Preparation process of acellular matrix particles

Abstract

The invention discloses a preparation process of acellular matrix particles, and relates to the technical field of biomedical material processing. The invention provides an acellular matrix particle preparation process based on a biological enzyme method and a fluidized bed principle. The acellular matrix particle preparation process comprises the following steps: alkali treatment, acellular dehydration, grinding and screening. The step of screening is based on the fluidized bed screening principle, the acellular matrix particles are input into a fluidized bed layer, the particles suspended on the fluidized bed layer can settle rapidly under the action of fluid, and the particles with small particle sizes can rise along with the fluid and enter a discharging system. The preparation process can realize accurate screening of acellular matrix particles, significantly reduce the particle size fluctuation range, can directionally prepare particles in a specific particle size interval, and has the characteristics of high efficiency, continuous production, simple operation and strong applicability.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical material processing, and particularly relates to a preparation process of acellular matrix microparticles. Background Art

[0002] Acellular Matrix (AM) is a biomaterial widely used in tissue engineering and regenerative medicine. It is obtained by removing the cellular components in tissues, retaining the natural extracellular matrix structure, and having good biocompatibility and biomechanical properties. In practical applications, biological cell raw materials are processed through processes such as alkali treatment, decellularization, dehydration, grinding, and sieving to obtain acellular matrix microparticles, which are used for mixing with other materials or directly for tissue repair.

[0003] The particle size of acellular matrix microparticles has an important influence on their biological properties and application effects. Too small microparticles may lead to a decrease in the mechanical properties of the material, while too large microparticles may affect their penetration and distribution in tissues. Therefore, it is very necessary to accurately screen the particle size of acellular matrix microparticles.

[0004] Chinese Patent CN119034010A (publication date: November 29, 2024) discloses an injectable acellular matrix microparticle implant and its preparation method and uses. The acellular matrix particles are prepared through processes such as inactivation, decellularization, freeze-drying, microparticle preparation, sub-packaging, and sterilization; the microparticles are obtained by screening with any two sieves within the range of a 40-mesh sieve (aperture 380 μm) and a 300-mesh sieve (aperture 48 μm).

[0005] Although the sieve method adopted in the above patent can achieve the screening of microparticles, the microparticles are prone to clogging the sieve holes, reducing the screening efficiency, and requiring frequent cleaning or replacement of the sieve, resulting in a decrease in screening accuracy.

[0006] In addition, common microparticle screening methods also include sedimentation method and centrifugation method, etc., which generally have some drawbacks, such as the microparticles are prone to clogging, the efficiency is low when processing a large number of microparticles, it is difficult to achieve continuous production, and the particle size fluctuation is large. In the commonly used particle size distribution D10 - D50 - D90 of 30 ± 15 μm - 115 ± 30 μm - 250 ± 50 μm, the particle size fluctuation is too large, which has many adverse effects on the subsequent microparticle sub-packaging, clinical use, and product efficacy.

[0007] Fluidized bed technology is a technology that uses a fluid (such as gas or liquid) to suspend solid particles and form a fluidized state. In a fluidized bed, there is a strong interaction between the particles and the fluid, and the movement state of the particles is similar to that of a fluid, so it has good mixing and mass transfer characteristics and is widely used in many industrial fields.

[0008] Chinese Patent CN116807893A (Publication Date: September 20, 2023) provides an application of fluidized bed side spray granulation technology in Shunqi Huatan granules. The steps include: S1 raw material ratio weighing, S2 fluidized bed setting and preheating, S3 raw material input and sealing, S4 dilution of Shunqi Huatan extract, S5 installation and sealing of peristaltic pump hose, S6 spray gun position adjustment and pressure setting, S7 granulation production, S8 particle screening and collection, and S9 particle packaging; By using fluidized bed side spray granulation technology, through precise raw material ratio and process parameter setting, including fluidized bed setting and preheating, installation and sealing of peristaltic pump hose, and spray gun position adjustment and pressure setting, etc., the efficient preparation of Shunqi Huatan granules is achieved. It utilizes the drying operation of fluidized bed technology in drug preparation, and the screening operation is carried out through an oscillating sieve, still having the aforementioned drawbacks.

