RGD-OGP bacteriophage nanofiber, difunctional RGD-OGP bacteriophage modified 3D printing porous titanium alloy stent and preparation method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering and 3D printing titanium alloy stent preparation, and particularly relates to RGD-OGP bacteriophage nanofibers, a bifunctional RGD-OGP-bacteriophage modified 3D printing porous titanium alloy stent and a preparation method and application of the bifunctional RGD-OGP-bacteriophage modified 3D printing porous titanium alloy stent. Based on a bacteriophage display technology, a genome of a filamentous bacteriophage is edited, bifunctional RGD-OGP polypeptide with osteogenesis promoting and adhesion promoting effects is displayed on pVIII protein of the bacteriophage, and the RO bacteriophage nanofiber with the surface containing thousands of RO polypeptides is prepared. On the basis, the RGD-OGP bacteriophage nanofiber is connected to the surface of the 3D printing porous titanium alloy stent in a filling mode, the bifunctional RO-bacteriophage modified 3D printing porous titanium alloy stent is prepared, the bone performance of the bifunctional RO-bacteriophage modified 3D printing porous titanium alloy stent is obviously improved compared with that of a conventional 3D printing porous titanium alloy stent, and a new choice is provided for solving the clinical bone defect repair problem.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and 3D printed titanium alloy stent preparation, and specifically relates to an RGD-OGP bacteriophage nanofiber, a bifunctional RGD-OGP-bacteriophage modified 3D printed porous titanium alloy stent, and a preparation method and application thereof. Background Art

[0002] In clinical practice, bone defects caused by trauma, infection, bone disease, and pathological fractures have always been a problem that plagues clinicians. Titanium alloys have been widely used in clinical practice as orthopedic implants due to their low density, high specific strength, good corrosion resistance, and good biocompatibility. However, the elastic modulus of solid titanium alloys does not match that of normal bone tissue, and the "stress shielding" effect is prone to occur, causing bone stress absorption around the implant, which ultimately leads to aseptic loosening of the implant.

[0003] At present, porous titanium alloy scaffolds prepared by 3D printing technology can well overcome the defect of mismatch between the mechanical properties of solid titanium alloy and bone tissue while ensuring sufficient support, and have been used in clinical practice with good results. However, no matter what form titanium alloy exists in, its biological inertness cannot be completely overcome, which makes it only play a supporting role to a large extent, but cannot achieve material-bone interface biofusion. Therefore, functionalizing the surface of 3D printed porous titanium alloy scaffolds to improve their osteogenic properties is currently a hot topic in clinical orthopedics.

[0004] Osteogenie Growth Peptide (OGP) is an endogenous 14-mer peptide present in serum. It was first isolated and purified from the culture medium of bone marrow regeneration after injury in the early 1990s. Over the years, people have conducted many studies on its biochemical and physiological properties. At present, the research on OGP in promoting osteogenesis and hematopoiesis is close to the clinical application stage. However, there is no relevant report in the field on how to apply OGP to 3D printing technology to improve the surface functionalization of titanium alloy scaffolds and improve the osteogenic performance of titanium alloy scaffolds. Summary of the invention

[0005] The purpose of the present invention is to provide an RGD-OGP phage nanofiber, a bifunctional RGD-OGP phage modified 3D printed porous titanium alloy scaffold, and a preparation method and application thereof. The osteogenic performance of the bifunctional RGD-OGP phage modified 3D printed porous titanium alloy scaffold prepared based on RGD-OGP phage nanofiber is significantly improved.

[0006] The present invention provides a method for preparing RGD-OGP bacteriophage nanofibers, comprising: displaying an RGD-OGP polypeptide on a pVIII protein of a filamentous bacteriophage through gene editing to obtain the RGD-OGP bacteriophage nanofibers, wherein the amino acid sequence of the RGD-OGP polypeptide is shown in SEQ ID NO.1.

[0007] Preferably, the filamentous phage comprises M13 phage.

[0008] Preferably, the step of displaying the RGD-OGP polypeptide on the pVIII protein of the filamentous phage through gene editing includes: inserting the nucleotide sequence encoding the RGD-OGP polypeptide into the gene VIII position of the M13 phage genome, expressing the obtained recombinant phage, and obtaining the RGD-OGP phage nanofiber.

[0009] The present invention also provides RGD-OGP bacteriophage nanofibers prepared by the preparation method described in the above technical solution.

[0010] The present invention also provides the use of the RGD-OGP bacteriophage nanofibers described in the above technical solution in modifying a 3D printed porous titanium alloy scaffold.

