CN115400270A

CN115400270A - Composite stent material for slowly releasing platelet-rich plasma and preparation method and application thereof

Info

Publication number: CN115400270A
Application number: CN202211092772.3A
Authority: CN
Inventors: 王淑芳; 周洁; 董云生
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2022-09-08
Filing date: 2022-09-08
Publication date: 2022-11-29

Abstract

The invention belongs to the technical field of biological materials and biomedical engineering, and particularly relates to a composite scaffold material for slowly releasing platelet rich plasma, a preparation method and application thereof, wherein the platelet rich plasma and an activator thereof are loaded on a scaffold carrier with a 3D porous structure, so that the platelet rich plasma is activated in macropores of the scaffold carrier to form gel and interpenetrates with a scaffold carrier network, and thus the composite scaffold material with the slowly releasing platelet rich plasma is formed; the composite stent material prepared by the method can slowly release PRP, prolong the acting time of the composite stent material in vivo, reduce the blood extraction times, provide a long-term effective treatment method for transplantation cases which can not be repeatedly operated, greatly relieve the pain of patients, endow the stent material with stronger biological activity by adding the PRP, fully exert the advantages of the composite stent material and the PRP, and realize the synergistic effect of adding one and more than two.

Description

Composite stent material for slowly releasing platelet-rich plasma and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials and biomedical engineering, and particularly relates to a composite stent material for slowly releasing platelet-rich plasma, and a preparation method and application thereof.

Background

PRP is a plasma concentrate rich in platelets that can be activated by calcium ions/thrombin and converted into a tightly packed polymeric fibrin network to form a PRP gel. PRP can secrete a plurality of high-concentration growth factors through the degranulation effect of activated platelets, the growth factors can induce specific gene expression in cells, and further promote cell proliferation, extracellular matrix formation, angiogenesis and the like. However, the yield of PRP is not high, and the PRP gel has poor mechanical properties, and the multienzyme environment at the wound site often causes rapid degradation, so that gel collapse and loss, growth factor burst release, and unstable existence at the wound site can play a role in each stage of wound repair, which greatly reduces the utilization efficiency of the growth factor and limits the clinical application. Therefore, a technique that enables the sustained release of PRP in the stent material is of great importance.

Patent CN105030826A discloses a composite platelet gel and a preparation method thereof, the composite platelet gel is obtained by activating a chitosan and PRP mixed solution, and the microstructure thereof is: the fibrin network structure obtained by PRP activation is connected with chitosan molecules to form a gel carrier bracket, and the cell factors derived from the platelet are adhered and anchored in the gel carrier bracket to form the composite platelet gel, and the specific process is as follows: firstly, chitosan and PRP freeze-thaw lysate are mixed, then an activating agent is added for standing, and fibrinogen in PRP is activated into a fibrin network structure. Compared with single PRP, the gel system can improve the problem of platelet-derived growth factor and active substance burst release, but has at least the following disadvantages:

(1) In the preparation process, the formation of the gel and the activation of the platelet-rich plasma are completed in one step, namely, in the formation process of the gel, the platelet-rich plasma also releases growth factors, when the gel in the system is not formed, a part of the growth factors are lost out of the gel system because no gel scaffold is adhered, so that the load of the growth factors in the finally formed gel system is less than the total amount of the released platelet-rich plasma;

(2) The patent only discloses that chitosan and PRP can be used in combination through the method, and because different materials have different gelling time, gelling strength and other properties, when the materials with different gelling properties from chitosan are used, the formed gel properties are different, and the slow release effect is different, the method is not necessarily suitable for the combination of other materials and PRP, and therefore the method has no universality.

Disclosure of Invention

In order to solve the technical problems, the invention provides a composite stent material for slowly releasing platelet-rich plasma and a preparation method and application thereof, firstly a stent carrier with a 3D porous structure is obtained, then PRP and an activating agent are added into the stent carrier, and after the PRP is activated, the released growth factors are directly adhered in the stent carrier, so that the loss of the growth factors is avoided, the load capacity is improved, and PRP gel and a stent carrier network are interpenetrating, so that a 'double-network' composite stent is formed, and the slow release effect is favorably improved; the mode allows PRP to be combined with various types of tissue engineering scaffolds, including hydrogel scaffolds, 3D printing scaffolds, novel material scaffolds and the like, and the selection of the preparation materials of the scaffolds can also be selected according to the required pertinence, so that the PRP composite scaffold has a wider application range.

