3D printing cavernous organ-like model capable of simulating erectile physiological process

Abstract

The invention discloses a 3D printing cavernous body organ-like model capable of simulating an erectile physiological process, and belongs to the technical field of tissue engineering and regenerative medicine. The hydrogel is formed by photo-curing 3D printing of a dual-network hydrogel material formed by blending a pig cavernous body acellular extracellular matrix pregel solution and polyethylene glycol diacrylate, and has a bionic sine curve expansion structure; micron-sized grooves for guiding the directional arrangement of smooth muscle cells are formed in the surface of the micro-sphere, and an elliptical cavity for providing structural support is formed in the micro-sphere. A pig cavernous body dECM pre-gel solution is used as a base material, so that a microenvironment for living of cells in a body can be effectively simulated; by regulating and controlling the proportion of PEGDA, the mechanical property of the model is matched with that of a natural cavernous body tissue. The organ model can reproduce the NO-mediated smooth muscle cell relaxation and tissue expansion physiological process in vitro, and provides an effective in-vitro platform for researching the pathological mechanism of erectile dysfunction, screening drugs and developing a novel treatment method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue engineering and regenerative medicine, in particular to a 3D-printed sponge organoid model that can simulate the physiological process of erection. BACKGROUND

[0002] Organoids are simple cell-based tissue engineering in vitro models that reproduce the various complex structures and functions of the corresponding tissues in vivo, and can be used to in-depth explore the basic mechanisms of human tissue development, regeneration and repair, and can also be applied in the fields of diagnosis, disease modeling, drug development and personalized medicine. Erectile dysfunction (ED) is defined as the repeated or persistent inability to obtain and / or maintain a penile erection sufficient for satisfactory sexual activity, and is a multidimensional but common male sexual dysfunction, which is generally divided into reflex factors and psychological factors. The former is caused by mental and psychological factors, and the latter may be caused by vascular, neurological, surgical and traumatic factors. Its incidence increases with age, and any change in the components of the erection response can lead to erectile dysfunction, so although it is not a fatal disease, interest in treating and remedying erectile dysfunction has existed since ancient times.

[0003] Nitric oxide (NO) is a gaseous signaling molecule and a key regulator of vascular homeostasis and adaptive responses, and is involved in many important physiological processes, such as vasodilation, neurotransmission, platelet aggregation, etc. The erection process mainly relaxes the vascular smooth muscle cells on the cavernosal sinus cavity through NO to allow the expansion of the sinus cavity, which in turn compresses the veins that transport blood, resulting in increased blood flow and decreased outflow, thereby completing the process. The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway is the most important inducer in this process. After receiving nerve stimulation, endothelial nitric oxide synthase (eNOS) in the endothelial cells of the cavernosal endothelium is activated, which decomposes L-arginine to release NO. After NO diffuses into the cavernosal smooth muscle cells, it stimulates soluble guanylate cyclase (sGC) to convert GTP to cGMP, thereby stimulating cGMP-dependent protein (PKG) to promote the relaxation of cavernosal smooth muscle cells.

[0004] Decellularized extracellular matrix (dECM) as a kind of biomaterials directly extracted from natural tissues has been widely used in tissue manufacturing. dECM has similar composition and components with its corresponding source tissue, and can better reproduce complex cell compatible microenvironment, thus helping to improve cell function and tissue morphogenesis. Therefore, the organoid model printed by using dECM as matrix material can better simulate the growth environment specific to the source tissue, and has high bionics. In conclusion, the sponge organoid model can well reproduce the physiological process of NO as a neurotransmitter relaxing smooth muscle cells in vitro, and provide help for exploring the process of erection and providing ideas for erectile dysfunction. SUMMARY

[0005] The purpose of the present application is to provide a 3D printed sponge organoid model that can simulate the physiological process of erection, which can reproduce the physiological process of NO as a neurotransmitter relaxing smooth muscle cells, and provide help for the treatment of erectile dysfunction.

[0006] To achieve the above-mentioned purpose, the present application provides a 3D printed sponge organoid model that can simulate the physiological process of erection, which is formed by a double network hydrogel material based on a porcine sponge decellularized extracellular matrix pre-gel solution and polyethylene glycol diacrylate PEGDA blend formed by light curing 3D printing; The model has a bionic sinusoidal expansion structure, the surface of the model is provided with micron-level grooves for guiding the directional arrangement of smooth muscle cells, and the inside of the model is provided with an elliptical cavity for providing structural support. The model can reproduce the physiological process of nitric oxide-mediated relaxation of smooth muscle cells and tissue expansion in vitro.

