Preparation method and application of photocuring autoplatelet concentrate hydrogel

Abstract

The invention is applicable to the technical field of biological materials, and provides a preparation method of photocuring autoplatelet concentrate hydrogel, which comprises the following steps: preparing methacrylated gelatin; preparing injectable platelet-rich fibrin; the preparation method comprises the following steps: preparing methacrylated iPRF hydrogel: weighing 500mg of a GelMA hydrogel precursor, placing the GelMA hydrogel precursor in 4ml of PBS, placing the PBS in a water bath box at 55 DEG C, and fully dissolving the GelMA hydrogel precursor to obtain a GelMA solution with the mass fraction of 10%; weighing 1mg of LAP photoinitiator, adding the LAP photoinitiator into the system solution, and stirring for 5 minutes; loading the liquid into a centrifugal tube, then adding 1ml of iPRF, fully and uniformly mixing the two components by using an injector, and irradiating for 30s under ultraviolet light of 405nm to crosslink the two components to obtain the iPRF-MA hydrogel. The method is mild in reaction condition and can be used at room temperature; the method is simple in experiment, good in repeatability and suitable for batch production. Meanwhile, the autoblood of the patient is taken as a raw material and is easy to obtain, low in cost and free of side effects, and real personalized medical treatment is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a preparation method and application of a photocurable autologous platelet concentrate hydrogel. Background Art

[0002] Tissue defects caused by tumors, trauma, inflammation and congenital diseases have always been a difficult problem in clinical treatment. Wound healing and tissue regeneration after defect formation is a dynamic and complex process. At present, the ideal means is to use regenerative medicine strategies to promote wound healing and tissue regeneration. The key to regenerative medicine is the three pillars of tissue engineering: scaffolds, cells and bioactive factors. Simply put, it is to promote the regeneration of new tissues by loading seed cells and bioactive factors on scaffold materials. There are some problems in this. Traditional biomaterial scaffolds lack biological activity, and the application of exogenous growth factors has problems such as high cost, poor safety and unpredictable effects. In this case, human autologous platelet concentrates (APC) have come into people's view. APC is obtained by centrifuging blood. In order to utilize the therapeutic potential of biomolecules secreted by platelets, blood deconstruction can produce autologous platelets and plasma-derived protein fibrin scaffolds. Autologous growth factors derived from platelets and plasma interact with fibrin, extracellular matrix and tissue cells in a combined, synergistic and multidirectional manner, thereby controlling the mechanism of tissue repair. As a type of biological product derived from the patient's blood, it naturally contains important elements in tissue engineering: Scaffold - a three-dimensional fibrin network formed by the polymerization of fibrinogen, whose unique intrinsic characteristics can directly promote wound healing by regulating cell behavior, and can also become a delivery system for bioactive molecules through spontaneous reactions in the body. Bioactive factors - a variety of growth factors released by platelet degranulation during the preparation process, because these growth factors are endogenous and have an optimal physiological ratio, they have a synergistic effect on tissue regeneration and have no side effects.

[0003] At present, various types of blood derivatives have been applied to different types of tissue defects, such as bone defects, periodontal tissue regeneration and soft tissue healing, and have shown good therapeutic effects. However, systematic reviews contradict the therapeutic effects, which is mainly due to the following disadvantages of autologous blood derivatives: ① Rapid degradation, unable to maintain long-term release of growth factors, and unable to ensure the long-term presentation of these biochemical signals. ② Poor mechanical properties, and poor matching between the mechanical properties and the complex environment of the tissue regeneration site, especially when it is used alone in bone defects, and it is difficult to maintain long-term structural integrity. ③ Limited clinical operation. Most of the APCs commonly used in clinical practice are already in a gel state after preparation, or uncontrollably form a gel spontaneously and need to be squeezed to remove water before being placed in the defect. Moreover, they have no adhesion to tissues, require complex suture fixation, and take a long time to operate. Therefore, it is of great clinical significance to develop an autologous platelet concentrate hydrogel with excellent mechanical properties, sustained release of growth factors, and controllable sol-gel conversion.

