CN112891630B - Implantable blood clot gel composition for bone repair and preparation method thereof - Google Patents

Abstract

The invention discloses an implantable blood clot gel composition, which comprises bone morphogenetic protein-2 and blood clot gel, wherein the bone morphogenetic protein-2 is encapsulated in the blood clot gel. The invention also discloses a preparation method of the implantable blood clot gel composition. The implantable blood clot gel composition has reliable source and lower cost, has excellent biocompatibility and the effect of promoting repair, can continuously release therapeutic drugs at the local part of bone defect, and has the therapeutic effect of promoting bone healing.

Description

Implantable blood clot gel composition for bone repair and preparation method thereof

Technical Field

The invention relates to the technical field of tissue repair, in particular to an implantable blood clot gel composition for bone repair and a preparation method thereof.

Background

Bone defects are a serious orthopedic disorder resulting in local dysfunction, delayed and nonunion of bones due to destruction of bone mass by congenital or acquired causes. Bone grafting is still currently the gold standard for clinical treatment, including autologous bone grafts (from the patient) and allogeneic bone graft implants (from the donor). However, there are many inherent drawbacks to these treatments in practical applications. For example, autologous bone grafting can cause unnecessary pain and trauma to the patient, and can easily cause complications at the donor site, resulting in injury, pain, and infection. Allograft has the potential for immune rejection and the risk of disease transmission. The limitations of bone defect treatment lead to a reduction in the quality of life of the patient. Conventional therapeutic agents for bone diseases in clinical use, including bone morphogenetic protein-2 and the like, are usually administered orally or by systemic injection, which is likely to cause systemic exposure, but at lower concentrations at local lesions.

Research finds that the advanced biological material shows excellent prospect in the aspects of promoting bone regeneration and repair, and provides a treatment method for replacing bone transplantation. Inorganic materials, natural and synthetic polymers, and inorganic and polymer composites have been used to construct bone treatment strategies, however, a number of problems have still hindered their clinical transformation. For example, most biomaterials are used as bone void fillers for fixation, and only a few are available for bone non-healing, with limited clinical indications. In view of the current strategy, drugs are more difficult to release from biomaterials in a sustained and controlled manner, and cannot be delivered locally for long periods of time. In addition, the long-term safety and effectiveness of existing biomaterial-based bone repair systems need to be further elucidated. In order to solve the bone repair problem, the development of a next generation bone repair strategy with osteoconductivity, osteoinductivity and high safety is urgently needed.

Bone healing is a regenerative process involving the involvement of a variety of active molecules and cells, including immune cells, precursor cells and stem cells. Many immune cells, such as neutrophils, monocytes/macrophages and T cells, infiltrate the local injury site with the concomitant production of various cytokines. Macrophages have high activity throughout bone repair due to their high diversity and plasticity. Therefore, there is a great potential for accelerating bone healing by preparing novel bone repair strategies based on biomaterials for regulating local microenvironment of bone injury.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an implantable clot gel composition which has reliable source and lower cost, has excellent biocompatibility and the effect of promoting repair, can continuously release therapeutic drugs at the local part of bone defect and has the therapeutic effect of promoting bone healing.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the invention provides an implantable clot gel composition comprising bone morphogenetic protein-2 and a clot gel, said bone morphogenetic protein-2 being entrapped in said clot gel.

Further, in the implantable blood clot gel composition, the content of the bone morphogenetic protein-2 is 0.01-3 mg/mL.

Further, the clot gel is formed by the coagulation of fresh blood.

Further, the clot gel is added with a procoagulant component or a crosslinking component, wherein the procoagulant component or crosslinking component comprises one or more of chitosan, polyamidoamine dendrimer, snake venom hemocoagulase, vitamin K, desmopressin acetate, blood coagulation factor VIII, prothrombin complex, thrombin, human fibrinogen, aminomethylbenzoic acid, tranexamic acid, aprotinin, aminocaproic acid and gelatin sponge. The addition of the procoagulant component or the crosslinking component solves the problems of long preparation time and insufficient viscoelasticity and toughness of the clot gel, and is beneficial to improving the mechanical strength, viscoelasticity, stability and durability of the clot gel. The proportion of the procoagulant component or the crosslinking component to the blood can be adjusted according to the performance of the blood clot gel, and the mass concentration of the procoagulant component or the crosslinking component to the blood can be 1-30%.

