CN114377203A - Preparation and application of injectable bone repair material capable of activating endogenous TGF beta 1 in situ - Google Patents

Abstract

The injectable bone repair material capable of activating endogenous TGF beta 1 in situ is formed by compounding two parts, namely a water-containing matrix and microspheres, wherein the microspheres are TypeA gelatin microspheres with uniform particle sizes and prepared by a microfluidic method, and the water-containing matrix is formed by dispersing gelatin molecules modified by laponite XLG and Polyethyleneimine (PEI) into a water phase. The injectable bone repair material can be prepared by adsorbing alkaline bicarbonate or neutral phosphate into gelatin microspheres by a diffusion loading method, and then mixing the gelatin microspheres with an aqueous matrix.

Description

Preparation and application of injectable bone repair material capable of activating endogenous TGF beta 1 in situ

Technical Field

The invention relates to the field of biomedical materials, in particular to preparation and application of an injectable bone repair material capable of in-situ activating endogenous TGF beta 1.

Background

One long-term goal of oromaxillofacial regenerative medicine is the treatment of defects, loss of function and aesthetic deformities of the oromaxillofacial bone tissue due to trauma, tumor resection, periodontal disease, alveolar ridge resorption, treatment-related osteonecrosis of the jawbone, and congenital deformities. When the damage exceeds the critical bone defect size, the bone tissue fails to heal spontaneously. At present, autologous bone grafting remains the "gold standard" for bone defect repair. However, the risks of pain, infection, nerve damage and loss of function associated with bone harvesting surgery limit its use. Allogeneic bone or xenogeneic bone transplantation is often accompanied by problems of immunological rejection or disease transmission and the like. Therefore, research for reconstructing a bone defect site by using a tissue engineering method has received great attention. It was reported that scientific research works with the topic of tissue engineering have been published in 41588 worldwide from 1999 to 2016. It is expected that by 2023, the total number of works will double; by 2027, the total number of the works is three times as large as that of the present days. This further illustrates the powerful and enormous potential of the field of tissue engineering science.

Cell transplantation (cell transplantation) and cell homing (cell homing), both of which are widely used in tissue engineering research. The former requires first obtaining a sample of stem cells from a patient; after in vitro amplification, injection into the defect site, either alone or with a scaffold material, is desired for the purpose of promoting tissue growth. The latter means that stem cells or progenitor cells migrate and move to the defect site under the induction of biological signals, thereby achieving tissue regeneration. The main scientific value of cell transplantation lies in that the cells can be subjected to gene modification and fluorescent labeling in vitro, and whether the cells participate in tissue regeneration or not can be conveniently and further researched; whether the cells keep dry or not is conveniently researched by means of continuous transplantation; and the influence of the pathological environment of the injury part on the transplanted cells is conveniently researched. However, in the process of cell transplantation, the difficulty of obtaining/separating living cells is high, the obtained cell types are uncertain, the cell maintenance and clinical operation process is complex and high in cost, injected cells are easy to die and lose, and isolated cells are easy to generate immune rejection, pathogen transmission, carcinogenicity and other problems, so that the clinical application of the isolated cells is limited. The method for promoting tissue regeneration by inducing the homing of Mesenchymal Stem Cells (MSCs) from extracellular matrix (ECM) at the edge of a defect or blood to the defect only by chemotaxis (chemotaxis) of biological signals (such as growth factors) can well avoid the problems caused by cell transplantation, is also a leading-edge hotspot of the current tissue engineering research, and is expected to be widely applied clinically in the future.

