65 over group was added with lentiviral transfected RAW246.7 expressing TLR2 and p65 mRNA simultaneously. When RAW246.7 reached 90% fusion, S.aureus or E.coli (500 ul, 1X 10) 6 CFUs/mL), co-culture at 37℃for 24 hours. Then, bacterial proliferation and metabolism indexes of the four groups are detected.
The results of the above experiments are shown in fig. 4 to 6.
After 3 days of coculture of LP01-PS with macrophages, the more LP01 was loaded on the surface of the material, the faster the macrophages migrated (FIG. 4A); the greater the number of macrophages migrating (fig. 4B). Meanwhile, LP01-PS promotes extracellular secretion of macrophages LL-37, mBD2, mBD3, mBD4 (FIG. 4C); mRNA expression of macrophages LL-37, mBD2, mBD, mBD4 was promoted (FIG. 4D). The above results suggest that LP01-PS enhances the antibacterial ability of macrophages themselves, and that antibacterial peptides secreted by macrophages can also enhance the antibacterial ability of other surrounding tissue cells.
In the LP01-PS, macrophage and bacteria co-culture system, the more LP01 was loaded, the fewer planktonic bacteria and LP01-PS surface adherent bacteria were measured by the dilution plating method (FIG. 4E). The quantitative analysis result of the LP01-PS surface adhesion bacteria mRNA shows that S.aureus biofilm formation specific genes fnbA and fnbB and expression of genes sucA and sucB related to TCA/PTS energy metabolism pathway are reduced. However, the s.aureus group stress sensing system increased expression of the major genes agrA and agrC, s.aureus sak and HtrA genes that promote biofilm degradation (fig. 4F). The expression level of the genes lacD and lacB related to the energy metabolism pathway of the coll TCA/PTS, the genes csgA, fliA and fimA related to the movement, the genes stx2 and ehaA related to the virulence, the genes ompA related to the synthesis of the biological membrane, pgaA and luxS, the genes ykgM, zinT, zunA related to the zinc steady state and Zun B mRNA is reduced (figure 4G), and the difference among groups is statistically significant. Scanning electron microscopy showed a progressive decrease in the number of s.aureus and e.coli on the surface of the LP01-PS group (fig. 4H). Bacterial live-dead staining showed an increasing proportion of death of E.coli on the surface of the LP01-PS group (FIG. 4I). The above results suggest that LP01-PS enhances the ability of macrophages to kill s.aureus and e.coli in vitro.
After 3 days of LP01-PS co-culture with macrophages, rt-PCR results showed a significant increase in the mRNA expression levels of macrophages TLR2 and P65 in the LP01-PS group (FIG. 5B). Meanwhile, as the surface-loaded LP01 was increased, immunofluorescence of macrophages showed that TLR2 and P65 protein signaling increased as well, and the partial content of P65 protein in the nucleus increased (fig. 5A). WB and its quantitative analysis also showed the same trend (fig. 5C). When macrophages were treated with TLR2 signaling pathway blockers oxapc and P65 signaling pathway blocker BAY11-7082, the extracellular secretion and mRNA expression levels of the antibacterial peptides LL-37, mBD2, mBD3 and mBD4 of macrophages were significantly reduced (fig. 5E), while the migration rate of macrophages was also significantly slowed down (fig. 5G). These results indicate that LP01-PS can enhance the antibacterial capacity of macrophages through TLR2/P65 signaling.