[0009] Chinese Patent CN116617170A (Publication Date: August 22, 2023) provides a method for preparing composite embedded structure α / β anhydrous lactose microparticles, which relates to the technical field of lactose preparation. Its fluidized bed unit includes a fluidized bed, and the bottom of the fluidized bed is connected through a heating system, a fan, and an air inlet. An air outlet is also provided at the top of the fluidized bed, and the heating system adopts adjustable temperature and flow rate. The aim is to coat α-lactose on the surface of β-lactose by using fluidized bed technology to maintain the stability of the whole lactose composite particles, and at the same time obtain composite particles with uniform properties and adjustable proportions. Its application purpose for the fluidized bed is to regulate the ratio of α-lactose / β-lactose and the water content of the particles, so as to adjust the dissolution rate, solubility, and compressibility of the lactose composite particles, rather than purposefully and precisely screening the particle size to improve the screening efficiency.

[0010] In view of this, the present application provides a preparation process for acellular matrix microparticles with better quality precision. Summary of the Invention

[0011] In order to solve the problems in the prior art such as low precision of particle screening, low screening efficiency, inability to continuously produce, and complex operation, the present invention provides the following technical solutions:

[0012] A preparation process for acellular matrix microparticles, comprising the following steps:

[0013] S1: Take acellular matrix raw materials, and treat the cell matrix raw materials with a sodium hydroxide solution with a concentration of 1% - 5% for 3 - 6 hours to obtain cell matrix materials;

[0014] S2: Place the cell matrix materials obtained through step S1 in a biological enzyme solution with a concentration of 0.1% - 0.15%, oscillate at 37°C for 4 - 6 hours, then dehydrate with an alcohol solution mixed in a certain proportion, and then freeze-dry to obtain well-air-dried acellular intermediates;

[0015] S3: Grind the acellular intermediate product obtained in step S2 using a cryogenic grinder for 3 - 4 hours to obtain microparticles with a particle size less than 850 μm.

[0016] S4: Precisely screen the microparticles obtained in step S3 according to their particle sizes using a fluidized bed device. This fluidized bed device consists of several parts: a fluidized bed reactor, a feeding system, a fluid conveying system, and a collection system.

[0017] Preferably, in step S1, a sodium hydroxide solution with a concentration of 2% - 3% is used for treatment for 4 hours.

[0018] As an option, in step S4, the fluidized bed reactor has a cylindrical structure and is internally provided with a fluidized bed layer. The fluidized bed layer is supported by a perforated plate, and a plurality of small holes are evenly distributed on the perforated plate for the entry of fluid and the suspension of microparticles. A microparticle outlet is provided at the top of the fluidized bed reactor for discharging the screened microparticles.

[0019] As an option, in step S4, the feeding system includes a feeding hopper and a feeding pipeline. The feeding hopper is used to store the acellular matrix microparticles to be screened, and the feeding pipeline connects the feeding hopper and the fluidized bed reactor to transport the microparticles above the fluidized bed layer.

[0020] As an option, in step S4, the fluid conveying system includes a fluid source, a pressure regulating valve, and a fluid distributor. The fluid source provides the fluid required for the fluidized bed according to the characteristics of the material to be processed. The fluid is transported from the fluid source to the bottom of the fluidized bed reactor through a fluid conveying pipeline, and a pressure regulating valve is installed on the conveying pipeline for regulating the intake pressure and measuring the flow rate. The fluid distributor is installed at the bottom of the fluidized bed reactor for evenly distributing the fluid onto the perforated plate so that the fluid can enter the fluidized bed layer evenly.

[0021] Preferably, in the fluid conveying system of step S4, high - purity nitrogen is selected as the fluid.

[0022] In step S4, the collection system includes a cyclone separator and a collection container. The cyclone separator is used to separate the microparticles and the gas, and the collection container is used to collect the acellular matrix microparticles within a specified particle size range. The tail gas is discharged from the gas outlet above the cyclone separator, and the collection container is sealed to prevent contamination and loss of the microparticles.

[0023] Further, the screening working principle of the fluidized bed reactor in step S4 is as follows: The fine particles obtained through step S3 enter above the fluidized bed layer of the fluidized bed reactor through the fluidized bed feeding system. The fluid delivery system delivers the fluid to the bottom of the fluidized bed reactor and evenly distributes it onto the perforated plate through the fluid distributor. After the fluid enters the fluidized bed layer, it interacts with the fine particles, causing the fine particles to suspend and form a fluidized state. In the fluidized bed layer, intense collisions and frictions occur between the fine particles and the fluid. According to the density and particle size of the fine particles, fine particles of different particle sizes will move at different speeds and trajectories. Larger-sized fine particles will settle faster, while smaller-sized fine particles will rise with the fluid. Fine particles within the specified particle size range pass through the fluidized bed layer and enter the collection system, where they are temporarily stored in the collection container.

[0024] Further, the fluidized bed reactor in step S4 controls the minimum fluidization velocity of the fine particle fluidization by adjusting the gas source pressure. The minimum output pressure of the key parameter gas source is 0.25 - 0.35 MPa.

