CN115252231A - Preparation method of support for bone defect repair - Google Patents

Abstract

The invention belongs to the technical field of medicine, and in particular relates to a preparation method of a scaffold for repairing bone defects. The invention combines image imaging and 3D printing technology, adopts the FDA-approved polymer material PLGA for implantation treatment, and constructs a personalized biphasic-loaded platelet-rich plasma and bisphosphonate scaffold according to the shape of the bone defect, so as to jointly promote the bone defect in the bone defect. Repair and osseointegration. The scaffold prepared by the method of the invention has a good application prospect in the clinical treatment of bone defects.

Description

A preparation method of a scaffold for bone defect repair

technical field

The invention belongs to the technical field of medicine, and in particular relates to a preparation method of a bracket used for bone defect repair.

Background technique

Nowadays, there are more and more patients with clinical bone defects. There are many factors for the formation of bone defects, including stress shielding, osteolysis, osteonecrosis, and infection. The clinical treatment of bone defects is difficult, and the current methods are limited, which has become one of the key factors that seriously affect the prognosis of surgery. The pathological mechanism of bone defects is extremely complex, and most of the non-traumatic bone defects have the characteristics of osteogenesis difficulty and localized osteoporosis.

Previous studies have confirmed that platelet-rich plasma (Platelet-rich plasma, PRP) can release a variety of high-concentration growth factors in the body, and the ratio of each growth factor is consistent with the normal ratio in the body. Disadvantages of poor osteogenesis stimulated by a single growth factor. PRP can also be coagulated into a gel by thrombin, adhere to the granular bone graft at the defect, prevent the loss of platelets, make the platelets secrete growth factors locally for a long time, and maintain a high concentration of growth factors.

Bisphosphonates (BPs), which are widely used clinically, can specifically combine with hydroxyapatite in bone, inhibit the activity of osteoclasts, promote the proliferation and differentiation of osteoblasts, and exert an osteogenic effect. However, oral bisphosphonate drugs cannot achieve high local concentrations, and often need to be applied in large doses, and there are risks of atypical femoral fractures and osteolithiasis.

Chinese Invention Patent Application "CN105854074A Composition and Method for Distraction Osteogenesis" The composition disclosed in this document includes a solution containing platelet-derived growth factor (PDGF) and bisphosphonates (BPs) and a biocompatible matrix, Wherein said solution is configured in the biocompatible matrix. The composition provided by this document has the effect of promoting and accelerating osteogenesis at the site of bone distraction. However, it has been found in practical applications that there are still many problems in the use of such materials. The type and ratio of the active ingredients (PDGF, BPs, etc.) The types and proportions of biocompatible substrates are prone to problems such as too fast or too slow release of active ingredients, and excessive biotoxicity. Seriously affect the application of such composite materials in bone repair.

Therefore, it is still necessary to further develop new drugs or scaffold materials with better effects of promoting bone repair and osseointegration.

Contents of the invention

Aiming at the defects of the prior art, the present invention provides a method for preparing a scaffold for repairing bone defects, with the purpose of preparing a biological scaffold that can better promote bone repair and osseointegration.

A method for preparing a scaffold for bone defect repair, comprising the steps of:

Step 1, based on the image data of the bone defect, perform three-dimensional modeling of the scaffold to be fabricated;

Step 2, making bisphosphonate/polylactic acid-glycolic acid copolymer scaffolds according to the modeling results of step 1;

Step 3, attaching the platelet-rich plasma to the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold obtained in step 2, to obtain a scaffold for bone defect repair;

Wherein, in step 2, the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold is made of the following components by weight:

0.01-2 parts of bisphosphonates;

Polylactic acid-glycolic acid copolymer 98-99.99 parts.

In step 3, the weight ratio of the platelet-rich plasma to the bisphosphonate/polylactic-co-glycolic acid scaffold is 5-10:90-95.

Preferably, in step 2, the method of making the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold is 3D printing.

