CN113244447B

CN113244447B - Controllably degradable porous magnesium phosphate bone cement and preparation method and application thereof

Info

Publication number: CN113244447B
Application number: CN202110553509.9A
Authority: CN
Inventors: 戴红莲; 赵雅楠; 余素春; 李文琴
Original assignee: Wuhan University of Technology WUT
Current assignee: Wuhan University of Technology WUT
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2022-06-07
Anticipated expiration: 2041-05-20
Also published as: CN113244447A

Abstract

The invention relates to the technical field of biomedical materials, in particular to controllable degradable porous magnesium phosphate bone cement and a preparation method and application thereof. The magnesium phosphate bone cement is compounded with gelatin microspheres, and the gelatin microspheres have sizes in multi-stage gradient distribution and cross-linking degrees in multi-stage gradient distribution. According to the invention, the gelatin microspheres with different crosslinking degrees and different sizes are combined and compounded into the magnesium phosphate bone cement, so that compared with the porous magnesium phosphate bone cement with single crosslinking degree and size range, the porous magnesium phosphate bone cement has a pore structure which is particularly suitable for cell attachment, bone and vascular tissue ingrowth and metabolic waste discharge and a degradation rate matched with the growth rate of new bones, the material degradation and the new bones are stably jointed, the bone repair process is regulated and optimized, and the bone repair efficiency is improved.

Description

Controllably degradable porous magnesium phosphate bone cement and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to controllable degradable porous magnesium phosphate bone cement and a preparation method and application thereof.

Background

Bone defects caused by trauma, bone tumors, car accidents, congenital diseases and the like are always important medical problems which are troubled and affect human health, are easy to cause bone nonunion, delayed healing or even nonhealing, and local dysfunction, and need a large amount of high-quality bone grafting materials for clinical repair. About 200 ten thousand bone transplants are performed worldwide every year, wherein autologous bone transplantation is the gold standard due to excellent osteoinduction and no immune rejection, but the source is limited, and other risks are easily brought by secondary operations; although the source of the allogeneic transplantation is wider, the pathogen transmission and the immunogenicity problems exist. Therefore, the artificial bone repair material has the advantages of unlimited sources, capability of repairing and regenerating body tissues, capability of continuously improving and the like, and is widely concerned by researchers.

Magnesium Phosphate Bone Cement (MPBC) is mainly prepared from Magnesium oxide (MgO), phosphate and a curing liquid according to a certain proportion. Because the coagulation time is controllable, the early strength is high, and the cured product has the adhesive property and the micro-expansion property, the MPBC has the following problems in application, but the MPBC is widely applied to the fields of fracture adhesion fixation, bone defect filling and repairing and the like: the bone tissue repair process is complex and is influenced by multiple factors such as biological effect, mechanical stimulation, nutrition supply, structure and characteristics of implant materials and the like. However, MPBC is mainly characterized by nanoscale and submicron pores inside, and lacks micron pores for the ingrowth of blood vessels, several hundred-micron macropores for the ingrowth of bone tissue, and through-pores for cell migration and transport of nutrients and metabolic wastes. The ideal bone repair process is a process that the scaffold material bears load in the early stage of implantation and then gradually degrades, and the process is accompanied with the continuous generation of new bone and gradually exerts mechanical and biological functions. The repair rate of bone defects is greatly related to the defect positions, the size of the defects and individual differences of patients, and the repair time is up to several months or even longer. Therefore, the MPBC degradation rate and the new bone growth rate are often difficult to match, cell adhesion and penetration are not facilitated, and a bony cladding surrounding the material is easily formed, so that the bone defect repair efficiency is reduced. And (III) the combination mode of the MPBC and surrounding tissues is mainly physical combination, so that the longitudinal depth of osseointegration is limited, and the bone repair process and the operation effect are influenced. And (IV) the MPBC hydration process is insufficient, so that MgO which does not participate in the reaction remains, and local pH is too high after hydrolysis, which is not favorable for biocompatibility.

Disclosure of Invention

In view of the above, the invention aims to provide a controllably degradable porous magnesium phosphate bone cement, and a preparation method and application thereof. The magnesium phosphate bone cement can form good bonding capacity with surrounding bone tissues, material degradation and stable connection with new bones are formed, the magnesium phosphate bone cement has good biocompatibility, and the treatment efficiency of bone defect repair and regeneration is improved.

The scheme adopted by the invention for solving the technical problems is as follows:

the controllably degradable porous magnesium phosphate cement is compounded with gelatin microspheres, and the gelatin microspheres have sizes in multi-stage gradient distribution and cross-linking degrees in multi-stage gradient distribution.