[0007] Preferably, the width of the micron-level groove is 20-400μm, and the wall thickness between the grooves is 50-500μm.

[0008] Preferably, the mass fraction of polyethylene glycol diacrylate PEGDA is 10%-40%, and the molecular weight is 1000-10000.

[0009] Preferably, 0.7mg / mL-1.5mg / mL citrine is added to the double network hydrogel material to improve the printing precision.

[0010] Preferably, the model is manufactured by using light curing 3D printing technology, and different parts are printed with different precision: the initial part is printed with a precision of 50μm, the middle groove and cavity part is printed with a precision of 10μm, and the groove part at the top of the model is printed with a precision of 20μm.

[0011] Preferably, the model is manufactured by using light curing 3D printing technology, and the model is printed with a printing precision of 20μm.

[0012] Preferably, the preparation method of the porcine corpus cavernosum acellular extracellular matrix pre-gel solution comprises: S1, after repeated freeze-thaw treatment of the porcine corpus cavernosum tissue, remove the foreskin and endothelial tissue, cut the corpus cavernosum tissue, and sequentially wash it with washing liquid A containing 0.5%-1.5% sodium dodecyl sulfate and 0.1%-0.5% penicillin, washing liquid B containing 1xPBS and 0.1%-0.5% penicillin, and deionized water containing 0.1%-0.5% penicillin; S2, vacuum freeze-drying the washed tissue at-80℃ for 3 days, crushing and sterilizing by 5-10kGy gamma ray irradiation; stirring and digesting the sterilized acellular extracellular matrix powder in 1-3mg / mL pepsin in 0.5M acetic acid solution for 2-4 days, adjusting the pH to 6.8-7.5 with 5M sodium hydroxide solution, and adding 10xPBS to adjust the ion concentration to obtain a porcine corpus cavernosum acellular extracellular matrix pre-gel solution.

[0013] In another aspect, the application also provides a use of the above-mentioned 3D printed corpus cavernosum organoid model capable of simulating the physiological process of erection in the research of the pathological mechanism of erectile dysfunction, drug screening or the development of treatment methods.

[0014] In another aspect, the application also provides a use of the above-mentioned 3D printed corpus cavernosum organoid model capable of simulating the physiological process of erection in the research of the pathological mechanism of erectile dysfunction, drug screening or the development of treatment methods.

[0015] Therefore, the 3D printed corpus cavernosum organoid model capable of simulating the physiological process of erection has the following beneficial effects: (1) The design scheme of the present application selects a variety of different specific surface area of corpus cavernosum organoid model structure, which has undergone fluid mechanics simulation and actual exploration, and has selected a kind of model with minimum surface stress and optimal expansion effect in the expansion process.

[0016] (2) The present application prepares a porcine corpus cavernosum acellular extracellular matrix pre-gel solution, which has good temperature sensitivity, plasticity and very low immunological rejection, and uses a mild treatment method in the whole decellularization process, realizing the high retention of collagen, laminin and glycosaminoglycan.

[0017] (3) The porcine corpus cavernosum acellular extracellular matrix pre-gel solution prepared by the present application as a substrate material printed organoid model can well reproduce the cell microenvironment specific to the source tissue, which is helpful to improve cell function and tissue morphogenesis; by doping a certain amount of PEGDA, the mechanical strength and tissue performance are matched.

[0018] (4) The cavernous body organoid model prepared by the present invention can reproduce the physiological process of NO as a neurotransmitter relaxing smooth muscle cells in vitro, which can provide ideas for the treatment of erectile dysfunction and help to carry out related drug screening in vitro.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A series of models with different specific surface areas were created for 3D modeling, where A is a sine curve; B is a schematic diagram of a three-dimensional expansion model. Figure 2 The changes in liquid flow velocity and stress on the surface of models with different specific surface areas when the volume expands to 3 times the original volume are shown. Among them, a is model 5-1, b is model 5-2, c is model 5-3, d is model 5-4, e is model 5-5, f is model 5-6, and g is model 5-7. Figure 3 The required flow rate and expansion rate for models with different specific surface areas to complete the expansion; Figure 4 Experimental route for preparing pregel solutions for decellularized extracellular matrix in porcine corpus cavernosum; Figure 5 For the quantitative characterization of the decellularization degree of the pregel solution, a is the standard curve of total protein concentration, b is the comparison of total protein content between natural tissue and pregel solution, c is the comparison of glycosaminoglycan content between natural tissue and pregel solution, d is the comparison of DNA electrophoresis bands between natural tissue and pregel solution, and e is the comparison of DNA content between natural tissue and pregel solution. Figure 6 Microscopic morphology images of printing inks with different formulations after photocuring; Figure 7 The rheological properties of the photocurable printing ink are given, where a is the storage modulus and b is a comparison between the storage modulus and the loss modulus. Figure 8 The tensile properties of the printing ink after photopolymerization are given, where a is the stress-strain curve, b is the breaking strength, and c is the elongation at break. Figure 9The expansion state of the model is shown when liquid is injected into the model at different flow rates. Among them, a is the change in expansion state of the uninoculated model at an injection rate of 7 μL / min; b is the change in expansion state of the model at an injection rate of 7 μL / min after cells are inoculated on the model surface; and c is the change in expansion state of the model at an injection rate of 6 μL / min after NONOates are added to the surface of the cell-inoculated model. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0025] Example 1 In 3ds Max, a smooth curve is obtained by controlling the number of segments of the sine curve from 60 to 100, while keeping the period of the sine curve constant at 5. The peak value of the sine curve is gradually increased from 0.01 mm to 0.45 mm to obtain a series of smooth sine curves of different lengths. This series of sine curves is then imported into Rhino software and made to flow in circles of the same size, connecting end to end to create a series of surfaces with different specific surface areas, such as... Figure 1 As shown in Part A of the diagram.

