CN115463264A - Temperature-sensitive interpenetrating polymer network hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of skin regeneration and soft tissue regeneration repair materials in tissue engineering, in particular to a temperature-sensitive interpenetrating polymer network hydrogel and a preparation method and application thereof, in-vitro fat is degreased, decellularized, denucleated, freeze-dried, ground and dissolved by pepsin hydrochloric acid solution to prepare fat-decellularized extracellular matrix pre-gel liquid, unactivated temperature-controlled platelet-rich plasma is collected from in-vitro blood, the fat-decellularized extracellular matrix pre-gel liquid and the unactivated temperature-controlled platelet-rich plasma are uniformly mixed under the low-temperature condition and are injected into a skin wound surface or soft tissue defect with an irregular shape, and the hydrogel is self-assembled into a gel state when the temperature is raised to about 37 ℃. The temperature-controlled injectable hydrogel disclosed by the invention is rich in collagen and fibrin to form, plays a good role in promoting tissue regeneration by releasing platelets and growth factors, and is suitable for treating irregular wound surfaces and filling soft tissue defects.

Description

Temperature-sensitive interpenetrating polymer network hydrogel and preparation method and application thereof

Technical Field

The invention relates to the field of skin regeneration and soft tissue regeneration repair materials in tissue engineering, in particular to a temperature-sensitive interpenetrating polymer network hydrogel and a preparation method and application thereof, and especially relates to a temperature-sensitive interpenetrating polymer network hydrogel based on fat-free extracellular matrix and platelet-rich plasma and a preparation method and application thereof.

Background

At present, acute and chronic wounds caused by various reasons, such as wounds, diabetic wounds and radioactive wounds, lack effective treatment means. In addition, severe acute and chronic wound formation is often accompanied by soft tissue defects of varying degrees. Delayed healing of the wound and loss of soft tissue after healing severely affect the quality of life, physiological and psychological health of the patient.

Platelet Rich Plasma (PRP) is widely used in clinical treatment of various acute and chronic wounds due to its autologous source, convenient acquisition, and good effect of promoting tissue regeneration. PRP is gel-like, but has poor mechanical properties, undesirable slow release of platelets and cytokines, high burst rate, and unstable therapeutic effect. At present, the effect of reducing burst release rate and strengthening sustained release function is achieved by mixing a chemical synthetic material such as sodium alginate with PRP, but the chemical synthetic material has weak biological property, and has the risks of difficult degradation, potential toxicity and the like, and difficult clinical application.

Interpenetrating Polymer Networks (IPNs) of collagen-fibrin hydrogels provided us with a hint to improve PRP. The principle of formation of fibrin network in PRP is similar to the fibrin hydrogel gel process, i.e. thrombin causes the conversion of fibrinogen to fibrin, which assembles into a fibrin gel. Fibrin hydrogels are reported to degrade rapidly and with short duration in vivo compared to collagen hydrogels. After the collagen-fibrin hydrogel is mixed with the collagen hydrogel in a prepolymerization mode, strong interaction exists between the collagen and the fibrin hydrogel to form the interpenetrating polymer network hydrogel. The addition of additional collagen during the formation of the fibrin network in PRP may improve the poor stability of the fibrin network and entrap growth factors in the interpenetrating polymer network, resulting in better sustained release.

However, the hydrogel prepared by the prior art method has poor temperature sensitivity and cross-linking stability, so that the effect of repairing the wound surface after injection is poor, and the application of the hydrogel is limited.

Disclosure of Invention

In order to solve the technical problems, the invention provides a temperature-sensitive interpenetrating polymer network hydrogel and a preparation method and application thereof. The collagen and the fibrin in the hydrogel are well combined to form an interpenetrating polymer network, and the hydrogel has the characteristics of temperature sensitivity and injectability, and can be used for treating various wound surfaces and filling soft tissue defects.

The first purpose of the invention is to provide a temperature-sensitive interpenetrating polymer network hydrogel, which is prepared by preparing fat acellular matrix powder from the body fat, preparing the fat acellular matrix pre-gel from the fat acellular matrix powder and pepsin, and mixing the fat acellular matrix pre-gel with the inactivated platelet rich plasma.

Preferably, the temperature-sensitive interpenetrating polymer network hydrogel contains collagen, glycosaminoglycan, growth factors, fibrin and platelets in a natural fat-free extracellular matrix.