The goal of inducing bone marrow mesenchymal stem cells (BMSCs) to home and promote bone regeneration can be achieved by releasing related growth factors through biological materials or other carriers at the bone defect. Yoon et al reported that exogenous transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein-2 (BMP-2) were added to chitosan hydrogel to promote repair of tibial defect in rabbits. However, the scheme for driving the release of exogenous growth factors at the bone defect has many disadvantages, such as easy degradation of the growth factors in the scaffold material, uncertain bioactivity and high cost. Therefore, many scholars have focused on studies to induce BMSC homing without direct addition of growth factors. Zhang et al, directly injecting adenovirus vector containing platelet-derived growth factor b (PDGF-b) and BMP-7 sequence together with scaffold material to the periodontal defect of beagle dog, the transfected cells can continuously generate corresponding growth factors, thereby promoting the repair of periodontal defect. This approach, while saving cost and avoiding the problem of protein instability, increases the likelihood of viral infection and immune rejection. In contrast, a protocol that utilizes endogenous growth factors to induce homing and osteogenesis of BMSCs would be a more attractive and safe and reliable option.

In mammals, endogenous TGF β 1 is widely present in organs, tissue matrices, and many cells. Among them, the bone matrix and platelets have the highest TGF β 1 content. TGF beta 1 plays an important role in organ development, damage repair, tumor inhibition, atherosclerosis and other aspects. In bone repair, TGF β 1 induces BMSC homing primarily through SMAD signaling pathways; it also increases osteoprogenitor cell proliferation and promotes chondrocyte differentiation (inhibits osteoblast differentiation) at high concentrations. Unlike other growth factors, TGF β 1 in the matrix and as secreted by the cells is present as a latent TGF β 1(LTGF β 1) complex and is unable to bind to its receptor. Activated TGF β 1(activated TGF β 1, aTGF β 1) can be obtained only after the non-covalent bond (electrostatic interaction) between the TGF β 1 protein dimer and latency-associated propeptide (LAP) is broken by a certain method. Therefore, how to safely and effectively activate endogenous LTGF beta 1 at the bone defect is a hot problem to be solved urgently in the research of bone tissue engineering.

LTGF β 1 may be activated under a variety of physicochemical conditions. LTGF beta 1 can be well activated in vitro by high-temperature heating (75-100 ℃) or ultrasonic oscillation, additional integrin combination, additional protease catalysis (such as matrix metalloproteinase and fibrinolysin) and other methods. But heat and ultrasound treatment at the defect is likely to cause secondary damage; the addition of macromolecular proteins to biomaterials is neither easy to preserve nor likely to elicit immune responses, and therefore neither of these methods is suitable for the activation of endogenous LTGF β 1. To date, only very limited literature has reported studies on how to promote tissue regeneration by activating endogenous LTGF β 1. The Mooney group applied low energy laser radiation to the residual tissue of dental pulp, and the resulting oxygen radicals activated endogenous LTGF β 1 released from the dentinal tubules. These aTGF β 1 induce migration of adult Dental Stem Cells (DSC) from apical foramen into root canals and achieve dental pulp regeneration. However, since the bone defect is usually large in range and often accompanied by bleeding, the low-energy laser can only act in a very small range, and the penetration force in blood is limited, so that the laser is not suitable for the research of bone tissue engineering.

The change in extracellular pH can serve a variety of regulatory roles. The non-physiological acidic (pH1-6) and basic (pH8-14) environments can disrupt the electrostatic interaction between LAP and TGF beta 1 protein dimer, thereby dissociating to obtain aTGF beta 1. After in vitro treatment of porcine cortical bone with concentrated hydrochloric acid, the acid solution was shown to contain aTGF β 1. After the acid solution is added into the culture medium, the expression of TGF beta 1 related target genes in fibroblasts can be detected. In addition, LTGF β 1 in serum could also be theoretically activated under non-physiological pH conditions. LTGF β 1 in serum is released from aggregated platelets during clot formation. In addition, the non-physiological pH environment can also significantly affect the process of bone repair. The alkaline pH environment is more beneficial to enhancing the activity of BMSC alkaline phosphatase and promoting the proliferation, differentiation and mineralization of osteoblasts; the acidic pH environment reduces the activity of alkaline phosphatase in osteoblasts and activates the activity of osteoclasts, thereby enhancing bone resorption. Thus, creating an environment of optimal alkaline non-physiologic pH at the bone defect and selecting the appropriate duration of action is likely to safely and effectively activate LTGF β 1 in the bone matrix and serum, thereby inducing BMSC homing and promoting bone regeneration.

