In-situ pore forming coenzyme-based bone adhesive as well as preparation method and application thereof

Abstract

The invention discloses an in-situ pore forming coenzyme-based bone adhesive as well as a preparation method and application thereof, bioactive glass, lipoic acid and lipoic acid salt are mixed, and a deep eutectic hot melt adhesive is formed through one-step heating by using strong hydrogen bonds between carboxyl and carboxylate radicals between LA and LA-Na. The adhesive disclosed by the invention is combined with in-situ pore formation and immune microenvironment regulation and control to promote repair of damaged bone tissues. In an in-vivo rabbit radius fracture repair model, the adhesive can firmly fix broken bone tissues and accelerate bone tissue regeneration. As the adhesive gradually degrades in the body, almost complete bone repair can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials, specifically relating to a polythioctic acid-coenzyme-based bone adhesive capable of in-situ pore formation, its preparation method, and its application. Background Technology

[0002] Fractures are a common orthopedic condition affecting millions of people. Currently, clinical fixation primarily relies on bone screws or plates. However, the introduction of these screws or plates not only leads to foreign body rejection and infection, but also requires removal after bone repair, significantly increasing the burden on both medical professionals and patients. Using bone adhesives to replace screws or plates represents an innovative revolution in orthopedic surgery. Commercially available bone adhesives include polymethyl methacrylate (PMMA), cyanoacrylate, and calcium phosphate cement. However, these adhesives suffer from drawbacks such as low adhesion strength to bone tissue, slow or no degradation in vivo, toxic degradation byproducts, and tendency to collapse in the moist environment of the body. Furthermore, these adhesives consist of highly chemically cross-linked, tightly packed structures lacking pores in the body, which restricts the ingrowth of bone-related cells and new bone tissue, severely hindering bone regeneration. In addition, none of these adhesives have the function of regulating the microenvironment of the bone injury area, which further limits their ability to promote the healing and repair of damaged bone.

[0003] Lipoic acid is widely used in adhesives due to its abundant terminal carboxyl groups. However, pure lipoic acid is unstable after polymerization and depolymerizes to form oligomers or monomers, resulting in a loss of adhesiveness. Although traditional methods can stabilize polylipoic acid by introducing monomers with multiple double bonds or polyvalent metal ions, this significantly reduces the biocompatibility of polylipoic acid adhesives. Furthermore, the inherent hydrophobic properties of lipoic acid limit its release in vivo. Sodium lipoate is a hydrophilic molecule that retains the anti-inflammatory, antioxidant, and antibacterial bioactivities of lipoic acid. In previous studies, we developed a polylipoic acid-based deep eutectic adhesive by utilizing the strong hydrogen bonds between the carboxyl and carboxyl groups of lipoic acid and lipoate. This achieved the stabilization of polylipoic acid without introducing any exogenous molecules and allowed for the in-situ release of lipoic acid-based active molecules. Based on the comprehensive properties of this adhesive, it holds promise for use in bone adhesives. However, the adhesive has insufficient adhesion strength to bone tissue and lacks the ability to form pores in situ, which limits the inward ingrowth of bone-related cells and new bone tissue, thus hindering bone regeneration. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a polylipoic acid-coenzyme-based bone adhesive capable of in-situ pore formation, its preparation method, and its application. Using lipoic acid, sodium lipoate, and bioactive glass as raw materials, the three powders are mixed in a certain mass ratio and thoroughly ground, followed by a hot-melt adhesive bonding process involving heating or deep eutectic melting. In this adhesive, the strong ionic hydrogen bonds formed between lipoic acid and sodium lipoate effectively reduce the melting temperature of the mixture, while the bioactive ratio effectively induces in-situ pore formation in the adhesive and regulates the release of lipoic acid-based active molecules, thereby controlling the microenvironment. The comprehensive properties of the obtained adhesive are demonstrated through investigation of its thermodynamic properties, physicochemical properties, bulk and adhesive strength, in vitro and in vivo bioactivity, and in vivo bone repair promotion capacity.

[0005] The technical objective of this application is achieved through the following technical solution.

[0006] An in-situ pore-forming coenzyme-based bone adhesive is composed of lipoic acid, lipoate, and bioactive glass. The mass ratio of lipoic acid to lipoate is 2:(1-1.2), and the mass of bioactive glass is less than 20% of the total mass of lipoic acid and lipoate. The bioactive glass is uniformly dispersed in the deep eutectic hot melt adhesive formed by heating the strong hydrogen bonds between the carboxyl and carboxylate groups of lipoic acid and lipoate.

