Infectious bone defect repair material and application

Abstract

The invention discloses a multifunctional infectious bone defect repair material and application thereof, the multifunctional infectious bone defect repair material comprises nano-hydroxyapatite as a carrier and a functional active substance loaded on the nano-hydroxyapatite, namely an antibacterial-immunoregulation-osteogenesis-promoting multifunctional peptide, the nano-hydroxyapatite is used as a common bone repair material carrier, and the nano-hydroxyapatite is used as a bone repair material carrier. The multifunctional peptide is loaded through electrostatic adsorption and calcium ion combination dual modes, and polypeptide functionalized nano-hydroxyapatite (P / nHA) is formed. Compared with the prior art, the preparation method has the advantages that the infectious bone defect repair material is provided, material construction is achieved in a simple mode and does not depend on compounding of multiple functional components, and multifunctional peptide modified nano-hydroxyapatite can achieve integration of multiple functions of infection control, immunoregulation and osteogenesis induction; the repair and regeneration of infectious bone defects are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of bone defect repair materials, and in particular to an infectious bone defect repair material and application thereof. Background Art

[0002] Maxillofacial infected bone defects (IBD) refer to the long-term exposure of jaw bone defects caused by trauma and other factors to the external environment, contamination by pathogens, or direct imbalance of immune homeostasis and bone structure destruction caused by oral pathogens and their metabolic mediators, including jaw osteomyelitis, apical periodontitis, periodontitis and peri-implantitis, which seriously affect the function and appearance of the patient's oral and maxillofacial system and may even become systemic lesions.

[0003] The onset of IBD includes three stages: microbial invasion and biofilm proliferation, host immune response to microorganisms, and osteoblastic / osteoclast metabolic disorders. Lesion debridement and bone transplantation techniques are currently commonly used clinical treatments. However, the oral and maxillofacial anatomical structure and microecological environment are complex, biofilms are difficult to completely remove, and bone filling materials such as hydroxyapatite (HA) lack antibacterial and immunomodulatory activity and have poor osteogenic induction effects. Therefore, the development of new bioactive preparations that are adapted to the pathological characteristics of IBD and have both infection control, immunomodulatory and osteogenic induction functions is an important scientific issue that needs to be urgently addressed in this field.

[0004] The high content of HA in bones and teeth gives it excellent tissue compatibility and osteoconductivity, making it a traditional preferred material in the field of bone repair. With the development of nanotechnology, nanohydroxyapatite (nHA) is gradually becoming a rising star in the field of bone repair because of its nanostructure that is closer to natural bone tissue and its enhanced physical, chemical and biological properties. However, neither pure HA nor nHA has antibacterial and immunomodulatory activity, and its clinical application effect is limited. In order to overcome the deficiencies of pure HA and nHA in antibacterial and immunomodulatory aspects, researchers are exploring the functional modification of them through protein drugs, aiming to give these materials multiple functions such as antibacterial, bone regeneration promotion and immune regulation, thereby broadening their clinical application range and improving therapeutic effects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above technical defects and realize the multifunctional integration of "infection control-immunomodulation-osteogenesis induction" by constructing a multifunctional peptide-modified nHA (P / nHA) to promote IBD bone repair and regeneration.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is: an infectious bone defect repair material, including a nano-hydroxyapatite carrier and a loaded multifunctional peptide.

[0007] Preferably, the multifunctional peptide includes a polypeptide having antibacterial, immunomodulatory and osteogenic effects, and the nano-hydroxyapatite and the multifunctional peptide are loaded by electrostatic adsorption and calcium ion binding.

[0008] Another aspect of the present invention discloses a method for preparing an infectious bone defect repair material, comprising the following steps:

[0009] S1: Prediction of calcium binding sites of multifunctional peptides based on the I-Tasser deep model to predict their binding potential;

[0010] S2: Mix the nano-hydroxyapatite and the multifunctional peptide in PBS and shake them; wash with ultrapure water and recover the precipitate by centrifugation, and freeze-dry at -80°C under vacuum overnight;

[0011] S3: After labeling the multifunctional peptide with rhodamine, S1-S2 was repeated and laser confocal microscopy was used to confirm the preparation of the infectious bone defect repair material.

