CN114377213A

CN114377213A - Novel selenium-enhanced bioactive bone cement and preparation method thereof

Info

Publication number: CN114377213A
Application number: CN202210177814.7A
Authority: CN
Inventors: 刘滔; 周全; 何帆; 刘昊; 侯明壮; 章君鑫
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-04-22

Abstract

The invention relates to the field of biological materials and medical application, and particularly discloses selenium-enhanced bioactive modified bone cement capable of promoting bone tissue repair and new bone growth and a preparation method thereof. The invention firstly discloses modified bone cement which comprises Silk Fibroin (SF), calcium phosphate bone cement (CPC) and sodium selenite (Na)₂SeO₃). Secondly, the invention also discloses a preparation method of the modified bone cement, which comprises the following steps: s1: na (Na)₂SeO₃Dissolving in SF liquid phase to obtain liquid mixture; s2: fully mixing the liquid mixture with CPC according to a liquid-solid ratio (0.4-0.6) mL/g; s3: and curing to obtain the modified bone cement. In reserving CPC-On the basis of the advantages of SF, Na with the capability of promoting the proliferation and osteogenic differentiation of mesenchymal stem cells is added into the SF solution₂SeO₃So as to accelerate the repair of the bone tissue defect, thereby having good clinical application prospect.

Description

Novel selenium-enhanced bioactive bone cement and preparation method thereof

Technical Field

The invention relates to the field of biological materials and medical application, and particularly discloses selenium-enhanced bioactive modified bone cement capable of promoting bone tissue repair and new bone growth and a preparation method thereof.

Background

One of the public health problems encountered in the world today is bone tissue damage due to various traumas, and the bone tissue defects therein seriously affect the normal healing of the bone tissue. Therefore, timely repair of bone tissue defects and reconstruction of the integrity of bone structure are the major means to cure this type of injury. For the repair of bone tissue defects, there are currently three methods that are more applied clinically: autologous bone grafting, allogeneic bone grafting and biomaterial filling repair. Since the autogenous bone is from the body of the patient, no immune rejection reaction exists, and the autogenous bone is accepted as the best choice, but the bone taking area is limited, and the autogenous bone causes additional injury to the patient, and increases unnecessary risks. Allogeneic bone transplantation, although an alternative to bone transplantation, is a scarce source of material and may involve immune rejection and the transmission of infectious diseases. Therefore, in view of the advantages and disadvantages of the above two bone grafts, the skilled person has to find a widely accepted biomaterial which can be applied more conveniently to the treatment of patients with bone tissue defects on a safe and effective basis.

Polymethyl methacrylate (PMMA) bone cement is currently the most widely used filling material in clinical practice, and its clinical value has been widely demonstrated in the treatment of vertebral compression fractures. However, PMMA still has some drawbacks that are difficult to completely overcome [ Belkoff SM, Molloy s. Temperature measurement polymerization for polymethyl methacrylate residue, Spine, 2003; 28: 1555-1559.]: the non-biodegradable polymer has no biological activity and can not be biodegraded, the non-polymerized monomer has certain toxicity, the surrounding tissues can be damaged due to heat release in the polymerization reaction process, and stress shielding is easily formed on the surrounding bone tissues due to overhigh mechanical strength. These disadvantages may lead to serious complications, and thus are difficult to be widely used in patients with bone tissue defects.

In recent years, Calcium Phosphate Cement (CPC) has attracted attention as an effective bone tissue defect filling material. Because the inorganic components of the material are very similar to the inorganic components of human bone tissues, the material has good biocompatibility, injectability, osteoconductivity and drug carrying property. However, many deficiencies of CPC have been observed through a series of clinical studies [ Xu HH, Simon CG Jr. Fast setting calcium-chitosan scaffold: mechanical properties and biological properties, Biomaterials, 2005; 26:1337T348 ]: 1. the collapse and leakage are easy to occur in the tissues containing liquid in the human body, and the organ and the blood vessel can be subjected to pressure embolism under severe conditions; 2. the mechanical strength performance is insufficient, so that the stable mechanical supporting strength is lacked; 3. it has no osteoinductive properties and thus has no effect on accelerating the healing rate of bone tissue defects. Therefore, in order to effectively apply such bone cement materials to the treatment of bone tissue defects, new bone cement materials having durable resistance to collapse, stable mechanical support, and a certain degree of accelerated bone tissue repair ability should be further sought. At present, the main method for meeting the requirements to a certain extent is to compound CPC and degradable high molecular polymer, thereby avoiding the defect of using CPC only.