[0007] The above-mentioned method for preparing bone adhesive involves mixing thioctic acid, thiocate salt, and bioactive glass as monomer powders, grinding them thoroughly, heating them to the melting temperature and holding them at that temperature to ensure that the uniformly mixed mixture of thioctic acid, thiocate salt, and bioactive glass is in a molten state, and then naturally cooling it to room temperature of 20-25 degrees Celsius to obtain the bone adhesive.

[0008] In the technical solution of this invention, thiocate is sodium thiocate.

[0009] In the technical solution of this invention, the mass ratio of thioctic acid and thiocate salt is 2:1.

[0010] In the technical solution of this invention, the mass of the bioactive glass is 10%-15% of the total mass of thioctic acid and thiocate.

[0011] In the technical solution of this invention, the heat preservation time is 1-2 hours.

[0012] The application of the bone adhesive of the present invention in the preparation of drugs for treating bone diseases.

[0013] Bioactive glass is a biocompatible and biodegradable active material that promotes bone regeneration. It contains abundant elements such as Ca, P, and Si, which have osteoinductive and osteoconductive functions. Upon contact with body fluids, bioactive glass forms a large amount of hydroxyapatite layer, which acts like collagen to promote osteogenic differentiation and regeneration. Furthermore, bioactive glass is weakly alkaline in aqueous solutions. This invention utilizes these properties of bioactive glass by mixing it with a thioctic acid / thiocate deep eutectic adhesive to construct a composite bone tissue adhesive. In this adhesive, in addition to its intrinsic bone repair-promoting effect, the bioactive glass effectively increases the bulk strength and adhesive strength of the adhesive, laying the foundation for effective and firm fixation of fractured tissue. Furthermore, the weak alkalinity of the bioactive glass promotes partial deprotonation of the surrounding polythioctic acid, forming sodium polythioate. Upon contact with body fluids, sodium polythioate gradually dissociates, acting as a pore-forming agent to induce in-situ pore formation in the adhesive and release thioctic acid-based active molecules. The formation of the porous structure facilitates the inward ingrowth of bone-related cells and new bone tissue, thereby efficiently promoting bone repair and regeneration. The released thioctic acid-based active molecules not only effectively regulate the immune microenvironment in the damaged bone tissue area but also promote osteogenic formation, further accelerating the repair and regeneration of damaged bone. This invention features a simple preparation method, widely available materials, and strong practicality.

[0014] The bone tissue adhesive of the present invention is a deep eutectic hot melt adhesive formed by mixing bioactive glass (BG), lipoic acid (LA), and lipoate (LA-Na) and utilizing the strong hydrogen bonds between the carboxyl and carboxylate groups of LA and LA-Na through a one-step heating process. In this adhesive, BG plays a crucial role. First, the addition of bone glycosides (BG) significantly increases the bulk and adhesive strength of the adhesive, laying a solid foundation for effective fixation of fractures. Second, BG contains abundant elements such as Ca, P, and Si, which can effectively promote osteoinduction and osteoconduction, accelerate bone regeneration, and form a large number of hydroxyapatite layers on its surface upon contact with body fluids. These hydroxyapatite layers act similarly to collagen, promoting the proliferation of bone-related cells and further accelerating bone regeneration. Finally, the weak alkalinity of BG can partially deprotonate the surrounding polylipoic acid (PolyLA) to form sodium polylipoate (PolyLA-Na). Upon contact with body fluids, PolyLA-Na gradually dissociates, acting as a foaming agent to promote in-situ pore formation in the adhesive and releasing LA-Na active small molecules. Furthermore, based on the anti-inflammatory, antioxidant, and antibacterial bioactivities of LA-based active small molecules, it can effectively regulate the microenvironment of damaged tissue areas. Moreover, related studies have demonstrated that LA-based active small molecules can also promote bone formation. Therefore, this adhesive combines in-situ pore formation and immune microenvironment regulation in a synergistic manner to promote the repair of damaged bone tissue. In an in vivo rabbit radius fracture repair model, this adhesive firmly fixes the fractured bone tissue and accelerates bone regeneration. As the adhesive gradually degrades in vivo, it can achieve almost complete bone repair. Attached Figure Description

[0015] Figure 1 This is a bar chart showing the test results of the melting temperatures of adhesives with different monomers and compositions.

[0016] Figure 2 These are infrared spectra of different monomers and adhesives with different compositions.

[0017] Figure 3 These are Raman spectra of thioctic acid monomers and binders with different compositions.

[0018] Figure 4 These are XRD patterns of different monomers and adhesives with different compositions.

