CN204971702U - Be used for prosthetic biological patch of tissue lesion - Google Patents

Abstract

The utility model discloses a be used for prosthetic biological patch of tissue lesion, including biomaterial stratum basale and cell matrix layer, the matrix layer is on attached to the stratum basale, is equipped with the parallel slot of a plurality of on the contact surface on stratum basale and cell matrix layer. The utility model discloses biological patch stratum basale has porous and ditch corrugation way structure simultaneously, not only can regulate and control the orientation of cell and grow and distribute, and mechanical properties that ditch corrugation way structure has can make adhesion that the cell matrix layer can be better on the stratum basale, avoids coming off. The product has the stable structure, and normal organizational structure can be simulated to suitable aperture, porosity and permeability, can provide the pipeline and the cell matrix of tissue growth to can bear and do benefit to repair of tissue and palingenetic cell.

Description

A biological patch for repairing tissue damage

technical field

The utility model relates to the field of medical equipment, in particular to a biological patch for repairing tissue damage.

Background technique

In the real world, traffic accidents, industrial accidents, sports accidents, and clinical operations will cause tissue damage. In clinical practice, materials are often used as biological patches to replace and repair human tissues. Biological patch refers to a material taken from the same kind of tissue, which is decellularized to remove various cells contained in the tissue while retaining the three-dimensional framework structure of the extracellular matrix and can be used to repair human soft tissue. According to the tissue source, it can be divided into allogeneic materials such as dermal acellular matrix, amniotic membrane, dura mater, etc., and heterogeneous materials such as porcine small intestinal submucosa, bovine, equine pericardium, bovine peritoneum, etc. Although this natural biological patch has been commercialized, its preparation is cumbersome and expensive. It is still a research hotspot to look for cheap, easy to obtain, and good biocompatibility synthetic bio-mesh to replace natural bio-mesh.

Invention content:

The purpose of the utility model is to provide a biocompatibility, low price, easy to obtain biological patch for repairing tissue damage.

The technical solution of the utility model is:

A biological patch for repairing tissue damage includes a biomaterial base layer and a cell matrix layer, the base layer is attached to the base layer, and several parallel grooves are arranged on the contact surface between the base layer and the cell matrix layer. The groove can be a convex groove protruding from the surface of the base layer, or a groove shape concave inward from the surface of the base layer. Grooves are arranged horizontally or vertically.

Preferably, the size of the trench is 10-50 μm wide and 10-30 μm deep.

In the biological patch mentioned above, the porosity on the base layer of the biological material is 50%-90%, and the pore diameter is 10 μm-100 μm.

In the aforementioned synthetic biological patch, the thickness of the base layer of the biological material is 0.1-3mm.

In the aforementioned synthetic biological patch, the base layer of the biological material is made of one or more of silk fibroin, chitosan, collagen, polylactic acid or polyglycolic acid.

In the above-mentioned biological patch, the cell matrix layer is obtained by decellularization after autologous or allogeneic source cells are secreted and formed, and the cells are Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal One or more of stem cells, umbilical cord blood stem cells or induced pluripotent stem cells.

The bio-patch described above does not contain foaming agents and/or cross-linking agents.

The utility model also discloses a preparation method of the above-mentioned biological patch,

(1) Design the texture of the PDMS elastic stamp, add the biomaterial solution to the stamp, and use the freeze-drying method to prepare the biomaterial base layer with grooves on the surface after molding and demoulding;

(2) culturing the base layer of the biomaterial and the cells to obtain the base layer of the biomaterial attached with cells;

(3) Decellularize the basal layer of the biological material on which the cells grow, and further freeze-dry it to obtain a biological patch.

A preferred version of the utility model is as follows:

According to the principle that the size of the micro-groove affects the shape and spreading behavior of cells, a groove-shaped micro-pattern structure that can guide the orientation distribution of nerve cells is designed.