In the coculture system of LP01-PS, macrophages and bacteria, scanning electron microscopy revealed that the S.aureus and E.coli numbers on the surface gradually increased in the group of macrophages after blocker treatment. Bacterial live-dead staining showed a gradual decrease in the mortality rate of e.coli (fig. 5D). Meanwhile, in the macrophage group after the blocker treatment, the mRNA expression amounts of e.coli TCA/PTS energy metabolic pathway-related genes lacD and lacB, movement-related genes csgA, fliA and fimA, virulence-related genes stx2 and ehaA, biofilm synthesis-related genes ompA, pgaA and luxS, zinc steady-state-related genes ykgM, zinT, zunA and ZunB were increased in the bacteria adhered to the LP01-PS surface (mRNA quantitative analysis) (fig. 5F). In S.aureus adhered to the surface of LP01-PS, the expression levels of the biofilm formation-specific genes fnbA and fnbB, and the TCA/PTS energy metabolic pathway-related genes sucA and sucB were also increased. While the expression levels of the agr population stress sensor system major genes agrA and agrC, and genes sak and HtrA promoting the degradation of the biofilm were decreased in s.aureus (fig. 5H). In addition, the number of blocking agent groups planktonic bacteria and material surface adherent bacteria measured by the dilution plating method increased (fig. 5I), and the differences between groups were statistically significant. Taken together, the results suggest that TLR2/P65 signaling pathway chemical blockers can reduce the antibacterial capacity of macrophages.
The results of in vitro changes in the antibacterial ability of macrophages after lentivirus silencing or high expression of the macrophage TLR2/p65 signaling pathway are shown in figure 6. LP01-PS was co-cultured with macrophages for 3 days, and after the addition of an equivalent amount of lentivirus with silencing of TLR2/p65 signaling pathway or high expression, the extracellular secretion (FIG. 6A) and mRNA expression (FIG. 6B) of the antibacterial peptides LL-37, mBD2, mBD3, mBD4 of macrophages showed different trends after treatment with lentivirus with silencing of TLR2/p65 signaling pathway or high expression. Specifically, these indicators decrease during silencing, while they increase during high expression. Taken together, these results suggest that silencing lentiviruses by TLR2/p65 signaling pathway can reduce the antibacterial capacity of macrophages, while high expression of lentiviruses by TLR2/p65 signaling pathway can enhance the antibacterial capacity of macrophages.
In the system of LP01-PS, macrophages and bacteria co-culture, the number of planktonic and material surface adhesion bacteria of e.coli and s.aureus was found to change as a trend of increasing numbers when TLR2/p65 signaling pathways were silenced and decreasing numbers when TLR2/p65 signaling pathways were highly expressed (fig. 6C). Meanwhile, the scanning electron microscope showed that the change trend of the number of S.aureus and E.coli on the surface of the material was the same, namely the number of silencing was increased and the number of high expression was decreased (FIG. 6D). By bacterial live-dead staining, it was found that the death rate of e.coli decreased when TLR2/p65 signaling was silent, while that increased with high expression (see fig. 6E). This indicates that silencing lentiviruses by TLR2/p65 signaling pathway can reduce the ability of macrophages to inhibit bacterial proliferation, whereas high expression of lentiviruses by TLR2/p65 signaling pathway can increase its ability to inhibit bacterial proliferation.
The quantitative analysis result of mRNA of the material surface adhesion bacteria shows that E.coli increases and high expression drops when mRNA expression amounts of TCA/PTS energy metabolism pathway related genes lacD and lacB, motion related genes csgA, fliA and fimA, virulence related genes stx2 and ehaA, biofilm synthesis related genes ompA, pgaA and luxS and zinc steady state related genes ykgM, zinT, zunA and Zun B are silenced (FIG. 6F). Whereas the expression trend of the S.aureus biofilm formation specific genes fnbA and fnbB, TCA/PTS energy metabolic pathway related genes sucA and sucB is that the expression trend is increased during silencing and the high expression is decreased. In addition, the trend of the expression change of the agr population stress sensor system main genes agrA and agrC of s.aureus, and the s.aureus sak and HtrA genes promoting the degradation of the biological membrane was that the silencing decreased and the high expression increased (fig. 6G). The results show that the ability of the TLR2/p65 signaling pathway silencing lentivirus-treated macrophages to inhibit bacterial metabolism is reduced, and the ability of the TLR2/p65 signaling pathway high-expression lentivirus-treated macrophages to inhibit bacterial metabolism is improved.