[0019] Figure 5 These are XPS graphs of adhesives with different compositions.

[0020] Figure 6 This is a bar chart showing the test results of tensile strength and tensile strain for adhesives with different compositions.

[0021] Figure 7The figures show the adhesion strength test results of different adhesive compositions to iron sheets, ceramics, PE boards, glass and bone fragments, as well as the adhesion test results of DESPG-15 to bone fragments and broken pig bones and the load-bearing capacity test results of the bonded bones.

[0022] Figure 8 These are SEM images of different adhesive compositions before and after immersion in simulated body fluids.

[0023] Figure 9 This is a schematic diagram showing the test results of monomer release behavior of adhesives with different compositions.

[0024] Figure 10 This is a bar chart showing the DPPH removal efficiency test results of different adhesive compositions at different times.

[0025] Figure 11 This is a schematic diagram showing the antibacterial efficiency of adhesives with different compositions and the results of bacterial plate culture.

[0026] Figure 12 This is a bar chart showing the cell compatibility test results of adhesives with different compositions.

[0027] Figure 13 These are photographs of osteogenic capacity tests using adhesives DESPG-0 and DESPG-15 with different compositions.

[0028] Figure 14 This is a characterization diagram of the results of subcutaneous implantation of the adhesive DESPG-15 of the present invention in rats.

[0029] Figure 15 This is a schematic diagram illustrating the results of repairing rabbit radius fractures using adhesives DESPG-0 and DESPG-15 with different compositions. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0031] Bone tissue adhesive is obtained by mixing these monomer powders in a certain mass ratio, grinding them thoroughly, heating them to their respective melting temperatures, and then allowing them to cool naturally to room temperature (20-25 degrees Celsius) after standing for 1 hour.

[0032] In the preparation process, the mass ratio of lipoic acid to sodium lipoate was kept constant at 2:1. By adjusting the mass of the bioactive glass, adhesives with different compositions were obtained. The resulting adhesives were named DESPG-x, where x represents the mass percentage of bioactive glass in the total mass of lipoic acid and sodium lipoate. For example, DESPG-15 is the adhesive obtained when the mass ratio of lipoic acid to sodium lipoate is 2:1 and the mass of bioactive glass is 15% of the total mass of lipoic acid and sodium lipoate. The adhesives were prepared according to the feed ratios specified in the preparation scheme, and the various properties of the obtained adhesives were characterized.

[0033] Figure 1 This is a bar graph showing the melting temperatures of adhesives with different monomers and compositions. It also represents the thermodynamic characteristics of adhesives containing different masses of bioactive glass. The graph shows that when only lipoic acid and sodium lipoate are present, the melting point of the mixture is approximately 42°C, significantly lower than that of lipoic acid and sodium lipoate alone. After adding bioactive glass, the melting point of the mixture increases, gradually rising with increasing bioactive glass content. This is because the weak alkalinity of the bioactive glass causes some lipoic acid to deprotonate, forming lipoate salts. Therefore, the increased lipoate salt content reduces the number of strong ionic hydrogen bonds between carboxyl groups and carboxylates, thus increasing the melting point of the mixture. The results also show that when the bioactive glass content is 15%, the melting temperature of the adhesive is 80°C, while when the bioactive glass content increases to 20%, the melting point of the adhesive increases to 116°C. Although the addition of bioactive glass increases the melting point of the adhesive, it is still far lower than that of sodium thiocate and bioactive glass alone.

[0034] Figure 2 The results show the infrared characterization of different constituent materials. From graph a, it can be seen that compared to pure lipoic acid and sodium lipoate, the adhesive exhibits peaks for both carboxyl and carboxylate groups, indicating that lipoic acid and sodium lipoate have complexed. Furthermore, the carboxylate peak in the adhesive shows a significant red shift compared to the carboxylate peak in sodium lipoate, indicating the formation of strong hydrogen bonds between lipoic acid and lipoate in the adhesive. From graph b, it can be seen that the adhesive exhibits infrared characterization at 1000 cm⁻¹. -1 The presence of a distinct Si-O-Si bond peak indicates that the bioactive glass has been successfully incorporated into the adhesive. Figure 3 The images show the Raman spectra of adhesives with different compositions. As can be seen from the figures, the peak of lipoic acid at 512 cm⁻¹ is split into two segments at 506 cm⁻¹ in all adhesives with different compositions. -1 and 526cm -1The two peaks indicate that thioctic acid underwent ring-opening polymerization in the adhesive, and the resulting polythioctic acid is stable within the adhesive. Furthermore, the introduction of bioactive glass does not affect the stability of the polythioctic acid.