Preferably, the skeleton components of the temperature-sensitive interpenetrating polymer network hydrogel are collagen derived from fat-free extracellular matrix and fibrin derived from platelet-rich plasma, and the temperature is raised from low temperature to 36-38 ℃ to form an interpenetrating polymer network through self-assembly.

The second purpose of the invention is to provide a preparation method of the temperature-sensitive interpenetrating polymer network hydrogel, which comprises the following steps:

a. defatting isolated fat, removing cells, removing nucleic acid, lyophilizing, and grinding to obtain fat-free extracellular matrix powder;

b. b, dissolving the fat acellular matrix powder prepared in the step a into HCl solution, adding pepsin, adjusting the pH of the solution to 7.35-7.45 after complete dissolution, and obtaining fat acellular matrix pre-gel;

c. taking a blood collection tube containing separation gel, adding in vitro blood at low temperature, centrifuging at 3500-4000r/min and 4 deg.C for 15-20min, and collecting the upper liquid of the separation gel as unactivated platelet-rich plasma;

d. uniformly mixing the fat acellular extracellular matrix pre-gel obtained in the step b with the platelet rich plasma which is not activated in the step c to prepare a temperature-sensitive interpenetrating polymer network pre-gel based on the fat acellular extracellular matrix and the platelet rich plasma;

e. and (d) heating the temperature-sensitive interpenetrating polymer network pre-gel prepared in the step (d) to 36-38 ℃, and keeping the temperature for more than 15min to obtain the temperature-sensitive interpenetrating polymer network hydrogel based on the fat-free extracellular matrix and the platelet-rich plasma.

Preferably, in the preparation method of the temperature-sensitive interpenetrating polymer network hydrogel, the in vitro fat and the in vitro blood are from allogeneic human sources or human self sources.

Preferably, in the preparation method of the temperature-sensitive interpenetrating polymer network hydrogel, the concentration of the HCl solution is 0.01mol/L.

Preferably, in the preparation method of the temperature-sensitive interpenetrating polymer network hydrogel, in the step b, 10mg of pepsin is added into 10mL of liquid after the fat-free extracellular matrix powder is dissolved in HCl.

Preferably, the preparation method of the temperature-sensitive interpenetrating polymer network hydrogel comprises the steps of taking a blood collection tube which does not contain an anticoagulant and contains a separation gel;

the fat-free extracellular matrix pre-gel and the inactivated platelet-rich plasma are uniformly mixed at a low temperature according to a mass ratio of 1.

Preferably, in the preparation method of the temperature-sensitive interpenetrating polymer network hydrogel, step b further comprises: dissolving 1000IU of thrombin in 2mL of 2wt% ₂ Preparing a thrombin solution in the solution, uniformly mixing the thrombin solution with the fat acellular matrix pre-gel prepared in the step b, and mixing the two substances according to the mixing ratio of mixing 1mL of the fat acellular matrix pre-gel with 80 mu L of the thrombin solution;

correspondingly, a blood collection tube containing an anticoagulant and separation gel is adopted in the step c;

and d, precooling at low temperature, and then uniformly mixing the fat-free extracellular matrix pre-gel with the inactivated platelet-rich plasma.

The third purpose of the invention is to provide an application of the temperature-sensitive interpenetrating polymer network hydrogel, and the temperature-sensitive interpenetrating polymer network hydrogel is used for preparing a therapeutic agent for irregular wound surfaces and a soft tissue defect filler.

Compared with the prior art, the invention has the following beneficial effects:

1. the fat acellular extracellular matrix (DAM) comes from a human body, is medical waste in liposuction surgery, is rich in fat tissue source, belongs to waste recycling by adopting redundant fat tissue after the liposuction surgery, and is wide in source, low in cost and easy to obtain. The fat acellular extracellular matrix and the platelet rich plasma are both from human bodies (from allogeneic human sources or autologous sources), and have strong biological activity and small rejection risk. The fat-free extracellular matrix has biological activity of promoting tissue regeneration, the main component of the skeleton of the fat-free extracellular matrix is collagen, and the crosslinking principle of the fat-free extracellular matrix (DAM) hydrogel is based on the preparation principle of the collagen hydrogel. The DAM hydrogel can play a good treatment effect in wound healing, and the mechanism is to promote angiogenesis and adjust macrophage polarization.