[0012] Preferably, the parameters of the PBS in S2 are mixing and shaking at 37° C. for 24 hours, washing with ultrapure water for 3 times, and centrifuging at 10,000 g for 3 minutes to recover the precipitate.

[0013] Another aspect of the present invention discloses the use of an infectious bone defect repair material in the implantation of bone filling materials.

[0014] Another aspect of the present invention discloses the use of an infectious bone defect repair material in maxillofacial infectious bone defects.

[0015] Another aspect of the present invention discloses the use of an infectious bone defect repair material in osteogenic induction.

[0016] Another aspect of the present invention discloses the use of an infectious bone defect repair material in infection control and immune regulation.

[0017] The advantages of the present invention compared with the prior art are: in the present invention, a functionalized bone defect repair material is formed by a dual-mode combination of a multifunctional peptide and nHA, which has excellent osteogenesis and angiogenesis potential, and is used for infection control and immunomodulation after filling; in addition, the multifunctional material is simple to prepare, and only requires the commonly used nHA and multifunctional peptide two-component construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a diagram of the structure of a multifunctional peptide;

[0019] Among them: (A) molecular structure of multifunctional peptide; (B) three-dimensional molecular structure of multifunctional peptide; (CD) secondary structure of multifunctional peptide.

[0020] Figure 2 It is the synthesis and characterization of multifunctional peptide-modified nHA;

[0021] Among them: (A) Prediction of the calcium binding domain of the multifunctional peptide; (B) Detection of the binding ability of the multifunctional peptide to nHA, the multifunctional peptide was labeled with red fluorescence, scale bar: 100μm; (C) Scanning electron microscope images of nHA and P / nHA, scale bar: 5μm; (D) Detection of the anti-biofilm formation ability of nHA and P / nHA, live bacteria were marked in green, and dead bacteria were marked in red, scale bar: 100μm.

[0022] Figure 3 This is a schematic diagram of the establishment of the rat mandibular IBD model and the implantation of bone filling materials.

[0023] Figure 4 It is the in vivo evaluation of the antimicrobial and immunomodulatory abilities of P / nHA;

[0024] Among them: (A) Colony formation of Staphylococcus aureus collected from granulation tissues of nHA group and P / nHA group 4, 7 and 14 days after surgery; (B) Expression of pro-inflammatory genes Il-1β, Il-6 and Tnf-γ in granulation tissues of blank group, nHA group and P / nHA group 14 days after surgery; (C) Expression of CD86 and CD206 in granulation tissues of blank group, nHA group and P / nHA group 14 days after surgery. + M1 and CD206 + M2 macrophages are labeled in brown, and cell nuclei are labeled in blue; H: nHA or P / nHA; NB: new bone; Scale bar: 50 μm; (D) Quantitative analysis of CD86 and CD206 immunohistochemical staining. ****p<0.0001, ***p<0.001, *p<0.05.

[0025] Figure 5 It is the in vivo evaluation of the osteoinductive ability of P / nHA;

[0026] Among them: (A) micro-CT images of bone defect area at 4 and 8 weeks after surgery; (BC) quantitative analysis of new bone volume and density; (D) H&E staining images of defect area at 4 and 8 weeks after surgery; H: nHA or P / nHA; NB: new bone; PDL: periodontal ligament; scale bar: 200μm or 100μm; (E) Masson staining images of defect area at 4 and 8 weeks after surgery; scale bar: 100μm; (F) immunohistochemical staining images of defect area at 8 weeks after surgery; OCN + 、OPN + and CD31 +Cells are marked in brown, and nuclei are marked in blue. Scale bar: 50 μm; (G) Quantitative analysis of immunohistochemical staining. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0028] Example 1

[0029] Methods: Multifunctional peptide functionalized nanohydroxyapatite P / nHA was prepared and characterized. I-Tasser software was used to predict the calcium binding ability of the peptide. 50 mg nHA (Engineering For Life, China) and 10 mL multifunctional peptide (32 μg / mL) were mixed and shaken in PBS at 37°C for 24 h to prepare P / nHA. The mixture was washed three times with 2 mL ultrapure water, centrifuged at 10000 g for 3 min, and the precipitate was recovered and freeze-dried at -80°C under vacuum overnight.