[0035] Figure 4 The XRD patterns of different monomers and adhesives with different compositions show that, compared to the monomers, all adhesives are in an amorphous state, indicating that the polythioctic acid in the adhesive is stable and has not undergone depolymerization. Furthermore, no crystallization peaks of bioactive glass appear in the XRD patterns, indicating that the bioactive glass is uniformly dispersed in the adhesive. Figure 5 The XPS graphs show different compositions of the adhesive. As can be seen from the graphs, after introducing bioactive glass into the adhesive, the adhesive exhibits a significant presence of Si elements, indicating the successful introduction of bioactive glass.

[0036] Figure 6 The figures reflect the tensile strength and tensile strain of adhesives with different compositions. As can be seen from the figures, the addition of bioactive glass significantly increases the bulk strength of the adhesive, providing a basis for increasing its adhesive strength. Although the addition of bioactive glass increases the tensile strength of the adhesive, the tensile strain remains unaffected, exhibiting high stretchability. This indicates that the addition of bioactive glass does not affect the movement of polymer chains within the adhesive, and the multiple hydrogen bonds between lipoic acid and sodium lipoate within the adhesive facilitate rapid exchange of dynamic bonds.

[0037] Figure 7 The results reflect the adhesion strength of different adhesive compositions to different matrices. ae correspond to the adhesion strength of different adhesive compositions to iron sheets, ceramics, PE boards, glass, and bone fragments, respectively, while f corresponds to the adhesion of DESPG-15 to bone fragments and fractured porcine club bones, and the load-bearing capacity of the bonded club bones. Regardless of the matrix, the adhesion strength of the adhesive increases with the increase of the bioactive glass content in the adhesive, because the addition of bioactive glass enhances the bulk strength of the adhesive. Based on the thermal properties, bulk properties, and adhesion properties of the adhesive, a component with a bioactive glass content of 15% was selected for subsequent animal experiments. Furthermore, as shown in the figure, the DESPG-15 adhesive can be remelted after heating, and can be extruded through a syringe and directly applied to the surface of bone fragments and fractured bone tissue. It enables rapid splicing of bone fragments and rapid fixation of fractured bone, and the bonded bone tissue can withstand a weight of 5 kg. This indicates that the DESPG-15 adhesive has excellent operability and adhesion to bone tissue.

[0038] Figure 8The images show SEM images of different adhesive compositions before and after immersion in simulated body fluid. It can be seen that without immersion in simulated body fluid, the adhesive surface is dense and non-porous. However, after immersion, porous structures appear on the surfaces of DESPG-5 and DESPG-15 adhesives, and the size and content of the pores increase with the amount of bioactive glass, indicating that the bioactive glass is key to inducing pore formation in the adhesive. In addition to in-situ pore formation, the adhesive also exhibits a large number of hydroxyapatite structures adhering to the pore surface, indicating that the simulated body fluid induces the formation of hydroxyapatite structures on the surface of the bioactive glass, which is crucial for promoting bone repair.

[0039] Figure 9 The reaction reflects the monomer release behavior of different adhesive compositions. Compared with DESPG-0, the release amount and release rate of thioctic acid-based active molecules in DESPG-15 are significantly higher, indicating that the addition of BG significantly promotes the release of thioctic acid-based active molecules. This is because the weak alkalinity of bioactive glass causes some polythioctic acid to be deprotonated to form sodium polythiocate, thus promoting the release of sodium polythiocate. Figure 10 The results reflect the DPPH removal efficiency of adhesives with different compositions. All adhesives, regardless of their composition, exhibited excellent DPPH removal effects, indicating that the release of small, active molecules from the thioctic acid groups in the adhesive effectively eliminates reactive oxygen species.

[0040] Figure 11 The results reflect the antibacterial efficiency of different adhesive components and the results of bacterial plate culture. a and b correspond to the antibacterial efficiency of different adhesive components against Staphylococcus aureus and Escherichia coli, while c corresponds to the macroscopic inhibition of Staphylococcus aureus and Escherichia coli by different adhesive components. All adhesive components exhibited good antibacterial effects against Staphylococcus aureus and Escherichia coli, and the plate culture results showed that the adhesives effectively inhibited bacterial proliferation, indicating that the adhesives have excellent antibacterial effects. Furthermore, the addition of bioactive glass did not affect the antibacterial efficiency of the adhesives.