According to the invention, by adding the collagen, the internal crosslinking degree of PRP is enhanced, and the skeleton structure is enhanced, so that a PRP product with enhanced slow release and better mechanical property is obtained, and the polarization of macrophage to M2 type is enhanced. In addition, the fat-free extracellular matrix has natural fat-forming signals due to the fact that the fat-free extracellular matrix is derived from human fat, can be used as a filling material for treating soft tissue defects, is low in rejection risk and high in bioactivity compared with existing heterogeneous collagen and chemical synthetic fillers, and is an ideal soft tissue filling material. Therefore, the invention prepares the temperature-sensitive interpenetrating polymer network hydrogel based on the fat-free extracellular matrix and the platelet-rich plasma based on the DAM hydrogel and the PRP.

Compared with the pure DAM hydrogel, the thermosensitive interpenetrating polymer network hydrogel prepared by the technical scheme of the invention based on the fat acellular extracellular matrix and the platelet-rich plasma has strong biological activity, is rich in fresh growth factors, and has better effects in promoting angiogenesis and macrophage polarization. In the filling treatment of soft tissue defect, the effect is superior to that of a pure DAM hydrogel due to the biological activity signal of self-generated lipid and the functions of promoting vascularization and promoting macrophage polarization. In conclusion, the hydrogel has good application prospects in various types of wound treatment and soft tissue defect filling.

2. Compared with the traditional PRP and pure DAT hydrogel, the interpenetrating polymer network structure in the hydrogel prepared by the invention is more stable and has stronger biological activity than a fibrin network in the PRP and a collagen network in the DAT, thereby being beneficial to the slow release of growth factors and the tissue regeneration treatment.

3. The temperature control PRP without external thrombin and anticoagulant is combined with the DAT hydrogel, and the interpenetrating polymer network hydrogel has the characteristics of double temperature sensitivity, is activated by temperature control, is formed by temperature control, and is suitable for irregular wound treatment and soft tissue filling. At 4 deg.C, PRP is not activated, and the hydrogel is in liquid state. When the temperature is increased to 37 ℃, fibrin in the PRP and collagen in the DAT form a stable interpenetrating polymer network hydrogel, and the PRP is simultaneously activated to release growth factors.

4. The platelet-rich plasma component is prepared by a low-temperature preparation method without adding an exogenous anticoagulant. The blood sampling and centrifuging process adopts low temperature of 4 ℃, thrombin in blood has low activity in low temperature, so that thrombin is not coagulated in preparation, and inactivated platelet-rich plasma is obtained after centrifugation. When the fat acellular pre-gel and the inactivated platelet rich plasma are fully and uniformly mixed at low temperature, the mixture is injected to the irregular wound surface or soft tissue defect, and the temperature-sensitive interpenetrating polymer network pre-gel based on the fat acellular extracellular matrix and the platelet rich plasma is still fluid for a short time, so that the irregular wound surface or soft tissue defect is fully filled. After contacting with the body, the temperature of the pre-gel is gradually raised to 37 ℃, and when the temperature is more than or equal to 15min, due to the self-assembly characteristic of the collagen and the fibrin, an interpenetrating polymer network of the collagen and the fibrin is formed, thereby playing the therapeutic role of wound surfaces and soft tissue defects.

5. The hydrogel contains collagen, glycosaminoglycan and bioactive factors related to tissue regeneration of extracellular matrix of natural adipose tissue; the hydrogel contains growth factors such as fibrin VEGF, PDGF, EGF, IGF and the like in platelet rich plasma. The main components of the hydrogel skeleton are an interpenetrating polymer network formed by self-assembling collagen derived from fat decellularized extracellular matrix and fibrin derived from platelet rich plasma in the process that the temperature is increased from low temperature to 37 ℃. The hydrogel has the excellent characteristics of same species source, slow release of platelets and growth factors, injection through a 27G needle, temperature control crosslinking and activation when being applied to tissue engineering skin regeneration and soft tissue filling.

Drawings

FIG. 1 shows the appearance and electron microscopic structure of example 1 (c) and comparative examples 1 (a) and 2 (b) of the present application. In FIG. 1, each electron micrograph is 80 μm in scale.

FIG. 2 shows the general effect of example 1 (c) and comparative examples 1 (a) and 2 (b) on the treatment of wound surface in nude mouse model for 14 days.

FIG. 3 shows Masson staining evaluation of the treatment effect of example 1 (c) and comparative examples 1 (a) and 2 (b) in a wound treatment nude mouse model for 14 days.