Preferably, the gelatin microspheres for compounding have a size of 50-400 μm and a degree of crosslinking of 15-45% in the completely dried state, wherein the ratio of the size of 50-100 μm, 100-200 μm, 200-300 μm, 300-400 μm is 1: (1-7): (1-7): (1-7); the proportion of the crosslinking degree of 0, 15-25%, 25-35% and 35-45% is 1: (1-7): (1-7): (1-7). The degree of crosslinking is determined by ninhydrin color development.

Preferably, the gelatin microspheres for compounding have a size of 50-400 μm and a degree of crosslinking of 15-45% in the completely dried state, wherein the ratio of the size of 50-100 μm, 100-200 μm, 200-300 μm, 300-400 μm is 1: (2-4): (2-4): (2-4); the proportion of the crosslinking degree of 0, 15-25%, 25-35% and 35-45% is 1: (2-4): (2-4): (2-4).

Preferably, the gelatin microspheres are swollen and equilibrated in an aqueous solution of citric acid at a concentration of 0.005-0.2g/ml before being compounded with the magnesium phosphate cement material.

Preferably, the controllably degradable porous magnesium phosphate bone cement comprises the following raw materials in percentage by mass: 15-35% of magnesium oxide powder, 25-65% of monopotassium phosphate, 5-30% of monocalcium phosphate, 0-20% (preferably 5-20%) of gelatin microspheres and 10-40% of citric acid aqueous solution.

Preferably, the particle size of the magnesia powder is 0.5-5 μm, and the specific surface area is 0.5-4m²(ii)/g; the concentration of the citric acid aqueous solution is 0.005-0.2 g/ml.

The invention also aims to provide a preparation method of the above controlled degradation porous magnesium phosphate bone cement, which comprises the following steps:

a. preparing gelatin microspheres with different crosslinking degrees and different sizes by adopting an emulsion polymerization method, and cleaning and drying the obtained gelatin microspheres for later use;

b. screening the gelatin microspheres prepared in the step a, grouping according to different sizes and crosslinking degrees, and uniformly mixing the microspheres with different sizes and crosslinking degrees;

c. b, adding the gelatin microspheres obtained in the step b into a citric acid aqueous solution for full swelling to serve as a bone cement liquid phase;

d. fully mixing and stirring the magnesium oxide powder, the potassium dihydrogen phosphate and the calcium dihydrogen phosphate with the bone cement liquid phase obtained in the step c to obtain bone cement paste;

e. and pouring the obtained slurry into a mold for molding, and maintaining the mold at the temperature of 37 ℃ in an environment with the relative humidity of 100% to obtain the controllably degradable porous magnesium phosphate bone cement.

Preferably, the preparation method of the gelatin microsphere comprises the following steps: dispersing 10-60% gelatin water solution in oil phase, stirring for some time, changing ice water bath, dripping crosslinking agent, crosslinking, centrifuging, cleaning, drying, and controlling stirring speed and crosslinking agent addition to obtain gelatin microspheres with different sizes and different crosslinking degrees.

Preferably, the oil phase is one or more of liquid paraffin, span 20, span 40, span 80, tween 20, tween 60 and tween 80; the cross-linking agent is one or more of glutaraldehyde, genipin, carbodiimide hydrochloride and N-hydroxysuccinimide.

The invention also aims to provide the application of the controllably degradable porous magnesium phosphate bone cement or the controllably degradable porous magnesium phosphate bone cement prepared by the preparation method on bone grafting materials.

Gelatin (Gelatin) has good compatibility and bioactivity, is a natural degradable high polymer material, is easy to absorb degradation products without inflammatory reaction, and is widely applied to the field of biological materials such as tissue engineering scaffolds, wound dressings, novel drug release carriers and the like. Biodegradable Gelatin Microspheres (GM) are doped into the MPBC, in-situ pore forming can be realized in bone cement, and abundant pore structures can guide cell attachment, growth of bone and vascular tissues and discharge of metabolic waste; the GM serves as an aggregate in the bone cement, so that the mechanical property is improved, and the compressive strength and the bearing capacity are improved; the GM contains an arginine-glycine-aspartic acid peptide sequence and a large number of hydrophilic groups, so that the recognition sites of cells can be increased, the adhesion and growth of the cells on the scaffold can be promoted, the bonding capability with surrounding bone tissues can be improved, and good biological combination can be formed; the degradation of GM is the hydrolysis of protein, which can neutralize the alkaline environment generated by the degradation of bone cement and improve the biocompatibility. Therefore, based on the excellent performances of MPBC and GM, the MPBC and GM are compounded to prepare the controllably degradable porous magnesium phosphate bone cement, and the controllably degradable porous magnesium phosphate bone cement can be used as the controllably degradable porous biological bone cement in the biomedical field.