Example 7
In vivo antibacterial experiments
Establishment of bacterial infection model related to subcutaneous implant material on back of mouse
A subcutaneous implant material related infection model is established on the back of a BALB/c mouse, and the specific steps are as follows: after anesthesia of mice by intraperitoneal injection of 3% sodium pentobarbital, shaving the backs, sterilizing, making 1cm cuts in the median portion, and dividing into five groups, each group of mice was implanted with different materials of Con (example 4), PS (comparative example 1), 1LP01-PS (example 3), 5LP01-PS (example 2) and 25LP01-PS (example 1), 3 groups each, and 100ul of self-fluorescent Staphylococcus aureus S.aureus (1×10) was simultaneously injected around the backs of the implanted prosthetic materials 7 CFU/ml) and then carefully suturing the wound. Another 5 mice were selected as homologous background samples, con group discs were implanted on the backs, respectively, and 100ul PBS was injected under the median skin on the backs of the discs. In the absence of specific pathogensThe mice were allowed free access to water and food under Somatic (SPF) conditions and kept for 14 days.
Bioluminescence detection of skin surrounding mouse back material
To observe the time course of anti-infective effects in the 5 groups of mice, the BALB/c mice were first anesthetized with 2% isoflurane, and then back photographed on days 0, 1, 3, 7, 14 post-operatively by IVIS luminea XRMS imaging system. Each mouse was imaged for 60s, and a mouse Hu Guangzi signal was acquired, generating a pseudo-color image. The corresponding bioluminescent Photon Intensities (PI) are expressed as bioluminescent fluxes: photons/second/cm 2 In addition, the bioluminescence region on the back of the mice was designated as the region of interest (ROIs) for quantification of bacterial content, and the bioluminescence flux of the ROI was then calculated by IVIS software (Caliper Life Sciences, perkinElmer) provided by the orthopedics institute of Shanghai market at the ruijin hospital.
Analysis of skin pathological tissue around mouse back material
Soft tissues surrounding each set of implant material were collected, fixed in 10% formalin buffer at 4 ℃ for 3 days, rinsed 3 times with fresh PBS, dehydrated in graded concentration alcohol solution, and embedded in paraffin. The samples were sectioned using a microtome (Leica, hamburg, germany). Finally dewaxed in xylene solution and examined for histological changes by light microscopy using May-Gru nwald Giemsa and hematoxylin and eosin (H & E) stainings. Meanwhile, collecting soft tissues around each group of materials, extracting RNA (ribonucleic acid) by a Trizol method, and detecting the change of the levels of the mouse Cathelicidin LL-37, the beta-defensin 2 (mBD-2), the beta-defensin 3 (mBD-3) and the beta-defensin 4 (mBD-4) in the mRNA in the tissues around each group by an rt-PCR method; taking subcutaneous soft tissues around the materials, putting the soft tissues into liquid nitrogen for freezing, grinding the soft tissues into powder in a high-speed grinder, weighing the powder, dissolving the powder in PBS, detecting changes of Cathelicidin LL-37, beta-defensin 2 (mBD-2), beta-defensin 3 (mBD-3) and beta-defensin 4 (mBD-4) at the tissue protein level by using an Elisa method, and carrying out statistical analysis.
Detection of bacterial count in skin surrounding mouse back material
To quantitatively evaluate the amount of bacteria in animal models, each group of subcutaneous insert pads was removed under aseptic conditions, and the samples were first gently washed twice with fresh PBS to remove zoobacteria. They were then immersed in 1ml PBS, sonicated at 150kHz for 10 min, then vortexed for 2 min to remove adherent bacteria, serially diluted in PBS solution, and 100 μl of diluent was evenly distributed on SBA in triplicate. In addition, soft tissue surrounding the implant was obtained, weighed, and homogenized in sterile tubes containing 1mL of PBS using a high-speed homogenizer (Shanghai Jingxin, inc., china), the soft tissue homogenate was serially diluted in PBS solution, 100 μl of diluent was uniformly distributed on the SBA, in triplicate. Finally, these plates were incubated overnight at 37℃and the number of bacterial colonies was counted and the surrounding tissue and the surface of the material were quantitatively analyzed, respectively.
