CN111067665A - Porous artificial leather and preparation method and mold thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种多孔人工真皮及其制备方法和模具，该多孔人工真皮包括生物材料层，所述生物材料层在厚度方向上设置有若干贯穿孔，若干所述贯穿孔的孔内壁具有凹陷或凸起。本发明的多孔人工真皮通过在贯穿孔的孔内壁设置凹陷或凸起，利于引导内皮细胞径向粘附、生长，有利于加快人工真皮的血管化进程，更快地形成微血管网络，缩短创面治疗周期。

The invention discloses a porous artificial dermis, a preparation method and a mold thereof. The porous artificial dermis comprises a biological material layer, the biological material layer is provided with a plurality of through holes in the thickness direction, and the inner walls of the holes of the plurality of through holes have depressions or raised. The porous artificial dermis of the present invention is beneficial to guide the radial adhesion and growth of endothelial cells by setting depressions or protrusions on the inner wall of the through-hole, and is beneficial to accelerate the vascularization process of the artificial dermis, form a microvascular network faster, and shorten the wound treatment. cycle.

Description

Porous artificial leather and preparation method and mold thereof

Technical Field

The invention relates to the field of medical instruments and the field of tissue engineering regeneration, in particular to porous artificial dermis and a preparation method and a mold thereof.

Background

The artificial dermis is prepared from biological materials such as collagen, hyaluronic acid, gelatin, polylactic acid and the like as main components by in vitro artificial treatment or preparation, and is used for repairing and reconstructing skin tissue defects. The artificial dermis bracket is widely applied in clinic since the first time of coming into the market in the 90 th 20 th century, and the composite transplantation of the artificial dermis and the autologous skin blade thick skin becomes one of important means for treating deep skin soft tissue defects. However, artificial dermis has problems of delayed vascularization, lack of anti-infection ability, lack of skin appendages, etc., wherein the delayed vascularization is an important factor affecting the success rate of artificial dermis grafting.

Angiogenesis is an important process in wound healing. The rapid vascularization plays a crucial role in promoting the regeneration of dermis, transferring nutrients and nutrients, clearing metabolic waste, reducing infection and the like, and is one of the great challenges faced by the current artificial dermis. After the autologous skin is transplanted, the revascularization is a dynamic process of the fusion of the autologous skin and the wound surface and the angiogenesis. Most of the capillaries in the transplanted autologous skin degenerate and are replaced by the capillaries grown from the wound surface to finally form a chimeric capillary network, and the transplanted autologous skin obtains enough oxygen again for oxygenation. After the autologous skin is transplanted, new blood vessels generally enter 3 days, and the vascularization can be completed and the autologous skin can survive for about 1 week. The artificial dermis is vascularized primarily from four weeks, and it usually takes 2-4 weeks to form a stable microvascular network after implantation. Therefore, there is a need for an artificial dermis capable of being vascularized rapidly after transplantation, which can further improve the survival rate of artificial dermis transplantation, shorten the hospitalization period and reduce the burden of patients.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides the porous artificial dermis and the preparation method and the mould thereof, and the porous artificial dermis can accelerate the vascularization process, form a microvascular network more quickly and shorten the treatment period of the wound surface.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a porous artificial dermis including a biomaterial layer, the biomaterial layer being provided with a plurality of through holes in a thickness direction, and inner walls of the plurality of through holes having recesses and/or protrusions.

According to the porous artificial dermis according to some embodiments of the present invention, the depth of the recesses or the height of the protrusions is 3 to 6 μm.

According to the porous artificial dermis according to some embodiments of the present invention, the plurality of through holes are regularly arranged.

According to the porous artificial dermis according to some embodiments of the present invention, the through-hole has a cross-section of a circular hole or a rectangular hole.