According to the invention, the gelatin microspheres with different crosslinking degrees and different sizes are combined and compounded into the magnesium phosphate bone cement, so that compared with the porous magnesium phosphate bone cement with single crosslinking degree and size range, the porous magnesium phosphate bone cement has a pore structure which is particularly suitable for cell attachment, bone and vascular tissue ingrowth and metabolic waste discharge and a degradation rate matched with the growth rate of new bones, the material degradation and the new bones are stably jointed, the bone repair process is regulated and optimized, and the bone repair efficiency is improved. The combination of the crosslinking degree and the size of the gelatin microspheres is adjusted within a certain range, so that the effect of obviously regulating the degradation rate of the bone cement can be achieved, the preparation process is simple and easy to operate, the adjustment can be conveniently carried out according to the individual difference of different patients, and the gelatin microspheres have good application prospect in the field of biomedical materials.

Drawings

FIG. 1 is a scanning electron micrograph (high power) of the magnesium phosphate cement prepared in example 1 degraded in PBS for 7 days;

FIG. 2 is a scanning electron micrograph (lower magnification) of the magnesium phosphate cement prepared in example 1 degraded in PBS for 7 days;

FIG. 3 is a graph showing the pH change of magnesium phosphate cement degraded in PBS for 60 days;

FIG. 4 is a graph showing the change of weight loss rate of magnesium phosphate bone cement degraded in PBS for 60 days;

FIG. 5 is a graph of staining of CD31 in Mg-phosphate cement implanted subcutaneously in SD rats for 14 days (arrows indicate blood vessels);

FIG. 6 is a micro-CT image of magnesium phosphate cement implanted into femoral defect of SD rat for 45 days;

FIG. 7 is a scanning electron micrograph of magnesium phosphate cement prepared in example 2 degraded in PBS for 30 days;

FIG. 8 is a graph showing cell proliferation of magnesium phosphate cement cultured together with osteoblasts for 5 days.

Detailed Description

The following examples are provided to further illustrate the present invention for better understanding, but the present invention is not limited to the following examples.

Example 1

The preparation process of this example includes:

a. dispersing 25% gelatin aqueous solution in liquid paraffin and span 80, stirring at a rotation speed of 200 r/min-400 r/min for 10min respectively, then changing ice water bath, dropwise adding carbodiimide hydrochloride to enable the concentration of the carbodiimide hydrochloride in the mixed solution to be 0-1 mol/L, crosslinking for 1h, centrifuging, cleaning, and airing in a ventilated place. The gelatin microspheres with different sizes and different crosslinking degrees are obtained by controlling the stirring speed and the addition amount of the crosslinking agent. Specifically, when the concentration of the crosslinking agent carbodiimide hydrochloride is 0.1M, the crosslinking degree of the obtained gelatin microspheres under the condition of different rotating speeds is 15-25% by adopting a ninhydrin color development method; when the concentration of the cross-linking agent is 0.5M, the cross-linking degree of the obtained gelatin microspheres under the condition of different rotating speeds is 25-35%; when the concentration of the cross-linking agent is 1M, the cross-linking degree of the obtained gelatin microspheres under the condition of different rotating speeds is 35-45%.

b. Sieving the gelatin microspheres prepared in the step a, wherein the gelatin microspheres are sieved according to the proportion of 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm of the size of 1: 4: 3: 2 and the proportion of the crosslinking degree of 0, 15-25%, 25-35% and 35-45% is 1: 4: 3: 2, mixing the components.

c. Weighing 18% of magnesium oxide powder, 36% of monopotassium phosphate, 12% of monocalcium phosphate, 36% of citric acid aqueous solution (with the concentration of 0.03g/ml) and 10% of the gelatin microspheres prepared in the step b according to the mass percentage.

d. C, adding the gelatin microspheres weighed in the step c into a citric acid aqueous solution according to the proportion in the step c, and fully swelling for 10 minutes to serve as a bone cement liquid phase;

e. fully mixing and stirring the magnesium oxide powder, the potassium dihydrogen phosphate and the calcium dihydrogen phosphate which are weighed in the step c and the bone cement liquid phase prepared in the step d;

f. and e, pouring the slurry prepared in the step e into a mold for molding, and maintaining the mold at 37 ℃ in an environment with the relative humidity of 100% to obtain the controllably degradable porous magnesium phosphate bone cement.