Preferably, the bisphosphonate is selected from at least one of alendronate sodium and risedronate sodium.

Preferably, the molecular weight of the polylactic acid-glycolic acid copolymer is 5500-6500.

Preferably, in the polylactic acid-glycolic acid copolymer, the molar ratio of lactic acid units to glycolic acid units is (70-80):(20-30).

Preferably, in step 3, the method of attaching the platelet-rich plasma to the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold is to use thrombin to coagulate the platelet-rich plasma on the bisphosphonate/polylactic acid -glycolic acid copolymer scaffold.

Preferably, the ratio of platelet-rich plasma to thrombin is 85-90:10-15.

The invention provides a method for preparing a biological scaffold for promoting the repair and integration of bone defects. The scaffold made by the invention uses the composition of bisphosphonate and polylactic acid-glycolic acid copolymer as the basic material, and adheres to platelet-rich plasma . By optimizing the raw materials and their proportions, the scaffold of the present invention has good physical and chemical properties and cell biocompatibility, and achieves the best effect of promoting osteogenesis and osseointegration.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 is the BPs/PLGA bone default repair scaffold prepared by 3D printing and the SEM photo (sample prepared by 3D printing, the thickness of the sample is 2mm, and the diameter is 8mm).

Fig. 2 is the test result of the degradation rate in Experimental Example 1.

Figure 3 is the release kinetics curve of BPs in Experimental Example 1.

Fig. 4 is the light microscope pictures of PRP/BPs/PLGA scaffold and osteoblasts co-cultured in Experimental Example 1 for 1 (A), 3 (B) and 5 (C) days.

Fig. 5 is a graph of cell viability of PRP/BPs/PLGA scaffold and osteoblasts co-cultured for 1, 3, and 5 days in Experimental Example 1.

Figure 6 is a picture of the PRP/BPs/PLGA scaffold implanted in the femoral condyle (A) and the staining results of tissue sections at 4 weeks (B), 8 weeks (C) and 12 weeks (D) after implantation in Experimental Example 2.

Detailed ways

The reagents and materials used in the following examples and experimental examples are commercially available unless otherwise specified.

Example 1 A preparation method for a scaffold for bone defect repair

This embodiment provides a scaffold for bone defect repair, the preparation method of which is as follows:

(1) 0.2 part of powdered bisphosphonate sodium alendronate is mixed with 99.8 parts of polylactic acid-glycolic acid copolymer (PLGA, molecular weight 5500, Jinan Daigang Biological Engineering Co., Ltd.) to obtain BPs/PLGA mixed powder, BPs/PLGA mixed powder is used as the raw material for subsequent 3D printing; among them, the type of bisphosphonate selected is alendronate sodium, the molecular weight of polylactic acid-glycolic acid copolymer is 6500, and the molar ratio of lactic acid unit and glycolic acid unit for 75:25;

(2) Obtain a porous structure model by modeling;

(3) Use a biopolymer printer to print BPs/PLGA mixed powder to make a BPs/PLGA scaffold; the scaffold prepared in this example is shown in Figure 1 . The pore diameter and porosity of the support were measured, and the pore diameter of the support was 250 microns, and the 200 porosity was 65%.

(4) The platelet-rich plasma is coagulated on the BPs/PLGA scaffold by thrombin to obtain a bio-scaffold (PRP/BPs/PLGA scaffold) for implantation into the bone defect site. The ratio of platelet-rich plasma to thrombin is 90:10, and the ratio of platelet-rich plasma to BPs/PLGA scaffold is 5:95.

The technical effects of the present invention will be further described below through experimental examples.

Experimental example 1 BPs/PLGA dosage ratio screening

1. Experimental method

The amount of BPs was adjusted, and the scaffold for bone defect repair was prepared according to the method in Example 1.

The weight ratios of BPs and PLGA in the three experimental groups are:

Experimental group 1: BPs/PLGA=0.02:99.98,

Experimental group 2: BPs/PLGA=0.2:99.80,

Experimental group 3: BPs/PLGA=2:98.