The invention is realized by the following technical scheme.

The invention provides a composite scaffold material for slowly releasing platelet rich plasma, which is prepared by loading PRP and an activator thereof onto a scaffold carrier with a 3D porous structure, so that the PRP is activated in macropores of the scaffold carrier to form gel and interpenetrates with a scaffold carrier network, thereby forming the composite scaffold material with the slowly releasing platelet rich plasma.

The macropores of the porous scaffold give PRP gel barrier and support effects, and the PRP loading carried out by using the method has the effect of slowly releasing the PRP, can be used for long-term maintenance of the treatment effect after in vivo transplantation of the biological scaffold, and preferentially solves the problem of burst release after PRP loading.

Preferably, the activator includes, but is not limited to, ca ²⁺ And prothrombin.

Preferably, the scaffold carrier includes, but is not limited to, hydrogel scaffold material, 3D printed scaffold material.

More preferably, the hydrogel scaffold material includes, but is not limited to, hyaluronic acid, polylysine, fibroin, gelatin, sodium alginate, collagen-based materials.

Preferably, the preparation method of the hydrogel scaffold material comprises the following steps:

preparing the material into a solution, adding a cross-linking agent or performing self-crosslinking in a mould, placing the solution at the temperature of minus 20 ℃ for reaction for 24 hours, and then performing freeze drying to obtain the hydrogel scaffold material with the porous structure.

The second purpose of the invention is to provide a preparation method of the composite scaffold material for slowly releasing platelet rich plasma, which comprises the following steps:

s1, preparing a scaffold carrier with a 3D porous structure;

obtaining a PRP;

and S2, uniformly mixing the PRP obtained in the S1 with an activating agent, injecting the mixture into the S1 scaffold material before the platelet-rich plasma is gelatinized (within 1 min), and standing at room temperature.

Preferably, in S2, the mixture is allowed to stand at room temperature for at least 5min.

The third purpose of the invention is to provide the application of the composite scaffold material for slowly releasing the platelet rich plasma in the preparation of a chronic wound repair material.

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

1. the present invention utilizes Ca ²⁺ Activating the platelets to release growth factors, so that fibrinogen in the plasma is converted into a fibrin network to form PRP gel; macroporous barrier utilizing scaffold material simultaneouslyUnder the action, the PRP forms gel in the macropore of the stent and is interpenetrated with the stent carrier network, thereby achieving the purpose of slowly releasing the PRP. Specifically, the method comprises the steps of firstly obtaining a scaffold carrier with a 3D porous structure, then adding PRP and an activator into the scaffold carrier, and directly adhering the released growth factors into the scaffold carrier after the PRP is activated, so that the loss of the growth factors is avoided, and the load capacity is improved; the PRP gel and the stent carrier network are interpenetrated to form a 'double-network' composite stent (the 'double-network' means two layers of cross-linked networks of the stent and the PRP gel network), which is beneficial to improving the PRP slow release effect, prolonging the action time of the PRP gel in vivo, reducing the blood extraction times, providing a long-term effective treatment method for transplantation cases which can not be operated repeatedly, and greatly relieving the pain of patients; meanwhile, the addition of the PRP endows the scaffold material with stronger biological activity, and the composite scaffold material gives full play to the advantages of the PRP and the scaffold material, thereby realizing the synergistic effect of adding one to more than two.

2. The preparation method can select naturally-occurring Ca in organism for PRP activation ²⁺ The PRP composite scaffold is prepared by a method of activating, wherein other chemical cross-linking agents are not introduced, the PRP composite scaffold can respond to a wound microenvironment for slow release, the biological activity of the wound microenvironment can not be damaged, the PRP composite scaffold is allowed to be combined with various tissue engineering scaffolds, including hydrogel scaffolds, 3D printing scaffolds, novel material scaffolds and the like, and the preparation materials of the scaffold can also be selected according to the required pertinence, so that the PRP composite scaffold has a wider application range.