Silk Fibroin (SF) is a natural polymer protein secreted by silkworms and has a composition including 18 amino acids essential to the human body and a wide medical value because of its excellent biocompatibility, degradability, modifiability and stable mechanical properties [ Li MZ, Wu ZY, Zhang CS, et al. In recent years, some studies have been made to apply SF to the study of bone tissue defect repair, and SF in combination with CPC has been used to fill bone tissue defects with effective repair effect [ dawn, sword, Zhao Beacon, etc.. silk fibroin/calcium phosphate cement external reinforced bone defect vertebral body [ J ]. study of chinese tissue engineering, 2010(47):45-48 ] [ Chen Ying Jun, Wang Nanxiang, Zhou Lei, etc.. experiment study of silk fibroin fiber/calcium phosphate cement composite material to repair rabbit radial bone defects [ J ]. China trauma journal of orthopedics, 2014, 16(1):62-66 ]. However, CPC-SF may require further investigation in order to achieve satisfactory results in terms of accelerating the repair of bone tissue defects. The inventor notices that CPC and SF have good characteristics of loading various medicaments and biological factors, and therefore, the inventor conducts related research to fully exert the advantages.

Selenium (Se) is an important non-metallic trace element, exists in vivo in the form of selenase and selenoprotein, is a preventive antioxidant in vivo, can block the formation of free radicals and eliminate free radicals and peroxides, thereby protecting mesenchymal stem cells (BM-MSCs) from damage caused by oxidative stress, maintaining normal bone metabolism, and the activity of the selenium depends on the supply of selenium [ zhui. At present, the Effect of Se on bone metabolism has been partially studied and all show that an appropriate Se concentration has a significant Effect on promoting bone tissue repair [ Belma T, Murat A, Belgin C, et al, Effect of selected great treatment on an ultra structural change in experimental biological rates bones [ J ]. Bio Trace Element Res, 2005, 107(2):167-180 ] [ Weijian, Luhan, Duaiping, etc.. pathological study of the Effect of selenium on the experimental fracture healing process in rabbits [ J ]. China journal of bone injury, 1999, 3, 12(2), 17-19 ].

However, sodium selenite (Na)₂SeO₃) The research on the modified bone cement is only reported, so that a new modified bone cement is urgently needed to be invented and solve the existing technical problems.

Disclosure of Invention

The main purpose of the invention is to add Na which can promote the proliferation and osteogenic differentiation of BM-MSCs on the basis of retaining the advantages of CPC-SF₂SeO₃. The novel modified bone cement has good biocompatibility, durable collapsibility resistance and stable mechanical support strength, and has the effect of accelerating bone tissue repair to a certain degree.

The invention uses sodium selenite (Na)₂SeO₃) Dissolving in SF liquid phase, mixing with CPC at a ratio of (0.4-0.6) ml/g, and mixing to obtain Na-carrying solution₂SeO₃Whether the CPC-SF modified bone cement has good repairing effect when being filled into a rat model with bone tissue defect or not.

In order to achieve the above purpose, the technical solution of the present invention is as follows:

the invention discloses a modified bone cement in a first aspect, which comprises SF, CPC and Na₂SeO₃。

The second aspect of the invention discloses a preparation method of modified bone cement, which comprises the following steps:

S1：Na₂SeO₃dissolving in SF liquid phase to obtain liquid mixture;

s2: fully mixing the liquid mixture with CPC according to a liquid-solid ratio (0.4-0.6) mL/g;

s3: and curing to obtain the bone cement.

Preferably, in S1, silk fibroin fiber is extracted from raw silk of bombyx mori, and after degumming and dissolution, SF liquid phase is obtained.

More preferably, the concentration of the SF liquid phase is 4% to 6%.

In some embodiments of the invention, the concentration of the SF liquid phase is 5%.