[0041] Figure 12 The results reflect the cell compatibility of different adhesive components, specifically the DESPG-0 and DESPG-15 adhesives. After co-culturing cells with both adhesives, cell viability remained comparable to the control group. Furthermore, with prolonged time, the DESPG-0 adhesive group also showed a cell proliferation-promoting effect, indicating that the lipoic acid active molecules can promote osteoblast proliferation, and that the bioactive glass did not affect the cell compatibility of the adhesives.

[0042] Figure 13The results reflect the osteogenic effects of DESPG-0 and DESPG-15 adhesives co-cultured with osteogenic cells. The results showed that DESPG-15 exhibited greater osteogenic activity compared to the control group and the DESPG-0 group, indicating that the formation of porous structures and the introduction of bioactive glass can significantly accelerate osteoogenesis.

[0043] Figure 14 The results reflect the subcutaneous implantation of DESPG-15 in rats. a corresponds to the degradation of DESPG-15 after different implantation times in rats; b corresponds to the remaining mass percentage of DESPG-15 after different implantation times in rats; c corresponds to the HE staining characterization of tissue and material after different implantation times in rats. The red arrows indicate newly grown tissue ingrained into the material. The adhesive gradually degrades with prolonged implantation time in vivo, forming a porous structure. The staining results show that new tissue gradually grows into the adhesive within this porous structure, indicating that DESPG-15 forms a porous structure in vivo and promotes the inward ingrained tissue growth.

[0044] Figure 15 This data reflects the results of DESPG-0 and DESPG-15 adhesives in repairing rabbit radius fractures. Image a corresponds to the rabbit radius fracture repair model; image b shows CT scans of the repaired rabbit radius fractures treated with different methods; images c and d show the bone volume and bone mineral density test results after different treatments; image e shows the mechanical strength test results of the repaired bone; image f shows the maximum load test results of the bone treated with different methods; and image g shows the mechanical strength curves of the bone treated with different methods. The CT images of the radius fracture sites treated with different methods show that, compared to the blank control, the bone growth of the adhesive-treated groups was more complete and coherent, and the bone volume and bone mineral density were significantly better than the control group. The DESPG-15 group showed the best healing effect. Furthermore, mechanical testing of the repaired bone revealed that the bone tissue repaired with DESPG-15 had the highest mechanical strength, approaching that of the normal group, followed by the DESPG-0 group. This indicates that the in-situ generation of the porous structure of DESPG-15 and the introduction of bioactive glass significantly accelerated the healing and regeneration of bone defects.

[0045] Adjusting the process parameters according to the present invention can achieve the preparation of adhesives, and testing has shown that they exhibit performance substantially consistent with that of the present invention. The present invention has been described above as exemplary. It should be noted that any simple modifications, alterations, or other equivalent substitutions that can be made by those skilled in the art without creative effort, without departing from the core of the present invention, fall within the protection scope of the present invention.

Claims

1. An in-situ pore-forming coenzyme-based bone adhesive, characterized in that, It is composed of lipoic acid, lipoate and bioactive glass. The mass ratio of lipoic acid to lipoate is 2:(1-1.2). The mass of bioactive glass is less than 20% of the total mass of lipoic acid and lipoate. The bioactive glass is uniformly dispersed in the deep eutectic hot melt adhesive formed by heating the strong hydrogen bonds between the carboxyl groups and carboxylate groups between lipoic acid and lipoate.

2. The in-situ pore-forming coenzyme-based bone adhesive according to claim 1, characterized in that, Thioctate is sodium thiocate.

3. An in-situ pore-forming coenzyme-based bone adhesive according to claim 1 or 2, characterized in that, The mass ratio of lipoic acid to lipoate is 2:

1.

4. An in-situ pore-forming coenzyme-based bone adhesive according to claim 1 or 2, characterized in that, The bioactive glass comprises 10%–15% of the total mass of lipoic acid and lipoate.

5. A method for preparing an in-situ pore-forming coenzyme-based bone adhesive as described in any one of claims 1-4, characterized in that, The bone adhesive is obtained by mixing thioctic acid, thiocate salt, and bioactive glass as monomer powders, grinding them thoroughly, heating them to the melting temperature and holding them at that temperature, so that the uniformly mixed mixture of thioctic acid, thiocate salt, and bioactive glass is in a molten state, and then naturally cooling it to room temperature of 20-25 degrees Celsius.

6. The method for preparing an in-situ pore-forming coenzyme-based bone adhesive according to claim 5, characterized in that, The heat preservation time is 1-2 hours.

7. The use of an in-situ pore-forming coenzyme-based bone adhesive as described in any one of claims 1-4 in the preparation of a drug for treating bone diseases.

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