Drawings

FIG. 1 shows the surface topography of PRP gel and PRP loaded stents before and after;

FIG. 2 shows the PRP release before and after loading;

FIG. 3 is a graph of type II diabetic rat skin wound repair;

FIG. 4 shows the expression of the indexes related to the inflammation and angiogenesis at the wound after the treatment of type II diabetic rats.

Detailed Description

In order to make the technical solutions of the present invention better understood and enable one skilled in the art to practice the present invention, the present invention is further described below with reference to specific examples and drawings, but the examples are not intended to limit the present invention.

The experimental methods and the detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The scaffold material has very wide application in the field of tissue engineering, and the tissue engineering scaffold has generally good biocompatibility and proper degradation performance matched with the regeneration speed of new tissues; the high porosity and 3D porous structure are beneficial to the migration of cells, the infiltration of blood vessels, the transportation of nutrient substances, the discharge of metabolic waste and certain mechanical strength, and can meet the complex mechanical environment in vivo; the source of the PRP clinical treatment is usually autologous blood, non-immunogenic; the multiple growth factors in the PRP are combined in a physiological proportion under a normal state of a human body, can be mutually associated in different stages in the wound healing process respectively to play a more superior synergistic effect, and are unique advantages which are difficult to achieve by a single recombinant growth factor; according to the invention, the PRP and the activator thereof are loaded on the composite stent by utilizing the macroporous barrier effect in the 3D porous structure stent, so that the PRP is activated in the macropores of the stent to form gel and interpenetrates with the stent carrier network, thereby forming the composite stent with the slow-release PRP, and the loading mode fully ensures the activity of the PRP. The sources of the above-mentioned PRP include humans and animals, and the preparation method thereof is not limited.

The composite scaffold material for sustained release of platelet rich plasma according to the present invention will be specifically described by the following examples.

Example 1

The preparation method comprises the steps of taking hyaluronic acid and polylysine as raw materials, firstly preparing a hyaluronic acid/polylysine hydrogel stent, injecting PRP and calcium gluconate solution into the hydrogel stent, activating the PRP in macropores of hydrogel to form gel, and interpenetrating the gel with a hydrogel stent carrier network to prepare the composite stent material for slowly releasing the PRP.

The preparation method of the composite scaffold material for sustained-release of PRP specifically comprises the following steps:

1) Preparing a scaffold material: at room temperature, 5% hyaluronic acid (HA,1.42×10 ⁶ da) and adding a chelating agent EDC into the aqueous solution, dissolving and stirring for 2h to fully activate carboxyl, thereby obtaining a reaction solution I. Preparing a 2% polylysine (epsilon-PLL) aqueous solution, adding an auxiliary chelating agent NHS into the aqueous solution, and adjusting the pH to 8.0 by using a NaOH solution to obtain a reaction solution II. And adding the reaction liquid II into the reaction liquid I, uniformly stirring, pouring into a mold, and crosslinking at room temperature for 1h to obtain the HA/epsilon-PLL hydrogel. After gelling, the gel is put into a temperature of minus 20 ℃ to react for 24h, and is fully pre-frozen, and then is transferred to a freeze drier to be frozen and dried for 24h, so as to obtain the HA/epsilon-PLL hydrogel scaffold with a porous structure.

2) Acquisition of PRP suspension: obtaining fresh sterile anticoagulation, and preparing PRP by a secondary centrifugation method: (1) centrifuge at 3500rpm for 15 minutes at room temperature. (2) After centrifugation, the blood can be observed to be divided into 3 layers, namely a plasma layer, a buffer coat layer and a red blood cell layer from top to bottom, the plasma layer, the buffer coat layer and part of red blood cells are collected and put into a sterile separation tube with serum separation gel, centrifugation is carried out for 15 minutes at 3500rpm, after centrifugation, the visible plasma and the inactivated PRP are positioned at the upper part of the separation gel, the red blood cells are positioned at the lower part of the separation gel, the red blood cells at the lower layer are discarded, and the PRP on the separation gel layer is collected. All the steps are operated in a clean bench. After being collected and mixed uniformly, the blood is analyzed and identified to determine the content of the blood platelet. Platelets were diluted 4-fold with plasma to give a suspension of inactive PRP.