Detection of bacterial Metabolic Capacity in skin around mouse Back Material
The subcutaneous insert discs of each group in the above experiments were collected, and the samples were first gently washed twice with fresh PBS to remove zoobacteria. Then, they were immersed in 1ml pbs, sonicated at 150kHz for 10 minutes, and then vortexed for 2 minutes to remove adherent bacteria. Extracting each group of bacterial RNA by centrifugation and PBS rinsing for 3 times, and detecting mRNA level changes of specific genes fnbA and fnbB formed by a biological film of staphylococcus aureus S.aureus by an rt-PCR method, wherein the changes of genes sucA and sucB are related to energy metabolism paths of staphylococcus aureus TCA/PTS; the agr quorum sensing system of staphylococcus aureus mainly comprises the changes of agrA and agrC, and the changes of sak and HtrA of the staphylococcus aureus for controlling the degradation of the biological membrane.
The results are shown in FIG. 7. After establishment of the mouse back subcutaneous infection model, the in vivo fluorescence detection system showed a gradual decrease in the mouse back fluorescence intensity at days 0, 1, 3, 7, 10, 14 as the number of LP01-PS surface load increased (fig. 7A and B). On day 14, the material of each group was removed from the back of the mice and the surfaces of the LP01-PS were sonicated, and S.aureus was isolated for each group. The quantitative analysis of the LP01-PS surface adhesion bacteria mRNA showed that S.aureus biofilm formation specific genes fnbA and fnbB, TCA/PTS energy metabolic pathway related genes sucA and sucB were expressed less, while the S.aureus agr population stress sensing system major genes agrA and agrC, S.aureus sak and HtrA genes promoting biofilm degradation were expressed more (FIG. 7C). The differences between groups are statistically significant. In addition, as the number of LP01 loaded on the surface of LP01-PS increased, the number of s.aureus in both the material surface adhesion bacteria and the skin tissue surrounding the material measured by the dilution plating method decreased (fig. 7D). The above results suggest that LP01-PS has a pronounced anti-S.aureus effect in vivo.
When the number of LP01 loaded on the surface of LP01-PS is increased, the protein secretion and mRNA expression of the antibacterial peptides LL-37, mBD2, mBD3, mBD4 are increased in the skin tissue around the material, and the difference is statistically significant (fig. 7E, fig. 7F). In addition, the scanning electron microscope results showed a reduced number of S.aureus for the LP01-PS group surface (FIG. 7G). HE staining, MPO immunohistochemistry, giemsa staining, and the results indicate that the more LP01 loaded on the surface of LP01-PS, the fewer inflammatory cells the surrounding skin infiltrates, the less inflammatory exudates, and the fewer bacteria. In the skin surrounding the 25LP01-PS group of materials, there was little inflammatory reaction and bacterial residue (fig. 7H). These results suggest that LP01-PS can increase the antibacterial ability of the skin surrounding it.
The results show that the LP01-PS has remarkable double antibacterial capability in vitro and in vivo. In vitro, the LP01-PS not only has direct inhibition effect on proliferation, metabolism, diffusion and virulence of S.aureus and E.coli, but also indirectly improves the killing effect of macrophages on the S.aureus and E.coli. In vivo, LP01-PS can promote increased secretion of LL-37, mBD-2, mBD-3, mBD-4 by surrounding skin tissue. It can be seen that LP01-PS not only directly antibacterial at the surface, but also enhances the immune antibacterial ability of macrophages or surrounding tissues, thereby effectively killing bacteria hidden in the surrounding tissues of the prosthesis.