[0025] Compared with the prior art, the technical solution of the present application has the following advantages and effects:

[0026] 1. High screening accuracy: By adjusting the fluid pressure of the fluidized bed, precise screening of the acellular matrix fine particles is achieved. Different gradients of fine particles with particle size ranges can be obtained through multiple screenings, such as 15μm - 50μm - 90μm, 110μm - 150μm - 200μm, to meet the requirements of different applications;

[0027] 2. High screening efficiency: The fluidized bed has good mixing and mass transfer characteristics, and can quickly disperse and screen the fine particles, greatly improving the screening efficiency;

[0028] 3. Continuous production: The process of the present invention can achieve continuous screening of the acellular matrix fine particles, without frequent shutdowns and equipment replacements, improving the production efficiency;

[0029] 4. Simple operation: The device has a simple structure, is easy to operate, and is easy to control and maintain;

[0030] 5. Strong applicability: By adjusting the fluid selected for the fluidized bed, it is applicable to the screening of acellular matrix fine particles from different sources and treatment methods, and has a wide application prospect.

[0031] The above is only an overview of the technical solution of the present application. To enable the technical means of the present application to be implemented in accordance with the content of the specification, and to make the above and other purposes, features, and advantages of the present application clearer and more understandable, the following embodiments of the present application are introduced in detail in conjunction with the accompanying drawings. According to the detailed description of the specific embodiments of the present application below in conjunction with the drawings, those skilled in the art will be more clear about the above and other purposes, features, and advantages of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly introduce the technical solutions of the present invention and its embodiments, the accompanying drawings required for the description of the embodiments of the present invention will be briefly introduced below.

[0033] Figure 1 It is a schematic flow chart of the preparation process of a decellularized matrix particle of the present invention;

[0034] Figure 2 It is a schematic structural diagram of the fluidized bed reactor in Embodiment 1 of the present invention.

[0035] Reference numerals: 1, fluidized bed reactor; 2, feed hopper; 3, feed pipeline; 4, fluid source; 5, fluid pipeline; 6, fluid distributor; 7, perforated plate; 8, particle outlet; 9, cyclone separator; 10, collection container; 11, pressure regulating valve. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] In order to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, those of ordinary skill in the art can make several improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0037] Embodiment 1

[0038] This embodiment provides a preparation process for decellularized matrix particles, and its main process flow is shown in Figure 1 , and the specific steps are as follows:

[0039] S1: Take the decellularized matrix raw material, and treat the cell matrix raw material with a sodium hydroxide solution with a concentration of 1% to 5% for 3 to 6 hours to obtain a cell matrix material;

[0040] S2: Place the cell matrix material obtained in step S1 in a biological enzyme solution with a concentration of 0.1% to 0.15%, oscillate at 37°C for 4 to 6 hours, dehydrate with an alcohol solution mixed in a certain ratio, and then freeze-dry to obtain a well-dried decellularized intermediate product;

[0041] S3: Grind the decellularized intermediate product obtained in step S2 with a low-temperature grinder for 3 to 4 hours to obtain particles with a particle size less than 850 μm;

[0042] S4: Precisely screen the particles obtained in step S3 according to the particle size by using a fluidized bed device. The fluidized bed device includes: a fluidized bed reactor; a feeding system; a fluid delivery system; a collection system.

[0043] Preferably, in step S1, a sodium hydroxide solution with a concentration of 2% - 3% is used for treatment for 4 hours.

[0044] In some embodiments, the structure of the fluidized bed reactor in step S4 is shown in Figure 2 . It is a cylindrical structure with a fluidized bed layer inside. The fluidized bed layer is supported by a perforated plate 7, and a plurality of small holes are evenly distributed on the perforated plate 7. A particle outlet 8 is provided at the top of the fluidized bed reactor 1.

[0045] In some embodiments, the feeding system in step S4 includes a feeding hopper 2 and a feeding pipeline 3. The feeding hopper 2 is used to store the acellular matrix particles to be screened, and the feeding pipeline 3 connects the feeding hopper 2 and the fluidized bed reactor 1 to transport the particles above the fluidized bed layer.

[0046] In some embodiments, the fluid delivery system in step S4 includes a fluid source 4, a fluid delivery pipeline 5, and a fluid distributor 6. The fluid source 4 provides the fluid required for the fluidized bed. After the fluid is adjusted to the appropriate pressure through a pressure regulating valve 11 on the fluid delivery pipeline, it is transported from the fluid source 4 to the bottom of the fluidized bed reactor 1. The fluid distributor 6 is installed at the bottom of the fluidized bed reactor 1 and is used to evenly distribute the fluid onto the perforated plate 7.

