CN115414533B

CN115414533B - A tissue engineering scaffold that simulates the physiological structure of the bladder wall and its preparation method

Info

Publication number: CN115414533B
Application number: CN202211142027.5A
Authority: CN
Inventors: 符伟军; 赵健; 肖树伟; 王鹏超; 凌争云; 安子彦; 符舟洋; 邵金鹏
Original assignee: First Medical Center of PLA General Hospital
Current assignee: First Medical Center of PLA General Hospital
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2024-02-23
Anticipated expiration: 2042-09-20
Also published as: CN115414533A

Abstract

The invention relates to a tissue engineering scaffold simulating a physiological structure of a bladder wall and a preparation method thereof. The tissue engineering scaffold comprises a silk fibroin film inner layer, a silk fibroin sponge layer and a silk fibroin film outer layer which are sequentially arranged from inside to outside; wherein: the silk fibroin film inner layer is used for realizing time sequence release of growth factors; the silk fibroin sponge layer is used for promoting the adhesion of cells and playing a supporting role; the silk fibroin film outer layer plays a role in protecting and preventing the tissue engineering scaffold from being damaged. The invention organically combines the silk fibroin with good biocompatibility and the time-series released pro-angiogenic growth factors to construct a complete delivery system, and fully considers the physiological level of the bladder wall in the system preparation process to carry out bionic construction. The system is applied to bladder repair reconstruction and shows good vascularization promotion effect.

Description

Tissue engineering scaffold simulating physiological structure of bladder wall and preparation method thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a tissue engineering scaffold simulating a physiological structure of a bladder wall and a preparation method thereof.

Background

Bladder tissue engineering is a product of the cross-birth of medicine, bioengineering and materials, and initially provides a new method for solving the problem of bladder replacement. The most common use in clinic is the replacement of the urinary bladder by the intestinal tract, but due to the inherent properties of the intestinal mucosa and intestinal wall tissue, complications such as infection, lithiasis, urological fistula and even cancer are often caused. Tissue engineering bladder is expected due to its good histocompatibility, tough biomechanical properties, and rich in various cells and bioactive components, and is once considered as the most potential organ replacement method.

The existing tissue engineering bladder stents are various in variety and different in function, but have the following two defects:

first, most tissue engineering bladder stents are single-layer structures, and do not mimic the physiological structure of the bladder wall so well that the expected functional index cannot be achieved after reconstruction. The reason is that complications such as stone formation, urinary fistula, smooth muscle hypoplasia, and imperfect contraction function can occur after the bladder tissue engineering scaffold with a single-layer structure is transplanted into a body, and the complications are related to unreasonable scaffold structure design.

Secondly, the tissue engineering bladder stent is not sufficiently vascularized, and the vascularization promoting component is used as an important component of the stent at the beginning of stent design. Studies have shown that the limit of diffusion of oxygen and nutrients around capillaries in tissues is 200 μm, and that after tissue engineering bladder grafts enter the body, ischemia hypoxia and metabolite accumulation are caused by insufficient vascularization, ultimately leading to graft atrophy and necrosis. Therefore, the realization of rapid and effective vascularization of tissue engineering bladder grafts is an important premise for ensuring survival, and even subsequent morphological structure and physiological function.

More specifically, although researchers have recognized the significance of vascularization in tissue engineering bladder survival, there have been few studies on ways to achieve vascularization. The traditional method for promoting vascularization of tissue engineering grafts by large omentum incubation has two disadvantages. Firstly, the incubation of the large omentum takes 1-2 weeks to form a layer of tissue membrane with capillaries around the implant, which is time-consuming; secondly, the large omentum is incubated, the implant is required to be wrapped in the large omentum, and secondary wounds are caused to the subject or patient when the stent embedded in the large omentum is taken out. The application of pro-angiogenic factors to tissue engineering grafts to achieve vascularization is now the most recent study. Most of the current studies use a pro-angiogenic factor, but the pro-angiogenic effect is not ideal. Angiogenesis is a complex and dynamic interaction between vascular endothelial cells and extracellular matrix, including initiation of angiogenesis and vascular maturation processes. Studies have shown that vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) alone can only form immature blood vessels during tissue engineering graft preparation, which are very fragile and prone to leakage. Under normal physiological conditions, angiogenesis is a complex process under the stimulation of a variety of cells, cytokines, and the external environment. Tissue engineering organ vascularization thus requires the combined use of multiple growth factors and takes into account the chronology of growth factor action.