Example 8
In vitro osteogenesis assay
LP01-PS promotes osteogenic differentiation of rBMSCs in vitro
A total of 5 groups of 1 10mm X1 mm discs per well, 3 complex wells per group, were filled with Con, PS, 1LP01-PS, 5LP01-PS and 25LP01-PS, respectively, in 24 well plates. According to 5X 10 per hole 5 rBMSCs were inoculated and each well contained 100U/ml penicillin500ul of DMEM cell culture solution containing 100U/ml streptomycin and 10% fetal bovine serum, and placed in 37 ℃ and 5% CO 2 Is cultured in a cell culture box. After 1 day of inoculation, most cells successfully grew on the wall, and then fresh cell culture medium was changed. Continuing to culture the cells, changing fresh cell culture medium every 2-3 days, collecting mesenchymal stem cells of each group, collecting cells at the time point of 14 th day, and detecting the change of the osteogenic related genes ALP, OCN, COL-1, BMP-2, OPN, RUNX2, BSP and OC in mRNA level by rt-PCR. Each group of cells was tested for mineralization capacity by ALP staining. Alizarin Red (ARS) staining was performed to assess extracellular calcium deposit formation. mRNA and protein level changes of Wnt/beta-catenin signal pathway related genes LRP-5, LRP-6, axin2 and beta-catenin of rBMSCs are detected by using rt-PCR, western blot and immunofluorescence method.
LP01-PS promotes osteogenic differentiation of rBMSCs in vitro through Wnt/beta-catenin signaling pathway
25LP01-PS was loaded into 24-well plates and divided into 3 groups of 1 10mm 1mm discs per well, 3 multiple wells per group. According to 5X 10 per hole 5 rBMSCs were inoculated with 500ul of DMEM cell culture medium containing 100U/ml penicillin, 100U/ml streptomycin and 10% fetal bovine serum per well, placed at 37℃in 5% CO 2 Is cultured in a cell culture box. After 1 day of inoculation, most cells successfully grew on the wall, and then fresh cell culture medium was changed. No chemical blocking agent was added to the 25LP01-PS group, and 20uM FH535,25LP01-PS+40uM FH535 was added to the 25LP01-PS+20uM FH535 group and 40uM FH535 group. Cells were collected on day 14 after establishment of the co-culture system, and changes in mRNA levels of osteogenic related genes ALP, OCN, COL-1, BMP-2, OPN, RUNX2, BSP, OC were detected by rt-PCR. Each group of cells was tested for mineralization capacity by ALP staining. Alizarin Red (ARS) staining was performed to assess extracellular calcium deposit formation.
In vivo osteogenesis experiments
In vivo study of LP01-PS to enhance osteogenic differentiation of rBMSCs
SD rat femur intercondylar implantation LP01-PS material for establishing animal bone formation model
According to the previous study, an animal osteogenic model was established between the femoral condyles of SD rats. The method comprises the following specific steps: after the mice are anesthetized by injecting 3% pentobarbital sodium into the abdominal cavity, shaving the lower limbs of the mice, thoroughly sterilizing, cutting a 1cm incision in the middle of the inner side of the right knee, punching a small hole with the diameter of 2mm between the femoral condyles of the right knee by an electric drill and a Kirschner wire, then respectively planting Con, PS, 1LP01-PS, 5LP01-PS and 25LP01-PS of different materials between the femoral condyles of each group, wherein the materials are cylinders with the diameter of 2mm multiplied by 10mm, 3 of each group, and then carefully suturing the wounds. The rats were kept free to obtain water and food for 8 weeks under Specific Pathogen Free (SPF) conditions.