3) Mixing the inactive PRP suspension and 10% calcium gluconate solution at a ratio of 10: 1, immediately injecting into a freeze-dried porous support to activate the PRP in the pores of the support to form gel, standing at room temperature for 5min to load the PRP into the porous support, wherein the surface appearances before and after loading are shown in FIG. 1.

As shown in fig. 1, the prepared hydrogel is a porous structure, and after the PRP is injected into the hydrogel, the PRP can be compounded with the hydrogel, and the released active substance can be directly adhered to the hydrogel matrix, thereby avoiding the loss of the active substance, improving the loading capacity, and facilitating the slow release of the active substance.

Example 2

The silk fibroin porous scaffold is prepared by taking silk as a raw material, PRP and calcium gluconate solution are injected into the scaffold, and the PRP is activated in macropores of the scaffold to form gel and interpenetrate with a scaffold carrier network to prepare the composite scaffold material for slowly releasing the PRP.

The preparation method of the composite stent material for sustained-release PRP comprises the following steps:

1) Preparing a scaffold material: weighing silk, dissolving in 9.3M lithium bromide solution to obtain 20% (w/v) silk solution, heating in water bath at 60 deg.C for 4 hr, and dialyzing with 3500Da dialysis bag for 48 hr; subsequently, dialysis was continued for 6-10h replacing 20% g/mL of PEG solution to obtain concentrated silk fibroin solution; centrifuging at 10000rpm and 4 deg.C for 20 min, removing impurities, determining the concentration of concentrated silk fibroin by weighing method, and adding appropriate amount of distilled water to make the concentration be 6%; adding 0.25% glutaraldehyde, reacting at-20 deg.C for 24h, transferring to a freeze drier, and freeze drying for 24h to obtain porous fibroin scaffold;

2) Acquisition of PRP suspension: obtaining fresh sterile anticoagulation, and preparing PRP by a secondary centrifugation method: (1) centrifuge at 3500rpm for 15 minutes at room temperature. (2) After centrifugation, the blood can be observed to be divided into 3 layers, a plasma layer, a buffer coat layer and a red blood cell layer are respectively arranged from top to bottom, the plasma layer, the buffer coat layer and part of red blood cells are collected and put into an aseptic separating tube with serum separating gel, centrifugation is carried out for 15 minutes at 3500rpm, after centrifugation, the plasma and the inactivated PRP are positioned at the upper part of the separating gel, the red blood cells are positioned at the lower part of the separating gel, the red blood cells at the lower layer are discarded, and the PRP on the separating gel layer is collected. All the steps are operated in a clean bench. After being collected and mixed uniformly, the blood is analyzed and identified to determine the content of the blood platelet. Platelets were diluted 4-fold with plasma to give a suspension of inactive PRP.

3) Mixing the inactive PRP suspension and 10% calcium gluconate solution at a ratio of 10: 1, immediately injecting into the freeze-dried porous scaffold to activate PRP in pores of the scaffold to form gel, standing at room temperature for 5min, and loading PRP into the porous scaffold.

Example 3

The preparation method comprises the steps of preparing a scaffold by taking collagen as a raw material, injecting PRP and calcium gluconate solution into the scaffold, activating the PRP in macropores of the scaffold to form gel, and interpenetrating the gel with a scaffold carrier network to prepare the composite scaffold material for slowly releasing the PRP.