FIG. 4 is a HE staining evaluation of soft tissue neogenesis effect in a soft tissue-filled rat model of example 1 (c) and comparative example 2 (b) of the present application.

FIG. 5 shows the evaluation of the sustained release effect of growth factors in example 1 (c) and comparative example 2 (b) of the present application.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described with reference to the following specific embodiments and the accompanying drawings. In the description of the present invention, reagents used are commercially available and methods used are conventional in the art, unless otherwise specified.

Example 1

A preparation method of temperature-sensitive interpenetrating polymer network hydrogel based on fat acellular extracellular matrix and platelet rich plasma comprises the following steps:

a. selecting isolated fat, and preparing the fat-free extracellular matrix powder by degreasing, decellularizing, removing nucleic acid, freeze-drying and grinding. The isolated fat is of allogeneic human origin. It should be noted that in a hospital or beauty institution, many of the aspirated fat is discarded, and the present invention only collects the excised fat, so step a of this embodiment is not a surgical method. In addition, "defatted, decellularized, de-nucleated, lyophilized, ground" reference "Zhao Y, fan J, bai s.biocompatibility of injectable hydrogel from decellularized human adoptase tissue in vitro and in vivo.j Biomed Mater Res B Appl biometer.2019jul; 107 (5): 1684-1694.Https:// doi. Org/10.1002/jbm. B.34261".

b. And (b) taking 200mg of the fat extracellular matrix-removed powder prepared in the step (a), dissolving the fat extracellular matrix-removed powder in 0.01mol/L HCl, and adding 10mg of pepsin into 10mL of the fat extracellular matrix-removed powder. After completion of the lysis, the pH of the solution was adjusted to 7.4 using 0.1mol/L NaOH and 10 XPBS (concentration of 0.1 mol/L) to obtain an adipose acellular matrix pre-gel, and the adipose acellular matrix pre-gel concentration was adjusted to 16mg/ml using 1 XPBS (concentration of 0.01 mol/L).

c. Placing a blood collection tube which does not contain anticoagulant and contains separation gel in an ice box, taking in vitro allogeneic human blood, loading 10mL of blood collection tube, centrifuging at 3500r/min at 4 ℃ for 15min, and taking 1mL of liquid on the upper layer of the separation gel to obtain the unactivated platelet-rich plasma.

d. And (c) uniformly mixing the fat acellular extracellular matrix pre-gel obtained in the step (b) with the platelet rich plasma which is not activated in the step (c) according to a mass ratio of 1.

e. When in use, the temperature-sensitive interpenetrating polymer network pre-gel prepared in the step d is heated to 37 ℃, and the temperature is preserved for 15min, so that the temperature-sensitive interpenetrating polymer network hydrogel based on the fat-free extracellular matrix and the platelet-rich plasma is obtained.

Example 2

A preparation method of temperature-sensitive interpenetrating polymer network hydrogel based on fat acellular epimatrix and platelet rich plasma comprises the following steps:

a. selecting isolated fat, and preparing fat-free extracellular matrix powder by degreasing, decellularizing, removing nucleic acid, freeze-drying and grinding. The preparation of fat-free extracellular matrix powder was the same as in example 1.

b. And (b) taking 200mg of the fat-free extracellular matrix powder prepared in the step (a), dissolving the fat-free extracellular matrix powder in 0.01mol/L HCl, and adding 10mg of pepsin into 10mL of the fat-free extracellular matrix powder. After completion of the lysis, the pH of the solution was adjusted to 7.4 using 0.1mol/L NaOH and 10 XPBS (concentration of 0.1 mol/L) to obtain an adipose acellular matrix pre-gel, and the adipose acellular matrix pre-gel was adjusted to 16mg/ml using 1 XPBS (concentration of 0.01 mol/L).

c. Dissolving 1000IU thrombin in 2mL of 2wt% CaCl ₂ And (c) preparing a thrombin solution in the solution, uniformly mixing the thrombin solution with the fat acellular extracellular matrix pre-gel prepared in the step (b), and mixing 1mL of the fat acellular extracellular matrix pre-gel with 80 mu L of thrombin solution to obtain the fat acellular matrix pre-gel containing thrombin.