[0004] At present, the use of biomaterials to modify APC is an emerging strategy. Most APCs, such as PRF and CGF, are directly in a colloidal state after preparation, and it is difficult to effectively combine with biomaterials. As a type of APC, injectable platelet-rich fibrin (iPRF) is in a liquid state at the beginning of preparation, and then slowly gels through coagulation in about 7 minutes. This characteristic is used to mix iPRF with biomaterials at the beginning of preparation, so that it can be evenly combined with hydrogels, and the chemical bonds used for coagulation can be combined with the chemical bonds in the hydrogel. On the one hand, it can block its spontaneous gelation process, and then control the gelation process through specific groups in the hydrogel (such as photoinitiator groups, thermosensitive groups) to achieve controllable sol-gel conversion. On the other hand, it can form a new network system based on the network of the hydrogel and use the characteristics of the hydrogel to improve the shortcomings of too fast growth factor release and poor mechanical properties. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a method for preparing and applying a photocurable autologous platelet concentrate hydrogel, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The embodiment of the present invention is implemented as follows: a method for preparing a photocurable autologous platelet concentrate hydrogel comprises the following steps:

[0007] Step 1, preparation of GelMA;

[0008] Step 2, preparation of iPRF;

[0009] Step 3: Preparation of iPRF-MA hydrogel:

[0010] Step 3.1: Weigh 500 mg of GelMA hydrogel precursor and place it in 4 ml of PBS, place it in a 55°C water bath, and wait for it to fully dissolve to obtain a 10% GelMA solution. Weigh 1 mg of LAP photoinitiator and add it to the above system solution, and stir for 5 minutes.

[0011] Step 3.2: Pour the above liquid into a centrifuge tube, then add 1 ml of iPRF, use a syringe to fully mix the two components, and irradiate with 405 nm ultraviolet light for 30 seconds to cross-link them to obtain iPRF-MA hydrogel.

[0012] According to a further technical solution, step 1 comprises the following specific steps:

[0013] Step 1.1, 5 g of type A pig skin gelatin was added to 50 ml of phosphate buffered saline (PBS) in portions. The system was placed in a 50°C water bath and stirred continuously at 400 rpm until completely dissolved, thereby obtaining a GelMA hydrogel precursor solution with a concentration of 10% (w / v).

[0014] Step 1.2: The GelMA hydrogel precursor solution was stirred at 400 rpm in a 50°C environment, and 1.5-3 ml of methacrylic acid was added dropwise at a rate of 0.5 ml / min. After the reaction was continued for 3 hours, NaOH solution or powder was added until the pH was adjusted to 7.2.

[0015] Step 1.3, pour the GelMA hydrogel precursor solution into 300ml of mild PBS solution at 50℃, mix well and transfer the liquid to 14000KD dialysis membrane. Keep it dialyzed in a 50℃ distilled water bath for 3-7 days. During this period, change the distilled water every 2 hours for the first two days, and the frequency of water change can be reduced to 3-4 times / day in the later period until equilibrium is reached.

[0016] Step 1.4: After the dialysis is completed, the GelMA hydrogel precursor solution is filtered using a filter with a pore diameter of 0.22 μm.

[0017] Step 1.5: The GelMA hydrogel precursor solution after filtration was aspirated out in batches and transferred to a large dish for freeze drying for 2 days to finally obtain a white porous foam-like GelMA hydrogel precursor.

[0018] According to a further technical solution, step 2 comprises the following specific steps:

[0019] Step 2.1. Take 5 ml of whole blood and put it into a plastic blood collection tube without anticoagulant.

[0020] Step 2.2: Centrifuge at 300 g for 3 minutes at room temperature and collect the upper light yellow liquid, which is iPRF.

[0021] Another purpose of an embodiment of the present invention is to provide an application of a photocured autologous platelet concentrate hydrogel, based on the iPRF-MA hydrogel prepared by the above method, and to use the iPRF-MA hydrogel as a scaffold to treat wound healing and tissue regeneration after defect formation.

