CN111116964A

CN111116964A - Biological functional surface modified polyether-ether-ketone material and preparation method and application thereof

Info

Publication number: CN111116964A
Application number: CN201911301351.5A
Authority: CN
Inventors: 聂彬恩; 岳冰; 霍市城; 王友
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2020-05-08

Abstract

The invention carries out biological functional modification on the orthopedic biomaterial PEEK sheet by combining a strong acid acidification method, and endows the orthopedic biomaterial PEEK sheet with biological activity of promoting osteogenic differentiation and bone mineralization of bone marrow mesenchymal stem cells and inhibiting secretion of inflammatory factors. The PEEK surface modification method is simple and low in cost, and the prepared PEEK implant has multiple biological functions, and is expected to promote in-vivo osseointegration of the PEEK orthopedic implant and reduce related complications of loosening caused by early excessive inflammatory reaction after the PEEK sheet is implanted into a body.

Description

Biological functional surface modified polyether-ether-ketone material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical polymer materials, and particularly relates to a bio-functional surface modified polyether-ether-ketone material, and a preparation method and application thereof.

Background

A perfect orthopaedics implant is implanted into the implant, and the occurrence of complications is avoided. Goodman (GoodmanSB, Biomaterials 2013,34(13):3174-3183) proposed orthopaedic biocoating implants to emphasize enhanced osteointegration and reduce chronic inflammatory responses in the future of coatings. Korean (Han, Biomaterials, 2010.31 (13): p.3465-70.) suggests that excellent orthopaedic prostheses not only have the potential to promote bone tissue growth, but also have good biocompatibility. In conclusion, the functional modification of the orthopedic implant surface is required not only to inhibit the inflammatory response around the implant, but also to enhance the osseointegration of the implant.

At present, in-vitro experiments for constructing the orthopaedic prosthesis are mostly limited to the research on bone formation promoting performance, but the reaction of an organism to the prosthesis is very complex, the bone formation promoting effect of in-vitro experiments for the modified orthopaedic prosthesis is better, but in-vivo bone integration experiments are not ideal, and the main reason is that an in-vivo environment is difficult to simulate in vitro. The inflammatory response of the body to endophytes is considered to be an important factor affecting the in vivo performance of the prosthesis, and the design based on this biomaterial should be based on avoiding the inflammatory immune response of the host. Meanwhile, the existing bone union research for evaluating the internal plant material mainly adopts a single-factor culture model of the material and bone marrow mesenchymal stem cells or osteoblasts stimulated by osteogenic induction liquid to evaluate through osteogenic differentiation indexes (expression of osteogenic related genes such as alkaline phosphatase, alizarin red, bone morphogenetic protein-2 and the like), and does not consider the inherent inflammatory reaction of the internal implant and the biologically functionalized internal implant to a host. The host inflammatory response to endophytes can greatly affect the in vivo performance of the implant, particularly osteointegration, i.e., osteogenic differentiation of mesenchymal stem cells.

Polyetheretherketone (PEEK) is a semi-crystalline linear polycyclic aromatic thermoplastic that was first developed by England scientists in 1978 (Ma, Int J Mol Sci. 2014; 15(4): 5426-. The molecular structure of PEEK takes aromatic molecules as a main body and ketone and ester functional groups are connected between aromatic rings, and the special chemical structure ensures that the PEEK shows stable physical and chemical properties, such as high temperature resistance, wear resistance, combustion resistance, hydrolysis resistance, chemical resistance and the like (Luo, J Mech Behav Biomed Mater.2014; 29: 103-113.12-15; Kurtz SM, biomaterials.2007; 28(32): 4845-, lack of biological activity, and can not be well integrated with host bone after being implanted into the body, thus greatly limiting the wide application of the traditional Chinese medicine in clinic.

Therefore, the surface modification of the polyetheretherketone biomaterial should promote the polyetheretherketone osseointegration and inhibit the inflammatory reaction, but most of the current functional materials applied to orthopedics have single performance of promoting the osseointegration or inhibiting the inflammatory reaction. Few orthopedic materials take into account the intrinsic inflammatory response of the host, not to mention the multiple functional materials that promote osteogenic differentiation, osteointegration and inhibit inflammatory response.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the biological inertia problem of a polyether-ether-ketone (PEEK) material, endowing the PEEK material with various biological functions through surface modification, and providing a bio-functional surface-modified PEEK material, and a preparation method and application thereof.