d. 10mL of allogeneic human blood (in vitro blood) is taken from each tube under the room temperature condition by using a blood collection tube containing anticoagulant and separation gel, and is centrifuged at 3500r/min,4 ℃ and 15min, and 1mL of liquid on the upper layer of the separation gel is taken, so that the inactivated platelet-rich plasma is obtained.

e. And (d) uniformly mixing the fat acellular matrix pre-gel containing thrombin obtained in the step (b) with the platelet rich plasma which is not activated in the step (d) according to a mass ratio of 1.

f. When in use, the temperature-sensitive interpenetrating polymer network pre-gel prepared in the step e is heated to 37 ℃, and the temperature is preserved for 15min, so that the temperature-sensitive interpenetrating polymer network hydrogel based on the fat-free extracellular matrix and the platelet-rich plasma is obtained.

Comparative example 1

A method for preparing temperature-controlled activated platelet rich plasma, comprising:

a. 10mL of isolated human whole blood was drawn using a 4 ℃ pre-cooled syringe and transferred to a 15mL 4 ℃ pre-cooled centrifuge tube.

b. And e, centrifuging 200g of whole blood in the centrifuge tube obtained in the step a for 10min at 4 ℃, and transferring the plasma to a new 4 ℃ precooling centrifuge tube.

c. And (c) centrifuging 1550g of the plasma obtained in the step b for 10min, and taking 1mL of lower layer liquid as the inactivated platelet rich plasma.

d. When in use, the temperature of the inactivated platelet rich plasma prepared in the step c is raised to 37 ℃, and the temperature is preserved for 15min, so that the temperature-controlled activated platelet rich plasma can be obtained.

Comparative example 2

A method for preparing an adipose decellularized extracellular matrix hydrogel comprises the following steps:

a. selecting isolated fat, and preparing the fat-free extracellular matrix powder by degreasing, decellularizing, removing nucleic acid, freeze-drying and grinding. The fat-free extracellular matrix powder was prepared in the same manner as in example 1.

b. Taking 100mg of the fat extracellular matrix-removed powder prepared in step a, dissolving the fat extracellular matrix-removed powder in 0.01mol/L HCl, and adding 10mg of pepsin into 10mL of the fat extracellular matrix-removed powder. After completion of the lysis, the pH of the solution was adjusted to 7.4 using 0.1mol/L NaOH and 10 XPBS (concentration of 0.1 mol/L) to obtain an adipose acellular matrix pre-gel, and the adipose acellular matrix pre-gel was adjusted to 8mg/ml using 1 XPBS (concentration of 0.01 mol/L).

c. When in use, the temperature of the pre-gel prepared in the step b is raised to 37 ℃, and the temperature is kept for 15min, so that the fat acellular extracellular matrix hydrogel can be obtained.

Performance study of hydrogels prepared in each example and comparative example:

1. gross appearance and microstructure

The scanning electron microscope analysis method comprises the following steps: for Scanning Electron Microscope (SEM) analysis, the hydrogel (fresh sample without lyophilization or gold plating) was placed on a temperature-controlled sample holder and cooled from room temperature to-20 ℃. Photography was performed using a Phenom Pro desktop microscope (Phenom-World, phenom Pro, the Netherlands) under 15kV conditions.

The general appearance and microstructure of the gel are shown in FIG. 1a (comparative example 1), FIG. 1b (comparative example 2) and FIG. 1c (example 1), the appearance is obtained by photographing the gel under different temperature conditions, as shown in the figure, the structure of the comparative example 1 is unstable, and a large amount of liquid is separated out and cannot be stably crosslinked; comparative example 2 was able to stabilize crosslinking; example 1 is more three-dimensional and stable in shape than comparative examples 1 and 2. The microstructure is observed by a Phenom Pro electron microscope, and a sample is placed in a temperature control sample cup and then is shot. Compared with comparative examples 1 and 2, the pore diameter of example 1 is more uniform, and the crosslinking degree is better.

2. Verification of therapeutic effects

The treatment effects are respectively the effect of promoting wound healing and the effect of regenerating soft tissues as filling materials.

