Three-dimensional porous scaffold and sustained-release gel system for pulmonary alveolar regeneration

Abstract

The invention relates to the field of bioengineering, in particular to a three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration, which is composed of a three-dimensional porous hydrogel scaffold and temperature-sensitive hydrogel loaded with FGF-7. The three-dimensional porous hydrogel scaffold is prepared from hydroxyethyl methylacrylate-sodium alginate-gelatin frozen gel, and the temperature-sensitive hydrogel loaded with the FGF-7 is chitosan / beta-glycerophosphoric acid / gelatin temperature-sensitive hydrogel loaded with the FGF-7. The invention constructs an effective pulmonary alveolar regeneration promoting system, which not only can promote the repair of damaged pulmonary alveoli and improve the life quality of patients, but also provides new strategies and hopes for the treatment of lung diseases, and has wide clinical transformation prospects.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, in particular to a three-dimensional porous scaffold and a sustained-release gel system for alveolar regeneration, and a preparation method and application thereof. Background Art

[0002] The alveoli are the basic functional units of the lungs. They have a hemispherical vesicle structure. The size can vary depending on the depth of breathing, and the average diameter is about 0.2 mm. Adults have about 300 million to 400 million alveoli, with a total area of ​​up to 100 square meters. The alveolar wall is very thin and is composed of a single layer of epithelial cells, mainly including type I and type II alveolar epithelial cells. Type I cells cover most of the surface of the alveoli and are the main cells that perform gas exchange functions. Type II cells are responsible for secreting surfactant, reducing the surface tension of the alveoli during breathing and maintaining alveolar stability. In addition, type II alveolar cells also serve as stem cells in the lungs. When the alveoli are damaged, they can proliferate and differentiate into type I alveolar cells. These characteristics of the alveoli make them play a vital role in physiology. They not only transport oxygen to the body, but also expel carbon dioxide produced by metabolism. They are an indispensable part of maintaining life activities.

[0003] Lung trauma can be caused by a variety of reasons, such as trauma, iatrogenic injury, spontaneous injury (such as rupture of bullae), etc. After lung trauma, the alveolar capillary membrane barrier is destroyed, leading to edema, inflammation and fibrosis. After alveolar damage, gas exchange efficiency decreases, which may cause a series of respiratory diseases, such as chronic obstructive pulmonary disease, pulmonary fibrosis, acute respiratory distress syndrome, etc. In severe cases, it may even lead to respiratory failure, posing a huge threat to human health.

[0004] The regeneration process after alveolar injury is full of challenges. Due to the special structure and function of alveoli, their self-repair ability is limited. Regeneration after alveolar injury involves the fine regulation of multiple cell types and molecular signals, including the self-renewal and differentiation of type II alveolar epithelial cells, and the possible participation of alveolar stem cells. In recent years, with the development of single-cell sequencing technology and lineage tracing technology, scientists have discovered new types of cells and stem cell states in the study of alveolar development and regeneration, providing a new perspective for understanding the mechanism of alveolar regeneration.

[0005] Despite this, the research on alveolar regeneration still faces many challenges, including how to simulate the alveolar microenvironment, how to promote the directional differentiation of stem cells, and how to improve the function of regenerated alveoli. Currently, researchers are trying to promote alveolar regeneration through various means such as tissue engineering technology, biomaterials science, and stem cell therapy. For example, porous scaffolds constructed with biomaterials can simulate the three-dimensional structure of alveoli and provide a platform for cells to attach and grow; while sustained-release gels containing bioactive factors can provide the necessary nutrients and signal molecules for alveolar regeneration, while also playing a sealing role to prevent wound leakage and bleeding.

[0006] In this research context, it is particularly important to develop a porous scaffold + sustained-release gel system for alveolar regeneration. This system is designed to provide a suitable microenvironment to promote the proliferation and differentiation of alveolar epithelial cells and accelerate the repair and regeneration of alveoli. The pore structure of the porous scaffold should mimic the size and morphology of natural alveoli to facilitate cell migration, adhesion and exchange of nutrients; while the sustained-release gel should contain bioactive factors that promote cell growth and differentiation, such as growth factors, cytokines, etc. In addition, the biocompatibility and degradability of the system are also important factors that need to be considered during design to ensure its safety and effectiveness in vivo.