[0011] The present invention also provides a dual-functional RGD-OGP bacteriophage modified 3D printed porous titanium alloy stent, comprising a 3D printed porous titanium alloy stent and RGD-OGP bacteriophage nanofibers loaded onto the surface of the 3D printed porous titanium alloy stent;

[0012] The RGD-OGP bacteriophage nanofiber is the RGD-OGP bacteriophage nanofiber described in the above technical solution.

[0013] The present invention also provides a method for preparing the bifunctional RGD-OGP phage modified 3D printed porous titanium alloy stent described in the above technical solution, comprising the following steps:

[0014] The 3D printed porous titanium alloy stent is subjected to ultrasonic cleaning and surface plasma treatment, and then mixed with poly-lysine for a first incubation to obtain a poly-lysine titanium alloy stent;

[0015] The poly-lysine titanium alloy scaffold is incubated in the RGD-OGP bacteriophage nanofiber solution for a second time to obtain the bifunctional RGD-OGP bacteriophage modified 3D printed porous titanium alloy scaffold.

[0016] Preferably, the concentration of the RGD-OGP bacteriophage nanofiber solution is 1 mg / mL.

[0017] Preferably, the conditions of the first incubation and the second incubation are respectively incubation with shaking at room temperature for 12-24 hours.

[0018] The present invention also provides the use of the RGD-OGP phage nanofibers described in the above technical scheme, the bifunctional RGD-OGP phage-modified 3D printed porous titanium alloy scaffold described in the above technical scheme, or the bifunctional RGD-OGP-phage-modified 3D printed porous titanium alloy scaffold prepared by the preparation method described in the above technical scheme in the preparation of medical devices for treating bone defects.

[0019] Beneficial effects:

[0020] The present invention provides a method for preparing RGD-OGP phage nanofibers. The present invention is based on phage display technology. By editing the genome of filamentous phage, a bifunctional RGD-OGP polypeptide (abbreviated as RO) with osteogenesis and adhesion promoting effects is displayed on the pVIII protein of the phage, and RO phage nanofibers with thousands of RO polypeptides on the surface are prepared, namely RO-phage.

[0021] On this basis, the present invention also provides a method for preparing a bifunctional RGD-OGP-phage modified 3D printed porous titanium alloy scaffold based on RGD-OGP phage nanofibers, that is, the RGD-OGP phage nanofibers are filled and connected to the surface of the 3D printed porous titanium alloy scaffold to prepare a bifunctional RO-phage modified 3D printed porous titanium alloy scaffold, and the bone performance of the scaffold is significantly improved compared with that of conventional 3D printed porous titanium alloy scaffolds, providing a new option for solving the problem of clinical bone defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 Schematic diagram of the genetic engineering of the bacteriophage in Example 1;

[0024] Figure 2 This is a diagram of the gene sequencing results of RO-phage in Example 1;

[0025] Figure 3 TEM characterization results of RO-phage in Example 1;

[0026] Figure 4 Schematic diagram of the process of loading RO-phage onto a 3D printed porous titanium alloy stent in Example 2;

[0027] Figure 5 This is a typical morphology of the RO-phage loaded 3D printed porous titanium alloy stent in Example 2;

[0028] Figure 6 The in vitro osteogenic performance of the RO-phage loaded 3D printed porous titanium alloy scaffold in Example 3;

[0029] Figure 7 These are the results of the animal implantation test of the RO-phage loaded 3D printed porous titanium alloy scaffold in Example 4, wherein a is a diagram of the material implantation operation at the distal end of the rabbit femur, b is a gross diagram of the ex vivo femur of the rabbits in each material group at 6 weeks, and c is a micro-CT scan of the ex vivo femur of each material group at 6 weeks. DETAILED DESCRIPTION

[0030] The present invention provides a method for preparing RGD-OGP bacteriophage nanofibers, comprising: displaying an RGD-OGP polypeptide on a pVIII protein of a filamentous bacteriophage through gene editing to obtain the RGD-OGP bacteriophage nanofibers, wherein the amino acid sequence of the RGD-OGP polypeptide is shown in SEQ ID NO.1.