[0030] In order to confirm the binding of the multifunctional peptide to nHA, the multifunctional peptide was labeled with rhodamine, and the above binding experiment was repeated and observed using a laser confocal microscope (Olympus FV3000, Japan).

[0031] In order to observe the surface morphology of P / nHA, platinum was coated by vacuum sputtering, and the sample was scanned by a field emission scanning electron microscope (FEIQUANTA 200, USA).

[0032] In order to detect the anti-biofilm formation ability of P / nHA, the samples were mixed with Staphylococcus aureus (1×10 6 CFU / mL) were incubated at 37°C for 24 h and the cells were stained with a bacterial live / dead staining kit (LIVE / DEAD TM BacLight TM , USA) staining, and the fluorescent staining samples were observed by Olympus FV3000.

[0033] Results: Multifunctional peptide (molecular structure see Figure 1 ) and nHA in two ways: electrostatic adsorption and calcium ion binding. First, the net charge of the multifunctional peptide is +5, and the calculated charge of nHA is negative, so the two can form electrostatic adsorption; secondly, the I-Tasser depth model predicts the calcium binding site of the multifunctional peptide, and the peptide may form ion binding with nHA through the abundant calcium ions in nHA ( Figure 2 A). The peptide was labeled with rhodamine to confirm that the multifunctional peptide could bind to nHA ( Figure 2B). Scanning electron microscopy observation of P / nHA microscopic morphology showed small spherical clusters ( Figure 2 C). The multifunctional peptide is loaded on the nano-hydroxyapatite phase through classical adsorption and calcium ion binding to form peptide-functionalized nano-hydroxyapatite, which is simple to prepare and has low economic cost of materials and operation cost.

[0034] P / nHA can kill pathogens attached to the surface and resist the invasion of pathogens ( Figure 2 D) This indicates that the functional material retains good in vitro antibacterial effect.

[0035] Example 2

[0036] Methods: To evaluate the antibacterial, immunomodulatory and osteogenic abilities of P / nHA, a mandibular IBD model was established using male SD rats (6-8 weeks old, 200-220 g). Figure 3 First, the rats were anesthetized, and a full-thickness incision of approximately 2 cm was made along the lower edge of the mandible to separate and expose the bone surface. A sterile diamond fissure drill (SF-41; MANI, Japan) and a high-speed handpiece were used to prepare a mandibular defect of approximately 3 × 1.5 × 1 mm 3 The defect area was divided into two groups: the front edge of the defect was extended to the mesial root of the first molar, and the rear edge was extended to the mesial root of the second molar. Subsequently, 10 μL of Staphylococcus aureus solution (1×10 6 CFU / mL) to induce infection. The rats were randomly divided into a blank group, a nHA group, and a P / nHA group, with 8 rats in each group. The materials were implanted into the defect, and the muscles and skin were repositioned and sutured in turn.

[0037] Granulation tissues were collected 4, 7, and 14 days after surgery, placed in 10 mL of sterile PBS, shaken, and homogenized for 10 min. 100 μL of the homogenate was inoculated on an agar plate and incubated at 37°C for 24 h before observing colony formation.

[0038] Granulation tissue was collected 14 days after surgery, and total RNA was extracted and reverse transcribed using NucleoZOL reagent. The mRNA expression of inflammation-related genes was detected by qRT-PCR. In addition, the granulation tissue was fixed, embedded in paraffin, and sliced ​​for immunohistochemical staining. The specific steps were as follows: dewaxing, blocking with 5% goat serum at room temperature for 30 minutes, incubation with primary antibodies for CD86 (1:500; 13395-1-AP, Proteintech Group) and CD206 (1:1000; 18704-1-AP, Proteintech Group) at 4°C overnight, rinsing with PBS three times, incubation with secondary antibodies of the corresponding species at room temperature for 1 hour, and scanning with a slice scanner. Four fields of view were randomly selected for each slice, and the ratio of CD86 and CD206 positive areas to total tissue area was measured using ImageJ 1.8.0 software.