The porous artificial dermis according to some embodiments of the invention, the diameter of the circular holes is 100-300 μm, and the hole pitch of the circular holes is 1-3 mm; the length of rectangular hole is 1 ~ 3mm, the width of rectangular hole is 1mm, the hole interval of rectangular hole is 1 ~ 5 mm.

According to the porous artificial dermis according to some embodiments of the present invention, the biomaterial layer is made of a raw material including collagen and mucopolysaccharide through a cross-linking reaction. The biological material layer has good biocompatibility and degradability, and is suitable for wound repair.

The porous artificial dermis according to some embodiments of the present invention, the raw material further includes at least one of VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), and polyphosphate.

Further according to some embodiments of the invention, the raw material comprises 85 to 98 wt% of collagen and 2 to 15 wt% of mucopolysaccharide.

In a second aspect of the present invention, there is provided a mold for preparing the above porous artificial dermis, comprising a bottom plate and a cofferdam surrounding the bottom plate, wherein the bottom plate is provided with a plurality of cylindrical structures, and the surfaces of the plurality of cylindrical structures are provided with protrusions and/or depressions.

According to the die provided by the embodiment of the invention, the thickness of the bottom plate is 2-5 mm, the wall thickness of the cofferdam is 2-5 mm, and the height from the cofferdam to the bottom plate is 1-3 mm.

According to the mold of some embodiments of the present invention, the depth of the depression or the height of the protrusion is 3 to 6 μm.

According to the mold of some embodiments of the invention, the columnar structure is a cylinder or a prism.

According to the die of some embodiments of the invention, the diameter of the cylinders is 100-300 μm, and the distance between the cylinders is 1-3 mm; the prism is the quadrangular prism, the length of quadrangular prism is 1 ~ 3mm, the width of quadrangular prism is 1mm, interval between the quadrangular prism is 1 ~ 5 mm.

According to the mold of some embodiments of the present invention, the surface of the mold is further coated with polyacrylamide. The surface of the mould is coated with polyacrylamide, so that the subsequent demoulding of the artificial dermis is facilitated.

According to the mold of some embodiments of the invention, the surfaces of a plurality of the columnar structures are provided with stripe-shaped protrusions and/or depressions; or spiral shaped protrusions and/or indentations.

Further in accordance with the dies of some embodiments of the invention, the striped or spiral pattern has a width of 10-40 μm and a pitch of 10-40 μm.

In a third aspect of the present invention, there is provided a method for preparing the porous artificial dermis, comprising the steps of:

preparing the mould;

preparing a biological material solution: mixing the raw materials including mucopolysaccharide solution and collagen solution, centrifuging, collecting precipitate, adding acetic acid, and dissolving again to obtain biological matrix solution;

preparing porous artificial dermis: pouring the biological material solution into the mold, freeze-drying, placing in a vacuum environment for thermal crosslinking, demolding, and placing in a crosslinking agent for crosslinking reaction to obtain the porous artificial dermis.

According to some embodiments of the invention, the process of preparing the mold is: and (3) designing the graph of the mold by using a computer, taking the polymer solution, printing, curing and molding to obtain the mold.

According to some embodiments of the invention, the polymer solution is a mixed solution of polydimethylsiloxane and a curing agent.

According to some embodiments of the invention, the freeze-drying process is in particular: freezing the mixture in an environment with the temperature of-50 to-70 ℃, annealing for 5 to 10min, and then carrying out freeze drying.

The embodiment of the invention has the beneficial effects that:

the embodiment of the invention provides a porous artificial dermis, which is characterized in that the inner wall of a through hole is provided with a recess or a bulge, so that the adhesion and proliferation of cells on the surface of the inner wall of the through hole are promoted, the radial adhesion and growth of endothelial cells are favorably guided, the arrangement of the endothelial cells in a normal tissue is approached, radial capillaries are further formed, the artificial dermis is vascularized more quickly, a capillary network can be established with an autologous skin transplanted above the artificial dermis, a nutritional support is provided, and the autologous skin can better survive. Compared with the mode that a concave or convex structure is arranged in the transverse direction of the plane where the artificial dermis is in contact with the wound surface, the transverse arrangement mode can only guide endothelial cells to grow on the transverse plane, cannot form radial capillaries and has no effect of accelerating vascularization of the artificial dermis.

