CN116099055A

CN116099055A - Preparation method and application of injectable guided bone regeneration composite material with biphase function

Info

Publication number: CN116099055A
Application number: CN202310049922.0A
Authority: CN
Inventors: 周艺群; 史洋; 柯志威; 顾静怡; 邵长鹆; 谢志坚
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-02-01
Filing date: 2023-02-01
Publication date: 2023-05-12

Abstract

The invention discloses a preparation method and application of an injectable guided bone regeneration composite material with a biphase function, and the preparation of the guided bone regeneration composite material with different pore diameters is realized through the biphase design of a common main body of the composite material. Wherein, the lower layer large aperture bone tissue scaffold material loaded with bone morphogenetic protein and calcium phosphate can induce bone tissue regeneration, and the upper layer small aperture soft tissue barrier layer can block competitive growth of fibroblast and epithelial cells, and the integrated biphasic scaffold material is obtained after light solidification, and the gradient porous structure of the scaffold ensures blood circulation and nutrition exchange, and finally realizes the dual functions of barrier membrane and induced bone regeneration in the bone regeneration guiding operation.

Description

Preparation method and application of injectable guided bone regeneration composite material with biphase function

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method and application of an injectable guided bone regeneration composite material with a biphase function.

Background

Bone tissue defects with different degrees of oral and maxillofacial regions can be caused by tooth extraction, trauma, periodontitis and the like, and the defects not only affect the appearance of a patient and prevent the oral cavity from performing normal physiological functions, but also possibly affect implantation repair. Studies have shown that the probability of bone augmentation surgery being required in implant surgery is nearly 50%. The most common clinical treatment for the above problems is currently directed bone regeneration (Guide bone regeneration, GBR). GBR is to implant bone substitute material into affected area, to induce or conduct bone to grow cells with bone formation potential around to promote bone regeneration in defect area, and then to cover soft tissue barrier membrane to prevent fast growth of gum soft tissue into bone defect and to provide ideal stable space for bone regeneration below.

The most commonly used bone substitute materials at present comprise autogenous bone, allogeneic bone and xenogeneic bone powder, but autogenous bone and allogeneic bone are not accepted by most patients because of the need to open up a second operation area and the risk of virus transmission. The heterogeneous bone powder has become the main strategy for clinically repairing bone tissues at present, however, certain limitations exist. Firstly, due to the limitation of an oral cavity operation space, bone meal-like structural materials need to be taken and put for multiple times in the use process, so that the clinical operation process is greatly influenced; secondly, the shaping capability is poor, the shape is not easy to maintain in operation and is scattered in other areas of an operation area, and the application of the vertical bone increment operation is limited; moreover, most clinical researches show that as most of the heterogeneous bone powder is hydroxyapatite or tricalcium phosphate materials, the substitution rate is low, and the ingrowth of new bone is greatly limited; meanwhile, the cost is very high, so that the injection bone repair material with the matched domestic degradation speed and new bone formation speed is developed, the new bone regeneration can be induced ideally, and a good shaping function can be realized, thereby providing convenience for clinical operation.

In GBR, in addition to the implantation of bone substitute materials, it is also necessary to cover soft tissue membranes, mainly collagen membranes (e.g., sea-Oak membranes), heterogeneous pericardium membranes (e.g., bio-Gide membranes), etc., which are currently most commonly used, to prevent the growth of gingival fibroblasts and epithelial cells, and these soft tissue membranes have a controllable degradation rate, can coordinate with the regeneration of surrounding tissues, and have a good barrier effect. However, the main component is collagen, so that the collagen is softer, is easy to shift during or after operation, loses barrier effect and affects the curative effect of the operation. Therefore, the invention aims to realize the biphasic effect of bone tissue repair and soft tissue barrier in hard tissue defect by using the same main material, thereby providing convenience for oral GBR operation, and the domestic GBR product can lighten the economic burden of patients and bring economic benefit to society.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a preparation method and application of an injectable guided bone regeneration composite material with a biphase function. The present invention includes a bone tissue scaffold layer for inducing hard tissue regeneration and a soft tissue barrier layer for blocking soft tissue ingrowth; the two are solidified into a whole by photoinitiation, thereby realizing the ideal guiding bone regeneration function.