Further, when the implantable blood clot gel composition is used, the implantable blood clot gel composition is directly placed at a bone defect part to be repaired, and in-situ release of the bone morphogenetic protein-2 is realized.

Further, the bone defect site includes skull, frontal bone, ethmoid bone, sphenoid bone, occipital parietal bone, temporal bone, nasal bone, lacrimal bone, maxilla bone, inferior turbinates, zygomatic mandible, hyoid bone, plow bone, spine, sternum, rib, coccyx, sacrum, scapula, clavicle, humerus, ulna, radius, carpal bone, metacarpal bone, phalangeal bone, hip bone, femur, patella, tibia, fibula, tarsal bone, metatarsal bone, phalanges of phalanges, and lower extremity bone.

Further, the bone morphogenetic protein-2 is slowly released through the clot gel.

Further, the administration dosage of the implantable blood clot gel composition is 0.5-15 mL/kg.

The implantable blood clot gel composition has the photo-thermal conversion efficiency and can generate mild thermal effect under the irradiation of near-infrared laser. In addition, the implantable clot gel composition may also modulate the immune microenvironment at the site of the bone defect.

In a second aspect, the present invention provides a method for preparing the implantable clot gel composition, comprising the steps of:

(1) mixing the bone morphogenetic protein-2 with fresh blood, and injecting the mixed solution into a mold;

(2) placing the mixed solution at 20-30 ℃ for 5-15 min for preliminary coagulation;

(3) drying the preliminarily coagulated composition at 35-40 ℃ to obtain the implantable clot gel composition.

Further, in step (1), a procoagulant component or a crosslinking component comprising one or more of chitosan, polyamidoamine dendrimer, snake venom hemocoagulase, vitamin K, desmopressin acetate, factor viii, prothrombin complex, thrombin, human fibrinogen, aminomethylbenzoic acid, tranexamic acid, aprotinin, aminocaproic acid and gelatin sponge is added to the clot gel.

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

1. the blood clot gel is mainly prepared from blood, the blood is taken from a patient, and the blood clot gel has good biocompatibility and degradability, can be completely metabolized, and has no toxic or side effect. Therefore, the blood clot gel constructed by using the blood has good safety, can be loaded with the bone morphogenetic protein-2 with high efficiency and can be released continuously.

2. In the invention, because the blood clot gel is dark red, the photothermal technology based on the blood clot gel has the potential of being excited by near infrared light to generate heat and can generate mild heat effect at the defect part, thereby promoting the repair and regeneration of the bone defect part.

3. The implantable clot gel composition has excellent immunoregulation property, can stimulate macrophage differentiation, regulate and control a local bone defect microenvironment and accelerate bone regeneration.

Drawings

FIG. 1 is a topographical view of an implantable clot gel composition of the present invention;

FIG. 2 is a scanning electron micrograph of an implantable clot gel composition of the present invention;

FIG. 3 is a drug loading rate of an implantable clot gel composition of the invention;

FIG. 4 is a BMP-2 drug release profile of an implantable clot gel composition of the present invention;

FIG. 5 is a graph of the rheological properties of an implantable clot gel composition of the invention;

FIG. 6 is a graph showing the biodegradation profile of an implantable clot gel composition of the invention;

FIG. 7 is a graph showing the osteoblast differentiation promoting effect of the implantable clot gel composition of the present invention;

FIG. 8 is an in vitro photothermal effect of an implantable clot gel composition of the invention;

FIG. 9 illustrates in vivo photothermal effects of an implantable clot gel composition of the invention;

FIG. 10 is a graph showing the photothermal stimulation of osteoblast differentiation-promoting activity of an implantable clot gel composition in accordance with the present invention;