Drawings

FIG. 1 is a schematic sectional view of a through-hole in a porous artificial dermis in example 1;

FIG. 2 is a plan view of a through-hole in the porous artificial dermis in example 1;

FIG. 3 is a schematic view of the structure of a mold in example 1;

FIG. 4 is a partial enlarged view of portion A of FIG. 3;

FIG. 5 is a schematic sectional view of a through-hole in the porous artificial dermis in example 2;

fig. 6 is a schematic sectional view of a through-hole in the porous artificial dermis in example 3.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

The artificial dermis plays a role in tissue induction, repair and regeneration in the process of wound repair, and a great number of biomaterials capable of forming the artificial dermis, such as collagen, hyaluronic acid, gelatin, polylactic acid and the like, one of the purposes of the embodiment of the invention is to arrange a through hole with a recess or a protrusion on the inner wall of the hole on the biomaterial layer forming the artificial dermis.

In this embodiment, a circular through hole penetrating radially is formed in a biomaterial layer, a micro-topology structure with stripe-shaped protrusions is formed on the inner wall of the through hole, the height of the protrusions is 4 μm in this embodiment, the interface of the through hole is a circular hole, the diameter of the circular hole is 150 μm, the hole pitch between the circular holes is 2mm, the cross-sectional view of the through hole is shown in fig. 1, and the top view of the through hole is shown in fig. 2. The porous artificial dermis of this example was prepared according to the following procedure:

(1) preparing a mould: the computer aided design is used to produce the pattern required by the mould, as shown in fig. 3, the mould is provided with a bottom plate 1 and a cofferdam 2 surrounding the bottom plate 1, a plurality ofcolumn structures 3 are arranged on the bottom plate 1, and the surfaces of the plurality ofcolumn structures 3 are provided with concave structures corresponding to the stripe-shaped bulges. In this embodiment, the mold is square, the thickness of the bottom plate 1 is 2mm, the wall thickness of the cofferdam 2 is 2mm, and the height from the cofferdam 2 to the bottom plate 1 (i.e., the inner depth of the mold) is 3 mm. In order to more clearly see the surface structure of the columnar structure, see fig. 4, fig. 4 is a partially enlarged view of a portion a in fig. 3, and the surface of the columnar structure has a concave structure corresponding to the stripe-shaped protrusions. Mixing Polydimethylsiloxane (PDMS) and a curing agent in a mass ratio of 1:1, fully stirring to obtain a polymer solution, printing by using the polymer solution as printing ink, and curing for 2h on a hot bed at 80 ℃ to obtain the mold. In the embodiment, the mold is made by curing at 80 ℃, and the actual curing temperature of the PDMS on the hot bed can be 80-95 ℃. The solidified mould is washed clean by ethanol and distilled water, and then a layer of polyacrylamide film with the thickness of 50 mu m is sprayed on the surface of the mould.

(2) Preparing a biological material solution: weighing collagen, dissolving in water to prepare 200g of collagen solution with the mass concentration of 0.3%, weighing 0.066g of chondroitin sulfate, dissolving in water to prepare chondroitin sulfate solution with the mass concentration of 2.5 mg/mL. And (3) atomizing the chondroitin sulfate solution into the collagen solution through high-speed airflow, and continuously stirring for 2 hours to obtain the collagen-chondroitin sulfate compound. Centrifuging the collagen-chondroitin sulfate complex at 8000rpm for 15min, collecting precipitate, adding 0.05M acetic acid to dissolve the precipitate again to obtain collagen concentration of 0.7% (w/w), and stirring for 2.5 hr to disperse uniformly to obtain biological material solution.