[0026] Based on these surfaces, a series of three-dimensional inflated models were drawn. Grooves ranging from 20μm to 400μm wide were designed on the model surfaces to inoculate smooth muscle cells, helping the cells to orient themselves on the model surface and concentrate relaxed cellular forces. The wall thickness between the grooves ranged from 50μm to 500μm. Elliptical cavities were designed inside the models to provide sufficient strength support for the printing of the structures. The inflated models are as follows: Figure 1 As shown in Part B.

[0027] Fluid dynamics simulations were performed using COMSOL models with different specific surface areas, such as... Figure 2 As shown, the maximum stress on the surface of model 5-1 during expansion is 3.83 × 10⁻⁶. 3 kPa, model 5-2 is 3.64 × 10 3 kPa, model 5-3 is 2.96 × 10 3kPa, model 5-4 is 2.42 × 10 3 kPa, model 5-5 is 2.69 × 10 3 kPa, for models 5-6 it is 3.08 × 10 kPa. 3 kPa, for models 5-7 it is 3.5 × 10 3 The kPa results show a trend of first decreasing and then increasing, indicating that there is an optimal model 5-4 in the middle, which has the least surface stress during expansion, is the easiest to expand and not easy to burst.

[0028] Based on experimental verification, the model with the optimal specific surface area was selected: This model was then used in a photopolymerization 3D printer to... Figure 1 Seven models with different specific surface areas, as shown in Figure B, were constructed. Then, liquid was injected into the models at a specific flow rate using a syringe pump to explore the required flow rate and expansion rate for models with different specific surface areas to complete expansion. The results are as follows: Figure 3 As shown, it can be seen that as the specific surface area increases, the flow velocity required for model expansion first decreases and then increases. That is, model 5-4 requires the least force for expansion and has the best expansion effect, which is consistent with the simulation results.

[0029] Example 2 according to Figure 4 The steps shown in the diagram for preparing the porcine corpus cavernosum decellularized extracellular matrix pregel solution specifically include: After performing 1-3 freeze-thaw cycles on the purchased chilled pig penis tissue, remove the outer foreskin tissue and scrape away as much of the internal endothelial tissue as possible, selecting only the corpus cavernosum tissue and cutting it into 3cm pieces. 3 The small pieces of tissue were weighed and placed in washing solution A at a mass ratio of 300 g / L, and stirred at 900 r / min for 48 h.

[0030] Washing solution A consists of a mixture of 1% sodium dodecyl sulfate (SDS) and 0.1% penicillin. It is changed every 3 hours for the first 24 hours and every 6 hours for the next 24 hours to ensure optimal decellularization efficiency.

[0031] The resulting clean tissue block was transferred to washing solution B and washed with agitation for 24 hours to ensure complete removal of washing solution A. Washing solution B consisted of a mixture of 1× PBS solution and 0.1% penicillin solution, and was changed every 3 hours.

[0032] The tissue was then transferred to deionized water and washed for 24 hours. 1g of potassium penicillin was added to every 1L of deionized water for sterilization and disinfection. The water was changed every 3 hours to ensure that the decellularized ECM had minimal immunogenicity.

[0033] Finally, the tissue was freeze-dried under vacuum at -80°C for 3 days to obtain a tissue that possesses both toughness and firmness. After being placed in liquid nitrogen for 3-5 minutes, it was quickly removed and pulverized using a grinder to finally obtain a spongy, extracellular matrix resembling meat floss.