The control group of the experiment was a scaffold made of pure PLGA material according to the method of Example 1.

Run the following test:

1. Degradation rate

Accurately measure the weight of each sample and record it as initial weight Mi (i=1, 2, 3, 4; respectively represent PLGA, PLGA-0.02% AS, PLGA-0.2% AS, PLGA-2% AS samples). Put the weighed sample into deionized water according to the mass ratio of deionized water: sample = 30:1, put it into a centrifuge tube and seal it (change the soaking solution in the tube every day), and place the centrifuge tube in a constant temperature water bath oscillation box ( 37°C, 72rpm). When replacing the solution, take out the sample, soak it in absolute ethanol for 5 minutes, then air-dry it at room temperature, and then measure the weight Mni of each sample. Calculate the degradation rate of each experimental sample at each time point (soaking time points: 1, 2, 4, 8, 12 weeks): degradation rate Pni=(Mi-Mni)/Mi×100%.

2. The release kinetic curve of BPs

The liquid exchanged for the degradation rate test was collected and mixed. At 1, 2, 4, and 8 weeks, the concentration of phosphorus in each soaking solution was measured by inductively coupled plasma optical emission spectrometer (ICP-OES).

3. In vitro cell test evaluation

Osteoblasts were co-cultured with PRP/BPs/PLGA composite bio-scaffold in vitro, and the combination of cells and scaffolds was observed by light microscope; cell viability was detected by CCK8 method.

2. Experimental results

The degradation rate of the sample is shown in Figure 1. The sample swelled slightly after soaking for a week, and the surface of the stent appeared relatively smooth. The reason may be due to the fact that in the first week, the sample absorbed a lot of water, water molecules entered between the molecular chains, and the material swelled. After soaking for 2 weeks, it was observed that the sample became significantly softer, which may be the result of molecular chain breakage after the sample absorbs water. With the increase of immersion time, individual samples had gelled by the fourth week, and some samples had broken into small pieces by the sixth week. By the eighth week, the samples of the sample group had all broken into small pieces, indicating that the material degraded faster. In contrast to the samples of the control group, the softening phenomenon was not broken until the twelfth week. This indicates that the scaffold provided by the present invention has a faster degradation rate than the scaffold made of pure PLGA material.

The release curves of BPs are shown in Fig. 3, and the samples released higher concentrations of bisphosphonates in the first week, showing higher phosphorus concentrations. With the increase of immersion time, the changes of experimental group 1 and experimental group 2 were not obvious, while experimental group 3 increased slightly after the second week. Analysis of the reason may be due to the material degradation process, due to the existence of material water absorption and material dissolution balance, so under the condition of degradation rate decline, phosphate is still released. For the samples of experimental group 3, the content of bisphosphonates in the samples was higher, which affected the crystallization of scaffold polymers, and showed faster material dissolution, thereby releasing more bisphosphonates. This result is consistent with the degradation experiment results.

The evaluation results of in vitro cell tests are shown in Figures 4 and 5. In Figure 4, the experimental group 2 was co-cultured with osteoblasts for 1, 3, and 5 days. The light microscope pictures showed that the number of osteoblasts gradually increased with time, and the appearance of osteoblasts normal. It shows that the scaffold of the present invention has the effect of promoting bone repair. The cell viability diagram in Figure 5 shows that the scaffolds of experimental group 1, experimental group 2, and experimental group 3 can all improve cell viability, but for experimental group 3 with a higher amount of BPs, the cell viability decreases with the increase of culture time .

The above experiments prove that the dosage ratio of BPs and PLGA is 0.02-0.2:99.8-99.98, and the more optimal one is 0.02:99.98.