[0007] In short, the structure and function of alveoli are crucial to human respiratory health, and the regeneration of alveoli after injury is a complex and challenging process. With the continuous advancement of science and technology, especially the development of tissue engineering technology and biomaterials science, new ideas and methods have been provided for alveolar regeneration. The development of an effective alveolar regeneration system will not only promote the repair of damaged alveoli and improve the quality of life of patients, but will also provide new strategies and hopes for the treatment of lung diseases. Summary of the invention

[0008] The purpose of the present invention is to develop an effective alveolar regeneration system, combining a three-dimensional porous hydrogel scaffold and a thermosensitive hydrogel loaded with fibroblast growth factor 7 (FGF-7) to promote in situ regeneration of alveoli at lung wounds.

[0009] The first aspect of the present invention provides a three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration, which is composed of a three-dimensional porous hydrogel scaffold and a thermosensitive hydrogel loaded with FGF-7; the three-dimensional porous hydrogel scaffold is made of hydroxyethyl methacrylate-sodium alginate-gelatin cryogel, and the thermosensitive hydrogel loaded with FGF-7 is a chitosan (CS) / β-glycerophosphate (β-GP) / gelatin (Gel) thermosensitive hydrogel loaded with FGF-7.

[0010] Furthermore, the method for preparing the three-dimensional porous hydrogel scaffold comprises the following steps:

[0011] Gelatin (from cold-water fish skin, molecular weight ~60,000) and alginate (3% w / v, sodium salt, from brown algae, viscosity ~250 cP at 25°C) were dissolved in degassed water to optimize the final ratio of polymer concentration (1:1 gelatin and alginate mass ratio, 300 mg each added to 10 mL water); hydroxyethyl methacrylate (HEMA) (0.7 mL) was added to this solution to a final concentration of 7% (volume fraction) and briefly cooled; 50 μL ammonium persulfate (APS) (10% w / v) and 20 μL N,N,N′,N′-tetramethylethylenediamine (TEMED) is used as an initiator, and 250 μL of poly(ethylene glycol) diacrylate (PEGda, molecular weight 575) and 50 μL of glutaraldehyde (25%, v / v) are used as cross-linking agents; the polymerization reaction is carried out in a liquid cryostat at -12°C overnight; the polymerized hydroxyethyl methacrylate-sodium alginate-gelatin gel is washed with deionized water at room temperature to remove unpolymerized monomers, and after complete thawing, it is vacuum dried in a freeze dryer to obtain a three-dimensional porous hydrogel scaffold, which is sterilized by plasma and stored at room temperature.

[0012] In the process of preparing three-dimensional porous scaffolds, polymer concentration and ratio are key factors affecting the properties of scaffolds. By changing the concentration and ratio of different polymers, the mechanical strength, elastic modulus, porosity and pore size of the scaffold can be controlled. In order to make the scaffold have physical properties similar to those of lung tissue, the above-mentioned optimal polymer concentration and ratio were explored and obtained.

[0013] Furthermore, the method for preparing the FGF-7 loaded thermosensitive hydrogel comprises the following steps:

[0014] Chitosan (CS) was dissolved in 0.1 mol / L HCl aqueous solution to prepare a 2% (w / v) chitosan (CS) solution; β-glycerophosphate (β-GP) was dissolved in deionized water to prepare a 56% β-GP solution, and the β-GP solution included 50 μg / mL FGF-7; gelatin (Da) was dissolved in deionized water to prepare a 0.5% gelatin aqueous solution; the volume ratio of CS solution, β-GP solution and gelatin aqueous solution was 40:8:1; the CS solution was magnetically stirred, and then the β-GP solution was added dropwise in an ice bath, and when the solution became turbid, the gelatin aqueous solution was added and magnetically stirred until the solution was completely mixed; the pH value was adjusted to 7.2 with 0.1 mol / L NaOH to obtain a CS / β-GP / Gel thermosensitive hydrogel loaded with FGF-7.