[0047] In some embodiments, the collection system in step S4 includes a cyclone separator 9 and a collection container 10 to collect acellular matrix particles within a specified particle size range.

[0048] Further, in step S4, first, the acellular matrix particles to be screened are placed into the feeding hopper 2, and the particles are transported above the fluidized bed layer through the feeding pipeline 3. Then, the fluid delivery system is started, and the fluid is transported from the fluid source 4 through the fluid delivery pipeline to the bottom of the fluidized bed reactor 1 and evenly distributed onto the perforated plate 7 through the fluid distributor 6. After the fluid enters the fluidized bed layer, it interacts with the particles, causing the particles to suspend and form a fluidized state. In the fluidized bed layer, the particles move at different speeds and trajectories according to their particle sizes. When the particles pass through the fluidized bed layer, they enter the collection system. The cyclone separator 9 captures the particles within the specified particle size range. Finally, the particles are collected into the collection container 10.

[0049] After the collection of the particles within the specified particle size range is completed, replace the collection container 10 and adjust the intake air pressure through the pressure regulating valve 11 to obtain particles within another particle size range.

[0050] The key to this process is to control the minimum fluidization velocity that enables the particles to be fluidized, and this velocity is controlled by adjusting the gas source pressure. The steps to calculate the minimum gas source pressure output by the pressure regulating valve based on the average density of the particles to be screened are as follows:

[0051] (1) Calculate the minimum fluidization velocity of the gas using the Wen-Yu empirical formula:

[0052] u mf = C1 * μ f / (ρ f * d)((ρ s - ρ f ) / ρ f )^C2

[0053] u mf —— is the minimum fluidization velocity (m / s)

[0054] μ f —— is the dynamic viscosity of the gas (Pa·s)

[0055] ρ f —— is the density of the gas (kg / m 3 )

[0056] ρ s —— is the density of the particles (kg / m 3 )

[0057] d —— is the diameter of the particles (m)

[0058] C1 and C2 are empirical constants. Usually, C1 = 33.67 and C2 = 0.0408.

[0059] u mf = 7.12 m / s

[0060] (2) By combining the cross-sectional area of the fluidized bed reactor and the cross-sectional area of the fluid delivery pipeline, the gas velocity of the gas source can be calculated, which is approximately 15 m / s.

[0061] (3) Calculate the gas source pressure using the Bernoulli equation:

[0062] P1 + 1 / 2ρ1u1 2 + ρ1gh1 = P2 + 1 / 2ρ2u2 2 + ρ2gh2

[0063] P —— gas pressure (Pa)

[0064] ρ —— gas density (kg / m 3 )

[0065] u —— gas velocity (m / s)

[0066] g —— acceleration due to gravity (m / s 2 )

[0067] h —— height of the gas (m)

[0068] In this formula, u2 = umf For the system pressure drop, 50% is taken. The minimum pressure of the key parameter gas source output is 0.25 - 0.35 MPa.

[0069] The acellular matrix microparticles in different particle size ranges can re-enter the fluidized bed system for secondary treatment to achieve more refined screening.

[0070] Table 1 Particle sizes obtained under different pressures

[0071]

[0072] The specific definitions of D10, D50, and D90 are as follows:

[0073] D10: It represents the particle size value corresponding to when the cumulative particle size distribution reaches 10%, that is, 10% of the particle sizes in the sample are less than or equal to this value. D10 is usually used to characterize the particle size range of the finer part in the particle population.

[0074] D50: Also known as the median diameter or median particle size, it represents the particle size value corresponding to when the cumulative particle size distribution reaches 50%, that is, 50% of the particle sizes in the sample are less than or equal to this value, and the other 50% of the particle sizes are greater than this value. D50 is usually used as a reference value for the average particle size of the powder.

[0075] D90: It represents the particle size value corresponding to when the cumulative particle size distribution reaches 90%, that is, 90% of the particle sizes are less than or equal to this value. D90 reflects the distribution of the coarser particles in the particle population and is usually used to control product quality or evaluate the proportion of coarse particles.

[0076] Example 2

[0077] This example is based on Example 1. Through laboratory experiments, acellular matrix microparticles are prepared when the key parameter gas source output pressure in step S4 is 0.3 MPa. Taking the existing technology electric sieve preparation method after grinding as a comparative example, the particle size range and screening efficiency data of the acellular matrix microparticles obtained in Example 2 and the comparative example are compared as follows.