Disclosure of Invention

The invention aims to provide preparation and application of an injectable bone repair material capable of activating endogenous TGF beta 1 in situ, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

the injectable bone repair material capable of activating endogenous TGF beta 1 in situ is formed by compounding two parts, namely a water-containing matrix and microspheres, wherein the microspheres are Type A gelatin microspheres with uniform particle sizes and prepared by a microfluidic method, and the water-containing matrix is formed by dispersing gelatin molecules modified by Laponite XLG and PEI into a water phase. The injectable bone repair material can be prepared by adsorbing alkaline bicarbonate or neutral phosphate into gelatin microspheres by a diffusion loading method, and then mixing the gelatin microspheres with an aqueous matrix.

Furthermore, the Type A gelatin microspheres are prepared by a microfluidic method, the diameter of the gelatin microspheres is about 120 mu m, and the matrix part is composed of 4.5 wt% of laponite and 2 wt% of PEI modified gelatin solution.

Further, the preparation method of the aqueous matrix comprises the following steps: dissolving PEI modified gelatin in water, shaking and dispersing laponite in water for 30s, and then uniformly mixing the laponite and the laponite to obtain an aqueous matrix, wherein the final concentration of the laponite hydrogel is 4.5 wt%, and the concentration of the PEI modified gelatin is 2 wt%.

Further, the PEI modified gelatin molecule preparation method comprises the steps of grafting PEI molecules and Type A gelatin molecules together through condensation reaction of amino-carboxyl catalyzed by carbodiimide, dialyzing and freeze-drying to obtain a dry sample.

Further, the aqueous solution of the basic bicarbonate has a pH value of 10, and the aqueous solution of the neutral phosphate has a pH value of 7.4.

The invention also provides a preparation method of the injectable bone repair material, which is characterized by comprising the following steps:

(1) preparing TypeA gelatin microspheres by a microfluidic method: the core component of the microfluidic system is a microfluidic chip made of PMMA material, the thickness of the microfluidic chip is 3mm, and the width of an internal pore channel is 150 micrometers. When the equipment is operated, the oil phase liquid cuts the TypeA gelatin solution of the water phase, and the obtained gelatin liquid drops with uniform size are finally subjected to chemical crosslinking, cleaning and freeze-drying treatment to form gelatin microspheres with the particle size of 120 mu m;

(2) PEI modified gelatin molecule: respectively dissolving PEI molecules and TypeA gelatin molecules in MES (4-morpholine ethanesulfonic acid, 2- (N-morpholinyl) ethanesulfonic acid) buffer solution with the pH value of 6, linking the PEI molecules and the TypeA gelatin molecules through amino-carboxyl condensation reaction catalyzed by carbodiimide, dialyzing and freeze-drying to obtain products;

(3) preparation of the aqueous matrix: dissolving PEI modified gelatin in water, vibrating and dispersing laponite in water, and then uniformly mixing the materials to obtain an aqueous matrix, wherein the final concentration of laponite hydrogel is 4.5 wt%, and the concentration of PEI modified gelatin is 2 wt%;

(4) the gelatin microspheres are loaded with alkaline bicarbonate or neutral phosphate by a diffusion loading method.

The invention provides application of an injectable bone repair material in inducing BMSC homing and promoting bone regeneration.

Further, the injectable bone repair material can effectively activate LTGF β 1 in serum.

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

(1) activation of LTGF beta 1 at bone defects by biomaterials

1) Controlling the alkaline pH of biological materials

The normal pH value of human blood is mainly maintained in the range of neutral alkali, namely 7.35-7.45 by carbonate buffer system and the like. In human body, toxic alkaline substances are injected, and the human tissue and electrolyte balance can be damaged by the short-term (less than 24 hours) strong alkaline environment and the long-term (more than 24 hours) alkaline environment. In this context, therefore, a buffer system of carbonate is used to adjust the pH of the material. The pH value of the sodium carbonate-sodium bicarbonate buffer solution can be controlled to be 9-11. In the control group, the pH of the material was adjusted to 7.4 using neutral Phosphate Buffered Saline (PBS).