The elastic stamp used in the utility model is polydimethylsiloxane (polydimethylsiloxane, PDMS), and its preparation method is after mixing the PDMS liquid prepared at room temperature and the cross-linking agent in a ratio of 10:1, and casting it into On the silicon wafer-based master template with horizontal or vertical groove-like micro-patterned distribution obtained by mask exposure in advance, then put it in a vacuum drying oven to remove air bubbles, dry and cure, and finally cure Peel off to get a flat PDMS elastic stamp.

Dissolve chitosan with acetic acid to prepare a solution with a certain concentration (1%, 2% and 5%), then add the solution dropwise on PDMS, place at -60°C--90°C, freeze for 2-4 hours, and freeze After that, the chitosan ice body is placed in an alkaline solution, fixed, and washed with water; the shaped chitosan base layer is freeze-dried to obtain a porous chitosan base layer with a porous structure and groove-like distribution on the surface.

The porous chitosan base layer is subjected to three-dimensional cell culture to obtain the chitosan base layer with cells attached to the surface and taken out. Decellularize the chitosan base layer with cells, and further freeze-dry it to obtain the biological patch of the utility model.

The pore diameter of the pores of the biological patch described in the utility model is 10 μm-100 μm detected by a scanning electron microscope.

The base layer of the biological patch of the utility model has both porous and groove texture structures, which can not only regulate the directional growth and distribution of cells, but also have mechanical properties of the groove texture structure to enable the cell matrix layer to better adhere to the base layer. On the bottom layer, avoid falling off. The product described in the utility model has a stable structure, suitable pore size, porosity and permeability, can simulate normal tissue structure, can provide tissue growth channels and cell matrix, and can carry cells that are beneficial to tissue repair and regeneration. The material used in the product of the utility model is a natural degradable material, which has good biocompatibility with the human body, and the prepared product does not contain foaming agents and crosslinking agents with exogenous toxicity and side effects brought in by the preparation process. The utility model has good tensile strength and has a cell matrix layer, which is more conducive to transporting the nutrients required for the cell growth process and providing a good growth environment for the cells. It works well. The method of the utility model is simple and convenient.

Description of drawings

Fig. 1 is a schematic diagram of the vertical groove pattern on the surface of the base layer of the biological patch described in the present invention.

Fig. 2 is a schematic diagram of the transverse groove pattern on the surface of the base layer of the biological patch described in the present invention.

Fig. 3 is a schematic diagram of the oriented growth of cells on the surface patterned base layer of the biological patch described in the present invention.

Fig. 4 is a schematic diagram of the biological patch structure of the convex groove described in the present invention (1 is the base layer, 2 is the groove, and 3 is the cell matrix layer).

Fig. 5 is a schematic diagram of the biological patch structure of the concave groove described in the present invention (1 is the base layer, 2 is the groove, and 3 is the cell matrix layer).

detailed description

Below in conjunction with embodiment the utility model is further described.

The terms used in the present invention generally have the meanings commonly understood by those skilled in the art unless otherwise specified.

Below in conjunction with specific embodiment and with reference to data further describe the utility model in detail. It should be understood that these embodiments are only for illustrating the utility model, but not limiting the scope of the utility model in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1

(1) Culture and purification of Schwann cells

One-day-old SD rats were taken from newborns, killed and disinfected with alcohol, and the bilateral sciatic nerves were taken, placed on an ice-bath operating table to remove the epineurium and adhesion tissue, after the collagenase/trypsin mixed digestion was complete, the supernatant was discarded and the complete medium Resuspend the cells and inoculate them into culture dishes coated with PDL. Replace with complete medium containing cytarabine (10μM) for 24 hours and culture for 48 hours, then replace with complete medium containing HRG (50ng/ml) and Forsklin (2μM) to continue culturing, change the medium every 3 days until the cells are confluent . After the cells are fused, trypsinize and centrifuge to obtain the cell pellet, resuspend the cells with 1ml Thy1.1 complete medium (1:1000), incubate on ice for 2 hours; centrifuge to discard the supernatant, and use a mixture of DMEM and complement (3:1) Resuspend the cells, incubate at 37°C for 1 hour, wash with complete medium twice after centrifugation, reseet in the culture dish, change the medium every other day, and use it after the cells are full.

