WO2011050622A1

WO2011050622A1 - Granular biomaterial for tissue repairing and preparation method thereof

Info

Publication number: WO2011050622A1
Application number: PCT/CN2010/074562
Authority: WO
Inventors: 彭代智; 左海斌; 郑必祥
Original assignee: 中国人民解放军第三军医大学第一附属医院
Priority date: 2009-10-29
Filing date: 2010-06-27
Publication date: 2011-05-05
Also published as: CN101695583A; CN101695583B

Abstract

A granular biomaterial for tissue repairing and the preparation method thereof are provided. A film biomaterial prepared from one or at least two selected from cellular tissue matrices, collagen, chondroitine sulfate, sodium hyaluronate, chitosan, polylactic acid, polyglycolic acid and alginate is frozen-dried, and then is cut into strips under preset parameters and normal temperature. At last, the biomaterial is cut into granules which have a diameter range of 60µm-6000μm before being packaged and sterilized. The method is simple and ensures that the grain diameter distribution concentration of the granules is more than or equal to 85%. The granular biomaterial meets the requirements for repairing multiple tissues or organs.

Description

Granular biomaterial for tissue repair and preparation method thereof

The invention belongs to a prosthetic material or a prosthetic covering material in the field of tissue engineering, in particular to a granular biological material for tissue repair and a preparation method thereof. Background technique

In the field of skin tissue repair, how to use the limited normal skin tissue of patients to maximize the scope of tissue transplantation is an important problem that needs to be improved in the repair of large-area skin defects. The normal skin tissue or prosthetic coating material is granulated, and the above purpose can be achieved by spraying and spreading. It has been widely used in the treatment of large-area burn patients. Injectable or refillable granulated tissue biomaterials are also widely used in orthopedics, neurosurgery, urology, and oral orthopedic surgery to achieve optimal repair with minimal trauma. At present, the hotspots of tissue repair and regeneration research are also looking for suitable granular tissue biomaterials for tissue engineering seed cells as a carrier for cell transplantation of tissues and organs, such as cells or stem cells of skin, nerves, cartilage and heart muscle. Loaded on granular tissue biomaterials for corresponding tissue repair and the like. It can be seen that how to prepare tissue biomaterial particles suitable for different tissue repair needs and uniform size has always been the focus of research and development of tissue repair and regeneration materials.

The method of granulating biological materials has been reported less frequently in the prior art. United States

LifeCell has a patent for invention published on April 15, 2008, published as US 7,358,284. The preparation method is summarized as follows: The dried AlloDerm® is milled into thin strips using a modified Zimmer mesh preparation, and then At ultra-low temperature, the thin strips are minced with a grouting machine, and after being twisted into granules, different screens are used to separate them at a low temperature. Finally, the granules are lyophilized, packaged, and sterilized; the particle concentration distribution of the prepared particles is 68%. Particle size range is 58 μπι ~ 593 μ m [Sclafani AP, Romo T, Jacono A. Evaluation of acel lular dermal graft in sheet (AlloDerm) and injectable (micronized AlloDerm) forms for soft tissue augmentation: clinical observations and histological analysis. Arch Facial Plast Surg 2000; 2: 130- 6.]. There is also CN101195044A, which was released on June 11, 2008. It is a small cell that has been smashed into granules by cryogenic freezing, repeated freezing and thawing, and sieving. It is 50 μ πι ~ 500 μ m. However, the above prior art still has the following disadvantages: (1) The preparation conditions are harsh, requiring ultra-low temperature and low temperature; (1) The preparation steps are numerous, requiring low-temperature freezing and repeated freezing and thawing; (3) the prepared particles have irregular shape; (4) The prepared particles are not concentrated even after sieving; (5) Particles with too small particle size (less than 52 microns) will cause damage to the immune cells of the body. Summary of the invention

The object of the present invention is to provide a granular biological material for tissue repair and a preparation method thereof according to the defects of the prior art, and the preparation condition is mild, the preparation method is simple, the prepared particle shape is regular, and the prepared particle can be prepared. The large diameter range and the single size distribution of the particle size are not only suitable for direct application, but also can load the corresponding tissue cells or related molecules that promote tissue regeneration to meet the needs of various tissue or organ repair.