The specific technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides a method for preparing an injectable bone regeneration guiding composite scaffold material with a biphasic function, which specifically comprises the following steps:

s1: dissolving large-aperture porous methacrylic acylated gelatin (GelMA, substitution rate 60%) with normal saline to obtain a pre-polymerized solution with a final concentration of 15%; then, the pH value of the prepolymer is regulated to be neutral, and the aseptic prepolymer is obtained after filtration;

s2: dissolving photoinitiator, hydroxyapatite (HAP) and Bone Morphogenetic Proteins (BMPs) in the sterile prepolymerization solution to obtain a final concentration of 15 percent of bone tissue scaffold layer prepolymerization solution;

s3: dissolving small-aperture porous methacrylic acylated gelatin (GelMA, substitution rate 30%) with normal saline to obtain a pre-polymerized solution with a final concentration of 15%; then, the pH value of the prepolymer is regulated to be neutral, and the sterile soft tissue barrier prepolymer is obtained after filtration;

s4: then placing the bone tissue scaffold layer prepolymer liquid into an injector, extruding and injecting the bone tissue scaffold layer prepolymer liquid into a bone defect area through a nozzle, trimming the outline by using a shaping knife, and repairing the bone tissue defect after photo-curing;

s5: and (3) dropwise adding the aseptic soft tissue barrier prepolymer onto the surface of the cured bone tissue scaffold material, flattening and shaping the surface by using a transparent film, curing the surface and the surface by using ultraviolet light, curing the surface and the surface into a whole by photoinitiation, and removing the transparent film to complete the guided bone regeneration operation.

Simultaneously, the film with the soft tissue barrier layer can be trimmed by scissors according to the defect range so as to meet the defect shape;

preferably, the pore diameter of the large-pore porous GelMA hydrogel is 300-500 mu m; in the large-aperture porous bone tissue repair precursor liquid, the substitution rate of the large-aperture porous GelMA hydrogel is 60%, the mass concentration is 15%, the mass concentration of the photoinitiator is 0.5%, and the pH value of the solution is 7.4. The substitution rate and concentration can be adjusted according to the actual blood supply condition.

Preferably, the light-resistant water bath stirring means magnetic stirring for 1h at 37 ℃ in a light-resistant water bath.

Preferably, the photoinitiator is Irgacure2959 ultraviolet photoinitiator, and ultraviolet lamp curing means curing for 1-2min by using an ultraviolet lamp with the wavelength of 405 nm.

Preferably, the pore diameter of the small pore diameter porous GelMA hydrogel is 100-300 mu m; in the small-aperture porous GelMA hydrogel precursor solution, the substitution rate of the small-aperture porous GelMA hydrogel is 30%, the mass concentration is 15%, the mass concentration of a photoinitiator is 0.5%, and the pH value of the solution is 7.4. The substitution rate and concentration can be adjusted according to the actual blood supply condition.

Preferably, the concentration of the HAP solution is 5% -30%, preferably 15%.

Preferably, in the large-aperture porous bone tissue repair precursor liquid, the mass ratio of HAP to GelMA is 1:1 or 2:1, and the mass ratio can be adjusted according to the bone defect shape and the actual shaping requirement; HAP uses deproteinized bone graft material of bovine bone.

Preferably, in the large-aperture porous bone tissue repair precursor solution, the concentration of the BMPs solution is 600-800ng/mL, preferably 600ng/mL.

In a second aspect, the present invention provides an injectable guided bone regeneration composite material with biphasic function prepared by the preparation method according to any one of the first aspect.

In a third aspect, the present invention provides a use of the injectable guided bone regeneration composite material with biphasic function according to the second aspect for preparing an injectable guided bone regeneration composite scaffold.