Multicolor continuous fluorescent labeling method and Van Gieson staining method for determining SD rat osteogenesis
Those newly formed bone tissues were marked using a polychromatic continuous fluorescence marking method. Alizarin red S (30 mg/kg, AL, sigma-Aldrich, USA) and calcein (20 mg/kg, CA, sigma-Aldrich, USA) were injected intraperitoneally in sequence at weeks 4 and 6 post-operatively. 8 weeks after surgery, the rats were euthanized, the right femur with the rod removed, sawed to the appropriate size, and the femur was then fixed in 10% formaldehyde buffer for subsequent analysis. Next, these external femur were dehydrated with ethanol and embedded in methacrylate esters without decalcification. The sections were then fabricated on a sawing microtome (Leica SP1600, munich, germany) and polished to a thickness of about 50um and then observed by Confocal Laser Scanning Microscopy (CLSM). The sections were then histologically visualized by staining with van Gieson's picrofuchsin
Microscopic CT evaluation
The above prepared femur was scanned using Micro-CT (Skyscan 1172,Bruker Micro-CT, munich, germany) to study bone tissue changes around the built-in material of each group. The scan is performed at a maximum X-ray energy and 360 deg. range, 50kV, with a resolution of 18um. Three-dimensional (3D) images were reconstructed from Nrecon (Bruker micro CT, skyscan, munich, germany) and analyzed using the CTAn program (Bruker micro CT, skyscan, munich, germany). The surrounding area of the rod with a 0.5mm radius in the bone marrow cavity was selected to quantify newly formed bone. Then the Bone-material contact area percentage (Bone-implant contact,%) Bone Mineral Density (BMD), bone volume fraction (Bone volume/total volume, BV/TV,%) Bone small for new Bone Liang Shuliang (Tb.N, mm) -1 ) Statistical analysis was performed on bone trabecular thickness (tb.th, μm), bone trabecular separation (tb.sp).
Pull-out test (Pull-out test)
3 samples were taken per group 8 weeks after surgery, fixed on steel apparatus with steel wire, the test was performed at a loading rate of 1mm/min, and then the load-deflection curve during pullout was recorded. We define the failure load as the maximum load.
Real-time fluorescent quantitative PCR
Total RNA was isolated from the skin of the back of the experimental mice, RAW246.7, bacteria or rBMSCs using Trizol (12183555, invitrogen, california, USA), respectively, and cDNA was reverse transcribed according to the manufacturer's instructions (A48571, thermo Fisher Scientific Inc., waltham, USA). RT-PCR was performed on 96-well plates using SYBR Premix Ex Taq kit (RR 001B, taKaRa, dalia, china) at a final volume of 20uL using the rapid RT-PCR system (ABI 7500,Applied Biosystems,Carlsbad,USA). The PCR conditions were 94℃initial denaturation for 5min,36 cycles of 94℃denaturation for 30s, 40s annealing, 72℃extension for 40s, and finally 72℃extension for 5min. Obtaining a cycle threshold, normalizing the data to processing by GAPDH and according to 2 -ΔΔCt The relative expression level was calculated by the method. Each experiment was repeated three times. All primers were obtained from Sangon (Shanghai Shengong Biological Engineering Co., LTD. Shanghai, china).
Western blot analysis
The differently treated RAW246.7 or rBMSCs cells were lysed in Radioimmunoprecipitation (RIPA) buffer and PhosSTOP (4906837001, roche, basel, switzerland). Protein concentration was determined by BCA protein assay (P0010S, beyotime, shanghai, china), an equivalent amount of protein (20 μg) was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to PVDF membrane (GVHP 04700, millipore, massachusetts, USA). Immunostaining was performed using specific antibodies after blocking with 5% nonfat milk powder for 2 hours, including rabbit NF-kB/p65 polyclonal antibody (1:100dilution,ab209216,Abcam,San Francisco,USA), rabbit TLR2 monoclonal antibody (1:100dilution,ab32536,Abcam,San Francisco,USA, rabbit β -catenin monoclonal antibody (1:100 dilution, abcam, san francisco, USA), rabbit action polyclonal antibody (1:100 dilution, abcam, san francisco, USA) and rabbit GAPDH polyclonal antibody (1:500 dilution, abcam, san francisco, USA), and gently shaking overnight at 4 ℃, after washing, treatment with specific secondary antibody (1:100dilution,ab205718,Abcam,San Francisco,USA) was performed at 37 ℃ for 2 hours, immunolabeling detection was performed by enhancing chemiluminescent reagents from company Thermo Scientific, and quantitative analysis of signal intensity was performed using Scion Image beta4.02 (Scion Corporation, NIH, maryland, USA).