In order to solve the technical problems, the invention provides a preparation method of a bio-functional surface modified polyetheretherketone material, which is characterized by comprising the following steps: the method comprises the following steps: and soaking the polyether-ether-ketone material in strong acid for acidification, then soaking in deionized water to remove redundant acid, then ultrasonically cleaning with deionized water, and drying to obtain the surface-modified polyether-ether-ketone material.

Preferably, the strong acid is hydrofluoric acid (HF), nitric acid (HNO)₃) Or HF and HNO₃One of mixed acid in equal proportion, the HF and the HNO₃Are all AR grade.

Preferably, the preparation method of the surface modified polyetheretherketone material specifically comprises the following steps: soaking the polyether-ether-ketone material in a strong acid solution for 0.5h, then soaking in deionized water for 24h to remove redundant acid, then ultrasonically cleaning with deionized water for 3 times, each time for 20min, and drying to obtain the surface-modified polyether-ether-ketone material.

The invention also provides the bio-functional surface modified polyetheretherketone material prepared by the method.

Preferably, the bio-functional surface modified polyetheretherketone material has the properties of promoting osteogenic differentiation and bone mineralization and inhibiting inflammatory reactions.

More preferably, the promotion of osteogenic differentiation and bone mineralization is the promotion of differentiation of bone marrow mesenchymal stem cells into osteoblasts and mineralization of an extracellular matrix of bone.

More preferably, the inhibition of the inflammatory response is inhibition of macrophage inflammatory response, specifically inhibition of the expression of inflammatory factors tumor necrosis factor (TNF- α), interleukin-1 β (IL-1 β), Inducible Nitric Oxide Synthase (INOS) and related inflammatory genes.

Further, the macrophage is a macrophage obtained by culturing human peripheral blood mononuclear cells in vitro for one week or under the induction of macrophage colony stimulating factor, or inducing a human peripheral blood mononuclear cell line (THP-1 cell line) for 1 day by propylene glycol monomethyl ether acetate (Phorbol-12-christate-13-acetate, PMA).

The invention also provides application of the bio-functionalized surface modified polyether-ether-ketone material in preparation of an orthopaedic prosthesis.

The multifunctional biofunctionalized orthopaedic prosthesis is characterized by being prepared from the polyether-ether-ketone material with the biofunctionalized surface modified, and has the following biological activities: promoting osseointegration while inhibiting inflammatory responses.

Compared with the prior art, the invention has the beneficial effects that:

(1) the PEEK material is modified in biological activity, namely the surface appearance of the PEEK material is changed through strong acid acidification, the PEEK material has anti-inflammatory reaction and can promote osteogenic differentiation of bone marrow mesenchymal stem cells.

(2) The invention provides a PEEK prosthesis with various biological activities and a preparation method thereof, belonging to the technical field of medical materials.

(3) The acid used in the invention is strong acid, and the invention has wide application in the field of material science, and the invention has simple preparation process, high efficiency and good repeatability. The PEEK material acidified by the combined strong acid provided by the invention has good stability and various biological properties, and can overcome the defects that the conventional PEEK material lacks biological activity in clinic application, cannot be well osseointegrated with host bones after being implanted into a body, and the like.

Drawings

FIG. 1: surface appearance change and water contact angle representation before and after PEEK acidification modification; wherein, fig. 1A is a scanning electron microscope image of surface morphology before and after PEEK acidification modification, fig. 1B is a measurement diagram of water contact angle before and after PEEK acidification modification, and fig. 1C is a bar graph of water contact angle measurement before and after PEEK acidification modification;

FIG. 2: after the PEEK is acidified and modified, the rat bone marrow mesenchymal stem cells are adhered and proliferated at different time points; wherein 2A is 2h, 4h and 6h adhesion assay, 2B is 1d, 3d and 5d cell proliferation assay;

FIG. 3: CLSM observation of the expansion condition of rat mesenchymal stem cells on the surface of the material after 24h of co-culture;

FIG. 4: biological performance experiments of PEEK before and after strong acid acidification modification: an alkaline phosphatase staining chart of osteoblast differentiation of rat bone marrow mesenchymal stem cells and quantitative comparison thereof; wherein, FIG. 4A is a staining chart, and FIG. 4B is a bar chart;