2) Preparing hydrogel material with injectable performance

In this project, each component of the material to be prepared has good biocompatibility, and it is formed by mixing an entity and a hydrogel. The entity is Type A gelatin microsphere, which supports cell migration and attachment and is degraded slowly; the hydrogel part is formed by mixing Type A gelatin solution with

XLG is combined, mainly adjusts the viscosity of the material, and degrades quickly.

3) The viscosity of the material is well controlled, so that the material can resist the dilution effect of liquid

Laponite (A)

XLG) is a disc-shaped nanoparticle (diameter 25nm, thickness 0.92nm) which itself has a very good osteogenesis promoting effect. In pure water, the edges of the nanodiscs are positively charged, while the two sides of the disks are negatively charged. It has been reported in the literature that at certain concentrations, when it comes into contact with positively charged Type A gelatin molecules in pure water, the whole system will become a shear-thinning biomaterial (STB) by strong electrostatic interactions, which is very resistant to liquid dilution (Sci Transl Med,2016.8(365):365ra 156). Due to Type A gelatin moleculeThe isoelectric point is at pH7-9, and at pH values above 9 it will only carry a negative charge. Therefore, in this item, Polyethyleneimine (PEI) molecules with polyamino structures are grafted onto Type a gelatin molecules, so that the Type a gelatin molecules carry positive charges under any pH condition, thereby maintaining the viscosity of the hydrogel material.

4) Well promotes the activation of TGF beta 1 in rat serum, promotes cell migration and bone repair in vivo

After adjusting the pH of the material by using PBS and phosphate buffer, we found that alkaline material can effectively activate TGF β 1 in rat serum. Activated serum can promote cell migration. In addition, the biological material is injected into the skull defect of the rat, and the alkaline material shows better bone repair capacity.

Drawings

Fig. 1 is a diagram for preparing gelatin microspheres with uniform particle size, wherein a is a design diagram of a microfluidic chip; B. gelatin microsphere liquid drops are formed in the micro-fluidic chip pipeline; C. and (3) preparing a gelatin microsphere microscope photo by utilizing a microfluidic mode.

FIG. 2 shows the ELISA method for detecting the activation of TGF beta 1 in rat serum by sodium bicarbonate solution with different pH values at different time points.

FIG. 3 shows the effect of different groups of hydrogels on the pH value of rat serum and the activation of TGF beta 1 of rat serum at different time points, wherein A. the activation experiment is shown schematically; B. a TGF beta 1 quantitative result in material activation serum; C. the result of pH change after incubation of the material with serum.

FIG. 4: in vivo experiments verify the osteogenesis of different groups of hydrogel, wherein A, a rat skull defect model is constructed, and different groups of hydrogel are injected into a defect area; B. three months later, the osteogenic effect of the hydrogels of the different groups was analyzed by μ CT (a. blank control group; b.ph7 MGel group; c.ph10 MGel group).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.

In order to facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings, in which several embodiments of the invention are shown, but which can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1 preparation of injectable bone repair Material capable of in situ activation of endogenous TGF beta 1

The hydrogel material is divided into two parts of a matrix and gelatin microspheres. Wherein the gelatin microspheres are prepared by adopting a microfluidic mode. In order to ensure the injectability of the final hydrogel material, the diameter of the prepared gelatin microsphere needs to be controlled to be about 120 μm.

(1) Preparation of TypeA gelatin microspheres: the core component of the microfluidic system is a microfluidic chip. The chip is designed by Solidworks CAD software and then is cut in a numerical control mode, and is made of PMMA resin, the thickness of the chip is 3mm, and the width of an inner pore channel is 150 micrometers. Preparing a 5 percent TypeA gelatin solution as a water phase, mineral oil containing 20 percent by weight of a surfactant Span80 as an oil phase, and respectively setting the injection speeds of the oil phase and the water phase to be 18.8ul/min and 3.08 ul/min. The collection device is placed in a water bath at 15 ℃ to coagulate the gelatin drops flowing out into colloid state, and then 50ml of 5% glutaraldehyde aqueous solution is added, and the mixture is vigorously stirred for 5 hours at 4 ℃ to crosslink and fix the gelatin microspheres. And (3) washing away mineral oil by using cyclohexane, washing away residual cyclohexane by using absolute ethyl alcohol, washing away the absolute ethyl alcohol by using deionized water, dispersing the microspheres into sufficient glycine aqueous solution of 25mM, and oscillating for 1h to seal the free aldehyde group tail end. Finally, deionized water is repeatedly washed, and the gelatin microspheres with the particle size of 120 mu m are obtained by freeze-drying (figure 1).