Preferably, in S2, the preparation method of CPC includes: preparing hydroxyapatite crystal seed, and mixing with Alfa-tricalcium phosphate powder and calcium hydrogen phosphate powder to obtain CPC.

More preferably, in S2, the formulation of CPC has a weight ratio of Alfa-tricalcium phosphate, calcium hydrogen phosphate and hydroxyapatite of 87:10: 3.

Preferably, in S3, the mixture obtained in S2 is left to cure at 37 ℃ in a saturated humidity environment for 2 to 5 days to obtain the bone cement.

Preferably, in S2, the liquid mixture is mixed with CPC in a liquid-solid ratio of: 0.5mL/g was mixed well.

In some embodiments of the invention, the formulation of the liquid mixture is: SF (5%) + (100. mu.M) Na₂SeO₃Solutions of

The third aspect of the invention discloses a product for promoting bone tissue repair and new bone growth, and the material of the product is the modified bone cement or the modified bone cement prepared by the method.

The fourth aspect of the invention discloses the application of the modified bone cement or the method in the fields of biological materials and medical application.

Compared with the prior art, the invention has the following advantages:

1. na-loaded catalyst prepared by the invention₂SeO₃The CPC-SF modified bone cement is an organic/inorganic compound, and can fully improve the interface compatibility between an organic phase and an inorganic phase in the CPC-SF, enhance the bonding performance of the bone cement and bone tissues and further improve the mechanical support strength of the bone cement. In addition, the CPC-SF has good drug loading characteristics, and can give full play to the drug release mechanism of the CPC in a dispersion regulation type.

2. SF is used as a curing liquid, so that the compression strength, the collapsibility resistance, the solidification time, the biocompatibility and the like of the CPC can be effectively improved.

3. Na₂SeO₃As a supplementary medicament of selenium element, the selenium element is dissolved in SF according to the planned concentration, and is fully blended with CPC until the selenium element is solidified, and then the selenium element is filled into the defect part of the bone tissue, so that BM-MSCs are protected from oxidative stress damage through the medicament property, the proliferation and osteogenic differentiation potential of the BM-MSCs are improved, and the repair of the defect part of the bone tissue is promoted.

Drawings

FIG. 1 is a scanning electron microscope image of the novel modified bone cement of the present invention.

FIG. 2 shows the injectability analysis of the novel modified bone cement of the present invention.

FIG. 3 shows the analysis of the compressive strength of the novel modified bone cement of the present invention.

FIG. 4 shows the collapsibility degradation resistance analysis of the novel modified bone cement of the present invention.

FIG. 5 shows the setting time analysis of the novel modified bone cement of the present invention.

FIG. 6 shows the biocompatibility analysis of the novel modified bone cement of the present invention.

FIG. 7 is a diagram showing an example of the application of the novel modified bone cement of the present invention.

FIG. 8 shows the experimental results of the novel modified bone cement of the present invention.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

The embodiment discloses a preparation method of CPC-SF modified bone cement, which comprises the following steps:

(1) preparation of CPC

Prepared by a liquid phase precipitation method: mixing Ca (N0)₃)₂And (NH)₄)₂HP0₄Respectively dissolving in steamed stuffing water to prepare 1mol/L solution. Mixing the prepared Ca (N0)₃)₂Solution and (NH)₄)₂HP0₄The solution is subjected to liquid phase precipitation reaction under alkaline conditions according to the Ca/P molar ratio of 1.67 to generate HA precipitate with low crystallinity, and then the HA precipitate is washed, filtered, dried at 80 ℃ and finally calcined for 3h at 800 ℃ T to obtain HA with high crystallinity.

Commercially available analytically pure raw material powder of α -TCP and DCPD was thoroughly mixed with the HA prepared above in a weight ratio of 87% α -TCP, 10% DCPD and 3% HA, and then wet-milled in a ball mill at 464rpm for 24h with anhydrous ethanol as a medium, wherein the weight ratio was: powder, agate balls and ethanol =4:30: 9. After ball milling, the CPC powder was dried in an oven at 80 ℃ and ground in an agate mortar to obtain CPC powder, which was finally stored in a vacuum drying oven.