[0034] After being packaged according to a certain weight, the product is sterilized by gamma irradiation at a dose of 5-10 kGy to obtain sterile porcine corpus cavernosum decellularized extracellular matrix powder.

[0035] Example 3 The sterile porcine corpus cavernosum decellularized extracellular matrix powder obtained in Example 2 was added at a ratio of 200 mg of powder per 10 mL of digestion solution C and stirred for 3 days at a stirring rate of 900 r / min. The entire process was carried out in a sterile environment.

[0036] Digestive solution C was prepared by mixing 50 mL of 0.5 M acetic acid solution with 100 mg of pepsin to obtain a pepsin solution with a concentration of 2 mg / mL. After filtration and sterilization through a 0.22 μm filter, sterile digestive solution was obtained.

[0037] After digestion, a homogeneous milky white solution was obtained, with virtually no insoluble tissue fragments. After centrifugation using an ultracentrifuge, the pH of the solution was adjusted to around 7 using 5M sodium hydroxide solution to terminate the digestion. Subsequently, one-tenth of the solution volume of 10×PBS solution was added to adjust the ion concentration, ultimately yielding a porcine corpus cavernosum decellularized matrix pregel solution, which can form a gel at 37°C and possesses the basic properties of decellularized matrix (dECM).

[0038] The degree of decellularization of the pregel solution was quantitatively characterized, and the results are as follows: Figure 5 As shown, a is the standard curve of total protein concentration, b is the comparison of total protein content between natural tissue and pre-gel solution, c is the comparison of glycosaminoglycan content between natural tissue and pre-gel solution, d is the comparison of DNA electrophoresis bands between natural tissue and pre-gel solution, and e is the comparison of DNA content between natural tissue and pre-gel solution. Figure 5 As shown in Figure b, the protein content of natural tissue is approximately 0.5 mg / mL, while the protein content of decellularized CCdECM hydrogel is 4.4 mg / mL, an increase of nearly 9 times. This indicates that the decellularization process effectively preserved the protein components. Furthermore, the GAG ​​content also increased from 1504 ng / mL to 4760 ng / mL, an increase of approximately 3 times. Figure 5 c). From DNA electrophoresis experiment ( Figure 5 Quantitative analysis of DNA content (d) Figure 5As shown in e), there is virtually no DNA residue inside the CCdECM hydrogel after decellularization, with a DNA content of approximately 32 ng / mg, which is far below the immune rejection threshold of 50 ng / mg, meeting the application standards for low immunogenicity biomaterials.

[0039] Example 4 PEGDA with a molecular weight of 6000 was added to the porcine corpus cavernosum decellularized extracellular matrix pregel solution at a mass ratio of 10%, 20%, and 30%. After mixing, 20 μL of Ru / SPS initiator and 1 mg of lemon yellow were added to each 1 mL of solution. After dissolving evenly, the final printing ink was obtained.

[0040] The microstructure of printing inks with different formulations after photocuring is as follows: Figure 6 As shown in the figure, the microstructure of the hydrogel indicates that the addition of PEGDA6000 provides a more porous structure for the hydrogel, which is more conducive to cell adhesion and the exchange of substances between the gel scaffold and the external environment.

[0041] The rheological properties of printing inks with different formulations after photopolymerization are as follows: Figure 7 As shown in the figure, where a represents the storage modulus and b represents the comparison between the storage modulus and the loss modulus. Rheological results indicate that the addition of dECM can form a dual-network structure inside the photocured material, effectively improving the storage modulus and enhancing the mechanical properties of the material after photocuring.

[0042] Tensile properties such as Figure 8 As shown, a represents the stress-strain curve, b represents the tensile strength, and c represents the tensile strength. Uniaxial tensile results indicate that the 20% PEGDA+dECM printing ink exhibits the most suitable mechanical properties, with a low tensile modulus of approximately 44 kPa and an elongation at break of approximately 78%, completely covering the strain range (≤50%) of erectile strain in the corpus cavernosum organoid model.

[0043] Example 5 Using a BMF nanoArch S140 printer and printing ink with a porcine corpus cavernosum decellularized extracellular matrix pregel solution as the matrix material, selected 3D expanded models were photopolymerized and 3D printed. The printing process was divided into three parts: the initial part was printed with a precision of 50 μm, the middle groove and cavity part with a precision of 10 μm, and the final groove part at the top of the model with a precision of 20 μm. This approach ensured the required precision of the model while improving printing efficiency.