Therefore, the technical scheme of the invention is provided based on the above.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a tissue engineering scaffold simulating a physiological structure of a bladder wall and a preparation method thereof. The invention organically combines the silk fibroin with good biocompatibility and the time-series released pro-angiogenic growth factors to construct a complete delivery system, and fully considers the physiological level of the bladder wall in the system preparation process to carry out bionic construction. The system is applied to bladder repair reconstruction and shows good vascularization promotion effect.

The invention provides a tissue engineering scaffold for simulating a physiological structure of a bladder wall, which comprises a silk fibroin film inner layer, a silk fibroin sponge layer and a silk fibroin film outer layer which are sequentially arranged from inside to outside; wherein:

the silk fibroin film inner layer is used for preventing urine from damaging deep structures and realizing time-series release of growth factors;

the silk fibroin sponge layer is used for promoting the adhesion of cells and playing a supporting role;

the silk fibroin film outer layer plays a role in protecting and preventing the tissue engineering scaffold from being damaged.

Preferably, the inner silk fibroin film layer comprises silk fibroin microspheres.

Preferably, the silk fibroin microsphere is internally coated with vascular endothelial cell growth factor.

Preferably, the silk fibroin microsphere has platelet-derived growth factors entrapped therein.

Preferably, the particle size of the silk fibroin microsphere coated with vascular endothelial growth factor is 4.429+ -1.096 μm.

Preferably, the particle size of the silk fibroin microsphere coated with platelet-derived growth factor is 315+ -87 nm.

In order to facilitate understanding of the present invention, the structural design concept of the tissue engineering scaffold will be described:

the bladder wall mainly comprises four layers in tissue structure: mucosal layer, submucosa, myolayer, and serosal layer. Wherein the mucous membrane layer is a waterproof layer composed of urothelial cells, and can form a mucous membrane barrier to prevent urine from damaging deep structures; submucosa comprises blood vessels and extracellular matrix, and plays a supporting and nutritional role on the submucosa and the myolayer; the muscular layer is rich in smooth muscle and is a main structure for finishing the function of bladder comfort and contraction; the serosa layer is positioned on the outermost layer of the bladder and plays a role in protecting the bladder.

The invention uses silk fibroin to construct a three-layer bracket, the innermost layer is composed of a layer of waterproof silk fibroin film, the mucosa layer of the bladder wall is simulated, microspheres which encapsulate the vascular growth factors are added in the preparation process, and the time-series release of the growth factors is realized by controlling the particle size of the microspheres, wherein the layer is the inner layer of the silk fibroin film. The middle layer is composed of silk fibroin sponge, simulates a urinary bladder wall submucosa and a muscle layer, has a porous structure capable of promoting cell adhesion, and provides a reserved channel and a support structure for regeneration of blood vessels, and the layer is the silk fibroin sponge layer. The outermost layer is composed of a layer of water-impermeable silk fibroin film, which simulates the serosa layer of the bladder wall, and is the outer layer of the silk fibroin film.

Based on the same technical conception, the invention also provides a preparation method of the tissue engineering scaffold simulating the physiological structure of the bladder wall, which comprises the following steps:

firstly preparing a silk fibroin film outer layer, then preparing a silk fibroin sponge layer on the upper layer of the silk fibroin film outer layer, then cutting the silk fibroin sponge layer to be smooth, and finally preparing a silk fibroin film inner layer on the upper layer of the silk fibroin sponge layer, thus obtaining the tissue engineering scaffold after completion.

Preferably, the method for preparing the silk fibroin film outer layer comprises the following steps:

and dripping the silk fibroin solution into a porous plate, and drying to obtain the silk fibroin film outer layer.

Wherein the temperature of the drying is 55-60 ℃ and the drying time is 10-12 h.

Preferably, the method for preparing the silk fibroin sponge layer comprises the following steps:

(1) Dripping silk fibroin solution into a porous plate, adding sodium chloride, standing until the silk fibroin solution is fully coagulated after the completion of the steps to obtain a prefabricated silk fibroin sponge layer;

(2) Rinsing the prefabricated silk fibroin sponge layer by deionized water, and obtaining the silk fibroin sponge layer after the rinsing is completed.