(2) Preparation of SF liquid phase

1) Degumming of domestic silk fiber

Placing the raw silkworms in Na with the concentration of 0.02mol/L₂CO₃Boiling the solution for 40min, then rubbing with hot water for three times, and then rubbing with deionized water for three times to remove sericin in the silk. And drying in an oven at 37 ℃ overnight to finally obtain the refined and sorted silk fibroin fibers.

2) Dissolving fibroin fiber

Preparing a lithium bromide (LiBr) solution with the concentration of 9.3mol/L, and putting the silk fibroin fiber at 60 ℃ for dissolving for 4 hours. Dialyzing the fibroin fiber for one week by using a dialysis bag with the molecular weight cutoff of 3500, and removing fibroin aggregate by high-speed centrifugation to finally prepare SF solution with the concentration of 5%.

(3) SF-CPC bone cement blending and setting

Mixing the prepared CPC powder with SF solution according to a liquid-solid ratio: 0.5ml/g (namely, 0.5ml SF solution is added into 1g CPC powder), blending and finally curing at room temperature to obtain the SF-CPC modified bone cement.

Example 2 carrying Na₂SeO₃Preparation step of CPC-SF modified bone cement

(1) Formulation of CPC

Prepared by a liquid phase precipitation method: mixing Ca (N0)₃)₂And (NH)₄)₂HP0₄Respectively dissolving in steamed stuffing water to prepare 1.00mol/L solution. Mixing the prepared Ca (N0)₃)₂Solution and (NH)₄)₂HP0₄The solution is subjected to liquid phase precipitation reaction under alkaline condition according to the Ca/P molar ratio of 1.67 to generate HA precipitate with low crystallinity, and then the HA precipitate is washed, filtered, dried at 80 ℃, and finally calcined at 800 ℃ T for 3h,obtaining HA with high crystallinity.

(2) Preparation of SF liquid phase

1) Degumming of domestic silk fiber

2) Dissolving fibroin fiber

LiBr solution with the concentration of 9.3mol/L is prepared, and silk fibroin fiber is placed at 60 ℃ for dissolving for 4 hours. Dialyzing the fibroin fiber for one week by using a dialysis bag with the molecular weight cutoff of 3500, and removing fibroin aggregate by high-speed centrifugation to finally prepare SF solution.

（3）Na₂SeO₃Preparation of the solution

Na to be purchased₂SeO₃Dissolving the powder in deionized water to prepare Na with the concentration of 100 mu M₂SeO₃And (3) solution.

(4) Novel modified bone cement blending and solidification

Mixing the above Na₂SeO₃Adding the solution into SF, and adding the prepared CPC powder and SF solution according to the liquid-solid ratio: 0.5ml/g, blending, and finally curing at room temperature to obtain Na-loaded₂SeO₃The CPC-SF modified bone cement.

Example 3

This example investigates the characterization of the novel selenium-enhanced bioactive bone cement obtained in example 2, and the modified bone cement obtained in example 1 is a control group.

(1) Surface topography observation

Breaking the solidified modified bone cement, drying, and performing surface gold spraying treatment on a copper round table, wherein the gold spraying time is 90S, the thickness is 20-30nm, and after the gold spraying is finished, scanning observation of the pore structure and microstructure of the material is performed by using a Scanning Electron Microscope (SEM) of a model S4800 at room temperature.

The modified bone cements obtained in example 1 and example 2 were subjected to electron microscope scanning, and the results of the electron microscope scanning are shown in fig. 1. The two modified bone cements have no obvious difference in microstructure of 100um and 50um, and a plurality of flake hydroxyapatite are concentrated on the agglomerate.

(2) Injectability

The injectability of the modified bone cement was tested by extrusion using a standard 1ml syringe with an internal diameter of 2 mm. Will contain Na₂SeO₃The fibroin solution and CPC powder are mixed and then immediately filled into an injector. The paste was then extruded from the syringe tip by applying a force to the plunger at a rate of 10mm/min, and extrusion was terminated when the force reached 150 newtons. Injectability (I) was calculated from the following formula: i = { (M)₀-M₁)/M ₀100%. Wherein M is₀Is the initial mass of cement, M₁Is the mass of cement remaining in the syringe after extrusion.