FIG. 5: carrying out strong acid acidification modification on bone mineralization alizarin red staining patterns of PEEK before and after modification and carrying out quantitative comparison on the staining patterns; wherein, FIG. 5A is a staining chart, and FIG. 5B is a bar chart;

FIG. 6 shows the secretion levels of inflammatory factors IL-1 β -6, TNF- α, and IL-8 after co-culture of PEEK with macrophages before and after modification by strong acid acidification;

wherein PEEK which has not been acid-treated is designated as Bare PEEK; nitric acid treatment of PEEK is designated PEEK-AN; the hydrofluoric acid treated polyetheretherketone is designated PEEK-AF; a mixed acid treatment of polyetheretherketone with nitric acid and hydrofluoric acid is known as PEEK-AFN.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

The invention provides a preparation method of a surface modified polyether-ether-ketone material, which specifically comprises the following steps:

soaking PEEK (10 mm in diameter and 2mm in thickness; Jiangsu Junhuate engineering plastics Co., Ltd.) in different acids for 0.5h for acidification treatment, then soaking in deionized water for 24h to remove redundant acid, then ultrasonically cleaning with deionized water for 3 times, each time for 20min, and airing at room temperature to obtain the surface modified polyether-ether-ketone material.

The nitric acid treated polyetheretherketone is designated PEEK-AN, the hydrofluoric acid treated polyetheretherketone is designated PEEK-AF, the nitric acid and hydrofluoric acid treated polyetheretherketone is designated PEEK-AFN, and Bare PEEK is not acid treated.

Characterization of PEEK surface morphology after strong acid modification:

the experiment is divided into four groups, namely Bare PEEK (common PEEK), PEEK-AF (PEEK acidified by hydrofluoric acid), PEEK-AN (PEEK acidified by nitric acid), PEEK-AFN (PEEK acidified by mixed acid of hydrofluoric acid and nitric acid in equal proportion);

each group of PEEK was sterilized and placed in a 24-well plate with 3 multiple wells per group, and PEEK was fixed overnight with 2.5% glutaraldehyde for SEM characterization; each PEEK was dehydrated through a series of graded ethanol solutions (30%, 50%, 70%, 80%, 90%, 100% and 100%); the PEEK samples were subsequently freeze dried, sputter coated with platinum, and observed using a scanning electron microscope (JEOL JSM-6700F, japan).

As shown in FIG. 1A, all PEEK materials have slightly different surface morphologies, and the ordinary PEEK has the smoothest surface.

Measurement of water contact angle of PEEK after modification by strong acid acidification:

static contact angles were measured on un-acidified PEEK and strongly acid treated PEEK by titration with 2mL double distilled water at room temperature.

The reduction in PEEK contact angle to different levels after acidification with strong acid, as shown in fig. 1B, 1C, indicates that the combination with strong acid acidification significantly increases the hydrophilicity of PEEK.

The cells adopted in the experiment are rat bone marrow mesenchymal stem cells, but other rat adult stem cells (adipose mesenchymal stem cells, endothelial progenitor cells and the like) have osteogenic differentiation potential and are also suitable for the experiment.

The study on the adhesion and proliferation of the mesenchymal stem cells:

bone marrow mesenchymal stem cells (rBMSCs) collected from rat tibia and femur were suspended in a minimal essential medium containing 10% fetal bovine serum and cultured at 37 ℃ in a humidified incubator with 5% CO2 and 95% air. Growth medium was changed every three days and third generation rbmscs were used for the experiment.

after disinfection and sterilization, all groups of PEEK are placed in a 24-hole plate, and each group is provided with 3 multiple holes; third generation Rat bone marrow mesenchymal stem cells (rBMSC) were seeded on each group of PEEK with controlled cell density (5X 10)⁴Per mL), 1mL per well; at 2h, 4h, 6h, 1d, 3d and 5d post inoculation, each PEEK sample was washed with PBS; adhesion and proliferation of cells were studied at predetermined time points using the CCK-8 method.

As shown in fig. 2A, adhesion tests were performed at 2h, 4h and 6h, respectively, and the cell adhesion efficiency of different PEEK materials was similar; as shown in FIG. 2B, PEEK-AFN group promoted cell proliferation at various time points in the cell proliferation experiments performed at 1d, 3d and 5d, respectively.