The bone cement is degraded in PBS for 7 days, and the gelatin microspheres and the calcium-phosphorus material form good bonding (figure 1), and uniform pores are formed inside the bone cement (figure 2); degradation in PBS for 60 days, pH 7.38-7.6 (FIG. 3), weight loss rate 28% (FIG. 4); the red blood cell is implanted under the skin of an SD rat, and the SD rat draws materials after 14 days and is stained with CD31, so that the red blood cell can promote angiogenesis (figure 5); the material is implanted into femoral defects of SD rats, and micro-CT scanning is carried out on the material obtained after 45 days, so that the material is uniformly degraded and generates new bones (figure 6).

Example 2

The preparation method differs from example 1 in that:

the proportion of the size of the gelatin microsphere obtained in the step b is 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm is 1: 3: 3: 3, the proportion of the cross-linking degree of 0, 15-25%, 25-35% and 35-45% is 1: 3: 3: 3.

the obtained bone cement is degraded in PBS for 30 days, and a large amount of calcium-phosphorus deposits are formed on the surfaces of the gelatin microspheres and in-situ formed holes (figure 7); degradation in PBS for 60 days with a weight loss rate of 22% (fig. 4); after 5 days of co-culture with osteoblasts, it was found that the bone cement significantly promoted osteoblast proliferation compared to the control group (osteogenic medium group) (fig. 8).

Example 3

The preparation method differs from example 1 in that:

the proportion of the size of the gelatin microsphere obtained in the step b is 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm is 1: 2: 3: 4, and the proportion of the cross-linking degree of 0, 15-25%, 25-35% and 35-45% is 1: 2: 3: 4.

the obtained bone cement was degraded in PBS for 60 days with a weight loss rate of 19% (fig. 4).

Example 4

The preparation method differs from example 1 in that:

the proportion of the size of the gelatin microsphere obtained in the step b is 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm is 1: 1: 1: 7, and the proportion of the cross-linking degree of 0, 15-25%, 25-35% and 35-45% is 1: 1: 1: 7.

the obtained bone cement was degraded in PBS for 60 days with a weight loss of 13% (fig. 4).

Example 5

The preparation method differs from example 1 in that:

the proportion of the size of the gelatin microsphere obtained in the step b is 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm is 1: 7: 1: 1, and the proportion of the cross-linking degree of 0, 15-25%, 25-35% and 35-45% is 1: 7: 1: 1.

the bone cement is degraded in PBS for 60 days, and the weight loss rate is 30 percent (figure 4); after 5 days of co-culture with osteoblasts, it was found that the bone cement significantly promoted osteoblast proliferation compared to the control group (osteogenic medium group) (fig. 8).

Example 6

The preparation method differs from example 1 in that:

the proportion of the size of the gelatin microsphere obtained in the step b is 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm is 1: 0.5: 0.5: 8, and the proportion of the cross-linking degree of 0, 15-25%, 25-35% and 35-45% is 1: 0.5: 0.5: 8.

the bone cement was degraded in PBS for 60 days with a weight loss rate of 8% (fig. 4).

Example 7:

the preparation method differs from example 1 in that:

the proportion of the size of the gelatin microsphere obtained in the step b is 50-100 μm, 100-200 μm, 200-300 μm and 300-400 μm is 1: 8: 0.5: 0.5, and the proportion of the cross-linking degree of 0, 15-25%, 25-35%, 35-45% is 1: 8: 0.5: 0.5.

the obtained bone cement was degraded in PBS for 60 days with a weight loss rate of 32% (fig. 4).

Example 8:

the preparation process of this example is:

a. weighing 18 percent of magnesium oxide powder, 36 percent of monopotassium phosphate, 12 percent of monocalcium phosphate and 36 percent of citric acid aqueous solution (the concentration is 0.03g/ml) according to the mass percent.

b. B, fully mixing and stirring the magnesium oxide powder, the potassium dihydrogen phosphate, the calcium dihydrogen phosphate and the citric acid aqueous solution which are weighed in the step a;

c. and c, pouring the slurry prepared in the step b into a mold for molding, and maintaining the mold at 37 ℃ in an environment with the relative humidity of 100% to obtain the controllably degradable porous magnesium phosphate bone cement.

The obtained bone cement is degraded in PBS for 60 days, the pH value is 7.82-8.19 (figure 3), and the weight loss rate is 6.5% (figure 4); the cells were implanted under the skin of SD rats, and after 14 days, the cells were stained with CD31, and only a small amount of angiogenesis was observed (FIG. 5); implanting the material into femoral defect of SD rat, taking materials after 45 days, and carrying out micro-CT scanning to find that the material is slowly degraded (figure 6); after 5 days of co-culture with osteoblasts, the cells were found to be in good growth state without significant toxicity, but without significant proliferation compared to the control group (osteogenic medium group) (fig. 8).