[0022] The preparation method and application of a photocurable autologous platelet concentrate hydrogel provided in an embodiment of the present invention, wherein injectable platelet-rich fibrin is obtained by low-speed centrifugation of autologous blood, and GelMA hydrogel solution is added when it is still in a liquid state at the initial stage. In this process, the fibrinogen of iPRF combines with GelMA through hydrogen bonds and electrostatic interactions, thereby blocking its spontaneous coagulation reaction, and then forms a cross-linked network by activating the photosensitive groups in the hydrogel, thereby realizing the light-controlled gelation of iPRF. The iPRF-MA hydrogel scaffold synthesized by the present invention, while improving its mechanical properties, is endowed with the characteristics of light-controlled gelation, so that it can fill irregular tissue defects in an injectable manner, and then form a hydrogel with good mechanical properties in situ in the tissue by light irradiation, which greatly facilitates clinical use. At the same time, through the encapsulation effect of the hydrogel and the affinity of the GelMA component in the hydrogel for the growth factor, the problem of explosive release of the initial growth factor of the traditional APC is overcome, and the sustained release of the growth factor is realized, so that it can play a role in the process of tissue regeneration for a long time. The growth factors in iPRF give the hydrogel strong biological activity, promote cell migration, adhesion and proliferation, induce cell positioning, adhesion and colonization while providing a microenvironment suitable for cell growth, and guide cell proliferation and differentiation. In addition, the various growth factors in the hydrogel can greatly promote vascularization and achieve rapid regeneration of tissues. The iPRF-MA hydrogel constructed by this method has shown ideal mechanical properties in relevant characterizations, and has been shown to play a key role in the process of tissue defect repair in in vitro and in vivo experiments, and has greatly promoted the expression of angiogenesis-related genes. Therefore, the present invention is expected to be applied to clinical treatments related to tissue defect repair in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 are scanning electron micrographs of GelMA and iPRF-MA;

[0024] Figure 2 is the macroscopic morphology of iPRF-MA;

[0025] Figure 3 The stress-strain curve and storage modulus and loss modulus curves of iPRF-MA;

[0026] Figure 4 is the release curve of growth factors in iPRF and iPRF-MA;

[0027] Figure 5 The growth of fibroblasts in iPRF and iPRF-MA;

[0028] Figure 6 Transwell results and cell migration rates of the blank group, GelMA group, and iPRF-MA group;

[0029] Figure 7 The scratch test results and cell migration rate of human umbilical vein endothelial cells (HUVECs) in the blank group, GelMA group and iPRF-MA group;

[0030] Figure 8 This is the result of the tube formation experiment of iPRF-MA;

[0031] Fig. 9 Schematic diagram of animal experiments of iPRF-MA;

[0032] Fig.10 H&E staining and Masson staining of skin tissue using iPRF-MA in a rat full-thickness wound model. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0035] An embodiment of the present invention provides a method for preparing a photocurable autologous platelet concentrate hydrogel, comprising the following steps:

[0036] Step 1, preparation of GelMA:

[0037] Step 1.1, 5 g of type A pig skin gelatin was added to 50 ml of phosphate buffered saline (PBS) in portions. The system was placed in a 50°C water bath and stirred continuously at 400 rpm until completely dissolved, thereby obtaining a GelMA hydrogel precursor solution with a concentration of 10% (w / v).

[0038] Step 1.2: The GelMA hydrogel precursor solution was stirred at 400 rpm in a 50°C environment, and 1.5-3 ml of methacrylic acid was added dropwise at a rate of 0.5 ml / min. After the reaction was continued for 3 hours, NaOH solution or powder was added until the pH was adjusted to 7.2.

[0039] Step 1.3, pour the GelMA hydrogel precursor solution into 300ml of mild PBS solution at 50℃, mix well and transfer the liquid to 14000KD dialysis membrane. Keep it dialyzed in a 50℃ distilled water bath for 3-7 days. During this period, change the distilled water every 2 hours for the first two days, and the frequency of water change can be reduced to 3-4 times / day in the later period until equilibrium is reached.

[0040] Step 1.4: After the dialysis is completed, the GelMA hydrogel precursor solution is filtered using a filter with a pore diameter of 0.22 μm.

[0041] Step 1.5: The GelMA hydrogel precursor solution after filtration was aspirated out in batches and transferred to a large dish for freeze drying for 2 days to finally obtain a white porous foam-like GelMA hydrogel precursor.

[0042] Step 2: Preparation of iPRF:

[0043] Step 2.1. Take 5 ml of rat whole blood and put it into a plastic blood collection tube without anticoagulant.

[0044] Step 2.2: Centrifuge at 300 g for 3 minutes at room temperature and collect the upper light yellow liquid, which is iPRF.

[0045] Step 3: Preparation of iPRF-MA hydrogel:

[0046] Step 3.1: Weigh 500 mg of GelMA hydrogel precursor and place it in 4 ml of PBS, place it in a 55°C water bath, and wait for it to fully dissolve to obtain a 10% GelMA solution. Weigh 1 mg of LAP photoinitiator and add it to the above system solution, and stir for 5 minutes.