(2) Culture of skin-derived fibroblasts

Take SD rats born 1 day old, kill them, and disinfect them with alcohol. Take out the back skin and put it in pre-cooled dissecting solution to carefully remove the subcutaneous tissue (fat, subcutaneous fascia layer, blood vessels, etc.); wash with PBS for 3 times, and use a scalpel blade Cut into small pieces (<1mm×1mm), digest completely with type I collagenase (1mg/ml), centrifuge and discard the supernatant, resuspend the cells in the complete medium, inoculate them into culture dishes, and subculture after the cells are 90% confluent . Fibroblasts will be contaminated with epithelial cells during primary culture, and most of the miscellaneous cells (epithelial and endothelial cells) will gradually die after several passages. Therefore, the cells used in the utility model are cells passed on for more than 3 generations.

(3) Culture and orientation of skin stem cells along Schwann cell differentiation

The culture and differentiation of skin stem cells refer to the literature "Isolation of skin-derived precursors (SKPs) and differentiation and denrichment of their Schwanncell progeny" (Jeffrey A Biernskie, Ian AM McKenzie, Jean G Toma & Freda D Miller, NATURE PROTOCOLS, 2006 (1): 2803-2812), a brief description is as follows: After alcohol disinfection, take out the back skin and place it in pre-cooled dissection solution to carefully remove the subcutaneous tissue (fat, subcutaneous fascia layer, blood vessels, etc.); wash with PBS for 3 times, then cut into small pieces (<1mm×1mm) with a scalpel , digested with 0.1% trypsin or type XI collagenase (1mg/ml) at 37°C for 45–60min, terminated the digestion, centrifuged and discarded the supernatant, and used proliferation medium (DMEM/F12 (3:1) containing 0.1% Double antibody, 40μg/ml fungizone, 40ng/mlFGF2, 20ng/mlEGF, 2% B27supplement) for suspension culture. Suspension-cultured cell spheres can be subcultured to obtain a sufficient number of skin stem cells.

The obtained skin stem cells were differentiated and cultured to obtain Schwann cells, and the specific steps were as follows: the skin stem cells were cultured in differentiation medium I (DMEM/F12 (3:1) containing 0.1% double antibody, 40 μg/mlfugizone, 40ng/mlFGF2, 20ng /mlEGF, 2% B27supplement, 10% FBS) for 3 days, and then in differentiation medium II (DMEM/F12 (3:1) containing 0.1% double antibody, 5μmforskolin, 50ng/mlheregulin-1β, 50μg/ml, 2%N2supplement , 1% FBS) cultured for 2-3 weeks to obtain Schwann cell colonies. A large number of Schwann cells (SKP-SC) differentiated from skin stem cells can be obtained after the colonies are picked and expanded, and the immunohistochemical results show that they are S100 positive.

(4) Culture of bone marrow mesenchymal stem cells

Adult SD rats were sacrificed by dislocation, soaked in 75% alcohol for 5 minutes, femurs and tibias were removed under aseptic conditions to expose the bone marrow cavity, the bone marrow cavity was washed with IMDM basic culture solution, and the bone marrow was collected. The harvested bone marrow was repeatedly aspirated into a syringe to make a single-cell suspension. Filter through a 200-mesh sieve, centrifuge in a horizontal centrifuge at 1000 rpm for 5 min, and discard the supernatant. Inoculate in complete IMDM medium (containing 10% fetal bovine serum) at a density of 4×10 ⁵ /cm ² , and culture at 37°C. After 24 hours, the medium was changed in full to remove non-adherent cells, and half of the medium was changed every 3 days thereafter. Cell morphology and growth were observed daily under an inverted microscope. When the cells covered the bottom of the dish and reached 90% confluence, they were subcultured.