The general idea of the present invention is to control the size of the prepared particles by setting different cutting parameters by using the controllability of mechanical cutting parameters, so that the particle size range of preparing a certain size of particles is controllable and relatively concentrated, and The cleavable particle has a large particle size range; and further, the membrane-shaped biological material is directly cut into granular biological material at normal temperature to meet the needs of various tissue or organ repair.

In order to achieve the object of the present invention, the technical solution drawn is as follows.

A granular biomaterial for tissue repair, the raw material is acellular tissue matrix (such as acellular dermal scaffold), collagen, chondroitin sulfate, hyaluronic acid, chitosan, polylactic acid, polyglycolic acid, alginate Membrane-like biomaterial prepared by any one or at least two combinations; the shape of the finished particles is regular, and the shape is cubic or rectangular; the particle size ranges from 60 μ π Γ 6000 μ πι; Degree ≥ 85 %.

The preparation method comprises the three-step process of lyophilization of the membrane-shaped biological material, preparation of the thin strip of biological material, and preparation of the granular biomaterial.

1) Lyophilization of membrane-like biomaterials

The membrane-shaped biological material is placed in a lyophilizer and lyophilized for 3 hours to 12 hours at a vacuum of 0.04 mbar - 0. 4 mbar at a temperature of - 30 ° C to _ 70 ° C. Remove moisture and dry completely.

2) Preparation of thin strips of biological materials

At room temperature, set the width cutting parameter in the range of 60 μ πι ~ 6000 μ πι to apply the patch-shaped creature The material is cut into strips of biological material with the set width parameters.

3) Preparation of granular biomaterials

At room temperature, the particle size cutting parameter is set in the range of 60 μ πι ~ 6000 μππ, and the thin strip biomaterial is cut into the granular biomaterial of the set particle size parameter; and the obtained granular biomaterial is divided into 1 g ~ 100g directly immersed in 75% ethanol or PVC vacuum packed; finally treated with 10 kGy ~ 25kGy cobalt 60 irradiation or PVC vacuum packaging, ethylene oxide disinfection.

The granular biological material of the invention and the preparation method thereof have the following advantages: First, the cutting condition is normal temperature, no need for ultra-low temperature or low temperature; Second, the method is simple and convenient, and is convenient for production line operation; Third, the obtained product has a regular shape and a cubic shape or Cuboid particles; Fourth, no sieving, particle size distribution concentration of the same size ≥ 85 %; Fifth, the particle size range of 60 μ m fly 000 μ m, particles of different sizes can be mixed; Different sizes of granules can be applied to tissue or organ repair by injection, spraying, spreading, and tamping methods. Seven is a wide range of raw materials, which can be acellular tissue matrix, collagen, chondroitin sulfate, hyaluronic acid, chitosan. A membrane-like biomaterial prepared by combining any one or a combination of at least two of polylactic acid, polyglycolic acid, and alginate.

The granular biomaterial prepared by the invention is suitable for direct application, and can also be used after loading corresponding tissue cells or related molecules for promoting tissue healing and regeneration, and can meet the needs of various tissue or organ repair. DRAWINGS

Figure 1 is a general observation of the 1. 2 匪 size acellular dermal scaffold particles.

Figure 2 shows the particle size distribution of the acellular dermal scaffold with a size of 0.7 匪.

Figure 3 shows the results of particle size distribution measurement of 0.2% 脱 size acellular dermal scaffold.

Figure 4 is a microscopic photo taken 3 hours after inoculation of human keratinocytes on acellular dermal scaffold particles (400 times)

Figure 5 shows the biomechanical properties of wound healing skin at 20 weeks after decellularized dermal scaffold granule composite graft autologous tomography (STSG+MA Li).

detailed description

The following is a list of multiple implementations of preparing granular biomaterials using acellular dermal scaffolds as raw materials. For example, the granular biomaterial of the present invention and a method for preparing the same are further explained. It is emphasized that the invention is not limited to the illustrated embodiments.