[0015] Furthermore, the molecular weight of the chitosan is 310,000-375,000 Da; the molecular weight of the gelatin is 40,000-50,000 Da.

[0016] Furthermore, the preparation method of the thermosensitive hydrogel loaded with FGF-7 specifically includes the following steps: weigh 1g CS on weighing paper and irradiate under ultraviolet light for 1 hour (30min on each side). After ultraviolet sterilization, dissolve CS in 50mL 0.1mol / L HCl aqueous solution to prepare a 2% (w / v) CS solution. Dissolve 5.6g β-GP in 10mL deionized water to prepare a β-GP solution with a mass fraction of 56% (including 50μg / mL FGF-7). Subsequently, dissolve 0.2g gelatin in 40mL deionized water to prepare a gelatin aqueous solution with a mass fraction of 0.5%. Before use, both the β-GP solution and the gelatin solution are filtered and sterilized using a 0.22μm disposable filter. The volume ratio of CS solution, β-GP solution and gelatin solution is 40:8:1. Then, 20 mL of CS solution was transferred to a beaker and magnetically stirred for 10 min, and then 4 mL of β-GP solution was added dropwise in an ice bath. When the solution became turbid, 500 μL of gelatin aqueous solution was added and magnetically stirred until the solution was completely mixed. The pH value was adjusted to 7.2 with 0.1 mol / L NaOH to obtain CS / β-GP / Gel thermosensitive hydrogel loaded with FGF-7.

[0017] The ratio of chitosan, β-glycerophosphate and gelatin is a key parameter affecting the properties of the sustained-release gel. By adjusting the ratio of different components, the gelation time of the thermosensitive hydrogel at 37°C can be changed to meet the needs of different scenarios.

[0018] The second aspect of the present invention provides a use of the three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration as described above in the preparation of a product for treating alveolar damage.

[0019] Furthermore, the product for treating alveolar damage is a product that promotes the repair of damaged alveoli and promotes the in situ regeneration of alveoli.

[0020] When the present invention is used, the patient's lung wound is first evaluated in detail, including the size, position, depth of the wound, and whether there is active bleeding or air leakage. According to the size and depth of the wound, a three-dimensional porous stent of suitable shape and size (close to the shape and size of the wound) is prepared, and a thermosensitive hydrogel loaded with FGF-7 is pre-configured. Blood and foreign matter are removed from the wound site, and active bleeding and air leakage are initially pressed to stop bleeding. The three-dimensional porous stent is filled in the wound, and the thermosensitive hydrogel is injected into the gap between the stent and the wound to fill the gap, and then the thermosensitive hydrogel is covered with the stent surface and the adjacent lung tissue surface to completely close the lung wound. After waiting for the thermosensitive hydrogel to completely gel into a solid state, the chest cavity is closed, and the muscles and skin are sutured layer by layer. After the operation, the patient's vital signs and respiratory status are closely observed, and a chest CT scan is performed to assess whether the wound has bleeding and air leakage.

[0021] The present invention has the advantages that:

[0022] 1. The present invention constructs an effective alveolar regeneration promoting system, which is composed of a three-dimensional porous scaffold and a FGF-7-loaded thermosensitive hydrogel, and can promote the in situ regeneration of alveoli at the lung wound.

[0023] 2. The porous scaffold simulates the size and shape of natural alveoli, providing a suitable microenvironment for cells, which is conducive to cell migration, adhesion and exchange of nutrients. At the same time, it accelerates the repair and regeneration process of alveoli by providing necessary signal molecules.

[0024] 3. The sustained-release gel can act as a seal to prevent air leakage and bleeding in the wound. It can also isolate the external environment and provide a stable environment for alveolar regeneration.

[0025] 4. It can not only promote the repair of damaged alveoli and improve the quality of life of patients, but also provide new strategies and hopes for the treatment of lung diseases. It has broad prospects for clinical transformation and potential economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Schematic diagram of three-dimensional porous hydrogel scaffold.

[0027] Figure 2 .Schematic diagram of injectable thermosensitive hydrogel.