FIG. 11 is a graph of macrophage recruitment by the implantable clot gel composition of the invention;

FIG. 12 is an immunofluorescence analysis of an implantable clot gel composition of the present invention recruiting macrophages;

FIG. 13 is a graph showing macrophage polarization of an implantable clot gel composition of the present invention;

FIG. 14 is a graph of local cytokine secretion following implantation of an implantable clot gel composition in accordance with the invention;

FIG. 15 is a Micro-CT analysis of a defect site in a mouse after treatment with an implantable clot gel composition of the invention;

FIG. 16 is Micro-CT analysis of rat defect sites after treatment with an implantable clot gel composition of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.

Example 1: synthesis and characterization of implantable clot gel compositions

(1) Dissolving the bone morphogenetic protein-2, adding the bone morphogenetic protein-2 into fresh blood to obtain a mixed solution of the blood and the bone morphogenetic protein-2, and injecting the mixed solution into a special mold;

(2) placing the mixed solution obtained in the step (1) at the temperature of 20-30 ℃ for 5-15 min for primary coagulation;

(3) and (3) drying the preliminarily coagulated composition obtained in the step (2) at 35-40 ℃ to obtain the implantable blood clot gel composition.

Fig. 1 is a photograph of an implantable clot gel composition prepared in example 1, and fig. 2 is a scanning electron micrograph of the implantable clot gel composition.

The precipitated liquid outside the framework of the implantable clot gel composition was aspirated and quantified, and the drug loading efficiency of the clot gel for bone morphogenetic protein-2 was examined, with the results shown in fig. 3. As can be seen from the figure, the implantable clot gel composition can be highly loaded with bone morphogenetic protein-2 at drug loadings as high as 90-99%.

The implantable gel carrying the drug is placed in a certain amount of phosphate buffer solution at 37 ℃, the supernatant is taken out at corresponding time points and supplemented with equal amount of isothermal buffer solution, the drug content in the buffer solution is determined by using an enzyme-linked immunosorbent assay, the release characteristics of the drug in the gel system are researched, and the obtained result is shown in figure 4. As can be seen from the figure, bone morphogenetic protein-2 loaded in the clot gel composition is continuously released from the gel matrix.

The rheological properties of the implantable clot gel compositions were examined using a rheometer and the results are shown in figure 5. The results in fig. 5 show that the clot gel composition of the present invention has good mechanical properties.

Example 2: biocompatibility and degradability of implantable clot gel compositions

The in vivo degradation process of the implantable clot gel composition was studied, the implantable clot gel composition was labeled with the fluorescent dye DiD and observed on days 1, 4, 7, 10, and 13 after implantation using fluorescence imaging of small animals, with the results shown in fig. 6.

Fig. 6 shows that the residence time of the implantable clot gel composition is significantly longer locally than the free dye and that the local clot gel composition exhibits a decreasing trend. This demonstrates that the implantable clot gel compositions of the present invention have good biocompatibility and degradable properties.

Example 3: osteogenic differentiation of implantable clot gel compositions

Alkaline phosphatase (ALP) is an early osteogenic marker of cell maturation and calcification. MC3T3-E1 cells were cultured on 6-well plates, treated with different experimental stimuli, and fixed in 4% neutral formalin. The sample was washed several times to remove the remaining liquid. Staining working solution 3 ml of alkaline phosphatase staining buffer, 10. mu.l of BCIP solution (300X), and 20. mu.l of NBT solution (150X) were prepared by mixing to prepare 3.03 ml of BCIP/NBT staining working solution. An appropriate amount of staining working solution was added to the samples to ensure adequate coverage. The samples were incubated in the dark for 5-30 minutes or longer (up to 24 hours) until color developed to the desired depth, the reaction was stopped and the staining working solution was removed and washed 1-2 times.