2.1. Method of producing a composite material

(1) Nude mouse full-layer skin defect model

All animal experiments were approved by the ethical committee for animal research at the fourth university of military medical science. Nude mice were anesthetized by inhalation of isoflurane. After pre-operative sterilization, a circular full-thickness wound was formed on the dorsal skin of each mouse using an 8 mm biopsy punch. Treatments with different interventions were performed in the four groups, once a week, and photographs were recorded. Wound tissue samples of nude mice were collected on day 14 post-surgery and fixed in 4% paraformaldehyde. All tissues were embedded in paraffin blocks and cut into paraffin sections, and these sections were stained with MASSON, the staining procedure was as follows: 1) Paraffin section and dewaxing to water; 2) Chromizing; 3) Sequentially washing tap water and distilled water; 4) Staining the nucleus with Regaud hematoxylin staining solution or Weibert hematoxylin staining solution for 5min; 5) Washing with water, and differentiating with hydrochloric acid and ethanol; 6) Washing with distilled water; 7) Acid red solution of Masson's Lichun red for 5min; 8) Soaking and washing the fabric for a moment by using 2% glacial acetic acid aqueous solution; 9) Differentiating by 1% phosphomolybdic acid aqueous solution for 3min;10 Without washing with water, directly blue-dyeing with aniline for 5min;11 Soaking and washing with 0.2% glacial acetic acid aqueous solution for a while; 12 95% alcohol, absolute alcohol, xylene clear, neutral gum seal.

(2) Mouse soft tissue new model

All animal experiments were approved by the ethical committee for animal research at the fourth university of military medicine. Nude mice were anesthetized by inhalation of isoflurane. After preoperative sterilization, 200 μ L of fat acellular extracellular matrix hydrogel and 200 μ L of temperature-sensitive interpenetrating polymer network hydrogel are respectively injected to two sides of the back of a mouse. Injected hydrogels were collected on day 28 post-surgery and fixed in 4% paraformaldehyde. All tissues were embedded in paraffin blocks and cut into paraffin sections, and these sections were stained with HE, the staining procedure was as follows: 1) Xylene (I) for 15min; 2) Xylene (II) for 15min; 3) Xylene: absolute ethanol = 1; 4) 100% ethanol (I) for 5min; 5) 100% ethanol (II) for 5min; 6) 80% ethanol for 5min; 7) Distilled water for 5min; 8) Staining sappan wood semen for 5min; 9) Flushing with running water for 5min;10 1% ethanol hydrochloride for 30s;11 Water washing for 30s;12 ) 5s of distilled water washing; 13 0.5% eosin for 1min;14 Distilled water for 30s;15 80% ethanol for 30s;16 95% ethanol (I) for 1min;17 95% ethanol (II) for 1min;18 Absolute ethanol (I) for 3min;19 Absolute ethyl alcohol (II) for 3min;20 Xylene (I) for 3min;21 Xylene (II) for 3min;22 Neutral gum blocking.

2.2. As a result, the

As shown in fig. 2a (comparative example 1), fig. 2b (comparative example 2), fig. 2c (example 1), the general observation of 14 days in the nude mouse wound healing model, the small wound area epithelialization of the treatment of example 1 was more complete. In Masson staining evaluation, as shown in fig. 3a (comparative example 1), fig. 3b (comparative example 2) and fig. 3c (example 1), the wound length of the example 1 is smaller, the collagen arrangement is more regular, part of skin appendages are new, and the wound healing effect is better than that of the comparative example 1 and the comparative example 2.

As shown in FIGS. 4a (comparative example 2) and 4b (example 1), HE histological evaluation was performed after the hydrogel was injected into rats, and it was found that example 1 had more cell infiltration, more excellent angiogenesis degree, and more significant fat regeneration.

3. In vitro growth factor sustained release study

To explore the growth factor release behavior of the hydrogels, 500 μ L of temperature controlled activated platelet rich plasma of comparative example 2 (t-PRP line in FIG. 5) was incubated with 1mL of 1 XPBS at 37 ℃ and 1mL of temperature sensitive interpenetrating polymer network hydrogel of example 1 (containing 500 μ L of t-PRP, t-DPI line in FIG. 5) was incubated with 1mL of 1 XPBS at 37 ℃. At time points of 1, 2, 4, 6, 12, 24, 48, 72 and 96 hours, 250 μ L of 1 × PBS was collected, frozen and replaced with 250 μ L of fresh 1 × PBS. The VEGF enzyme-linked immunosorbent assay kit is used for determining the secretion condition of the growth factors. The results in fig. 5 show that the invention enhances the internal crosslinking degree of PRP and strengthens the skeleton structure by adding the form of collagen, thereby obtaining a PRP product with enhanced slow release and better mechanical property.