Example 2 Animal Experiments of PRP/BPs/PLGA Scaffold Used in Bone Defect Repair

1. Experimental method

1. Establishment of rabbit bone defect model: New Zealand white rabbits, 2.5-3.5Kg, were selected, fed independently, fed freely, and used for experiments after one week of feeding. Surgical procedure: general anesthesia, shaved hair, fixed on the operating table, iodophor disinfection. Make an arc-shaped incision about 5 cm in length parallel to the front edge from the lateral side of the knee joints of both hind limbs at 1.0-1.5 cm from the front edge; separate and expose the anatomical landmarks of the lateral femoral condyle layer by layer, and prepare a 6 mm diameter and 8 mm deep bone with a circular hollow bone drill Cylindrical bone defect. At a distance of 6 mm from the center of the bone defect, a titanium nail with a diameter of 2 mm was screwed into each side symmetrically for positioning. During the process, the flushing solution was used to cool down, and the blood vessels and nerve bundles should be protected.

2. In vivo study of the bone repair effect of PRP/BPs/PLGA on osteogenesis: the rabbit femoral condyle bone defect model was successfully established, with a defect size of 6 mm in diameter and 8 mm in depth. They were randomly divided into 5 groups: group A, implanted with porous PLGA scaffolds; group B, implanted with BPs/PLGA scaffolds; group C, implanted with PRP/PLGA scaffolds; group D, implanted with PRP/BPs/PLGA scaffolds; Blank control group. After the operation, the animals were intramuscularly injected with 40,000 U/day of gentamicin for 3 consecutive days. At 1, 2, and 3 months after operation, new bone formation and integration and ingrowth with surrounding bone tissue and materials were observed histologically.

2. Experimental results

As shown in Figure 6, at the 4th week, the material was significantly degraded, and a small amount of new bone was observed. At the 8th week, the scaffold degraded more, and the amount of new bone increased. At the 12th week, the scaffold was almost completely degraded, and the new bone became mature. Bone tissue shows that the PRP/BPs/PLGA scaffold of the present invention does have a good effect on bone repair.

It can be seen from the above examples and experimental examples that the present invention provides a scaffold for bone defect repair by optimizing the composition and ratio of raw materials, which has good physical and chemical properties and cell biocompatibility, and achieves the best Promote bone formation and osseointegration. The invention has good application prospect in bone repair.

Claims (7)

1. A preparation method for a support for bone defect repair, characterized in that, comprising the steps: Step 1, based on the image data of the bone defect, perform three-dimensional modeling of the scaffold to be fabricated; Step 2, making bisphosphonate/polylactic acid-glycolic acid copolymer scaffolds according to the modeling results of step 1; Step 3, attaching the platelet-rich plasma to the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold obtained in step 2, to obtain a scaffold for bone defect repair; Wherein, in step 2, the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold is made of the following components by weight: 0.01-2 parts of bisphosphonates; 98-99.99 parts of polylactic acid-glycolic acid copolymer; In step 3, the weight ratio of the platelet-rich plasma to the bisphosphonate/polylactic-co-glycolic acid scaffold is 5-10:90-95. 2. The preparation method according to claim 1, characterized in that: in step 2, the method of making the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold is 3D printing. 3. The preparation method according to claim 1, characterized in that: the bisphosphonate is selected from at least one of alendronate sodium and risedronate sodium. 4. according to the described preparation method of claim 1, it is characterized in that: the molecular weight of described polylactic acid-glycolic acid copolymer is 5500-6500. 5. according to the described preparation method of claim 1, it is characterized in that: in described polylactic acid-glycolic acid copolymer, the molar ratio of lactic acid unit and glycolic acid unit is (70-80):(20-30). 6. The preparation method according to claim 1, characterized in that: in step 3, the method of attaching the platelet-rich plasma to the bisphosphonate/polylactic acid-glycolic acid copolymer scaffold is to use thrombin to Platelet-rich plasma was clotted on the bisphosphonate/poly(lactic-co-glycolic acid) scaffold. 7. The preparation method according to claim 6, characterized in that: the dosage ratio of the platelet-rich plasma and thrombin is 85-90:10-15.

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