(2) PEI modified gelatin molecule: respectively dissolving PEI and TypeA gelatin in 0.1 mol/L2- (N-morpholino) ethanesulfonic acid buffer (MES) at pH 6; adding a catalyst carbodiimide (EDC) and a stabilizer N-hydroxysuccinimide (NHS), and stirring at 37 ℃ for reacting for 24h to obtain the PEI modified gelatin molecule. Preferably, 0.9g of TypeA gelatin, 180mg of PEI (MW 1800Da), 38.34mg of EDC and 23.02mg of NHS are dissolved in 100ml of MES as described above, reacted at 37 ℃ for 24 hours, dialyzed against a deionized water environment for three days, and lyophilized to give PEI-modified gelatin. Ninhydrin test can quantify amino, the amino content of PEI modified gelatin molecule is increased, and dispersion test and rheology result prove that PEI modified gelatin molecule has stronger binding force with laponite particles and is not easy to disperse in water.

(3) Aqueous base: dissolving PEI modified gelatin in water, shaking and dispersing laponite in water for 30s, and then uniformly mixing the materials to obtain an aqueous matrix, wherein the final concentration of laponite hydrogel is 4.5 wt%, and the concentration of PEI modified gelatin is 2 wt%.

(4) Activating LTGF β 1: SD rat whole blood is collected, kept still for half an hour until the blood is coagulated, and centrifuged at 5000rpm for 10min to obtain serum. The serum acid-base strength was adjusted to pH 9, 10, 11 and 12 using 1M sodium hydroxide and hydrochloric acid solutions, and after 0.5h, 2h and 20h, the pH of all serum samples was adjusted back to 7.4, after which the TGF β 1 content in rat serum was determined by ELISA (FIG. 2). The results prove that the pH10 has moderate acid-base strength and can effectively activate TGF beta 1 to about 10ng/ml in serum within 0.5 hour.

(5) Preparing an injectable bone repair material: firstly, adopting a diffusion loading method to enable the gelatin microspheres to respectively adsorb alkaline and neutral salts. The microspheres are respectively placed into a sodium bicarbonate solution with the pH value of 10 and a PBS solution with the pH value of 7.4 for 0.5h and then freeze-dried, the freeze-dried microspheres and PEI modified or PEI unmodified gelatin matrix are mixed according to the proportion of 5:1 to prepare four different injectable hydrogels, and the change of TGF beta 1 in rat serum is respectively activated by the four hydrogels through an ELISA method within 0.5 to 48 hours (figures 3A and B). At the same time, the change in the pH of the serum sample was measured using an electronic pH meter (fig. 3C). The results demonstrate that the injectable hydrogel base at pH10 is moderately strong and can effectively activate TGF β 1 in rat serum within 0.5, 10, 24 and 48 hours.

Example 2 use of injectable bone repair materials that can activate endogenous TGF β 1 in situ

First construct SAnd D, a rat skull defect model. Performing intraperitoneal injection anesthesia on 2% sodium pentobarbital, fixing the head of a rat, preparing the skin, sterilizing by 2% iodophor, cutting the skin and the muscle layer 5-8mm from the center of the cranial midline, and stripping the periosteum to expose the bone surface. Circular bone defects are made on two sides of a cranial central suture by trephines with the outer diameter of 5mm, gelatin matrix microsphere hydrogel modified by PH7 PEI (PH7 Mgel) and gelatin matrix microsphere hydrogel modified by PH10PEI (pH10 Mgel) are injected in the defect area, a muscle layer is sutured by absorbable threads of 5-0, and a skin layer is sutured by threads of 5-0. And (3) putting the postoperative rat on a 37 ℃ heat-insulating pad until the rat is awake and recovers consciousness, and normally feeding the rat in different cages. After three months, CO₂The rats are sacrificed in the box, and the skull is scanned by the micro CT after the materials are taken, and the three-dimensional reconstruction is carried out to analyze the osteogenesis effect. The results of the study preliminarily confirmed that the hydrogel of microspheres modified with PEI at pH10 had better osteogenesis (FIG. 4).