Morphological study of mesenchymal stem cells:

after disinfection and sterilization, all groups of PEEK are placed in a 24-hole plate, and each group is provided with 3 multiple holes; third generation Rat bone marrow mesenchymal stem cells (rBMSC) were seeded on each group of PEEK with controlled cell density (5X 10)⁴Per mL), 1mL per well; after 24 and 48 hours post-inoculation, each PEEK sample was washed with PBS and fixed with 4% paraformaldehyde. Subsequently, it was immersed in 0.1% Triton X-100 for 15 minutes, and then gently rinsed with PBS (pH 7.4). The PEEK samples were then immersed in a 4', 6' -dimido-2-phenylindole (DAPI) solution (5. mu.g/ml) for 15 minutes for nuclear staining. Cytoskeleton was stained with phalloidin for 30 minutes according to the manufacturer's protocol, and then the morphology of the cells was analyzed with a confocal microscope (Leica TCS SP 2; germany).

As shown in fig. 3, all groups showed the best cell attachment. Cell growth and diffusion on PEEK-AFN is more active than in the Bare-PEEK group. In addition, the cell morphology of the Bare-PEEK group was circular, while the cells of the PEEK-AN and PEEK-AF groups showed triangular or polygonal shapes. In addition, the cells of the PEEK-AFN group were polygonal, with cells stretching well.

Bone marrow mesenchymal stem cell osteogenic differentiation research:

after disinfection and sterilization, all groups of PEEK are placed in a 24-hole plate, and each group is provided with 3 multiple holes; third generation Rat bone marrow mesenchymal stem cells (rBMSC) were seeded on various groups of PEEK materials with controlled cell density (5X 10)⁴Per mL), 1mL per well; 37 ℃ and 5% CO₂Culturing in a cell culture box for 24 hours under the condition to reach cell adherence, and replacing the cell culture medium with alpha-MEM containing osteogenic inducing liquidThe culture medium (osteogenesis inducing liquid: 10% fetal bovine serum culture liquid added with 50 μ M vitamin C, 10mM β -sodium glycerophosphate and 100nM dexamethasone), then, changing liquid every two days, carrying out alkaline phosphatase staining to evaluate the early differentiation of rat bone marrow mesenchymal stem cells osteoblasts 7 days and 14 days later, and carrying out alizarin red staining to evaluate the mineralization of extracellular matrix to obtain the bone late differentiation mark after 14 days and 25 days later.

Alkaline phosphatase staining: after 7 days of co-culture of the PEEK materials of each group with the RBMMSCs, the osteogenic medium was removed and the PEEK of each group was removed and ALP stained according to the ALP staining kit (petun sky): after 3 times of PBS light washing, putting into a fixing solution and fixing for 30s at 4 ℃; washing with PBS for 3 times, adding working solution, and placing in 37 deg.C water bath for 45 min; finally, the working solution was aspirated, washed 3 times with PBS, and dried to take a picture.

As shown in FIG. 4A, ALP stains more deeply in PEEK-AFN than Bare PEEK. ALP activity was significantly higher on day 7 in combination with the strongly acidic acidized PEEK material than the non-acidified PEEK material. Fig. 4B is alkaline phosphatase quantification: at day 7, PEEK-AFN had higher alkaline phosphatase activity compared to barre PEEK and had a clear statistical significance (. about.p < 0.001). It is shown that treating PEEK material in combination with strong acid acidification can promote osteogenic early differentiation by enhancing the activity of alkaline phosphatase.

Alizarin red staining: the experiment was also divided into four groups and placed in a 24-well plate with three multiple wells per well, and the mineralization of the extracellular matrix of the cell osteoblasts was observed by alizarin red staining. Terminating the culture at 10d and 14d, sucking out the culture medium, performing PBS light washing for 3 times, fixing for 10min by using 95% ethanol, and performing light washing for 3 times; adding 0.1% alizarin red solution into 48-well plate, and placing in 37 deg.C water bath tank for 45 min; and (5) repeatedly carrying out light washing by double distilled water to remove redundant dye liquor, and finally airing and taking a picture.