Wherein the rinsing time is 70-72 h, and water is changed for 3-5 times every 24 h.

Preferably, the method for preparing the inner layer of the silk fibroin film comprises the following steps:

(S1) uniformly mixing a silk fibroin solution and vascular endothelial cell growth factor, then blending with polyvinyl alcohol, and drying after completion to obtain a dry film a;

(S2) mixing the dry film a with water, dissolving, and centrifuging to obtain a silk fibroin microsphere solution for coating vascular endothelial cell growth factors;

(S3) uniformly mixing the silk fibroin solution and the platelet-derived growth factor, then blending with polyvinyl alcohol, and drying after completion to obtain a dry film b;

(S4) mixing the dry film b with water, dissolving, and centrifuging to obtain a silk fibroin microsphere solution of the entrapped platelet-derived growth factor;

(S5) mixing the silk fibroin solution, the silk fibroin microsphere solution coated with vascular endothelial growth factors and the silk fibroin microsphere solution coated with platelet-derived growth factors, dripping into a porous plate, and drying to obtain the silk fibroin inner layer.

Wherein the rotational speed of the centrifugation is 10000-11000 r/min, the temperature of the centrifugation is 3-4 ℃, and the time of the centrifugation is 18-30 min. The temperature of the drying is 55-60 ℃, and the drying time is 10-12 h.

The beneficial effects of the invention are as follows:

1. compared with the tissue engineering bladder support constructed by the existing single-layer or double-layer composite material, the tissue engineering support simulating the physiological structure of the bladder wall disclosed by the invention has the advantages that the defects of poor biocompatibility and long degradation time of the composite material are avoided, the multi-layer structure and function of the bladder wall can be simulated, meanwhile, the material has good biocompatibility and high mechanical strength, and the material source is wide, the manufacturing method is simple, and the large-scale production is suitable.

2. The invention uses silk fibroin microspheres to realize the time-series release of VEGF and PDGF-BB growth factors, effectively promotes early vascularization after tissue engineering bladder support transplantation, and improves the survival rate of the graft. The time sequence release system of the growth factors constructed by the invention organically combines materials and the growth factors, and simulates the multi-layer structure of the bladder wall to the maximum extent in a construction mode. The diameter of the VEGF-coated silk fibroin microsphere is 4.429+/-1.096 mu m, the volume is large, the release speed of the growth factors is high, and the VEGF-coated silk fibroin microsphere can be quickly released for about 7 days. The PDGF-BB entrapped silk fibroin microsphere has the diameter of 315+/-87 nm, small volume and slow release speed of growth factors, and can be slowly released for more than 30 days. VEGF plays an important role in the initial stage of angiogenesis, PDGF-BB is a key factor in the mature stage of angiogenesis, and the time-series release of VEGF and PDGF-BB can effectively promote angiogenesis. In addition, the system does not need to be embedded by a large omentum, so that the time is saved and the occurrence of secondary wounds of the study objects is avoided. The invention organically combines the structure bionics with the promotion of tissue engineering bladder graft vascularization, greatly improves the graft survival rate, and provides a new method and a product for the clinical difficult problem of bladder repair and reconstruction.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic structural diagram of a tissue engineering scaffold simulating the physiological structure of the bladder wall according to the present invention.

Fig. 2 is a physical diagram of the tissue engineering scaffold simulating the physiological structure of the bladder wall, wherein: fig. 2A is a top view and fig. 2B is a side view.

FIG. 3 is a scanning electron micrograph and a particle size distribution diagram of VEGF-coated silk fibroin microspheres in example 1.

FIG. 4 is a scanning electron micrograph and a particle size distribution diagram of PDGF-BB-entrapped silk fibroin microspheres in example 2.

FIG. 5 is a cumulative release profile of 5% silk fibroin microspheres coated with VEGF and 0.5% silk fibroin microspheres coated with PDGF-BB in test example 1.

FIG. 6 is a comparative result of live/dead staining in test example 2.

FIG. 7 shows the result of comparison of CCK8 detection experiments in test example 3.