Compared with the prior art, the invention has the following beneficial effects:

1) The invention prepares the bone tissue scaffold layer and the soft tissue barrier membrane with different apertures by using the same main body, respectively induces bone tissue regeneration and soft tissue barrier by using the aperture size, simultaneously ensures the regeneration of blood vessels, and realizes the bidirectional function of maintaining the bone defect repair space by the barrier membrane and the bone substitute in the GBR technology. Wherein the small-aperture soft tissue barrier layer covering the defect surface ensures blood circulation and nutrient exchange while blocking the competitive growth of fibroblasts and epithelial cells; meanwhile, the bone tissue scaffold with large aperture can induce new bone regeneration, and the aperture can ideally induce cell osteogenesis to differentiate, so as to realize the function of promoting repair and regeneration of bone defect areas.

2) The invention optimizes the plasticity of the hydrogel by adding the hydroxyapatite into the main material, so that the hydrogel still has ideal mechanical property and shapeability in the unfavorable bone defect form, meets the requirements of personalized bone contours, has certain mechanical property and proper degradation time, and realizes the unfavorable bone defect form, such as vertical/horizontal bone increment.

3) The soft tissue scaffold can be trimmed according to clinical actual conditions, avoids related anatomical structures to cover defect areas in an ideal way, can form an integrated scaffold material with a bone tissue scaffold layer through photoinitiation, solves the displacement problem caused by inconsistent soft tissue and hard tissue body materials at present, breaks a clinical technical barrier, and provides a new thought for guiding bone regeneration repair materials in new times.

4) According to the invention, the barrier membrane function is realized through the small-aperture GelMA, so that a heterogeneous biological membrane is not required to be additionally covered in GBR operation, the operation mode of the traditional GBR operation is improved, and the operation efficiency is improved. Meanwhile, the membrane can obtain personalized forms through construction so as to meet the barrier requirements of different bone defect areas and meet the effective, efficient and personalized clinical treatment modes.

Drawings

FIG. 1 is a schematic diagram of a large pore size porous bone tissue repair precursor solution in an embodiment;

FIG. 2 illustrates implantation and shaping of a bone tissue scaffold according to an embodiment;

FIG. 3 is a clinical application of the biphasic functional integrated stent material of the example;

FIG. 4 is a schematic illustration of a method for preparing a soft tissue scaffold using a transparent film according to an embodiment.

Detailed Description

The invention is further illustrated and described below with reference to the drawings and detailed description. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.

Examples

This example provides a method for preparing an injectable guided bone regeneration composite with biphasic function, the following specific implementation of this example is illustrated by aiming at the vertical bone defect caused by trauma:

1) Preparation of large-aperture porous GelMA prepolymerization liquid

And (3) dissolving the large-aperture porous GelMA hydrogel by using normal saline, then uniformly dissolving the photoinitiator in the porous GelMA solution, and magnetically stirring in a water bath at 37 ℃ in a dark place for 1h to prepare GelMA prepolymer.

In the example, the pore diameter of the adopted large-pore porous GelMA hydrogel is 300-500 mu m, the adjustable range of the physical and chemical properties of the GelMA is large, and the requirements on the strength and stability of a bone tissue scaffold required by vertical bone defect are high, and the scaffold is required to maintain a bone grafting space in the initial stage of implantation, so that the substitution rate of the GelMA in the GelMA prepolymer solution is 30%, the mass concentration is 10%, the mass concentration of a photoinitiator is 0.5%, the photoinitiator is Irgacure2959 violet photoinitiator, and the pH of the solution is adjusted to 7.4.

2) Preparation of large-aperture porous bone tissue repair precursor liquid

And (2) respectively adding HAP and BMPs into the prepolymer of the large-aperture porous GelMA prepared in the step (1) and fully and uniformly mixing, wherein the mass ratio of the HAP to the GelMA is 2:1, and the concentration of BMP-2 is 600ng/ml, so as to obtain the large-aperture porous bone tissue repair precursor (shown in figure 1).

