CN105769381B - A kind of biological sticking patch for tissue damage reparation - Google Patents

Abstract

The invention discloses a biological patch for repairing tissue damage, which comprises a biological material base layer and a cell matrix layer, the base layer is attached to the base layer, and several parallel patches are arranged on the contact surface between the base layer and the cell matrix layer. groove. The base layer of the biological patch of the present invention has both porous and groove texture structures, which can not only regulate the oriented growth and distribution of cells, but also the mechanical properties of the groove texture structure can enable the cell matrix layer to better adhere to the base layer on, to avoid falling off. The product of the present invention 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.

Description

A biological patch for repairing tissue damage

technical field

The invention relates to the field of medical devices, 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 present invention is to provide a biological patch for repairing tissue damage with good biocompatibility, low price and easy availability.

Technical solution of the present invention 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 present invention 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 present invention 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 present invention is polydimethylsiloxane (polydimethylsiloxane, PDMS), and its preparation method is to mix the PDMS liquid prepared at room temperature and the cross-linking agent in a ratio of 10:1, and then cast it to the On the silicon wafer-based master template with horizontal or vertical groove-like micro-patterned distribution obtained by mask exposure, then put it in a vacuum drying oven to vacuumize to remove air bubbles, dry and cure, and finally peel off after curing That is, a planar PDMS elastic stamp is obtained.

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 lyophilize it to obtain the biological patch of the present invention.

The pore diameter of the pores of the biological patch of the present invention is 10 μm-100 μm detected by scanning electron microscope. The base layer of the biological patch of the present invention has both porous and groove texture structures, which can not only regulate the oriented growth and distribution of cells, but also the mechanical properties of the groove texture structure can enable the cell matrix layer to better adhere to the base layer on, to avoid falling off. The product of the present invention 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 materials used in the products are natural degradable materials, which have good biocompatibility with the human body. The products produced do not contain foaming agents and crosslinking agents with exogenous toxicity and side effects brought in by the preparation process. The invention 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 invention is simple and easy to implement.

Description of drawings

Fig. 1 is a schematic diagram of the groove texture on the surface of the base layer of the biological patch according to the present invention.

Fig. 2 is a schematic diagram of the oriented growth of cells on the patterned base layer on the surface of the biological patch according to the present invention.

Fig. 3 is the schematic diagram of the biological patch structure of the present invention (A is a schematic diagram of a biological patch with a convex groove, B is a schematic diagram of a biological patch with a concave groove, 1 is a base layer, 2 is a groove, and 3 is a cell basal layer).

Detailed ways

The present invention will be further described below in conjunction with embodiment.

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

The present invention will be further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only for illustration of the present invention, but not to limit the scope of the present invention 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 present invention are cells of more than three 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 enrichment of their Schwann cell progeny" (Jeffrey A Biernskie, Ian A McKenzie, Jean G Toma&Freda D Miller, NATURE PROTOCOLS, 2006(1): 2803-2812), and the brief description is as follows: 1-day-old newborn SD rats were sacrificed and alcohol-sterilized, the back skin was taken out and placed in pre-cooled dissecting fluid, and the subcutaneous tissue (fat, subcutaneous fascia layer, blood vessels, etc.) was carefully removed; After washing with PBS for 3 times, cut into small pieces (<1mm×1mm) with a scalpel blade, digest with 0.1% trypsin or type XI collagenase (1mg/ml) at 37°C for 45-60min, terminate the digestion and centrifuge The supernatant was cultured in suspension with proliferation medium (DMEM/F12 (3:1) containing 0.1% double antibody, 40 μg/ml fungizone, 40ng/ml FGF2, 20ng/ml EGF, 2% B27supplement). 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/ml fungizone, 40ng/ml FGF2 , 20ng/ml EGF, 2% B27supplement, 10% FBS) for 3 days and then in the differentiation medium II (DMEM/F12 (3:1) containing 0.1% double antibody, 5μm forskolin, 50ng/ml heregulin-1β, 50μg/ ml, 2% N2supplement, 1% FBS) 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 present invention is polydimethylsiloxane (polydimethylsiloxane, PDMS), and its preparation method is to mix the PDMS liquid prepared at room temperature and the cross-linking agent in a ratio of 10:1, and then cast it to the On the silicon wafer-based master template with horizontal or vertical groove-like micro-pattern distribution obtained by mask exposure, as shown in FIG. 1 . 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 and 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 a 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 2. The chitosan base layer with cells was decellularized. After washing with PBS, deionized sterile water was hypotonic at 37°C for 10 minutes; the cell extract was added to lyse the cells at 37°C for 10-15 minutes; after washing with PBS for 3 times, DNase was added I (4mg/ml) 37 DEG C digest 30min to remove DNA, it is further freeze-dried, obtains the biological patch of the present invention, as shown in Figure 3, A is the biological patch schematic diagram of convex groove, and B is concave Schematic diagram of a biopatch with grooves, 1 is the basal layer, 2 is the groove, and 3 is the 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 (9)

1. a kind of biological sticking patch for tissue damage reparation, including biomaterial basalis and cellular matrix layer, hypothallus it is attached On the base layer, basalis is equipped with several parallel grooves with the contact surface of cellular matrix layer, it is characterised in that described Biological sticking patch is prepared using following steps：

（1）PDMS elastomeric stamp lines is designed, biomaterial solution is added on seal, using lyophilized side after shaping and demoulding Method, which prepares surface, has the biomaterial basalis of ditch slot texture；

（2）Biomaterial basalis is cultivated with cell, obtains attaching the biomaterial basalis for growing and having cell；

（3) the biomaterial basalis that growth has cell is subjected to de- cell processing, it is further freezed, obtains biology Sticking patch.

2. biological sticking patch as claimed in claim 1, it is characterised in that the groove is laterally or longitudinally to be distributed.

3. biological sticking patch as claimed in claim 1, it is characterised in that the size of the groove is：It is 10-50 μm wide, deep 10- 30µm。

4. biological sticking patch as claimed in claim 1, it is characterised in that the biomaterial basalis connects with cellular matrix layer Abundant hole is distributed with contacting surface upper surface and inside.

5. biological sticking patch as claimed in claim 4, it is characterised in that the porosity on the biomaterial basalis is 50%- 90%, and aperture is 10 μm -100 μm.

6. biological sticking patch as claimed in claim 1, it is characterised in that the biomaterial base layer thickness is 0.1-3mm.

7. biological sticking patch as claimed in claim 1, it is characterised in that the biomaterial basalis is gathered by fibroin albumen, shell One or more in sugar, collagen, polylactic acid or polyglycolic acid are made.

8. the biological sticking patch as described in one of claim 1-7 item, it is characterised in that the cellular matrix layer is autologous or same Kind of xenogenic origin cell is secreted to be formed after take off cell and obtain.

9. biological sticking patch as claimed in claim 8, it is characterised in that the cell be schwann cells, skin-derived into fiber One or more in cell, skin progenitor cell, mesenchymal stem cell, navel blood stem cell or induced pluripotent stem cells.