[0031] The amino acid sequence shown in SEQ ID NO.1 of the present invention is specifically RGDYGFGG. The filamentous phage of the present invention preferably includes M13 phage. M13 phage is a nanofiber virus composed of 5 capsid proteins encapsulating a single-stranded circular genome. Capsid proteins include pill, pVI, pVII, pVIII and pIX, wherein 5 copies of pill and pVI proteins are at one end of the phage, 5 copies of pVII and pIX proteins are at the other end of the phage, and thousands of copies of pVIII proteins are on the side wall of the phage. The present invention edits the genome of M13 phage, displays the RGD-OGP polypeptide on the pVIII protein of the phage, and prepares RO phage nanofibers containing thousands of RO polypeptides on the surface, i.e., RO-phage.

[0032] The method of displaying the RGD-OGP polypeptide on the pVIII protein of the filamentous phage by gene editing of the present invention preferably comprises the following steps: inserting the nucleotide sequence encoding the RGD-OGP polypeptide into the gene VIII position of the M13 phage genome, expressing the obtained recombinant phage, and obtaining the RGD-OGP phage nanofiber. The present invention does not specifically limit the method for preparing the recombinant phage, and the conventional gene editing steps in the art can be used. The present invention does not specifically limit the expression method, and the steps in the conventional phage display technology in the art can be used. The nucleotide sequence encoding the RGD-OGP polypeptide of the present invention is preferably as shown in SEQ ID NO.2, specifically 5'-CGTGGCGACTACGGCTTCGGTGGC-3'.

[0033] The present invention also provides RGD-OGP bacteriophage nanofibers prepared by the preparation method described in the above technical solution. Through TEM characterization, it is found that the RGD-OGP bacteriophage nanofibers have a nanofiber-like morphology.

[0034] The present invention also provides the use of the RGD-OGP bacteriophage nanofibers described in the above technical solution in modifying a 3D printed titanium alloy stent. The 3D printed titanium alloy stent described in the present invention is preferably a 3D printed porous titanium alloy stent.

[0035] Specifically, the present invention also provides a dual-functional RGD-OGP bacteriophage modified 3D printed porous titanium alloy stent, comprising a 3D printed porous titanium alloy stent and RGD-OGP bacteriophage nanofibers loaded onto the surface of the 3D printed porous titanium alloy stent;

[0036] The RGD-OGP bacteriophage nanofiber is the RGD-OGP bacteriophage nanofiber described in the above technical solution.

[0037] The present invention also provides a method for preparing the bifunctional RGD-OGP-phage modified 3D printed porous titanium alloy stent described in the above technical solution, comprising the following steps:

[0038] The 3D printed porous titanium alloy stent is subjected to ultrasonic cleaning and surface plasma treatment, and then mixed with poly-lysine for a first incubation to obtain a poly-lysine titanium alloy stent;

[0039] The poly-lysine titanium alloy scaffold is incubated in the RGD-OGP bacteriophage nanofiber solution for a second time to obtain the bifunctional RGD-OGP-phage modified 3D printed porous titanium alloy scaffold.

[0040] The present invention ultrasonically cleans and plasma-treats the titanium alloy stent and then mixes it with poly-lysine for a first incubation to obtain a poly-lysine titanium alloy stent. The present invention preferably ultrasonically cleans the titanium alloy stent in ethanol and ultrapure water in sequence, and dries it after the ultrasonic cleaning. The present invention does not specifically limit the specific parameters of the ultrasonic cleaning and drying, and the parameters for conventional cleaning of titanium alloy stents in the art can be used. The average pore size of the 3D printed porous titanium alloy stent of the present invention is preferably 500-700μm, the pillar size is preferably 400μm, and the porosity is preferably 70%-80%.

[0041] The first incubation condition of the present invention is preferably incubated with shaking at room temperature for 12-24 hours, more preferably incubated overnight.

[0042] In the present invention, after the first incubation, the titanium alloy stent after the first incubation is preferably cleaned and dried with ultrapure water. The ultrapure water cleaning and drying process is not particularly limited and can be a conventional ultrapure water cleaning and drying process in the art.

[0043] After obtaining the poly-lysine titanium alloy stent, the present invention performs a second incubation of the poly-lysine titanium alloy stent in the RGD-OGP bacteriophage nanofiber solution to obtain the bifunctional RGD-OGP bacteriophage modified 3D printed porous titanium alloy stent. The final concentration of the RGD-OGP bacteriophage nanofiber solution of the present invention is preferably 1 mg / mL. The conditions of the second incubation of the present invention are preferably room temperature oscillation incubation for 12-24 hours, more preferably incubation overnight. The present invention of the present invention preferably performs ultrapure water washing and drying on the titanium alloy stent after the second incubation after the second incubation. The process of ultrapure water washing and drying is not particularly limited, and the conventional ultrapure water washing and drying process in the art can be used.