[0028] Figure 3 .Application diagram.

[0029] Figure 4 .General view of lung wound repair.

[0030] Figure 5 .CT images after lung repair.

[0031] Figure 6 HE staining pictures after lung repair

[0032] Figure 7 .AQP-5 immunofluorescence staining after lung repair.

[0033] Figure 8 . Immunofluorescence staining of SFTPC after lung repair. DETAILED DESCRIPTION

[0034] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0035] Embodiment 1:

[0036] The alveolar regeneration promoting system of the invention is composed of a three-dimensional porous scaffold and a thermosensitive hydrogel carrying fibroblast growth factor 7 (FGF-7).

[0037] 1. Three-dimensional porous scaffold

[0038] The preparation method of the three-dimensional porous scaffold is as follows:

[0039] Gelatin (from cold-water fish skin, molecular weight ~60,000) and alginate (3% w / v, sodium salt, from brown algae, viscosity ~250 cP at 25°C) were dissolved in degassed water, with a gelatin to alginate mass ratio of 1:1, and 300 mg of each was added to 10 mL of water. Hydroxyethyl methacrylate (HEMA) (0.7 mL) was added to this solution to a final concentration of 7% (volume fraction) and briefly cooled. 50 μL of ammonium persulfate (APS) (10% w / v) and 20 μL of N,N,N′,N′-tetramethylethylenediamine (TEMED) were added as initiators, as well as 250 μL of poly(ethylene glycol) diacrylate (PEGda, molecular weight 575) and 50 μL of glutaraldehyde (25% v / v) as crosslinkers. The polymerization reaction was carried out in a liquid cryostat at -12°C overnight. The polymerized hydroxyethyl methacrylate-sodium alginate-gelatin gel is washed with deionized water at room temperature to remove unpolymerized monomers, and then completely thawed and vacuum dried using a freeze dryer to obtain a three-dimensional porous hydrogel scaffold, which is sterilized with plasma and stored at room temperature.

[0040] The three-dimensional porous scaffold of the present invention has a porous structure similar in size and shape to natural alveoli. The three-dimensional porous scaffold is filled in the lung wound, which can provide surface and mechanical support for cell adhesion and migration, and the porous structure is conducive to the development of early alveolar-like structure. In addition, the in vivo degradation rate of the porous scaffold is compatible with the regeneration rate of the alveolar-like structure. The porous scaffold is made of hydroxyethyl methacrylate-sodium alginate-gelatin cryogel, has good biocompatibility, and the degradation products do not cause toxic reactions. The porosity of the three-dimensional porous scaffold is about 80±2%, and the diameter is (60±10) μm, which is similar to the porosity (68.88±3.63%) and diameter ((54.86±17.78) μm) of normal lung tissue, providing a structural basis for alveolar regeneration.

[0041] 2. Sustained-release gel

[0042] The synthesis method of the thermosensitive hydrogel loaded with FGF-7 is as follows:

[0043] 1g CS (molecular weight: 310000-375000Da) was weighed on weighing paper and irradiated under ultraviolet light for 1 hour (30min on each side). After ultraviolet sterilization, CS was dissolved in 50mL 0.1mol / L HCl aqueous solution to prepare a 2% (w / v) CS solution. 5.6g β-GP was dissolved in 10mL deionized water to prepare a β-GP solution with a mass fraction of 56% (including 50μg / mL FGF-7). Subsequently, 0.2g gelatin (molecular weight: 40000-50000Da) was dissolved in 40mL deionized water to prepare a gelatin aqueous solution with a mass fraction of 0.5%. Before use, both the β-GP solution and the gelatin solution were filtered and sterilized using a 0.22μm disposable filter. The volume ratio of CS solution, β-GP solution and gelatin solution was 40:8:1. Then, 20 mL of CS solution was transferred to a beaker and magnetically stirred for 10 min, and then 4 mL of β-GP solution was added dropwise in an ice bath. When the solution became turbid, 500 μL of gelatin aqueous solution was added and magnetically stirred until the solution was completely mixed. The pH value was adjusted to 7.2 with 0.1 mol / L NaOH to obtain CS / β-GP / Gel thermosensitive hydrogel loaded with FGF-7.