The results of the pushout and syringeability analysis performed with the modified bone cement are shown in fig. 2. With or without Na₂SeO₃The modified bone cement of (2) shows no difference in the extrapolated graph and the injectability analysis, and the injection coefficient of the two bone cements is between 75% and 80%. This is probably because CPC-SF absorbs water and dissolves and tightly binds to the deposited hydroxyapatite crystals when the solid and liquid phases are mixed, increasing the viscosity of the slurry, increasing the friction between the slurry and the inner wall of the injector and between particles in the slurry during injection, and resulting in not very high injection coefficient.

(3) Compressive strength

The mechanical properties of the modified bone cement were evaluated by uniaxial compression test in a universal mechanical testing machine (HY-1080) equipped with a 10 kN weighing cell and a crosshead speed of 0.5 mm/min. Respectively will be put inThe surface of the modified bone cement cured by a cylindrical mold (diameter 6mm, height 12mm) for 1 day, 3 days and 5 days is polished to be flat. During the compression test, the load and displacement were recorded until the sample failed and an engineering stress-strain curve was generated and the maximum stress value before failure was determined as the compressive strength. P =^4N/_ΠD ²P is the compressive strength of the sample, N represents the peak load value, and D represents the diameter of the sample.

The compressive strength results of the modified bone cement are shown in fig. 3. With or without Na₂SeO₃The modified bone cement has good compression resistance, and the time points of curing for 1 day, 3 days and 5 days are not obviously different. Since CPC is brittle, its mechanical strength is insufficient. After CPC powder and SF liquid phase are mixed, part of calcium phosphate salt is quickly dissolved, and the generated calcium ions generate strong binding force with hydroxyl and carboxyl on a SF side chain. In addition, the SF with the net structure tightly connects HA crystals, so that the bonding strength among HA particles is increased, and meanwhile, the composite material HAs good stress dispersion characteristics, so that the compressive strength of the bone cement is improved.

(4) Setting time

Will contain or not contain Na₂SeO₃The SF liquid and CPC powder are mixed, immediately filled into a cylindrical die and smoothed by a spatula. The initial setting time was determined where a light needle (weight 113.4g, diameter 2.12mm) could not be inserted 1.5 mm above the cement top surface; the final setting time was such that a heavy needle (weight 453.6g, diameter 1.06mm) did not leave a complete circular indentation in the cement surface.

The set time results for the modified bone cement are shown in fig. 4. With or without Na₂SeO₃The final setting time of the modified bone cement is within 30min, and good stability can be provided in the early stage; the initial setting time is about 7min, which is enough for operation. SF is used as an additive material with good biocompatibility, and can effectively improve the curing time of CPC. This is probably because the SF of the network structure provides a template for HA deposition when the solid and liquid phases are mixed, thus accelerating the hydration process and greatly improving the efficiency of coagulation.

(5) Resistance to collapsibility and degradation.

Mixing CPC powder with Na or not₂SeO₃The SF solution of (1) is mixed and then filled into a cylindrical mold. After the initial setting time is reached, demoulding, putting the mixture into deionized water, and continuously curing for 6 hours in a water environment at 37 ℃. The appearance of the bone cement was photographed to analyze its anti-collapse properties. In the same way, the prepared novel bone cement is put in deionized water, taken out at different time points, weighed, aired and weighed, and the mass loss condition of the novel bone cement is analyzed.

The collapsibility resistance and degradability of the modified bone cement are shown in fig. 5. With or without Na₂SeO₃The modified bone cement has consistent effect in the aspect of collapsibility resistance; there was also no significant difference in the mass loss of the two bone cements as seen in the 8-week degradation experiments. Firstly, the above results may be due to the fact that alpha-TCP is easily to generate HA when meeting water, so that the alpha-TCP is not easy to degrade; secondly, a large amount of HA is deposited on the surface of phosphate particles, and composite connection between HA and SF with a net structure is realized by hydrogen bonds and ionic bonds between calcium ions and carboxylate anions, so that the problems of high brittleness and easiness in collapse of pure CPC can be effectively solved.