[0078] Table 2 Comparison of screening particle sizes

[0079]

[0080] Table 3 Comparison of screening efficiencies

[0081]

[0082] The experimental data show that for the screening operation of the same particle size microparticles for the same time, the processing capacity of the example is much greater than that of the comparative example, and the collection efficiency is also higher than that of the comparative example; when preparing the same amount of microparticles with the same particle size, the total processing time of Example 2 is also much less than that of the comparative example.

[0083] In summary, compared with the traditional preparation process, the acellular matrix microparticles prepared by the fluidized bed screening process of the present invention have a larger single - processing capacity, greatly improving the screening accuracy and screening efficiency, and providing a more uniform microparticle raw material for the application of acellular matrix microparticles.

[0084] The above - mentioned are only exemplary embodiments of the present invention, not all embodiments. Those skilled in the art should understand that various changes and variations can be made to the exemplary embodiments of the present disclosure without departing from the spirit and scope of the present disclosure in essence, and all such changes and variations are included within the protection scope of the present disclosure defined by the claims. The protection scope of the present disclosure is defined by the appended claims, and equivalents of these claims are also included.

Claims

1. A preparation process of acellular matrix microparticles, characterized in that It includes the following steps: S1: Take the acellular matrix raw material and treat the cell matrix raw material with a sodium hydroxide solution at a concentration of 1% - 5% for 3 - 6 hours to obtain the cell matrix material; S2: Place the cell matrix material obtained in step S1 into a biological enzyme solution with a concentration of 0.1% - 0.5%, oscillate at 37°C for 4 - 6 hours, dehydrate with an alcohol solution mixed in a certain proportion, and then freeze-dry to obtain the well-air-dried acellular intermediate product; S3: Grind the acellular intermediate product obtained in step S2 with a low-temperature grinder for 3 - 4 hours to obtain microparticles with a particle size less than 850 μm; S4: Precisely screen the microparticles obtained in step S3 according to the particle size using a fluidized bed device; the fluidized bed device includes: a fluidized bed reactor, a feeding system, a fluid delivery system, and a collection system.

2. The preparation process of an acellular matrix microparticle according to claim 1, characterized in that, In step S4, the fluidized bed reactor has a cylindrical structure with a fluidized bed layer inside; the fluidized bed layer is supported by a perforated plate (7), and a plurality of small holes are evenly distributed on the perforated plate (7). A microparticle outlet (8) is provided at the top of the fluidized bed reactor (1).

3. The preparation process of an acellular matrix particle according to claim 2, characterized in that, In step S4, the feeding system includes a feeding hopper (2) and a feeding pipeline (3); the feeding hopper (2) is used to store the acellular matrix microparticles to be screened, and the feeding pipeline (3) connects the feeding hopper (2) and the fluidized bed reactor (1) to transport the microparticles above the fluidized bed layer.

4. The preparation process of an acellular matrix particle according to claim 1 or 2 or 3, characterized in that, In step S4, the fluid delivery system includes a fluid source (4), a fluid delivery pipeline (5), and a fluid distributor (6); the fluid source (4) provides the fluid required for the fluidized bed; after adjusting the pressure to a suitable pressure through a pressure regulating valve (11) on the fluid delivery pipeline (5), it is transported from the fluid source (4) to the bottom of the fluidized bed reactor (1); the fluid distributor (6) is installed at the bottom of the fluidized bed reactor (1) and is used to evenly distribute nitrogen onto the perforated plate (7).

5. The preparation process of an acellular matrix particle according to claim 4, wherein, In step S4, the collection system includes a cyclone separator (9) and a collection container (10).

6. A preparation process of acellular matrix microparticles according to claim 2 or 3 or 5, characterized in that: The screening workflow of the fluidized bed reactor in step S4 is: S41: The microparticles obtained in step S3 enter above the fluidized bed layer of the fluidized bed reactor through the fluidized bed feeding system; S42: The fluid delivery system transports the fluid to the bottom of the fluidized bed reactor and evenly distributes it onto the perforated plate through the fluid distributor; S43: After the fluid enters the fluidized bed layer, it interacts with the microparticles to make the microparticles suspended and form a fluidized state; S44: After the microparticles pass through the fluidized bed layer, they enter the collection system and are temporarily stored in the collection container.

7. A preparation process of acellular matrix microparticles according to claim 6, characterized in that: The fluidized bed reactor in step S4 controls the minimum fluidization velocity of the microparticle fluidization by adjusting the gas source pressure; the key parameter, the minimum output pressure of the gas source is 0.25 - 0.35 MPa.

8. A preparation process of an acellular matrix microparticle according to any one of claims 1, 2, 3, 5, and 7, characterized in that, In step S4, high-purity nitrogen is selected as the fluid.

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