[0044] The RGD peptide of the present invention has the function of promoting adhesion, and the OGP peptide has the function of promoting bone formation. The two peptides are combined for phage display to prepare RGD-OGP phage nanofibers. The RGD-OGP phage nanofibers are filled and connected to the surface of a 3D printed porous titanium alloy scaffold to prepare a bifunctional RO-phage modified 3D printed porous titanium alloy scaffold, and its bone formation performance is significantly improved compared with that of a conventional 3D porous titanium alloy scaffold.

[0045] Based on the above advantages, the present invention also provides the use of the RGD-OGP bacteriophage nanofibers described in the above technical solution, the bifunctional RGD-OGP bacteriophage modified 3D printed porous titanium alloy scaffolds described in the above technical solution, or the bifunctional RGD-OGP-bacteriophage modified 3D printed porous titanium alloy scaffolds prepared by the preparation method described in the above technical solution in the preparation of medical devices for treating bone defects. The bifunctional RGD-OGP-bacteriophage modified 3D printed porous titanium alloy scaffolds described in the present invention are preferably used as orthopedic implants to treat bone defects.

[0046] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1

[0048] A method for preparing RGD-OGP bacteriophage nanofibers, comprising the following steps:

[0049] 1. The M13 phage is a nanofiber virus composed of a single-stranded circular genome wrapped by five capsid proteins. The capsid proteins include pill, pVI, pVII, pVIII and pIX, of which five copies of pill and pVI proteins are at one end of the phage, five copies of pVII and pIX proteins are at the other end of the phage, and thousands of copies of pVIII protein are on the side wall of the phage. In this study, the genome of the M13 phage was edited, and the RGD-OGP polypeptide was displayed on the pVIII protein of the phage through gene editing, and RO phage nanofibers with thousands of RO polypeptides on the surface were prepared, namely RO-phage, in which Figure 1 Schematic diagram of genetic engineering of bacteriophage;

[0050] Specifically, the DNA sequence encoding the RGD-OGP polypeptide is inserted into the gene VIII position of the M13 phage genome.

[0051] The amino acid sequence of the RGD-OGP polypeptide is RGDYGFGG (SEQ ID NO. 1), and the DNA sequence encoding the RGD-OGP polypeptide is 5'-CGTGGCGACTACGGCTTCGGTGGC-3' (SEQ ID NO. 2).

[0052] 2. The RO-phage prepared in Example 1 was sequenced, and the results were as follows: Figure 2 As shown, it was found that the DNA sequence corresponding to the designed RGDYGFGG polypeptide was successfully inserted into the genome of the phage.

[0053] 3. Take 1 mg / mL RO-phage solution and drop it onto the carbon film copper grid, and then use uranyl acetate for negative staining. Observe it under a transmission electron microscope (TEM). The results are as follows: Figure 3 shown.

[0054] Depend on Figure 3 The successful preparation and nanofibrous morphology of engineered RO-phage were verified.

[0055] Example 2

[0056] A method for preparing a bifunctional RGD-OGP-phage modified 3D printed porous titanium alloy scaffold, the steps are as follows:

[0057] The 3D printed porous titanium alloy scaffold was ultrasonically cleaned with ethanol and ultrapure water and dried, and then surface plasma treated. It was then immediately incubated with a polylysine (PLL) solution overnight (room temperature, oscillation), washed with ultrapure water and dried to obtain a Ti-PLL scaffold, and then incubated with a RO-phage solution with a concentration of 1 mg / mL overnight (room temperature, oscillation), washed with ultrapure water and dried to obtain a Ti-PLL-RO-phage scaffold, that is, a bifunctional RGD-OGP-phage modified 3D printed porous titanium alloy scaffold. The schematic diagram of RO-phage loading onto the 3D printed porous Ti scaffold is shown in FIG. Figure 4 shown.

[0058] The prepared Ti-PLL-RO-phage scaffold was observed under a scanning electron microscope (SEM). Figure 5 As shown, it can be seen from the SEM characterization results of the porous Ti scaffold that there is no obvious difference in the morphology observed by SEM before and after phage loading.

[0059] Example 3

[0060] Four groups were set up, in which the Blank group was a 3D printed porous titanium alloy scaffold, the PLL group was a Ti-PLL scaffold prepared according to the steps in Example 2, the WT group was a scaffold obtained by incubating the Ti-PLL scaffold in M13 phage overnight according to the steps in Example 2, and the RO group was a 3D printed porous Ti scaffold prepared according to the preparation steps of Example 2 with RO-phage loaded thereon.