Claims (8)

1. An injectable bone repair material capable of activating endogenous TGF beta 1 in situ is characterized by comprising a water-containing matrix and microspheres, wherein the microspheres are TyPEA gelatin microspheres with uniform particle sizes prepared by a microfluidic method, the water-containing matrix is formed by dispersing gelatin molecules modified by laponite and Polyethyleneimine (PEI) into a water phase, and after alkaline bicarbonate or neutral phosphate is adsorbed into the gelatin microspheres by a diffusion loading method, the gelatin microspheres and the alkaline bicarbonate or neutral phosphate are mixed to prepare the injectable bone repair material.

2. The injectable bone repair material according to claim 1, wherein the Type A gelatin microspheres are prepared in a microfluidic manner, the diameter of the gelatin microspheres is about 120 μm, and the aqueous matrix part consists of 4.5 wt% of laponite gel and 2 wt% of PEI modified gelatin solution.

3. The injectable bone repair material according to claim 2, characterized in that the aqueous matrix is prepared by a method comprising: dissolving PEI modified gelatin in water, shaking and dispersing laponite in water for 30s, and then uniformly mixing the laponite and the laponite to obtain an aqueous matrix, wherein the final concentration of the laponite hydrogel is 4.5 wt%, and the concentration of the PEI modified gelatin is 2 wt%.

4. The injectable bone repair material according to claim 1, wherein the PEI modified gelatin molecule is prepared by grafting PEI molecule and Type A gelatin molecule together through carbodiimide catalyzed amino-carboxyl condensation reaction, dialyzing and freeze-drying to obtain a dry sample.

5. The injectable bone repair material according to claim 1, wherein the basic bicarbonate has a pH of 10 in aqueous solution and the neutral phosphate has a pH of 7.4 in aqueous solution.

6. The method for preparing an injectable bone repair material according to claim 1, comprising the steps of:

(1) preparing TypeA gelatin microspheres by a microfluidic method: the core component of the microfluidic system is a microfluidic chip and is made of polymethyl methacrylate (PMMA), the thickness of the microfluidic chip is 3mm, the width of an internal pore channel is 150 micrometers, when the equipment runs, the oil-phase liquid cuts the TypeA gelatin solution of the water phase, and the obtained gelatin liquid drops with uniform size are finally subjected to chemical crosslinking, cleaning and freeze-drying treatment to form gelatin microspheres with the particle size of 120 micrometers;

(2) PEI modified gelatin molecule: respectively dissolving PEI molecules and TypeA gelatin molecules in MES (4-morpholine ethanesulfonic acid, 2- (N-morpholinyl) ethanesulfonic acid) buffer solution with the pH value of 6, linking the PEI molecules and the TypeA gelatin molecules through amino-carboxyl condensation reaction catalyzed by carbodiimide, and dialyzing and freeze-drying to obtain products;

(3) preparation of the aqueous matrix: dissolving PEI modified gelatin in water, vibrating and dispersing laponite in the water, and then uniformly mixing the laponite and the laponite to obtain an aqueous matrix, wherein the final concentration of the laponite hydrogel is 4.5 wt%, and the concentration of the PEI modified gelatin is 2 wt%;

(4) the gelatin microspheres are loaded with alkaline bicarbonate or neutral phosphate by a diffusion loading method.

7. Use of the injectable bone repair material of claim 1 to induce BMSC homing and promote bone regeneration.

8. Use according to claim 7, wherein the injectable bone repair material is effective to activate latent TGF β 1 in serum.

Images