[0044] The sustained-release gel of the present invention is a thermosensitive hydrogel loaded with fibroblast growth factor 7 (FGF-7), and the thermosensitive hydrogel is mixed with chitosan (CS), β-glycerophosphate (β-GP) and gelatin (gel). The thermosensitive hydrogel has good injectability at room temperature and can change from a solution state to a gel state within about 5 minutes at 37°C. The thermosensitive property enables it to be injected around the porous scaffold to fill the gap, and can be transformed into a gel state under the action of body temperature, which can play a sealing role to prevent wound leakage and bleeding. FGF-7, also known as keratinocyte growth factor (KGF), plays a vital role in lung tissue development, homeostasis and regeneration. FGF-7 activates downstream signal transduction pathways by binding to FGF receptors, promoting cell proliferation, differentiation and migration. Studies have shown that FGF-7 plays an important role in the regeneration and repair of alveolar epithelial cells. In a 3D cell culture model, FGF-7 can promote the formation of lung spheres by lung epithelial stem / progenitor cells (LSPC) and induce the formation of lung organoids. These lung organoids contain alveolar-like areas, express alveolar type I and type II cell markers, and airway-like structures with club cells and ciliated cells. In addition, FGF-7 also shows differences in promoting different lung epithelial cell types, and can direct differentiation to distal lung lineages. FGF-7 is also involved in regulating the metabolism and energy supply of alveolar stem cells. The chitosan-β-glycerophosphate-gelatin thermosensitive hydrogel system can achieve the loading and sustained release of FGF-7, allowing FGF-7 to play a long-term role and promote alveolar regeneration.

[0045] Through the above steps, the present invention successfully prepared a three-dimensional porous hydrogel scaffold and a thermosensitive hydrogel loaded with FGF-7, which combined to form a porous scaffold + sustained-release gel system that can promote in situ regeneration of alveoli, and has broad prospects for clinical transformation.

[0046] Embodiment 2:

[0047] This system consists of a three-dimensional porous hydrogel scaffold and a thermosensitive hydrogel loaded with FGF-7, and is used to promote alveolar regeneration.

[0048] Rats were anesthetized by intraperitoneal injection of 2% pentobarbital (150 mg / kg). The skin, muscles, and intercostal muscles were cut layer by layer at the 4th and 5th intercostal spaces on the left side to expose the left lung. A wound with a diameter of 3 mm and a depth of 2 mm was made using an animal biopsy punch to complete the construction of the rat lung wound model. A three-dimensional porous scaffold of appropriate shape and size (close to the shape and size of the wound) was prepared, and the thermosensitive hydrogel loaded with FGF-7 was pre-configured. The blood at the wound site was cleared, and active bleeding and air leakage were initially pressed to stop bleeding. The three-dimensional porous scaffold was filled in the wound, and the thermosensitive hydrogel was injected into the gap between the scaffold and the wound to fill the gap. The thermosensitive hydrogel was then covered with the scaffold surface and the adjacent lung tissue surface to completely close the lung wound. After waiting for the thermosensitive hydrogel to completely gel into a solid state, the chest cavity was closed, and the muscles and skin were sutured layer by layer. After surgery, the vital signs and respiratory status of the experimental rats were closely observed, and chest CT scans were performed to assess whether there was bleeding and air leakage in the wound. The effect of alveolar regeneration was evaluated by histological examination, immunohistochemical staining, and pulmonary function tests.