The preparation of embodiment 2 biological patch

(1) Preparation of elastic stamp

The elastic stamp used in the utility model is polydimethylsiloxane (polydimethylsiloxane, PDMS), and its preparation method is after mixing the PDMS liquid prepared at room temperature and the cross-linking agent in a ratio of 10:1, and casting it into On the silicon wafer-based master template with horizontal or vertical groove-like micro-pattern distribution obtained by mask exposure in advance, as shown in FIG. 1 and FIG. 2 . Then put it into a vacuum drying oven to vacuum out air bubbles, dry and cure, and finally peel off after curing to obtain a planar PDMS elastic stamp.

(2) Preparation of micropatterned chitosan base layer

First dissolve chitosan with acetic acid to prepare a solution with a certain concentration (1%, 2% and 5%), then add the solution dropwise on PDMS, place at -60°C-90°C, freeze for 2-4 hours, and After freezing, the chitosan ice body is placed in an alkaline solution, fixed, and washed with water; the shaped chitosan base layer is freeze-dried to obtain a porous chitosan base layer with a porous structure and groove-like distribution on the surface.

(3) Construction of cell matrix layer

Slowly inject the complete medium into the culture container, then add 2.5×10 ⁷ cells and the aseptically treated chitosan base layer, then fill the entire container with the complete medium, and control the final cell density to 1×10 ⁵ / ml, after exhausting the air in the system, start rotating microgravity circulation perfusion culture; put the rotary bioreactor in a 37°C CO ₂ incubator, and the rotating speed is 10 rpm for the first 24 hours, so that the cells and the chitosan substrate layer Adequate contact and attachment; after 24 hours, adjust the rotation speed of the rotary bioreactor so that the chitosan base layer can be suspended in the culture medium; after continuing to cultivate for 2 days, replace it with a differentiation medium to promote the secretion of extracellular matrix; After 2 weeks, the culture was terminated, and the chitosan base layer with cells attached to the surface was taken out. Microscopy showed that the cells grew oriented on the surface patterned chitosan base layer, as shown in Figure 3. The chitosan base layer with cells was decellularized. After washing with PBS, deionized sterile water was hypotonic at 37°C for 10 minutes; cell extract was added to lyse the cells at 37°C for 10-15 minutes; after washing with PBS for 3 times, DnaseI was added (4mg/ml) 37 DEG C digest 30min to remove DNA, it is further freeze-dried, obtains the biological patch described in the utility model, as shown in Figure 4 or Figure 5, 1 is base layer, 2 is groove, 3 is cell matrix layer.

Scanning electron microscope results show that the extracellular matrix components secreted by supporting cells are evenly distributed on the surface of the chitosan base layer. The chitosan base layer containing the natural cell matrix layer can be stored at -80°C for future use, and can also be further freeze-dried save. Then freeze-dry to obtain the chitosan biological patch containing the cell matrix, the chitosan base layer surface and inside are distributed with abundant pores, and the cell matrix layer adheres to the base layer and the surface of the pores. The measured porosity is 89%, and the average pore diameter is 87-98 μm.

Claims (8)

1. A biological patch for repairing tissue damage, characterized in that it comprises a biomaterial base layer and a cell matrix layer, the base layer is attached to the base layer, and the contact surface between the base layer and the cell matrix layer is provided with several parallel patches. of the groove. 2. The biological patch according to claim 1, characterized in that the grooves are distributed horizontally or vertically. 3. The biological patch according to claim 1, characterized in that the size of the groove is: 10-50 μm wide and 10-30 μm deep. 4. The biological patch according to claim 1, characterized in that there are abundant pores distributed on the surface and inside of the contact surface between the biological material base layer and the cell matrix layer. 5. The biological patch according to claim 4, characterized in that the porosity of the biological material base layer is 50%-90%, and the pore diameter is 10 μm-100 μm. 6. The synthetic biological patch according to claim 1, characterized in that the thickness of the base layer of biological material is 0.1-3mm. 7. The biological patch according to any one of claims 1-6, characterized in that the cell matrix layer is obtained by decellularization after secretion and formation of autologous or allogeneic source cells. 8. The biological patch as claimed in claim 7, wherein said cells are Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells or induced pluripotent stem cells one or several.