[0039] Rats were killed 4 and 8 weeks after surgery, and the mandibles were collected and fixed in 4% paraformaldehyde for 24 hours. The scans were performed using micro-CT (SCANCO Medical, Switzerland). The relevant scanning parameters were set as follows: spatial resolution 34 μm, voltage 70 kV, current 200 μA, power 14 W. After scanning, three-dimensional images were reconstructed and bone parameters such as new bone mass and bone density were measured.

[0040] Subsequently, the samples were decalcified in 10% EDTA for 3 months, embedded in paraffin, and sectioned. Hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and immunohistochemical staining were performed. The specific steps of immunohistochemistry were as described above, and the primary antibodies used were as follows: OCN (1:50; 23418-1-AP, Proteintech Group), OPN (1:250; 22952-1-AP, Proteintech Group), and CD31 (1:50; #AF6191, Affinity Biosciences).

[0041] Results: At 4 and 7 days after surgery, the number of Staphylococcus aureus colonies in the P / nHA group was small, while that in the nHA group was obvious. As time extended to 14 days, Staphylococcus aureus disappeared in the P / nHA group, while the colony density in the nHA group remained unchanged ( Figure 4 A). In the infected state, excessive bacterial proliferation leads to immune cell dysregulation and imbalance of inflammatory factors, so we further studied the changes in inflammatory gene expression and macrophage polarization. Compared with the blank group and nHA group, the expression of pro-inflammatory genes Il-1β, Il-6 and Tnf-γ in the P / nHA group was significantly inhibited ( Figure 4 B); Meanwhile, CD86 in P / nHA group + The number of cells (M1 pro-inflammatory macrophages) was the smallest, while CD206 + The number of cells (M2 repair macrophages) increased significantly, while the number of M1 pro-inflammatory macrophages was higher in the blank group and nHA group, and the number of M2 repair macrophages was lower ( Figure 4 CD).

[0042] Bone regeneration was detected at 4 and 8 weeks after surgery. Micro-CT results showed that the P / nHA group had significant new bone formation, which may be attributed to the good antibacterial and anti-inflammatory ability of P / nHA in the early stage ( Figure 5 AC). H&E and Masson staining showed that there was almost no new bone formation in the blank group, and the defect area was filled with fibrous tissue. There was some bone regeneration at the edge of the nHA group, while the amount and morphology of the new bone tissue in the P / nHA group were good ( Figure 5DE). Immunohistochemical staining showed that the expression of OCN, OPN and CD31 in the P / nHA group was significantly increased, with superior osteogenic and angiogenic potential ( Figure 5 FG).

[0043] The above results indicate that the dual-mode loaded multifunctional peptide-modified nanohydroxyapatite prepared by this simple method has good antibacterial-immunomodulatory-osteogenic effects and can be used as a low-cost, easy-to-prepare, and multi-functional infectious bone defect repair material.

[0044] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A multifunctional infectious bone defect repair material, characterized in that: It includes a nano-hydroxyapatite carrier and multifunctional peptide molecules loaded via dual modes of electrostatic adsorption and calcium ion adsorption.

2. A multifunctional infectious bone defect repair material according to claim 1, characterized in that: The multifunctional peptide molecule is a polypeptide with antibacterial, immunomodulatory and osteogenesis effects, and the nano-hydroxyapatite is combined with the multifunctional peptide molecule through electrostatic adsorption and calcium ions.

3. Use of a multifunctional infectious bone defect repair material as claimed in claim 1 in implantation of bone filling materials.

4. Use of a multifunctional infectious bone defect repair material as claimed in claim 1 in maxillofacial infectious bone defects.

5. Use of a multifunctional infectious bone defect repair material as claimed in claim 1 in osteogenic induction.

6. Use of a multifunctional infectious bone defect repair material as claimed in claim 1 in infection control and immune regulation.

Images