Example 9

The preparation method differs from example 1 in that:

step a, adopting a rotating speed of 200r/min and a cross-linking agent concentration of 0.1M to obtain the gelatin microsphere with a single size and a cross-linking degree, wherein the size of the gelatin microsphere is 300-400 mu M, and the cross-linking degree is 15-25%. There is no step b grading process.

The obtained bone cement and osteoblasts are cultured for 5 days, and have no obvious toxicity (figure 8), but the degradation rate of the microspheres is too high, so that good framework support is difficult to form, and the microspheres are in grading defect, so that the comprehensive performance index of the bone cement is poor; the cells were implanted under the skin of SD rats, and after 14 days, the cells were stained with CD31, and a small amount of angiogenesis was observed, and the effect of angiogenesis was inferior to that of example 1 (FIG. 5).

Example 10

The preparation method differs from example 1 in that:

and a step a, adopting a rotating speed of 400r/min and a cross-linking agent concentration of 1M to obtain the gelatin microspheres with single size and cross-linking degree, wherein the size of the gelatin microspheres is 50-150 mu M, and the cross-linking degree is 35-45%. There is no step b grading process.

The obtained bone cement has incomplete microsphere grading inside, too small size and slow degradation, is difficult to form rich pore structures, is not beneficial to angiogenesis (figure 5), and has little difference from the embodiment 8 without gelatin microspheres in the effect of blood vessel growth.

While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

1. The controllably degradable porous magnesium phosphate bone cement is characterized in that gelatin microspheres are compounded in the magnesium phosphate bone cement, and the gelatin microspheres have sizes in multi-stage gradient distribution and crosslinking degrees in the multi-stage gradient distribution; the gelatin microsphere for compounding has the size of 50-400 μm and the crosslinking degree of 15-45% under the condition of complete drying, wherein the proportion of the size of 50-100 μm, the size of 100-200 μm, the size of 200-300 μm and the size of 300-400 μm is 1: (1-7): (1-7): (1-7); the proportion of the crosslinking degree of 0, 15-25%, 25-35% and 35-45% is 1: (1-7): (1-7): (1-7).

2. The controllably degradable porous magnesium phosphate bone cement according to claim 1, characterized in that the gelatin microspheres used for compounding have a size of 50-400 μm and a degree of crosslinking of 15-45% in the completely dried condition, wherein the ratio of the size of 50-100 μm, 100-: (2-4): (2-4): (2-4); the proportion of the crosslinking degree of 0, 15-25%, 25-35% and 35-45% is 1: (2-4): (2-4): (2-4).

3. The controllably degradable porous magnesium phosphate bone cement of claim 1, characterized in that the gelatin microspheres are in swelling equilibrium in aqueous citric acid solution at a concentration of 0.005-0.2g/ml before being compounded with magnesium phosphate bone cement material.

4. The controllably degradable porous magnesium phosphate bone cement of claim 1, which comprises the following raw materials by mass percent: 15-35% of magnesium oxide powder, 25-65% of monopotassium phosphate, 5-30% of monocalcium phosphate, 5-20% of gelatin microspheres and 10-40% of citric acid aqueous solution.

5. The controllably degradable porous magnesium phosphate bone cement according to claim 4, wherein said magnesium oxide powder particle size is 0.5-5 μm, specific surface area is 0.5-4 m/g; the concentration of the citric acid aqueous solution is 0.005-0.2 g/ml.

6. The method for preparing the controllably degradable porous magnesium phosphate bone cement according to any one of claims 1 to 5, characterized by comprising the following steps:

7. The method according to claim 6, wherein the gelatin microspheres are prepared by: dispersing 10-60% gelatin water solution in oil phase, stirring for some time, changing ice water bath, dripping cross-linking agent, fully cross-linking, centrifuging, cleaning, drying, and controlling stirring speed and cross-linking agent addition to obtain gelatin microspheres with different sizes and different cross-linking degrees.

8. The preparation method according to claim 7, wherein the oil phase is one or more of liquid paraffin, span 20, span 40, span 80, tween 20, tween 60 and tween 80; the cross-linking agent is one or more of glutaraldehyde, genipin, carbodiimide hydrochloride and N-hydroxysuccinimide.

9. The use of the controllably degradable porous magnesium phosphate cement according to any one of claims 1 to 5 or the controllably degradable porous magnesium phosphate cement obtained by the preparation method according to any one of claims 6 to 8 as a bone graft material.