Example 1

The specific preparation method of 1.2 匪 acellular dermal scaffold particles is as follows:

(1) Decellularized dermal scaffold freeze-dried: The prepared acellular dermal scaffold was placed in a lyophilizer, and the vacuum was 0.1 mbar, lyophilized for 6 hours, and the temperature was 45 ° C;

(2) Preparation of thin strip decellularized dermal scaffold: Set the cutting parameter to 1.2 匪, and cut the lyophilized acellular dermal scaffold into thin strips (width: 1.2 匪)-like biomaterial;

(3) Preparation of granular acellular dermal scaffold: Set the cutting parameter to 1.2 匪, and cut the prepared strip-shaped acellular dermal scaffold with a width of 1.2 切割 into pellets (specification: 1.2 匪). The prepared granules are regular and uniform in size. , the average particle size is 1261 μπι (see Figure 1);

(4) Packaging, Disinfection: The prepared acellular dermal scaffold particles were packed into 1 g and directly immersed in 75% ethanol for storage. Example 2

The specific preparation method of the acellular dermal scaffold particles with a specification of 0.7 如下 is as follows:

(1) Decellularized dermal scaffold freeze-dried: The prepared acellular dermal scaffold was placed in a lyophilizer, and the vacuum was 0.1 mbar, lyophilized for 6 hours, and the temperature was 55 °C;

(2) Preparation of thin strip acellular dermal scaffold: Set the cutting parameter to 0.7 匪, and cut the lyophilized acellular dermal scaffold into thin strips (width: 0.7 匪).

(3) Preparation of granular acellular dermal scaffold: Set the cutting parameter to 0.7 匪, and cut the prepared strip-shaped acellular dermal scaffold with a width of 0.7 切割 into pellets (size: 0.7 匪). The prepared granules are regular in shape and uniform in size. , the shape is nearly cubic;

(4) Packaging, disinfection: The prepared acellular dermal scaffold particles are packed into 2 g, and the PVC is vacuum-packed and sterilized by ethylene oxide.

(5) The particle size analyzer detects the average particle size of the acellular dermal scaffold particles as 875 μπι, and the particle concentration concentration in the range of 479 μπι~ 1096 μπι is 90.93% (see Figure 2). The results showed that the prepared acellular dermal particles had a small span of particle size and a high degree of concentration. Example 3:

The specific preparation method of the 0.2 匪 acellular dermal scaffold particles is as follows: (1) Acellular dermal scaffold freeze-dried: The prepared acellular dermal scaffold is placed in a lyophilizer, vacuumed at 0.1 mbar, lyophilized for 6 hours, and the temperature is 50 ° C;

(2) Preparation of thin strip decellularized dermal scaffold: Set the cutting parameter to 0.2 匪, and cut the lyophilized acellular dermal scaffold into thin strips (width: 0.2 匪)-like biomaterial;

(3) Preparation of granular acellular dermal scaffold: The cutting parameter was set to 0.2 匪, and the prepared strip-shaped acellular dermal scaffold having a width of 0.2 切割 was cut into granules (specification: 0.2 匪);

(5) The particle size analyzer detects the average particle size of the acellular dermal scaffold particles as 323 μm, and the particle concentration concentration in the range of 182 μm to 417 μπι is 87.86% (see Figure 3). The results showed that the particle size span of the prepared particles was significantly smaller than that of AlloDerm particles of 58 μπΓ593 μπι, and the particle distribution concentration was higher than that of AlloDerm particles (68%) by nearly 20 percentage points. Example 4:

Preparation of human keratinocyte decellularized dermal scaffold particles, the specific steps are as follows:

(1) Preparation of acellular dermal scaffold particles: 20 mg of the preserved acellular dermal scaffold particles were washed with physiological saline and washed 3 times. Place the granules in a 1.5 ml sterile centrifuge tube, add 1 ml of the cell culture solution and soak for 6 hours, then discard the culture solution for use.

(2) Preparation of human keratinocytes: Under sterile conditions, cultured human keratinocytes were digested with 0.25% trypsin at 37 ° C for 5 min and then pipetted; culture medium containing human keratinocytes was centrifuged at 1000 rpm. , 5 min; Discard the supernatant; add the culture solution, mix the cells, and count.