The reference numerals in the drawings are:

1-silk fibroin inner layer; 11-VEGF-entrapped silk fibroin microspheres; 12-PDGF-BB-entrapped silk fibroin microspheres; a 2-silk fibroin sponge layer; 3-silk fibroin film outer layer.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

Example 1

The embodiment provides a preparation method of a tissue engineering scaffold simulating a physiological structure of a bladder wall, which comprises the following steps:

(I) Preparation of silk fibroin film inner layer 1:

(1) 1mL of 5% (W/V) silk fibroin solution and 200ng of VEGF are uniformly mixed, then are mixed with 5% (W/V) polyvinyl alcohol according to a volume ratio of 1:4, ultrasonic 25% amplitude acts for 30s, and then are poured into a culture dish and placed in a 60 ℃ oven for overnight to obtain a dry film a;

(2) Taking off the dry film a from the culture dish, putting the dry film a into a 50ml centrifuge tube, adding 20ml deionized water, putting the centrifuge tube on a shaking table, shaking at room temperature for 30min to enable the centrifuge tube to be fully dissolved, and finally putting the centrifuge tube into a centrifuge for centrifugation for 20min under the conditions of 11000r/min and 4 ℃ to obtain a silk fibroin microsphere 11 solution for encapsulating VEGF; the diameter of the microsphere is 4.429+/-1.096 mu m measured by a scanning electron microscope, and a scanning electron microscope image and a particle size distribution diagram are shown in figure 3;

(3) 1ml of 0.5% (W/V) silk fibroin solution and 200ng PDGF-BB are uniformly mixed, then are mixed with 0.5% (W/V) polyvinyl alcohol according to a volume ratio of 1:4, ultrasonic 25% amplitude acts for 30s, and then are poured into a culture dish and placed in a 60 ℃ oven for overnight to obtain a dry film b;

(4) Preparing a PDGF-BB-entrapped silk fibroin microsphere 12 solution by adopting the same operation as that of the step (2); the diameter of the microsphere is 315+/-87 nm measured by a scanning electron microscope, and a scanning electron microscope image and a particle size distribution diagram of the microsphere are shown in figure 4;

(5) And (3) fully mixing 100 mu L of 8% (W/V) silk fibroin solution, 50 mu L of VEGF-coated silk fibroin microsphere 11 solution and 50 mu L of PDGF-BB-coated silk fibroin microsphere 12 solution, dripping into a 24-pore plate, uncovering, and placing into a 60 ℃ oven for drying overnight to obtain the silk fibroin film inner layer 1.

(II) preparation of silk fibroin sponge layer 2:

and (3) dripping 600 mu L of 8% (W/V) silk fibroin solution into a 24-hole plate, uniformly scattering 1.2g sodium chloride, covering a cover, standing at room temperature for 48 hours, placing into a large beaker filled with 2L deionized water for rinsing for 72 hours after the solution is fully solidified, changing water for 3-5 times a day, and obtaining the silk fibroin sponge layer 2 after completion.

(III) preparation of silk fibroin film outer layer 3:

200 mu L of 8% (W/V) silk fibroin solution is dripped into a 24-pore plate, uncapped and put into a 60 ℃ oven for drying overnight, and the silk fibroin film outer layer 3 can be obtained.

(IV) preparing a tissue engineering scaffold:

and (3) preparing an outer layer of the silk fibroin film according to the step (III), preparing a silk fibroin sponge layer on the upper layer of the outer layer of the silk fibroin film according to the step (II), cutting the silk fibroin sponge layer to be flat, and finally preparing an inner layer of the silk fibroin film on the upper layer of the silk fibroin sponge layer according to the step (I), thus obtaining the tissue engineering scaffold.

Example 2

On the basis of example 1, example 2 provides a tissue engineering scaffold which simulates the physiological structure of the bladder wall and is prepared by example 1, and referring to fig. 1, the scaffold comprises a silk fibroin film inner layer 1, a silk fibroin sponge layer 2 and a silk fibroin film outer layer 3 which are sequentially arranged from inside to outside; wherein:

the silk fibroin film inner layer 1 is used for realizing time sequence release of growth factors;

the silk fibroin sponge layer 2 is used for promoting the adhesion of cells and playing a supporting role;

the silk fibroin film outer layer 3 plays a role in protecting and preventing the tissue engineering scaffold from being damaged.