In conclusion, the thermosensitive interpenetrating polymer network hydrogel based on the fat acellular extracellular matrix and the platelet rich plasma is suitable for wound and soft tissue defect treatment and has good clinical application prospect.

It should be noted that, when the present invention relates to numerical ranges, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. The temperature-sensitive interpenetrating polymer network hydrogel is characterized in that the temperature-sensitive interpenetrating polymer network hydrogel is prepared by preparing fat from body fat into fat acellular matrix powder, preparing the fat acellular matrix powder with pepsin into fat acellular matrix pre-gel, and mixing the fat acellular matrix pre-gel with unactivated platelet rich plasma.

2. The temperature-sensitive interpenetrating polymer network hydrogel of claim 1, wherein the temperature-sensitive interpenetrating polymer network hydrogel comprises collagen, glycosaminoglycan, growth factors, fibrin, and platelets in a natural fat-free extracellular matrix.

3. The temperature-sensitive interpenetrating polymer network hydrogel according to claim 1, wherein the skeleton components of the temperature-sensitive interpenetrating polymer network hydrogel are collagen derived from fat-free extracellular matrix and fibrin derived from platelet-rich plasma, and are self-assembled to form an interpenetrating polymer network in the process that the temperature is increased from low temperature to 36-38 ℃.

4. The method of preparing a temperature-sensitive interpenetrating polymer network hydrogel of claim 1, comprising:

a. defatting isolated fat, removing cells, removing nucleic acid, lyophilizing, and grinding to obtain fat-free extracellular matrix powder;

b. b, dissolving the fat acellular matrix powder prepared in the step a into HCl solution, adding pepsin, adjusting the pH of the solution to 7.35-7.45 after complete dissolution, and obtaining fat acellular matrix pre-gel;

c. taking a blood collection tube containing separation gel, adding in vitro blood at low temperature, centrifuging at 3500-4000r/min and 4 ℃ for 15-20min, and taking the upper liquid of the separation gel to obtain unactivated platelet rich plasma;

d. uniformly mixing the fat acellular extracellular matrix pre-gel obtained in the step b with the platelet rich plasma which is not activated in the step c to prepare a temperature-sensitive interpenetrating polymer network pre-gel based on the fat acellular extracellular matrix and the platelet rich plasma;

e. and (d) heating the temperature-sensitive interpenetrating polymer network pre-gel prepared in the step (d) to 36-38 ℃, and keeping the temperature for more than 15min to obtain the temperature-sensitive interpenetrating polymer network hydrogel based on the fat-free extracellular matrix and the platelet-rich plasma.

5. The method for preparing the temperature-sensitive interpenetrating polymer network hydrogel according to claim 4, wherein the ex vivo fat and the ex vivo blood are derived from an allogeneic human source or a human body.

6. The temperature-sensitive interpenetrating polymer network hydrogel according to claim 4, wherein the concentration of HCl solution is 0.01mol/L.

7. The temperature-sensitive interpenetrating polymer network hydrogel according to claim 6, wherein in step b, 10mg pepsin is added to 10mL of liquid after dissolving fat-free extracellular matrix powder in HCl.

8. The temperature-sensitive interpenetrating polymer network hydrogel according to claim 4, wherein a blood collection tube containing no anticoagulant and separation gel is taken;

the fat-free extracellular matrix pre-gel and the inactivated platelet-rich plasma are uniformly mixed at a low temperature according to the mass ratio of 1.

9. The temperature-sensitive interpenetrating polymer network hydrogel of claim 4, wherein step b further comprises: dissolving 1000IU of thrombin in 2mL of 2wt% ₂ Preparing a thrombin solution in the solution, uniformly mixing the thrombin solution with the fat acellular extracellular matrix pre-gel prepared in the step b, and mixing the two substances according to the mixing ratio of mixing 1mL of the fat acellular extracellular matrix pre-gel with 80 mu L of thrombin solution;

correspondingly, a blood collection tube containing an anticoagulant and separation gel is adopted in the step c;

and d, precooling at low temperature, and then uniformly mixing the fat-free extracellular matrix pre-gel with the inactivated platelet-rich plasma.

10. The use of the temperature-sensitive interpenetrating polymer network hydrogel of claim 1, wherein said temperature-sensitive interpenetrating polymer network hydrogel is used for the preparation of a therapeutic agent for irregular wounds and a filler for soft tissue defects.

Images