3) Preparation of small-aperture porous soft tissue barrier precursor liquid

And (3) dissolving the small-aperture porous GelMA hydrogel by using normal saline, and then uniformly dissolving the photoinitiator in the small-aperture GelMA solution, and magnetically stirring in a water bath at 37 ℃ in a dark place for 1h to prepare the small-aperture porous soft tissue barrier precursor solution.

In this example, the pore size of the small pore size porous GelMA hydrogel used was 100-300. Mu.m. The barrier membrane material is shaped to a certain extent according to different defect forms, and is difficult to shift by maintaining a certain form in the early stage of defect recovery, so that the substitution rate of GelMA in the small-aperture porous soft tissue barrier precursor liquid is 30%, the mass concentration is 15%, the mass concentration of the photoinitiator is 0.5%, the photoinitiator is Irgacure2959 ultraviolet photoinitiator, and the pH of the solution is regulated to 7.4.

4) Implantation and shaping of bone tissue scaffold layer

Loading the large-aperture porous bone tissue repairing precursor liquid obtained in the step 2) into a sterile injector, pushing the sterile injector into a vertical bone defect area, curing the bone tissue by using a 405nm ultraviolet lamp for 1-2min to form a bone tissue bracket, and trimming the surface of the bracket to a normal contour by using a plastic cutter after curing. The bone tissue scaffold can be filled into each area of the defect by the injector, and the scaffold is tightly combined with the rest tissue of the affected area, wherein the diameter of the adopted needle is 150 mu m, and the injection flow rate is 5ml/min. The specific method is that the sterile needle is firstly and slowly injected into the low-level tissue of the bone tissue defect area, the lifting bracket is gradually injected and shaped, and finally the densification filling is completed.

In this embodiment, when the height of the vertical bone defect exceeds 10mm, the ultraviolet lamp curing mode may adopt layered curing, and curing is performed once every 3mm of height is injected until the required scaffold height is reached, so that the large-aperture porous bone tissue repair precursor solution at the vertical bone defect is cured, and the bone tissue scaffold strength is ensured, as shown in fig. 2. Here, since the vertical bone increment requires high molding ability for bone tissue material, the mass ratio of GelMA to HAP is 1:2.

5) Dual phase function integrated stent material formation

The small pore porous soft tissue barrier precursor in step 3) is dripped onto the bone tissue scaffold surface using a sterile instrument. As shown in fig. 3, the bone tissue scaffold and the barrier membrane were adhered by irradiation with 405nm uv light for 30min to obtain an integrated double-layered scaffold, and after curing, the scaffold was trimmed to a normal profile using a shaping blade, completing implantation of bone regeneration repair scaffold material and personalized GBR surgery.

In this embodiment, when the bone defect morphology is unfavorable and the soft tissue scaffold material easily flows away, the small-aperture porous soft tissue barrier precursor can be dripped on the surface of the soft transparent film, as shown in fig. 4, the film area is determined according to the defect size, the prepolymer with different area ranges is dripped on the film according to the defect size by using a sterile injector, and the soft tissue barrier layer is obtained by curing for 1-2min by using a 405nm ultraviolet lamp. And shaping the soft tissue barrier layer according to the actual bone defect range, removing redundant materials by scissors to ensure that the soft tissue barrier layer has a thickness of at least 2mm, enabling the edge to be tightly attached to the defect area without leaving a gap, obtaining a personalized soft tissue repair layer, and completing the implantation of the integrated double-layer bracket by combining the operations.

The barrier membrane material and the bone tissue scaffold are combined by opening double chains through GelMA under illumination, and in the embodiment, a layer of small-aperture porous soft tissue barrier precursor liquid can be coated on the surface of the bone tissue scaffold, and then the soft tissue scaffold is placed to enable the combination to be firmer, so that the integrated repair scaffold material is finally formed.

In addition, according to the bone defect shape and filling convenience, the large-aperture porous bone tissue repair precursor liquid with different GelMA concentration and HAP concentration can be selected to meet different defect requirements, so that personalized filling and repair can be realized.