(6) Biocompatibility

The sterilized modified bone cement was added to the α -MEM medium at 0.2 g/mL, and left to stand for 24 hours to extract the leachate. Culture of 5 × 10 per well in 96-well plates³Cells were removed for 24h, and the cell culture medium was removed and the leachate was added at a concentration of 100 μ L per well. At 37 deg.C, 5% CO₂The corresponding leachate is changed the next day and the fourth day. According to the instructions of the Cell Counting Kit-8 (CCK-8) Kit, 100. mu.L of serum-free medium containing 10% CCK-8 reagent was added to each well on the third and fifth days, respectively. At 37 ℃ 5% CO₂After incubation for 1 h in the incubator, absorbance at 450 nm was measured using a microplate reader. The absorbance values are linear with cell number.

The biocompatibility of the modified bone cement is shown in fig. 6. The result shows that the proliferation capacity of the BMMSCs cultured by adding the normal alpha-MEM culture medium is obviously better than the effect of culturing the bone cement leachate. On the third day, contain Na₂SeO₃Cultured with the modified bone cement extractThe proliferation of BMMSCs is not obviously better than that of the BMMSCs without Na₂SeO₃(ii) a But at day five, the two bone cements were significantly different. This indicates that Na was added₂SeO₃The modified bone cement can effectively promote the proliferation capacity of BMMSCs and improve the biocompatibility of CPC-SF bone cement.

EXAMPLE 4 specific examples

The modified bone cement obtained in example 2 was used in the concrete example, as shown in fig. 7. First, a rat is injected with 30mg/kg sodium pentobarbital of 3% for anesthesia, the distal lateral condyles of both femurs are shaved and sterilized, wounds of appropriate size are incised, then circular defects with the diameter of 4mm and the depth of 5mm are drilled, and the modified bone cement prepared in the embodiment 2 of the invention is implanted.

The results of the modified cement experiments after implantation into the femoral condyle defect of the rat are shown in fig. 8. The bilateral femurs of the modeled rats were removed at one and two months post-surgery, scanned using Micro-CT, and the nascent bone BV/TV (ratio of bone surface area to tissue volume) around the modified bone cement was analyzed by software. The experimental result indicates that the modified bone cement added with the sodium selenite can remarkably promote the formation of new bone around the defect and accelerate the repair of bone tissues.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The modified bone cement is characterized by comprising silk fibroin, calcium phosphate bone cement and sodium selenite.

2. A preparation method of modified bone cement is characterized by comprising the following steps:

s1: dissolving sodium selenite in the silk fibroin liquid phase to obtain a liquid mixture;

s2: fully mixing the liquid mixture with calcium phosphate bone cement according to a liquid-solid ratio (0.4-0.6) mL/g;

s3: and curing to obtain the modified bone cement.

3. The method as claimed in claim 2, wherein the silk fibroin liquid phase is obtained by extracting silk fibroin fiber from raw silkworms, degumming and dissolving in S1.

4. The method of claim 3, wherein the concentration of the silk fibroin liquid phase is 4% -6%.

5. The method according to claim 2, wherein in S2, the method for preparing the calcium phosphate cement comprises: preparing hydroxyapatite crystal seed, and mixing with Alfa-tricalcium phosphate powder and calcium hydrogen phosphate powder to obtain calcium phosphate cement.

6. The method of claim 5, wherein in S2, the weight ratio of Alfa-tricalcium phosphate, calcium hydrogen phosphate and hydroxyapatite in the formulation of the calcium phosphate cement is 87:10: 3.

7. The method of claim 2, wherein in S3, the mixture obtained in S2 is cured at 37 ℃ in a saturated humidity environment for 2-5 days to obtain the bone cement.

8. The method according to claim 2, wherein in S2, the liquid mixture is mixed with the calcium phosphate cement in a liquid-to-solid ratio: 0.5mL/g was mixed well.

9. A product for promoting bone tissue repair and new bone growth, wherein the material of the product is the bone cement of claim 1 or the modified bone cement prepared by the method of claims 2-8.

10. Use of the modified bone cement according to claim 1 or the method according to claims 2-8 in the fields of biomaterials and medical applications.