As shown in fig. 5, mineralized nodules were formed in each acidified PEEK material at each time point higher than in the other non-acidified PEEK groups. Alizarin red quantification: at day 14, PEEK-AFN, PEEK-AF, PEEK-AN compared to PEEK P < 0.05; at day 25, PEEK-AFN, PEEK-AN compared to PEEK. P < 0.05. The fact that the extracellular mineralization mechanism can be remarkably promoted by combining strong acid acidification treatment of the PEEK material is demonstrated.

Macrophage inflammatory response:

inducing human peripheral blood mononuclear cell line THP-1 cell with (phosphor-12-christate-13-acetate, PMA) for 1 day as macrophage source, sterilizing the materials, placing at the bottom of 24-well plate, and controlling cell density at 2 × 10⁴Inoculating on each group of surfaces in the presence of 5% CO₂Culturing in a 37 ℃ incubator, extracting cell RNA on the 1 st day and the 3 rd day respectively without adding a medicament for inducing macrophage inflammatory reaction in a culture medium, and detecting the expression of the gene level of the inflammatory factor by reverse transcription and real-time quantitative PCR technology.

FIG. 6 shows the secretion levels of inflammatory factors IL-1 β -6, TNF- α and IL-8 after co-culture of PEEK with macrophages in each group, and from the results, it can be seen that the non-acidified PEEK secretes more IL-1 β -6, TNF- α and IL-8, and the treatment of PEEK by acidification combined with a strong acid inhibits the secretion of IL-1 β -6, TNF- α and IL-8, PEEK-AFN has P < 0.05 compared to the Bare-PEEK group.

In conclusion, the PEEK acidified by the strong acid provided by the invention has various biological properties, promotes osteogenic differentiation of mesenchymal stem cells and inhibits macrophage inflammatory reaction, and can overcome the defect that the current PEEK is clinically applied. The invention has simple process, high efficiency and good repeatability, and the prepared orthopedic prosthesis not only has the potential of promoting the growth of bone tissues, but also has good biocompatibility.

Claims

1. A preparation method of a bio-functional surface modified polyetheretherketone material is characterized by comprising the following steps: and soaking the polyether-ether-ketone material in strong acid for acidification, then soaking in deionized water to remove redundant acid, then ultrasonically cleaning with deionized water, and drying at room temperature to obtain the surface-modified polyether-ether-ketone material.

2. Biological work as claimed in claim 1The preparation method of the surface-modified polyether-ether-ketone material is characterized in that the strong acid is HF and HNO₃Or HF and HNO₃One of mixed acid in equal proportion, the HF and the HNO₃Are all AR grade.

3. The method for preparing a bio-functionalized surface-modified polyetheretherketone material according to claim 1, wherein the method comprises: soaking the polyether-ether-ketone material in a strong acid solution for 0.5h, then soaking in deionized water for 24h to remove redundant acid, then ultrasonically cleaning with deionized water for 3 times, each time for 20min, and drying to obtain the surface-modified polyether-ether-ketone material.

4. A biofunctionalized surface-modified polyetheretherketone material prepared by the method of any one of claims 1 to 3.

5. The bio-functional surface modified polyetheretherketone material according to claim 4, wherein the surface modified polyetheretherketone material has properties to promote osteogenic differentiation and bone mineralization and to inhibit inflammatory reactions.

6. The bio-functional surface modified polyetheretherketone material of claim 5, wherein the promotion of osteogenic differentiation and bone mineralization is the promotion of differentiation of mesenchymal stem cells into osteoblasts and mineralization of the extracellular matrix of bone.

7. The bio-functional surface modified polyetheretherketone material according to claim 5 wherein the inhibition of inflammatory response is inhibition of macrophage inflammatory response, in particular inhibition of the expression of TNF- α -1 β.

8. The bio-functional surface-modified polyetheretherketone material of claim 7 wherein the macrophages are human peripheral blood mononuclear cells cultured in vitro for one week or induced by macrophage colony stimulating factor, or human peripheral blood mononuclear cells induced by propylene glycol monomethyl ether acetate for one day.

9. Use of the biofunctionalized surface-modified polyetheretherketone material of claim 4 in the preparation of an orthopaedic prosthesis.

10. A multi-biofunctionalized orthopaedic prosthesis, prepared from a biofunctionalized surface-modified polyetheretherketone material according to claim 4, having the following biological activities: promoting osseointegration while inhibiting inflammatory responses.