The invention aims to prepare the double-layer integrated repair bracket through the same main body material, thereby realizing synchronous personalized repair of oral clinical soft and hard tissues, avoiding displacement of a collagen membrane and providing great convenience for operation.

The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.

Claims

1. A method for preparing an injectable guided bone regeneration composite material with a biphasic function, which is characterized in that the material comprises a bone tissue scaffold layer for inducing bone tissue regeneration and a barrier layer for blocking soft tissue ingrowth; the barrier layer is attached to the bone tissue support layer, and after light irradiation curing, an integrated composite support material with the barrier film and the dual functions of inducing bone regeneration and guiding bone regeneration can be obtained;

the preparation method of the bone tissue scaffold layer comprises the following steps:

dissolving the large-aperture porous methacrylic acylated gelatin hydrogel with normal saline, then sequentially and uniformly dissolving the photoinitiator, the hydroxyapatite and the bone morphogenetic protein in the hydrogel, and uniformly stirring the mixture in a light-shielding water bath to obtain a large-aperture porous bone tissue repair precursor solution; injecting the large-aperture porous bone tissue repair precursor liquid into a affected area, and solidifying by an ultraviolet lamp after shaping to obtain a bone tissue scaffold layer;

the preparation method of the soft tissue barrier layer comprises the following steps:

dissolving small-aperture porous methacrylic acylated gelatin hydrogel with normal saline, uniformly dissolving a photoinitiator therein, and uniformly stirring in a light-resistant water bath to obtain small-aperture porous soft tissue barrier precursor liquid; and injecting the small-aperture porous soft tissue barrier precursor liquid into a affected area, and curing by an ultraviolet lamp after shaping to obtain the soft tissue barrier layer.

2. The method for preparing an injectable guided bone regeneration composite material with biphasic function according to claim 1, wherein the pore size of the large pore size porous methacryloylated gelatin hydrogel is 300-500 μm; in the large-aperture porous bone tissue repair precursor liquid, the substitution rate of hydrogel is 60%, the mass concentration is 15%, the mass concentration of photoinitiator is 0.5%, and the pH of the solution is 7.4; the concentration of bone morphogenic protein is 600-800ng/mL, preferably 600ng/mL.

3. The method for preparing the injectable guided bone regeneration composite material with the biphasic function according to claim 1, wherein the light-resistant water bath stirring is magnetic stirring for 1h at 37 ℃ in a light-resistant water bath.

4. The method for preparing the injectable guided bone regeneration composite material with the biphasic function according to claim 1, wherein the photoinitiator is an Irgacure2959 ultraviolet photoinitiator, and the ultraviolet lamp curing is performed for 1-2min by using a 405nm ultraviolet lamp.

5. The method for preparing an injectable guided bone regeneration composite material with biphasic function according to claim 1, wherein the pore size of the small pore size porous methacryloylated gelatin hydrogel is 100-300 μm; in the small-aperture porous methacrylic acylated gelatin hydrogel precursor liquid, the substitution rate of the hydrogel is 30%, the mass concentration is 15%, the mass concentration of the photoinitiator is 0.5%, and the pH of the solution is 7.4.

6. The method of preparing an injectable guided bone regeneration composite material with biphasic function according to claim 1, wherein the concentration of the hydroxyapatite solution is 5% -30%, preferably 15%.

7. The method for preparing the injectable guided bone regeneration composite material with the biphasic function according to claim 1, wherein the mass ratio of the hydroxyapatite to the macroporous porous methacryloylated gelatin hydrogel in the macroporous porous bone tissue repair precursor solution is 1:1 or 2:1; the hydroxyapatite is deproteinized bone grafting material of bovine bone.

8. The method for preparing an injectable guided bone regeneration composite material with a biphasic function according to claim 1, wherein the large-pore porous bone tissue repair precursor solution is injected by a sterile syringe.

9. An injectable guided bone regeneration composite material with biphasic function prepared by the preparation method of any one of claims 1 to 8.

10. Use of the injectable guided bone regeneration composite material with biphasic function according to claim 9 for the preparation of an injectable guided bone regeneration composite scaffold.