1) Preparing a scaffold material: placing the mixed solution of 2 percent collagen and 0.135 mu M sodium hydroxide at the temperature of minus 20 ℃ for reaction for 24 hours;

2) Acquisition of PRP suspension: obtaining fresh sterile anticoagulation, and preparing PRP by a secondary centrifugation method: (1) centrifuge at 3500rpm for 15 minutes at room temperature. (2) After centrifugation, the blood can be observed to be divided into 3 layers, a plasma layer, a buffer coat layer and a red blood cell layer are respectively arranged from top to bottom, the plasma layer, the buffer coat layer and part of red blood cells are collected and put into an aseptic separating tube with serum separating gel, centrifugation is carried out for 15 minutes at 3500rpm, after centrifugation, the plasma and the inactivated PRP are positioned at the upper part of the separating gel, the red blood cells are positioned at the lower part of the separating gel, the red blood cells at the lower layer are discarded, and the PRP on the separating gel layer is collected. All the steps are operated in a clean bench. And collecting, uniformly mixing, and performing blood analysis and identification to determine the content of the platelets. Platelets were diluted 4-fold with plasma to give a suspension of unactivated PRP.

3) Mixing the inactive PRP suspension and 10% calcium gluconate solution at a ratio of 10: 1, immediately injecting into the freeze-dried porous scaffold to activate PRP in pores of the scaffold to form gel, standing at room temperature for 5min, and loading PRP into the porous scaffold. Transferring to a freeze dryer for freeze drying for 24h to obtain the collagen porous scaffold with a porous structure.

The materials prepared in the above examples were tested for their properties as follows.

1. Total protein Release assay

The stent, the PRP and the PRP loading stent in the example 1 are respectively added into 4.0mL of simulated body fluid, the mixture is placed on a vertical suspension instrument, the release efficiency of the PRP is detected under the condition of 37 ℃, 200 mu L of reaction liquid is respectively taken out from 4h, 12h, 24h, 2d, 4d and 7d, and 200 mu L of the simulated body fluid is supplemented. After the reaction, the concentration of total protein in the reaction solution was measured by using the BCA kit. After the stent is loaded, the slow release of the PRP can be effectively realized, as shown in figure 2, the PRP release speed is higher as seen in figure 2, and the PRP is loaded on the hydrogel stent, the release speed is slower, which shows that the strategy of the invention can reduce the release speed of the active substance generated by the PRP and has the slow release effect.

2. Test for wound healing

In order to sufficiently reduce the onset condition of chronic skin wounds, SD rats were fed high-sugar and high-fat for 8 weeks and then injected with 2mg/mL Streptozotocin (STZ) at an injection dose of 20mg/kg to model type II diabetic rats. When the blood sugar is more than or equal to 10mmol/L and the polydipsia and diuresis are accompanied, the body weight is reduced, and the model is formed. Molded rats were selected for anesthesia with 3% sodium pentobarbital (i.v. 40 mg/kg), followed by preparation of a full-thickness skin defect of 8mm x 8mm on their backs using sterile skin trephines and implantation of empty stents, PRP and PRP loaded stents in example 1. Animals were sacrificed on

days

7, 14, and 21, respectively, and the defective tissues were collected and fixed and embedded in OCT, and the embedded samples were frozen at 6 μm and sectioned, and after the sections were rehydrated, HE staining was performed to observe the distribution of cells, and immunofluorescence staining was performed to observe the formation of specific proteins such as platelet-endothelial adhesion molecule (CD 31), α smooth muscle actin (α -SMA), inducible Nitric Oxide Synthase (iNOS), and macrophage arginase (Arg-1), thereby evaluating the regeneration status.

As a result, as shown in fig. 3 and 4, the scaffold-loaded PRP group was more effective in repairing skin defects, and histological staining analysis showed that the scaffold-loaded PRP group was more effective in epithelialization process and collagen deposition. Immunofluorescence staining shows that the bracket-loaded PRP group can obviously promote angiogenesis, shorten the transition time from the inflammatory phase to the proliferation phase of the wound and reduce inflammatory reaction. The results prove that the PRP load stent prepared by the invention has the sustained-release effect, prolongs the action time and has better treatment effect.

The results show that the method provided by the invention can be suitable for combining different tissue engineering scaffold materials with PRP, and the prepared composite scaffold material has a PRP slow-release effect and can effectively promote the healing of chronic skin wounds. In addition, it should be noted that, the invention, which adopts the steps of preparing the scaffold carrier with the 3D porous structure and then adding the PRP and the activator into the scaffold carrier, has the following advantages compared with the case that the PRP is mixed in the preparation process of the scaffold carrier:

(1) The PRP is mixed during the preparation of the stent material, and the cross-linking agent and other substances present in the system may destroy the activity of the PRP during stirring and mixing, resulting in the loss of PRP. The loading mode of direct activation of the invention is a loading mode of physical mode, and during the process of activating PRP, no interference of other chemical substances exists, and no damage is generated to the biological activity of the PRP.