(3) Culture of human keratinocyte-depleted dermal scaffold particles: 0.5 ml of a culture medium containing 2×10 ⁶ /ml human keratinocytes was added to a 5 ml sterile centrifuge tube with a spare 20 mg granule carrier. Incubate for 3 hours at 37 ° C in a 5% CO ₂ incubator.

(4) Microscopic observation of the decellularized dermal scaffold particles loaded with human keratinocytes: The culture solution was discarded, and the human keratinocyte decellularized dermal scaffold particles were washed with physiological saline for 2 times. Microscopically, human keratinocytes were attached to the granule scaffold, indicating that the acellular dermal scaffold is easily loaded with human keratinocytes (see Figure 4). Example 5

Animal experiment of acellular dermal scaffold particles for skin defect wound repair, specific steps ^ Under the mouth:

(1) Preparation of skin defect model: Anesthesia in SD rats (male, 220-260 g) (1% pentobarbital, 40 mg/kg, intraperitoneal injection), 4 cm X 6 cm in the middle of the back Full thickness skin.

(2) Preparation of autologous tomographic skin: Autologous full-thickness skin of 4 cm X 6 cm was made into autologous tomographic skin (STSG), which was wrapped with gauze moistened with PBS solution for use.

(3) Preparation of acellular dermal scaffold particles: Acellular dermal scaffold particles (specification: 0.5 匪, area expansion ratio of 2:1) soaked in 75% alcohol were placed in a funnel with filter paper, using PBS solution Rinse several times and wash off alcohol.

(4) Composite transplantation of autologous tomographic skin (STSG) and acellular dermal scaffold particles: The washed acellular dermal scaffold particles are spread on the dermis surface of STSG, and then covered with the wound surface, and the edge of the skin is sutured. The distance is 1 匪 ~ 2 缝. After the wound, the oil yarn was bandaged and fixed (bandage for 2 weeks).

(5) The biomechanical properties of the wounded wounds were measured at 20 weeks postoperatively (see Figure 5). The results showed that the acellular dermis was compared with the autologous tomographic skin grafted rat (STSG) without decellularized dermal granules. The wounded skin of the granule composite autologous tomography skin grafted rat (STSG+MA Li) has a higher skin tensile strength.

Claims

Rights request

1. A granular biomaterial for tissue repair, which is characterized in that: it is prepared by using a membrane-shaped biological material as a raw material; the shape of the finished particle is regular, and the shape is cubic or rectangular; the particle size ranges from 60 μm ~6{){){)μπι; Single particle size distribution concentration of ^ 85 %.

2. The particulate biomaterial according to claim 1, wherein said membrane-like biomaterial is acellular tissue matrix, collagen, chondroitin sulfate, hyaluronic acid, chitosan, polylactic acid, polyhydroxyl Prepared by any one or a combination of at least two of acetic acid and alginate.

3. A method of preparing a granular biomaterial for tissue repair according to claim 1, characterized in that it consists of the following three steps:

1) Lyophilization of membrane-like biomaterials

The membrane-shaped biological material is placed in a lyophilizer, and lyophilized for 3 hours to 12 hours under a vacuum of 0.04 mbar to 0.4 mbar at a temperature of 30 ° C to 70 ° C to remove moisture and completely dry;

2) Preparation of thin strips of biological materials

At room temperature, a width cutting parameter is set in a range of 60 μπι to 6000 μπι, and the membrane-shaped biomaterial is cut into a strip-shaped biomaterial having a set width parameter;

3) Preparation of granular biomaterials

At room temperature, the particle size cutting parameters are set in the range of 60 μπι to 6000 μπι, and the thin strip biomaterial is cut into the granular biomaterials with the set particle size parameters; and the obtained granular biomaterial is separately packed into lg ~100g directly Soak in 75% ethanol or PVC vacuum packaging; finally use lOkGy ~25kGy cobalt 60 irradiation treatment or PVC vacuum packaging, ethylene oxide disinfection.