(3) Preparing porous artificial dermis: pouring the biological material solution prepared in the step (2) into a mold, leveling, pre-freezing for 3h at-60 ℃, annealing for 5-10min, freezing for half an hour at-60 ℃, and transferring to a freeze dryer for freeze drying for 24 h. Then placing the collagen composite layer under the high-temperature vacuum condition of 100 ℃ for high-temperature crosslinking for 24 hours, and demoulding to obtain the collagen composite layer. Soaking the obtained collagen composite layer in 0.25% glutaraldehyde-acetic acid solution for 24h, and cleaning with purified water to obtain porous artificial dermis.

Example 2

The embodiment provides a porous artificial dermis, a rectangular through hole penetrating in the radial direction is arranged on a biomaterial layer, a micro-topological structure with stripe depressions is arranged on the inner wall of the through hole, the depth of the depressions in the embodiment is 6 μm, the interface of the through hole is a rectangular hole, the diameter of the rectangular hole is 2mm, the width of the rectangular hole is 1mm, the hole spacing between the rectangular holes is 3mm, and the schematic cross-sectional view of the through hole is shown in fig. 5. The porous artificial dermis of this example was prepared according to the following procedure:

(1) preparing a mould: and (3) producing the pattern required by the mold by using computer aided design, wherein the mold is provided with a bottom plate and a cofferdam surrounding the bottom plate, a plurality of prismatic structures are arranged on the bottom plate, and the surfaces of the prismatic structures are provided with convex structures corresponding to the striated depressions. Mixing polydimethylsiloxane and a curing agent in a mass ratio of 1:1, fully stirring to obtain a polymer solution, printing by using the polymer solution as printing ink, and curing for 2 hours on a hot bed at 80 ℃ to obtain the mold. The solidified mould is washed clean by ethanol and distilled water, and then a layer of polyacrylamide film with the thickness of 50 mu m is sprayed on the surface of the mould.

(2) Preparing a biological material solution: weighing collagen, dissolving in water to prepare 200g of collagen solution with the mass concentration of 0.3%, weighing 0.106g of chondroitin sulfate, dissolving in water to prepare chondroitin sulfate solution with the mass concentration of 3 mg/mL. And (3) atomizing the chondroitin sulfate solution into the collagen solution through high-speed airflow, and continuously stirring for 2 hours to obtain the collagen-chondroitin sulfate compound. Centrifuging the collagen-chondroitin sulfate complex at 8000rpm for 15min, collecting precipitate, adding 0.03M acetic acid to dissolve the precipitate again to obtain collagen concentration of 0.6% (w/w), and stirring for 2.5 hr to disperse uniformly to obtain biological material solution.

(3) Preparing porous artificial dermis: pouring the biological material solution prepared in the step (2) into a mold, leveling, pre-freezing for 3h at-60 ℃, annealing for 5-10min, freezing for half an hour at-60 ℃, and transferring to a freeze dryer for freeze drying for 24 h. Then placing the collagen composite layer under the high-temperature vacuum condition of 100 ℃ for high-temperature crosslinking for 24 hours, and demoulding to obtain the collagen composite layer. Soaking the obtained collagen composite layer in 0.1% glutaraldehyde-acetic acid solution for 24h, and cleaning with purified water to obtain porous artificial dermis.

Example 3

In this embodiment, a circular through hole penetrating in the radial direction is formed in the biomaterial layer, a spirally recessed micro-topology structure is formed on the inner wall of the through hole, the depth of the recess is 5 μm in this embodiment, the interface of the through hole is a circular hole, the diameter of the circular hole is 100 μm, the hole pitch between the circular holes is 3mm, and a schematic cross-sectional view of the through hole is shown in fig. 6. The porous artificial dermis of this example was prepared according to the following procedure:

(1) preparing a mould: and (3) producing the required graph of the mold by using computer aided design, wherein the mold is provided with a bottom plate and a cofferdam surrounding the bottom plate, a plurality of cylindrical structures are arranged on the bottom plate, and the surfaces of the cylindrical structures are provided with convex structures corresponding to the spiral depressions. Mixing polydimethylsiloxane and a curing agent in a mass ratio of 1:1, fully stirring to obtain a polymer solution, printing by using the polymer solution as printing ink, and curing for 2 hours on a hot bed at 80 ℃ to obtain the mold.

(2) Preparing a biological material solution: weighing collagen, dissolving in water to prepare 200g of collagen solution with the mass concentration of 0.3%, weighing 0.012g of chondroitin sulfate, dissolving in water to prepare chondroitin sulfate solution with the mass concentration of 2 mg/mL. And (3) atomizing the chondroitin sulfate solution into the collagen solution through high-speed airflow, and continuously stirring for 2 hours to obtain the collagen-chondroitin sulfate compound. Centrifuging the collagen-chondroitin sulfate complex at 8000rpm for 15min, collecting precipitate, adding 0.1M acetic acid to dissolve the precipitate again to obtain collagen concentration of 0.8% (w/w), and stirring for 2.5 hr to disperse uniformly to obtain biological material solution.

(3) Preparing porous artificial dermis: pouring the biological material solution prepared in the step (2) into a mold, leveling, pre-freezing for 3h at-60 ℃, annealing for 5-10min, freezing for half an hour at-60 ℃, and transferring to a freeze dryer for freeze drying for 24 h. Then placing the collagen composite layer under the high-temperature vacuum condition of 100 ℃ for high-temperature crosslinking for 24 hours, and demoulding to obtain the collagen composite layer. Soaking the obtained collagen composite layer in 0.3% glutaraldehyde-acetic acid solution for 24h, and cleaning with purified water to obtain porous artificial dermis.

Claims (10)

1. The porous artificial dermis is characterized by comprising a biological material layer, wherein a plurality of through holes are formed in the biological material layer in the thickness direction, and the inner walls of the through holes are provided with depressions and/or protrusions.

2. The porous artificial dermis according to claim 1, wherein the depth of the concavity or the height of the convexity is 3 to 6 μm.

3. The porous artificial dermis according to claim 1, wherein a plurality of the through holes are arranged regularly.

4. The porous artificial dermis according to claim 1, wherein the through-hole has a cross section of a circular hole or a rectangular hole.

5. The porous artificial dermis according to any one of claims 1 to 4, wherein the biomaterial layer is made from a raw material comprising collagen, mucopolysaccharide through a cross-linking reaction.

6. A mold for preparing a porous artificial dermis according to any one of claims 1 to 5, comprising a bottom plate and a cofferdam surrounding said bottom plate, wherein a plurality of columnar structures are arranged on said bottom plate, and the surface of a plurality of said columnar structures is provided with protrusions and/or depressions.

7. The mold according to claim 6, wherein the columnar structure is a cylinder or prism.

8. The mold of claim 7, wherein the surface of the mold is further coated with polyacrylamide.

9. The method for preparing porous artificial dermis according to any one of claims 1 to 5, characterized by comprising the steps of:

preparing a mold according to any one of claims 6 to 8;

preparing a biological material solution: mixing the raw materials including mucopolysaccharide solution and collagen solution, centrifuging, collecting precipitate, adding acetic acid, and dissolving again to obtain biological material solution;

preparing porous artificial dermis: pouring the biological material solution into the mold, freeze-drying, placing in a vacuum environment for thermal crosslinking, demolding, and placing in a crosslinking agent for crosslinking reaction to obtain the porous artificial dermis.

10. The method for preparing porous artificial dermis according to claim 9, wherein the freeze-drying process is specifically: freezing the mixture in an environment with the temperature of-50 to-70 ℃, annealing for 5 to 10min, and then carrying out freeze drying.

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