Alizarin red staining is another method for determining mineralized nodules in osteoblasts, in addition to alkaline phosphatase staining. MC3T3-E1 cells were induced and cultured for about 3 weeks using an implantable clot gel composition and cells were fixed in 4% neutral formalin. Alizarin red staining solution was dropped on the sample for 5 minutes. After gentle washing with water, the samples were rinsed for a few seconds with McGee-Russell differentiation solution. The nuclei were lightly stained with hematoxylin stain from Mayer for 1-2 minutes and then washed 3 times with water. The sample was dehydrated to transparency and sealed with resin media.

As shown in FIG. 7, the alkaline phosphatase activity of the cells treated with the implantable clot gel composition was significantly enhanced compared to the other controls, and calcium nodules and calcium salt deposits were detected in the implantable clot gel composition in higher numbers than in the control group, indicating that the implantable clot gel composition effectively promoted osteogenic differentiation of the cells.

Example 4: in vitro photothermal effects of implantable clot gel compositions

To investigate the effect of laser intensity on the heat transfer efficiency of the implantable clot gel composition, the implantable clot gel composition was applied at different powers (0W/cm)²、0.2W/cm²、0.3W/cm²And 0.4W/cm²) The temperature rise curve was recorded by an infrared camera after 808nm laser irradiation for about 10 minutes, and the result is shown in FIG. 8.

The results of fig. 8 show that implantable clot gel compositions having a reddish brown color can be used as novel photothermal agents, producing mild heat under laser irradiation, which has been shown to promote bone repair.

Example 5: in vivo photothermal effects of implantable clot gel compositions

The implantable clot gel composition was implanted subcutaneously in mice using 0.7W/cm²The temperature rise curve was recorded by an infrared camera after irradiating 808nm laser light for 10 minutes at a power, and the result is shown in FIG. 9.

As can be seen in fig. 9, the implantable clot gel composition also had excellent photothermal effects in vivo.

Example 6: photo-thermal stimulation of implantable clot gel compositions on osteoblast differentiation promoting effects

The implantable clot gel composition was used to photothermally stimulate MC3E3-T1 cells and protein expression was analyzed by western blot. The total protein amount of the sample was determined by BCA protein kit. 20-30. mu.g protein per well was used. Samples were incubated with primary anti-Runx 2, Osterix, Hsp 47 and β -actin, followed by secondary antibody, and enhanced chemiluminescence methods were used to detect protein signaling. Heat Shock Protein (HSP)47, a HSP in mammals, regulates collagen crosslinking, which is essential for the biosynthesis and molecular maturation of collagen type I associated with osteogenesis.

The results in FIG. 10 show that the implantable clot gel composition was effective in activating Hsp 47 expression after light exposure, and that the photothermal stimulation of Runx2 expression by the implantable clot gel composition was 5-fold and 2-fold higher than by NIR irradiation and the implantable clot gel composition alone, respectively, and that Ostetrix expression was also significantly increased compared to the control group.

Example 7: photothermal stimulation immune regulation effect of implantable blood clot gel composition

To assess local bone immunology after treatment, mice with skull defects were photothermal treated with PBS or an implantable clot gel composition. The heads were photographed contrastingly on the third, seventh and fourteenth days after treatment. Blood clots and adjacent local tissue in the skull defects of the mice were removed and prepared into single cell suspensions, which were analyzed by flow cytometry, and the results are shown in fig. 11-13.

As can be seen from fig. 11, the composition of the present invention significantly recruited macrophages compared to the control group, and fig. 12 shows that immunofluorescence also shows a large amount of macrophage infiltration. Referring to FIG. 13, by phenotyping the infiltrating macrophages, it was found that the macrophage types in the composition were more polarized in the early phase as pro-inflammatory M1 type and gradually shifted from pro-inflammatory M1 polarization to anti-inflammatory repair of M2 type macrophages in the later phase of bone treatment. The results show that the composition has an excellent bone immune microenvironment regulation effect and is beneficial to promoting the healing of local bones.