The results are shown in FIG. 8. After 14 days of coculture of LP01-PS with rBMSCs, the osteogenic differentiation major genes ALP, OCN, COL-1, BMP-2, OPN, RUNX2, BSP, OC and the like were significantly increased (FIG. 8A), the migration amount of rBMSCs to LP01-PS was increased (FIG. 8B), and ALP and ARS staining was also significantly deepened (FIG. 8C) with the increase of the enrichment amount of PDA and LP01 on the surface of the material. LP01-PS was implanted in the femur of the rat for 8 weeks, the maximum extraction force was increased (fig. 8D), micro-CT showed an increase in the surrounding new bone of the material (fig. 8E), sequential fluorescent staining of new bone showed an increase in calcium salt deposition at different time points (fig. 8F), and Van Gieson staining around the material showed an increase in new collagen fibers (fig. 8G). Further analysis showed that BMD, percent bone material contact area, BV/TV, tb.N, tb.Th increased and Tb.Sp decreased for the new bone (FIG. 8H). The above results suggest that LP01-PS promotes osteogenic differentiation of rBMSCs in vitro and the generation of new bone in vivo.
After 7 days of coculture of LP01-PS with rBMSCs, the cellular immunofluorescence intensity of β -catenin of rat rBMSCs was increased (FIG. 8I), WB protein expression was quantitatively increased (FIG. 8J), and mRNA expression of the main subunits LRP5, LRP6, axin2, β -catenin of WNT/β -catenin signaling pathway was increased (FIG. 8K). After the WNT/β -catenin signaling pathway chemical blocker treatment of rBMSCs, ALP of rBMSCs and ARS deposited with extracellular calcium salts became light (fig. 8L), the expression of osteogenic genes was decreased (fig. 8M). The above results suggest that LP01-PS promotes osteogenic differentiation in vitro through the WNT/beta-catenin signaling pathway of rBMSCs.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. An artificial joint prosthesis material LP01-PS, which is characterized in that the raw materials of the LP01-PS comprise polyether ether ketone, LP01 and polydopamine.
2. A method for preparing the prosthetic material LP01-PS of claim 1, comprising the steps of: sulfonating and hydrothermally treating polyether-ether-ketone to obtain sulfonated polyether-ether-ketone with porous surface; mixing sulfonated polyether-ether-ketone, polydopamine powder, LP01 powder and Tris solution to obtain a mixture, and shaking to synthesize the LP01-PS.
3. The method according to claim 2, wherein the polyetheretherketone is polyetheretherketone rod or polyetheretherketone disk.
4. A method of manufacture according to claim 3, wherein the polyetheretherketone rod has a diameter of 2mm and a length of 10mm.
5. A method of manufacture according to claim 3, wherein the polyetheretherketone disc has a diameter of 10mm and a thickness of 1mm.
6. The method of claim 2, wherein the steps of sulfonating and hydrothermally treating comprise: and (3) sulfonating the polyether-ether-ketone with 98% concentrated sulfuric acid at room temperature for 8min, and washing, and then, carrying out water bath at 120 ℃ for 4h to obtain the sulfonated polyether-ether-ketone with porous surface.
7. The method of claim 2, wherein the polydopamine concentration in the mixture is 1-25mg/ml and the LP01 concentration in the mixture is 1-25mg/ml.
8. The method according to claim 2, wherein the molar concentration of the Tris solution is 0.01M and the pH of the Tris solution is 8.5.
9. The method according to claim 2, wherein the rotational speed of the shaking synthesis is 5rpm for 1 hour.
10. The method of claim 2, wherein unbound and loosely bound particles are removed after shaking synthesis.
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