[0049] The present invention verifies the application effect of the system in a rat lung wound model. The biocompatibility of the system was preliminarily evaluated through gross photos of the lungs after surgery. The results showed that the lung wounds had no obvious inflammatory response and fibrosis, and no adhesion to the chest wall ( Figure 4 ). Then, chest CT scan was used to detect lung expansion after surgery and evaluate whether the wound was leaking. The results showed that the rats' lungs were well expanded, with no obvious atelectasis or pneumothorax, proving the safety of the system. ( Figure 5 ). Next, the changes in alveolar structure were evaluated by HE staining of paraffin sections of lung tissue. The results showed that there was no significant change in alveolar structure after the application of this system, no thickening of the alveolar wall, no obvious fibrosis and inflammatory cell infiltration, and a new alveolar-like structure was formed at the wound site ( Figure 6). Finally, we detected alveolar regeneration by immunofluorescence staining. AQP-5 is a specific marker for type I alveolar cells (AT1), and SFTPC is a specific marker for type II alveolar cells (AT2). The results of AQP-5 and SFTPC immunofluorescence staining showed that AT1 and AT2 cells were regenerated in the lung wound, proving that this system promoted the in situ regeneration of alveoli in the lung wound ( Figure 7 and Figure 8 ).

[0050] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration, characterized in that: It consists of a three-dimensional porous hydrogel scaffold and a thermosensitive hydrogel loaded with FGF-7; the three-dimensional porous hydrogel scaffold is made of hydroxyethyl methacrylate-sodium alginate-gelatin cryogel, and the thermosensitive hydrogel loaded with FGF-7 is a chitosan / β-glycerophosphate / gelatin thermosensitive hydrogel loaded with FGF-7.

2. The three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration according to claim 1, characterized in that: The method for preparing the three-dimensional porous hydrogel scaffold comprises the following steps: Gelatin and alginate were dissolved in degassed water, the mass ratio of gelatin to alginate was 1:1, and 300 mg of each was added to 10 mL of water; hydroxyethyl methacrylate was added to the solution to make its final volume fraction 7%, and the solution was briefly cooled; 50 μL of 10% (w / v) ammonium persulfate and 20 μL of N,N,N′,N′-tetramethylethylenediamine were added as initiators, and 250 μL of poly(ethylene glycol) diacrylate and 50 μL of 25% (v / v) glutaraldehyde were added as cross-linking agents; the polymerization reaction was carried out in a liquid cryostat at -12°C overnight; the polymerized hydroxyethyl methacrylate-sodium alginate-gelatin gel was washed with deionized water at room temperature to remove unpolymerized monomers, and after being completely thawed, it was vacuum dried in a freeze dryer to obtain a three-dimensional porous hydrogel scaffold, which was sterilized by plasma and stored at room temperature.

3. The three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration according to claim 1, characterized in that: The method for preparing the FGF-7 loaded thermosensitive hydrogel comprises the following steps: The chitosan solution was prepared by dissolving chitosan in a 0.1 mol / L HCl aqueous solution to prepare a 2% (w / v) chitosan solution; β-glycerophosphate was dissolved in deionized water to prepare a 56% β-glycerophosphate solution, wherein the β-glycerophosphate solution included 50 μg / mL FGF-7; gelatin was dissolved in deionized water to prepare a 0.5% gelatin aqueous solution; the volume ratio of the chitosan solution, the β-glycerophosphate solution and the gelatin aqueous solution was 40:8:1; the chitosan solution was magnetically stirred, and then the β-glycerophosphate solution was added dropwise in an ice bath, and when the solution became turbid, the gelatin aqueous solution was added and magnetically stirred until the solution was completely mixed; the pH value was adjusted to 7.2 with 0.1 mol / L NaOH to obtain a chitosan / β-glycerophosphate / gelatin thermosensitive hydrogel loaded with FGF-7.

4. The three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration according to claim 3, characterized in that: The molecular weight of the chitosan is 310000-375000Da; the molecular weight of the gelatin is 40000-50000Da.

5. The three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration according to claim 3, characterized in that: Chitosan solution was prepared after chitosan was sterilized by irradiation under ultraviolet light; β-glycerophosphate solution and gelatin aqueous solution were filtered and sterilized using a 0.22 μm disposable filter.

6. Use of the three-dimensional porous scaffold and sustained-release gel system for alveolar regeneration as claimed in any one of claims 1 to 5 in the preparation of a product for treating alveolar damage.

7. The use according to claim 6, characterized in that: The product for treating alveolar damage is a product that promotes the repair of damaged alveoli and promotes the in situ regeneration of alveoli.

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