[0061] The alkaline phosphatase kit (ALP) was used to detect the ALP concentration in osteoblasts MC3T3-E1 before treatment with each material. The treatment conditions were 37°C and 5% CO2 incubator. The results are shown in Figure 2. Figure 6 As shown, it was found that the bone performance of the phage-loaded group was significantly improved compared with the blank group, and the phage-loaded RO gene further promoted osteogenesis.

[0062] Example 4

[0063] The inventors constructed a rabbit distal femoral bone defect model. Figure 7 The New Zealand white rabbit distal femur bone defect model and material implantation experiment shown in a are used to observe the bone ingrowth around and inside the material after material implantation.

[0064] Four groups were set up: the Blank group was a 3D printed porous titanium alloy scaffold, the Control group was a Ti-PLL scaffold prepared according to the steps in Example 2, the WT group was a scaffold obtained by incubating the Ti-PLL scaffold in M13 phage overnight according to the steps in Example 2, and the RO group was a 3D printed porous Ti scaffold prepared according to the preparation steps of Example 2 with RO-phage loaded thereon.

[0065] The above groups were subjected to a material implantation experiment in New Zealand white rabbit distal femoral bone defect. Six weeks after the material implantation, the in vitro groups of this embodiment contained rabbit femoral samples such as the material. Figure 7 The in vitro image of the implanted material in the distal femoral bone defect of a New Zealand white rabbit shown in middle b shows that bone ingrowth was found around and inside the materials of each group through micro-CT scanning, among which the bifunctional RO-phage modified 3D printed porous titanium alloy scaffold group had the largest amount of bone around and inside the materials ( Figure 7 The in vivo experimental results show that the bifunctional RO-phage modified 3D printed porous titanium alloy scaffold prepared by the present invention has osteogenic performance, and the effect is better than the 3D printed porous titanium alloy scaffold not modified by RO-phage.

[0066] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing RGD-OGP bacteriophage nanofibers, It is characterized in that include: The RGD-OGP polypeptide is displayed on the pVIII protein of the filamentous phage through gene editing to obtain the RGD-OGP phage nanofiber. The amino acid sequence of the RGD-OGP polypeptide is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, It is characterized in that The filamentous phage includes M13 phage.

3. The preparation method according to claim 2, It is characterized in that The step of displaying the RGD-OGP polypeptide on the pVIII protein of the filamentous phage through gene editing includes: inserting the nucleotide sequence encoding the RGD-OGP polypeptide into the geneVIII position of the M13 phage genome, expressing the obtained recombinant phage, and obtaining the RGD-OGP phage nanofiber.

4. The RGD-OGP bacteriophage nanofibers prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the RGD-OGP bacteriophage nanofibers described in claim 4 in modifying 3D printed titanium alloy stents.

6. A bifunctional RGD-OGP phage-modified 3D printed porous titanium alloy scaffold, It is characterized in that It includes a 3D printed porous titanium alloy stent and RGD-OGP bacteriophage nanofibers loaded onto the surface of the 3D printed porous titanium alloy stent; The RGD-OGP bacteriophage nanofiber is the RGD-OGP bacteriophage nanofiber according to claim 4.

7. The method for preparing the bifunctional RGD-OGP phage modified 3D printed porous titanium alloy stent according to claim 6, It is characterized in that The steps include: The 3D printed porous titanium alloy stent is subjected to ultrasonic cleaning and surface plasma treatment, and then mixed with poly-lysine for a first incubation to obtain a poly-lysine titanium alloy stent; The poly-lysine titanium alloy scaffold is incubated in the RGD-OGP bacteriophage nanofiber solution for a second time to obtain the bifunctional RGD-OGP bacteriophage modified 3D printed porous titanium alloy scaffold.

8. The preparation method according to claim 7, It is characterized in that The concentration of the RGD-OGP bacteriophage nanofiber solution is 1 mg / mL.

9. The preparation method according to claim 7, It is characterized in that The conditions of the first incubation and the second incubation are respectively incubation with shaking at room temperature for 12-24 hours.

10. Use of the RGD-OGP phage nanofibers according to claim 4, the bifunctional RGD-OGP phage-modified 3D printed porous titanium alloy scaffold according to claim 6, or the bifunctional RGD-OGP-phage-modified 3D printed porous titanium alloy scaffold prepared by the preparation method according to any one of claims 7 to 9 in the preparation of medical devices for treating bone defects.

Images