[0044] Example 6 Using a syringe pump, liquid is injected into the printed model of Example 5 at a certain flow rate to cause it to expand. Figure 9As shown in Figure a, at an injection rate of 7 μL / min, the model initially showed little change. It began to expand after approximately 30 seconds and rapidly increased in size within 20 seconds. However, after seeding cells onto the model surface, at the same injection rate of 7 μL / min, the model struggled to expand, exhibiting minimal volume change. This indicates that after cell seeding, the cellular forces connecting the cells made expansion more difficult. Figure 9 (b)

[0045] Liquid was injected into the cell-inoculated model at an injection rate of 6 μL / min. After 2 min 50 s, it was observed that the model failed to expand. At this point, the injection was stopped, and a certain amount of NONOates was added to the surface of the model. After waiting for 3 min for the NO to completely decompose and release the smooth muscle cells, the liquid was injected into the model again at the same flow rate. Soon after, the model was observed to expand rapidly. Figure 9 (c) indicates that NO, through a series of physiological processes, enabled smooth muscle cells to relax, thus helping the model to complete its expansion.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A 3D-printed corpus cavernosum organoid model that can simulate the physiological process of erection, characterized in that: The model was 3D printed by photopolymerization of a double-network hydrogel material based on a pregel solution of decellularized extracellular matrix from porcine corpus cavernosum and polyethylene glycol diacrylate (PEGDA). The model features a biomimetic sinusoidal expansion structure, with micron-sized grooves on its surface to guide the directional alignment of smooth muscle cells, and an elliptical cavity inside the model to provide structural support. The model can reproduce the physiological processes of nitric oxide-mediated smooth muscle cell relaxation and tissue expansion in vitro.

2. The 3D-printed corpus cavernosum organoid model that can simulate the physiological process of erection according to claim 1, characterized in that: The width of the micron-level grooves ranges from 20μm to 400μm, and the wall thickness between the grooves ranges from 50μm to 500μm.

3. The 3D-printed corpus cavernosum organoid model that can simulate the physiological process of erection according to claim 1, characterized in that: The mass fraction of polyethylene glycol diacrylate (PEGDA) is 10%-40%, and the molecular weight is 1000-10000.

4. The 3D-printed corpus cavernosum organoid model that can simulate the physiological process of erection according to claim 1, characterized in that: The dual-network hydrogel material contains 0.7 mg / mL-1.5 mg / mL of lemon yellow to improve printing accuracy.

5. A 3D-printed corpus cavernosum organoid model capable of simulating the physiological process of erection according to claim 1, characterized in that, The model was manufactured using photopolymer 3D printing technology, with different parts printed with different precision: the initial part was printed with a precision of 50μm, the middle groove and cavity part was printed with a precision of 10μm, and the groove part on the top of the model was printed with a precision of 20μm.

6. A 3D-printed corpus cavernosum organoid model capable of simulating the physiological process of erection according to claim 5, characterized in that, The model was manufactured using photopolymer 3D printing technology, with a printing precision of 20μm for the entire model.

7. A 3D-printed corpus cavernosum organoid model capable of simulating the physiological process of erection according to claim 1, characterized in that, The preparation method of the porcine corpus cavernosum decellularized extracellular matrix pregel solution includes: S1. After repeated freeze-thaw treatment of porcine corpus cavernosum tissue, the prepuce and endothelial tissue are removed, the corpus cavernosum tissue is cut into small pieces, and washed sequentially with washing solution A containing 0.5%-1.5% sodium dodecyl sulfate and 0.1%-0.5% penicillin, washing solution B containing 1×PBS and 0.1%-0.5% penicillin, and deionized water containing 0.1%-0.5% penicillin. S2. The washed tissue was freeze-dried under vacuum at -80℃ for 3 days, pulverized, and then sterilized by irradiation with 5-10kGy gamma rays. The sterilized decellularized extracellular matrix powder was digested in 0.5M acetic acid solution with 1-3mg / mL pepsin for 2-4 days. The pH was adjusted to 6.8-7.5 with 5M sodium hydroxide solution, and 10×PBS was added to adjust the ion concentration to obtain a pregel solution of decellularized extracellular matrix from porcine corpus cavernosum.

8. The application of a 3D-printed corpus cavernosum organoid model as described in any one of claims 1-7, capable of simulating the physiological process of erection, in the study of the pathological mechanism of erectile dysfunction, drug screening, or the development of treatment methods.

9. The application of a 3D-printed corpus cavernosum organoid model as described in any one of claims 1-7 in simulating the erectile physiological process mediated by the NO-cGMP signaling pathway in vitro.

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