(2) The load mode adopted by the invention can ensure the sterility of the PRP composite bracket preparation process. After the stent material is obtained, the stent material can be sterilized by alcohol, ultraviolet and the like, and then the obtained sterile PRP and the activator thereof are directly injected into the stent. The aseptic property of the material cannot be guaranteed by directly mixing the PRP with the material, and in order to guarantee the activity of the PRP, the composite material obtained by adding the PRP into the material cannot be sterilized by alcohol, irradiation and other modes.

(3) The load mode adopted by the invention can ensure the current use of the PRP composite bracket. When not in use, PRP can be stored at-80 ℃, and the sterilized bracket can be sealed and stored; when needed, taking out the PRP, adding a proper amount of activating agent, mixing and injecting into the bracket. The preparation processes of the PRP and the stent material are not mutually interfered, the mixing process has no gap, and the PRP and the stent material are easy to store and obtain and have higher clinical feasibility.

(4) The load mode adopted by the invention can effectively realize the slow release of the PRP, and the macroporous barrier effect of the stent material can ensure that the PRP slowly releases growth factors to the wound part, thereby effectively serving each period of wound recovery. The PRP gel network and the stent material are not chemically crosslinked due to the pure physical load, so that the performance of the stent is not affected, the bioactivity of the stent is greatly enhanced, and the effect of 1+1 & gt 2 is realized.

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, it is intended that such changes and modifications be included within the scope of the appended claims and their equivalents.

Claims

1. The composite scaffold material for slowly releasing the platelet rich plasma is characterized in that the platelet rich plasma and an activating agent thereof are loaded on a scaffold carrier with a 3D porous structure, so that the platelet rich plasma is activated in macropores of the scaffold carrier to form gel and interpenetrates with a scaffold carrier network, and the composite scaffold material with the slowly releasing platelet rich plasma is formed.

2. The method for preparing a composite scaffold material for sustained release of platelet rich plasma according to claim 1, wherein said activator comprises Ca ²⁺ And prothrombin.

3. The method for preparing the composite scaffold material for sustained release of platelet rich plasma according to claim 1, wherein the scaffold carrier comprises hydrogel scaffold material and 3D printing scaffold material.

4. The method for preparing the composite scaffold material for slowly releasing platelet rich plasma according to claim 3, wherein the hydrogel scaffold material comprises hyaluronic acid, polylysine, fibroin, gelatin, sodium alginate and collagen-based materials.

5. The method for preparing the composite scaffold material for slowly releasing platelet-rich plasma according to claim 4, wherein the method for preparing the hydrogel scaffold material comprises the following steps:

preparing the material into a solution, adding a cross-linking agent or carrying out self-crosslinking in a mould, then placing the solution at the temperature of minus 20 ℃ for reaction, and then carrying out freeze drying to obtain the hydrogel scaffold material with a porous structure.

6. The method for preparing the composite scaffold material for sustained release of platelet rich plasma according to any one of claims 1 to 5, comprising the steps of:

s1, preparing a scaffold carrier with a 3D porous structure;

obtaining platelet rich plasma;

and S2, uniformly mixing the platelet rich plasma obtained in the step S1 with an activating agent, injecting the mixture into the S1 support carrier before the platelet rich plasma is gelatinized, and standing at room temperature.

7. The method according to claim 6, wherein the mixture is allowed to stand at room temperature for at least 5min in S2.

8. The method according to claim 6, wherein the concentration of the platelet-rich plasma in S2 is 4 times that of the physiological concentration, and the volume ratio of the platelet-rich plasma to the 10% by mass calcium gluconate solution is 10: 1.

9. The use of the composite scaffold material for sustained release of platelet rich plasma according to claim 1 in the preparation of a chronic wound repair material.