In addition, blood clots in the skull defects of the mice and adjacent local tissues are treated by tissue cytokine extracting solution, and then the secretion level of the local relevant proinflammatory and anti-inflammatory cytokines is measured by an enzyme-linked immunosorbent kit. As can be seen in FIG. 14, photothermal treatment with the implantable clot gel composition significantly stimulated a significant increase in the inflammatory cytokines TNF-a and IL-6 over the first 7 days, which was approximately two-fold and four-fold that of the control group. On day 14, the inflammatory factor concentration tended to decrease. In addition, the concentration of the anti-inflammatory cytokine IL-10 was gradually increased and the concentration of IL-4 was maintained at a higher level.

Example 8: analysis of effect of implantable clot gel composition on photothermal treatment of bone defects

A drill bit was used to make severe circular defects in the cranium of experimental Balb/c mice and SD rats, avoiding puncturing the meninges during the procedure. The free BMP-2 or clot gel/implantable clot gel composition is then injected into the site of the cranial defect. The surgical wound is closed carefully to avoid loss of the drug. The 24 experimental mice were randomly divided into 6 groups, 1 st group: a control group; group 2: free BMP-2; group 3: an illumination group; group 4: clot gel; group 5: implantable clot gel compositions and group 6: the photo-thermal treatment group can be implanted with the blood clot gel composition. The irradiation frequency was once every three days for a total of three times during the experiment. All experiments were performed in a sterile environment and the protocol was in compliance with animal ethical specifications. Skull bone from mouse and rat models were harvested 8 and 12 weeks post-surgery, respectively, and fixed in neutral formalin for subsequent computerized tomography (micro-CT) to evaluate regenerated new bone, with results shown in fig. 15, 16.

As can be seen from fig. 15-16, the implantable clot gel composition of the present invention significantly promoted bone growth and repair in the bone injury area, accelerating bone healing in both large and mouse models.

In conclusion, the implantable blood clot gel composition has excellent biocompatibility and the effect of promoting bone repair.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (7)

1. An implantable clot gel composition for bone repair comprising bone morphogenic protein-2 and a clot gel, said clot gel formed by the coagulation of fresh blood, said bone morphogenic protein-2 being entrapped in said clot gel;

when in use, the implantable blood clot gel composition is directly placed on a bone defect part to be repaired, and the bone defect part is irradiated by near infrared laser so as to promote bone growth and repair of the bone defect area.

2. The implantable blood clot gel composition for bone repair of claim 1, wherein the implantable blood clot gel composition comprises 0.01 to 3mg/mL of bone morphogenetic protein-2.

3. An implantable clot gel composition according to claim 1, wherein the clot gel further incorporates a procoagulant or cross-linking component comprising one or more of chitosan, polyamidoamine dendrimer, snake venom haemagglutinase, vitamin K, desmopressin acetate, factor viii, prothrombin complex, thrombin, human fibrinogen, aminomethylbenzoic acid, tranexamic acid, aprotinin, aminocaproic acid and gelatin sponge.

4. The implantable clot gel composition for bone repair of claim 1, wherein the bone defect site comprises a frontal bone, an ethmoid bone, a sphenoid bone, an occipital bone, an parietal bone, a temporal bone, a nasal bone, a tear bone, a palatine bone, a maxilla, a turbinate, a zygomatic bone, a mandible, a hyoid, a plow bone, a spine, a sternum, a rib, a coccyx, a sacrum, a scapula, a clavicle, a humerus, an ulna, a radius, a carpal bone, a metacarpal bone, a phalangeal bone, a hip bone, a femur, a patella, a tibia, a fibula, a tarsal bone, a metatarsal bone, a phalanges.

5. An implantable clot gel composition for bone repair according to claim 1, wherein the bone morphogenic protein-2 is slowly released through the clot gel.

6. An implantable clot gel composition for bone repair according to claim 5, wherein the implantable clot gel composition is administered in a dose of 0.5 to 15 mL/kg.

7. The method of preparing an implantable clot gel composition for bone repair of claim 1, comprising the steps of:

(1) mixing the bone morphogenetic protein-2 with fresh blood, and injecting the mixed solution into a mold;

(2) placing the mixed solution at 20-30 ℃ for 5-15 min for preliminary coagulation;

(3) and drying the primarily coagulated composition at 35-40 ℃ to obtain the implantable clot gel composition.

Images