Further, the silk fibroin film inner layer 1 comprises silk fibroin microspheres.

Further, the silk fibroin microsphere is internally coated with vascular endothelial cell growth factors.

Further, the silk fibroin microsphere is internally coated with platelet-derived growth factors.

Further, the particle size of the VEGF-coated silk fibroin microsphere 11 was 4.429.+ -. 1.096. Mu.m.

Further, the particle size of the PDGF-BB-entrapped silk fibroin microsphere 12 is 315+/-87 nm.

A physical view of the tissue engineering scaffold obtained in example 2 is shown in FIG. 2.

Test example 1

To verify the release properties of the VEGF-and PDGF-BB-entrapped silk protein microspheres, the cumulative release amounts of the VEGF-entrapped 5% and PDGF-BB-entrapped 0.5% silk protein microspheres 1d, 3d, 7d, 14d, 21d were measured using the Elisa kit, respectively, and the cumulative release curves were plotted. As shown in FIG. 5, it can be seen from the graph that VEGF is released mainly in 7d, and the release amount is more than about 10 times of PDGF-BB release amount, and two growth factors are released slowly and continuously after 7 days.

Test example 2

To verify the biocompatibility of the resulting tissue engineering scaffold simulating the physiological structure of the bladder wall of example 1, a live/dead staining experiment was performed. live/dead staining is a common method for testing the biocompatibility of materials, by which living cells exhibit green fluorescence and dead cells exhibit red fluorescence. The tissue engineering scaffolds (group A) and the control group (group B) obtained in example 1 were cultured with the scaffold extract and the normal medium, respectively. The tissue engineering scaffold obtained in example 1 has good cell growth and no obvious difference from the control group, which is shown by the fact that the tissue engineering scaffold obtained in example 1 has small cytotoxicity and good biocompatibility when the tissue engineering scaffold is respectively dyed in 1d, 3d and 5 d.

Test example 3

CCK8 detection is also a common method for testing the biocompatibility of materials. The principle is that the proliferation activity of the cultured cells is detected by using a cell counting kit (Cell Counting Kit-8), and the faster the proliferation of the cells is, the darker the color is; the greater the cytotoxicity, the lighter the color. The absorbance was measured by a microplate reader. The Scaffold and Control groups used stent extract and normal medium, respectively, to culture cells. The absorbance was measured at 0d, 1d, 3d, 5d, and 7d, respectively, and as can be seen from FIG. 7, the proliferation rate of the tissue engineering scaffold cell obtained in example 1 was good, which indicates that the tissue engineering scaffold obtained in example 1 was small in cytotoxicity, suitable for cell growth proliferation, and good in biocompatibility.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. The tissue engineering scaffold simulating the physiological structure of the bladder wall is characterized by comprising a silk fibroin film inner layer, a silk fibroin sponge layer and a silk fibroin film outer layer which are sequentially arranged from inside to outside; wherein:

the silk fibroin film inner layer is used for realizing time sequence release of growth factors; wherein:

the inner layer of the silk fibroin film comprises silk fibroin microspheres, the silk fibroin microspheres respectively encapsulate vascular endothelial cell growth factors and platelet-derived growth factors, the particle size of the silk fibroin microspheres encapsulating the vascular endothelial cell growth factors is 4.429+/-1.096 mu m, and the particle size of the silk fibroin microspheres encapsulating the platelet-derived growth factors is 315+/-87 nm;

2. The method for preparing the tissue engineering scaffold simulating the physiological structure of the bladder wall according to claim 1, which is characterized in that the preparation method comprises the following steps:

3. The method for preparing a tissue engineering scaffold simulating physiological structures of bladder wall according to claim 2, wherein the method for preparing the silk fibroin film outer layer comprises the following steps:

4. The method for preparing a tissue engineering scaffold simulating physiological structures of bladder wall according to claim 2, wherein the method for preparing a silk fibroin sponge layer comprises the following steps:

5. The method for preparing a tissue engineering scaffold simulating physiological structures of bladder walls according to claim 2, wherein the method for preparing an inner layer of a silk fibroin film comprises the following steps: