CN115429938A

CN115429938A - Double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration and preparation method thereof

Info

Publication number: CN115429938A
Application number: CN202211205833.2A
Authority: CN
Inventors: 樊李红; 刘爽; 黎明; 褚莹莹; 尤晨凯
Original assignee: Wuhan University of Technology WUT
Current assignee: Wuhan University of Technology WUT
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2022-12-06

Abstract

The invention discloses a double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration and a preparation method thereof.

Description

Double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration and a preparation method thereof.

Background

Skin is the biggest tissue organ of human body, and simultaneously with external area of contact the biggest, the condition of taking place the wound is also most common, and ordinary wound injury only needs simple the wrapping just can reach the effect of treatment, but to the skin injury of degree of depth, the treatment scheme of optimization at present is skin grafting operation, and the source of its skin is mainly three types at present: firstly, allogeneic skin transplantation has a plurality of problems of immunological rejection and the like; second, autologous skin transplantation, which is the best clinical method for treating dermal injury at present, has problems such as insufficient donor and secondary injury of normal skin tissue of a patient; third, artificial dermis products, such as Lando and Pelnac, currently have unsatisfactory effects of repairing dermal tissue wounds in clinical use, and finally, autologous skin grafting operations are required. In conclusion, the research on the dermal tissue repair inducing material for repairing the damaged dermal tissue, which has the effects of well promoting dermal regeneration, angiogenesis and functional recovery, to gradually replace the traditional skin grafting operation treatment has important research significance.

Acellular matrices (ECM) are prepared from tissues of various allogeneic or xenogeneic sources, including the dermis, bladder, small intestine, mesothelium, pericardium, heart valves, and the like, which are composed primarily of various types of proteins and carbohydrates, such as collagen, elastin, laminin, fibronectin, and glycosaminoglycans (GAGs), among others. The ECM prepared by the tissue decellularization technique has excellent biological activity and protein nutrients, can effectively induce migration, adhesion and proliferation of tissue cells, and is currently used in the fields of inducing tissue regeneration and the like. In the prior art, a porcine achilles tendon joint acellular matrix is commonly used, an acellular matrix material is used for in-situ transplantation repair of a muscle defect model, the result shows that newly formed muscle fibers are formed, the number of positive cells of a muscle differentiation factor (Myod) and a myosin heavy chain (MyHC) in a scaffold group is the highest at day 30, and a specific biological component of the acellular matrix has good induction and regeneration capacity on myoblasts, which fully shows that the acellular matrix has a remarkable promoting effect on induced migration, adhesion and proliferation of histiocytes, and the material prepared by using the acellular matrix has great application potential in the field of dermal tissue wound repair.

However, the artificial dermis has poor repair performance, is slow in healing speed, and is likely to form scars.

Disclosure of Invention

In view of this, the present application provides a double-layered artificial dermal scaffold material for self-piezoelectric induced tissue regeneration and a preparation method thereof, which improve a wound healing environment, and the piezoelectric membrane stimulates the tissue to heal rapidly with a low scar rate.

In order to achieve the technical purpose, the following technical scheme is adopted in the application:

in a first aspect, the application provides a double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration, which comprises an acellular matrix cross-linked freeze-dried scaffold layer and an electrostatic spinning piezoelectric film covering the surface of the acellular matrix cross-linked freeze-dried scaffold layer.

Preferably, the electrostatic spinning piezoelectric film is a left-handed polylactic acid electrostatic spinning piezoelectric film compounded by nano zinc oxide.

Preferably, the acellular matrix is that of the skin of a stinging pig.

Preferably, the thickness of the acellular matrix cross-linked freeze-dried scaffold layer is 3-6mm, and the thickness of the electrostatic spinning piezoelectric film is 0.3-0.5mm.

In a second aspect, the present application provides a method for preparing a double-layered artificial dermal scaffold material for self-piezoelectric induced tissue regeneration, comprising the steps of:

s1, sequentially soaking skin tissues of the dolphin in a surfactant solution, an acid solution and a digestive enzyme solution, dialyzing to obtain an extracellular matrix material, crosslinking the extracellular matrix material by using a crosslinking agent, and freeze-drying to obtain a decellularized matrix crosslinked freeze-dried scaffold material;

s2, dissolving nano zinc oxide in a dichloromethane solution of the L-polylactic acid, and preparing an electrostatic spinning piezoelectric film by using an electrostatic spinning process;

and S3, adhering the electrostatic spinning piezoelectric film to the surface of the acellular matrix crosslinked freeze-dried scaffold material to obtain the self-piezoelectric induced tissue regeneration double-layer artificial dermis scaffold material.

Preferably, the mass ratio of the nano zinc oxide to the levorotatory polylactic acid is 1-5.

Preferably, the voltage of the electrostatic spinning process is as follows: +12kv/-10kv, liquid ejection velocity: 1-3 mm/h.

Preferably, the concentration of the dichloromethane solution of the levorotatory polylactic acid is 5-15%.

Preferably, the concentration of the surfactant is 0.5% -2%.

Preferably, the cross-linking agent is a mixture of NHS and EDC.

The beneficial effects of this application are as follows:

1. the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration has high biological activity, is biodegradable, promotes dermis repair, can effectively avoid inflammation in the dermis repair process, effectively induces growth and repair of dermis cells, has a good tissue protein structure, contains a large amount of substances such as viscoelastic protein, polysaccharide, growth factors and the like, can induce the recruitment of surrounding fibroblasts from a wound part, promotes the rapid proliferation and adhesion of cells, and closes a wound; the outer layer is an electrostatic spinning piezoelectric film, and the generated voltage acts on the acellular matrix cross-linked freeze-dried scaffold material of the inner layer, so that the inflammation and cell expression microenvironment for wound healing are improved, the excessive deposition of collagen can be effectively inhibited, and the effects of quick healing and no scar are realized;

2. compared with the extracellular matrixes from other sources, the acellular matrix used by the method has the advantages that the acellular matrix is derived from the skin of the stinging dolphin, the excellent protein structure and strength of the skin of the stinging dolphin can realize rapid migration and adhesion of cells, meanwhile, the exchange of nutrient substances can be effectively realized, the capacity of inducing tissue regeneration is better, voltage stimulation generated by the electrostatic spinning piezoelectric film can be more easily obtained, and the cells rapidly move due to the piezoelectric stimulation have a good space structure;

3. the electrostatic spinning piezoelectric film used in the application is an electrostatic spinning film of ZnO/PLLA, has antibacterial property and piezoelectric property, the PLLA has piezoelectric property, the spinning film can be polarized in an electrostatic spinning mode, and meanwhile, the nano zinc oxide can also increase the beta-phase composition of a polymer to enhance the piezoelectric property;

4. the electrostatic spinning piezoelectric film can isolate the external environment, can also produce continuous electrical stimulation to promote wound healing, and meanwhile, the strong hydrophobic capacity of the polylactic acid can also prevent the adhesion of the film and tissues.

Drawings

Fig. 1 is a piezoelectric performance test result of a piezoelectric film;

FIG. 2 shows the result of the antibacterial performance test of the piezoelectric film.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Sodium ions distributed on the surface of human skin make the epidermis of the skin have electric potential, and the damage of the epidermis can cause the change of current to generate an electric field. Electrical Stimulation (ES) is used as an exogenous electric field to act on a skin wound surface, and can promote wound healing and tissue regeneration by enhancing intercellular signal conduction, accelerating blood circulation and promoting cell proliferation through electric current. Conventional ES therapy typically requires an external power source and electrodes to deliver small currents to the wound site, but these medical devices have limited flexibility and persistence, resulting in greatly limited application of electrical stimulation therapy in wound healing. Accordingly, an increasing number of researchers are working on developing wireless and non-invasive power sources for stimulating wound healing. The piezoelectric material can generate voltage through self mechanical deformation, has good capability of inducing the directional migration of tissue cells, and can be applied to the field of electrical stimulation treatment, in particular to wound repair.

Based on this, the present invention has been made.

The application provides a double-deck artifical dermal scaffold material from regeneration of piezoelectricity induced tissue, including acellular matrix crosslinked freeze-dried scaffold layer, cover in the electrostatic spinning piezoelectric film on acellular matrix crosslinked freeze-dried scaffold layer surface, the cover mode can be for bonding or pressfitting.

The mechanism that takes the acellular matrix crosslinked freeze-dried scaffold layer as the inner layer and the electrostatic spinning piezoelectric film as the outer layer to achieve the effects of quick healing and no scar is as follows: acellular matrix is a sponge material with a pore structure, and is used for the moist environment that the wound can not be guaranteed to continue for wound healing, but the electrostatic spinning membrane has a hydrophobic structure and micron-level pore diameter, the good healing environment of wound can be guaranteed to these performances, the healing speed is accelerated, and simultaneously, the outer membrane also has the piezoelectric property, the voltage effect of production and the acellular matrix cross-linking freeze-drying support layer effect of inner layer, the inflammation and the cell expression microenvironment for wound healing are improved, the excessive deposition of collagen can be effectively inhibited, and therefore the formation of scars is avoided.

The electrostatic spinning piezoelectric film is a left-handed polylactic acid electrostatic spinning piezoelectric film compounded by nano zinc oxide, and an electrostatic spinning film of A-ZnO/PLLA has antibacterial property and piezoelectric property, wherein the A-ZnO is the nano zinc oxide, the particle size of the A-ZnO is 1-50nm, the PLLA has the piezoelectric property, the spinning film can be polarized through electrostatic spinning, and meanwhile, the nano oxidizability can also increase the composition of a polymer beta phase to enhance the piezoelectric property.

The acellular matrix is the acellular matrix of the skin of the stinging dolphin, the skin protein of the stinging dolphin is high in strength, has a good tissue protein structure, contains a large amount of viscoelastic protein, polysaccharide, growth factors and other substances, can induce the recruitment of surrounding fibroblasts from a wound part, simultaneously promotes the rapid proliferation and adhesion of cells, closes a wound, has the commonality of fish skin, does not have immune antigenicity, is high in strength of a made dermal scaffold material, can realize rapid migration and adhesion of the cells, can also effectively realize the exchange of nutrient substances, is better compared with other collagen, is better used with a piezoelectric material between the dermis prepared by the skin of the stinging dolphin, has a good space structure for the rapid movement of the cells caused by piezoelectric stimulation, and is more beneficial to inducing the regeneration of tissues.

It should be noted that compared to the conductive film as the outer layer material, the electrospun piezoelectric film of the present application has the following advantages: piezoelectric film can be through the vibrations product voltage of human skin muscle, the hyperplasia and the differentiation of amazing wound cell, and the conducting film only has electric conductivity, and do not possess the ability of spontaneous production electro photoluminescence, current conducting film is simple can completely cut off wound and external environment, the follow-up destruction that still can lead to newborn granulation of partial membrane, and the piezoelectric film of this application not only can completely cut off external environment, the electro photoluminescence that can also the product lasts, promote wound healing, the powerful hydrophobic ability of polylactic acid also can prevent the adhesion of membrane and tissue simultaneously, the voltage that the piezoelectric film produced can act on the inlayer genuine leather support, thereby improve the wound healing environment, realize quick scar-free healing effect.

The acellular matrix cross-linked freeze-dried scaffold layer has the thickness of 3-6mm, dermal damage filling can be effectively realized within the range, the electrostatic spinning piezoelectric film has the thickness of 0.3-0.5mm, the piezoelectric performance within the range is the strongest, and the thickness is determined by a mould used for preparing each material or cutting after molding.

In addition, the present application provides a method for preparing a double-layered artificial dermal scaffold material for self-piezoelectric induced tissue regeneration, comprising the steps of:

s1, sequentially soaking skin tissues of dolphin in a surfactant solution with the concentration of 0.5-2%, an acid solution with the concentration of 0.5-1.5% and a digestive enzyme solution with the concentration of 100-900 ppm, dialyzing for 3 days to obtain an extracellular matrix material, crosslinking the extracellular matrix material by using a mixed solution of EDC with the concentration of 50mM and NHS with the concentration of 25mM, and freeze-drying to obtain a decellularized matrix crosslinked freeze-dried scaffold material; the surfactant is one or more of sodium dodecyl sulfate, sodium glycocholate, benzalkonium chloride, polysorbate and fatty glyceride; the acid solution is one or more of hydrochloric acid, sulfuric acid, acetic acid and phosphoric acid; the digestive enzyme is one or more of pepsin, trypsin and glutamic acid protease, the digestive enzyme can unwind partial protein chain segment, and the unwinding promoting effect is obvious in an acid environment;

s2, dissolving the nano zinc oxide in a dichloromethane solution of 5-15% L-polylactic acid, wherein the mass ratio of the nano zinc oxide to the L-polylactic acid is 1-5: +12kv/-10kv, liquid ejection speed: preparing an electrostatic spinning piezoelectric film under the condition of 1-3 mm/h;

and S3, adhering the electrostatic spinning piezoelectric film to the surface of the acellular matrix cross-linked freeze-dried scaffold material to obtain the self-piezoelectric induced tissue regeneration double-layer artificial dermis scaffold material.

The double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration prepared by the scheme can be directly laid outside a dermis injury part in a surgical operation and is used for repairing dermis injury.

The present invention is further illustrated by the following specific examples.

Example 1

A double-layer artificial dermis scaffold material for self-piezoelectric induction tissue regeneration comprises a acellular matrix cross-linked freeze-dried scaffold layer of dolphin skin, and a left-handed polylactic acid electrostatic spinning piezoelectric film compounded by nano zinc oxide and adhered to the surface of the acellular matrix cross-linked freeze-dried scaffold layer of the dolphin skin; the thickness of the acellular matrix cross-linked freeze-dried scaffold layer is 3mm, and the thickness of the electrostatic spinning piezoelectric film is 0.3mm;

the preparation method comprises the following steps:

s1, respectively soaking chopped skin tissues of the oplopanax elatus nakai in 0.8% sodium glycocholate, 0.7% sulfuric acid solution and 400ppm trypsin, dialyzing the treated solution for 3 days to obtain an extracellular matrix material, adding an EDC/NHS cross-linking agent, uniformly mixing, injecting into a mold for cross-linking, and freeze-drying to obtain a decellularized matrix cross-linked freeze-dried scaffold material;

s2, doping nano zinc oxide into a dichloromethane solution of the L-polylactic acid, wherein the concentration of the L-polylactic acid solution is 8%, and the mass of the nano zinc oxide and the L-polylactic acid is 5:100, at a voltage of: +12kv/-10kv, liquid ejection speed: preparing an electrostatic spinning piezoelectric film by using electrostatic spinning under the condition of 1-3 mm/h;

and S3, adhering the electrostatic spinning piezoelectric film to the surface of the acellular matrix cross-linking freeze-drying scaffold material to obtain the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration.

Example 2

The other contents of the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration are the same as those of the embodiment 1, except that the mass of the nano zinc oxide and the levorotatory polylactic acid is 1:100.

example 3

The other contents of the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration are the same as those of the embodiment 1, except that the mass of the nano zinc oxide and the levorotatory polylactic acid is 2:100.

example 4

A double-layered artificial dermis scaffold material for self-piezoelectric induced tissue regeneration, the other contents are the same as in example 1, except that the mass of the nano zinc oxide and the levorotatory polylactic acid is 3:100.

example 5

The other contents of the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration are the same as those of the embodiment 1, except that the mass of the nano zinc oxide and the levorotatory polylactic acid is 4:100.

example 6

A double-layered artificial dermal scaffold material for tissue regeneration induced by piezoelectric induction, which is otherwise the same as in example 1, except that the surfactant solution, the acid solution, and the digestive enzyme are, in order, 0.5% sodium dodecylsulfate, 0.5% hydrochloric acid solution, and 100ppm pepsin.

Example 7

A double-layered artificial dermal scaffold for inducing tissue regeneration by self-piezoelectric stimulation was prepared in the same manner as in example 1, except that the surfactant solution, the acid solution and the digestive enzyme were benzalkonium chloride (1%), acetic acid solution (0.8%) and glutamic acid protease (400 ppm) in this order.

Example 8

A double-layered artificial dermal scaffold material for inducing tissue regeneration by self-piezoelectricity is otherwise the same as in example 1, except that the acellular matrix of the skin of a stinging pig is replaced with the acellular matrix of the joint of the tendon of Achilles tendon of pig.

Example 9

The other contents of the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration are the same as those of the embodiment 1, except that the levorotatory polylactic acid electrostatic spinning piezoelectric film compounded by nano zinc oxide is replaced by the levorotatory polylactic acid electrostatic spinning piezoelectric film.

Example 10

The other contents of the double-layer artificial dermis scaffold material for self-piezoelectric induced tissue regeneration are the same as those of the embodiment 1, except that the levorotatory polylactic acid electrostatic spinning piezoelectric film compounded by nano zinc oxide is replaced by the levorotatory polylactic acid electrostatic spinning piezoelectric film compounded by zinc oxide.

Evaluation test

To be used commercially

Performance tests were performed on the artificial dermis material, the PELNAC artificial dermis material, and the acellular matrix cross-linked freeze-dried scaffold layer of the single-layer stinging dolphin skin as comparative materials with examples 2, 8, 9, and 10 of the present scheme, wherein the lower layer of the PELNAC artificial dermis material was the non-terminal collagen of the pig tendon, the upper layer was the silica gel membrane,

the lower layer of the artificial dermis material is a collagen layer, the upper layer is a silica gel layer, and the performance test comprises the following steps: skin defects with the diameter of 20mm at the back and the depth of 2mm of 30 groups of SD rats are selected as wound models, the average values of the dressing change times, the wound inflammation condition, the immunogenicity, the reducing substances, the healing time and the scar rate in the healing process are recorded, and the results are shown in table 1.

TABLE 1 wound healing Effect

As can be seen from table 1, the double-layered artificial dermis material prepared by the present scheme has a faster healing speed, less inflammation, a lower scar rate, and fewer dressing changes than the three comparative materials, and as can be seen from examples 2 and 8, the scar rate is lower, the healing time is faster, the dressing changes are fewer, and the acellular matrix of the dolphin skin is better than the acellular matrix of the pig tendon as the inner layer of the artificial dermis material, as can be seen from examples 2, 9, and 10, when the electrostatic spinning piezoelectric film does not contain nano zinc oxide, the antibacterial property, the healing time, and the scar rate are all affected, while when the nano zinc oxide is a common zinc oxide material, the healing time and the scar rate are not as good as those of example 2, and it can be seen that the nano zinc oxide and the l-polylactic acid in the electrostatic spinning piezoelectric film have a synergistic effect.

The use ratio of nano zinc oxide to l-polylactic acid in the electrostatic spinning piezoelectric films of examples 1 to 5 was studied, and the l-polylactic acid was used as a blank control, and as shown in fig. 1, the piezoelectric performance increased with the increase of the ratio of nano zinc oxide, and the mass of nano zinc oxide to l-polylactic acid was 5:100 is the best piezoelectric effect, which shows that the nano zinc oxide and the levorotatory polylactic acid have the function of synergistically enhancing the piezoelectric performance; the antibacterial performance (escherichia coli) in example 2 was tested by comparing the common zinc oxide/l-polylactic acid composite electrostatic spinning piezoelectric film and the l-polylactic acid electrostatic spinning piezoelectric film, and the results are shown in fig. 2, which indicates that the nano zinc oxide and the l-polylactic acid have synergistic antibacterial effect.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention.

Claims

1. A double-layer artificial dermis scaffold material for self-piezoelectric induction tissue regeneration is characterized by comprising a acellular matrix cross-linked freeze-dried scaffold layer and an electrostatic spinning piezoelectric film covering the surface of the acellular matrix cross-linked freeze-dried scaffold layer.

2. The double-layered artificial dermis scaffold material for self-piezoelectric induced tissue regeneration according to claim 1, wherein the electrospun piezoelectric film is a levorotatory polylactic acid electrospun piezoelectric film compounded by nano zinc oxide.

3. The double-layered artificial dermal scaffold material for self-piezoelectric-induced tissue regeneration according to claim 1, wherein the acellular matrix is that of dolphin's skin.

4. The double-layered artificial dermis scaffold material for self-piezoelectric induced tissue regeneration according to claim 1, wherein the thickness of the acellular matrix cross-linked freeze-dried scaffold layer is 3-6mm, and the thickness of the electrospun piezoelectric film is 0.3-0.5mm.

5. A method for preparing a double layered artificial dermal scaffold material for self-piezoelectric induced tissue regeneration according to any one of claims 1 to 4, comprising the steps of:

s1, sequentially soaking skin tissues of the trogopterus formosanus in a surfactant solution, an acid solution and a digestive enzyme solution, dialyzing to obtain an extracellular matrix material, crosslinking the extracellular matrix material by using a crosslinking agent, and freeze-drying to obtain a decellularized matrix crosslinked freeze-dried scaffold material;

6. The method for preparing the double-layered artificial dermis scaffold material for self-piezoelectric induced tissue regeneration according to claim 5, wherein the mass ratio of the nano zinc oxide to the L-polylactic acid is 1-5.

7. The method for preparing a double-layered artificial dermis scaffold material for self-piezoelectric induced tissue regeneration according to claim 5, wherein the voltage of the electrospinning process is as follows: +12kv/-10kv, liquid ejection speed: 1-3 mm/h.

8. The method for preparing a double-layered artificial dermal scaffold material from piezoelectric-induced tissue regeneration according to claim 5, wherein the concentration of the dichloromethane solution of the L-polylactic acid is 5 to 15%.

9. The method for preparing a double-layered artificial dermis scaffold material for self-piezoelectric induced tissue regeneration according to claim 5, characterized in that the concentration of the surfactant is 0.5% -2%.

10. The method for preparing a double-layered artificial dermal scaffold material for self-piezoelectric-induced tissue regeneration according to claim 5, wherein the cross-linking agent is a mixture of NHS and EDC.