[0047] Step 3.2: Pour the above liquid into a centrifuge tube, then add 1 ml of iPRF, use a syringe to fully mix the two components, and irradiate with 405 nm ultraviolet light for 30 seconds to cross-link them to obtain iPRF-MA hydrogel.

[0048] In the embodiment of the present invention, in step 2, during the preparation of iPRF, rat whole blood is taken as an example to facilitate subsequent physical and chemical research. In actual application, injectable platelet-rich fibrin can be obtained by low-speed centrifugation of the patient's autologous blood.

[0049] Performance evaluation:

[0050] Figure 1 For GelMA( Figure 1 A) and iPRF-MA( Figure 1B) Scanning electron microscopy image, the results show that iPRF-MA hydrogel has a uniform pore structure, which indicates that the hydrogel is conducive to cell migration and nutrient exchange.

[0051] Figure 2 This is the macroscopic morphology of iPRF-MA. The results show that iPRF-MA has good injectability and plasticity.

[0052] Figure 3 The stress-strain curve and storage modulus and loss modulus curve of iPRF-MA, where A is the stress-strain curve of iPRF-MA with different proportions; B is the storage modulus and loss modulus curve of iPRF-MA, and the red arrow indicates the sol-gel transition point triggered by light. The results show that iPRF-MA containing 25% iPRF has both good biological activity and mechanical strength, and compared with pure iPRF, the mechanical strength of iPRF-MA hydrogel is increased to 1500pa, which is about 15 times that of iPRF, indicating that the present invention has greatly improved the mechanical strength of traditional iPRF.

[0053] Figure 4 The release curves of growth factors (PDGF and VEGF) in iPRF and iPRF-MA show that obvious growth factor burst release can be found in traditional iPRF at the initial time, and the release of growth factors can only be maintained for 5-7 days. In contrast, the release rate of growth factors in iPRF-MA hydrogel is significantly slower than that of iPRF hydrogel, and the release time is as long as 14 days. This shows that iPRF-MA hydrogel can not only hinder the rapid degradation of iPRF through encapsulation, but also the GelMA component can provide high adhesion ability for growth factors. The above characteristics synergistically hinder the rapid release of growth factors in iPRF, allowing growth factors to play a role in tissue healing for a long time.

[0054] Figure 5 The live / dead cell fluorescence staining results of iPRF and iPRF-MA after 1, 4 and 7 days of cell culture. Live cells appear green due to calcein-AM, while dead cells appear red due to propidium iodide (PI) staining. As shown in the figure, the iPRF-MA hydrogel group showed the highest green fluorescence intensity at each time point. The above results show that iPRF-MA hydrogel is more conducive to cell migration and proliferation.

[0055] Figure 6 A is the transwell results of the blank group, GelMA group and iPRF-MA group. Figure 6B is the cell migration rate of the blank group, GelMA group and iPRF-MA group. The degree of vascularization determines the healing speed of chronic wounds. New blood vessels can deliver oxygen and nutrients to the wound site, which can accelerate tissue regeneration. As the key to vascular regeneration, the proliferation, migration and tube formation of vascular endothelial cells (HUVEC) determine the regeneration effect of tissues. The transwell experiment was used to verify the effect of hydrogel on the migration ability of HUVEC. As shown in the figure, iPRF-MA significantly enhanced the migration ability and migration rate of HUVEC.

[0056] Figure 7 A shows the results of scratch test of human umbilical vein endothelial cells (HUVECs) in blank group, GelMA group and iPRF-MA group. Figure 7 A is the cell migration rate of the scratch test in the blank group, GelMA group and iPRF-MA group. As shown in the figure, iPRF-MA and CurCDs@iPRF-MA significantly enhanced the migration ability and migration rate of HUVEC. After 48 hours of culture, iPRF-MA increased the cell migration rate from 53% to 99%.

[0057] Figure 8 The results of the tube formation experiment of iPRF-MA are shown in Figure 2. The tube formation assay was then performed to evaluate the effect of the hydrogel on the angiogenic potential of HUVEC. As shown in the figure, iPRF-MA showed higher efficiency in angiogenesis, characterized by a larger number of tubes and more obvious tubular structures. This shows that iPRF-MA can effectively promote cell migration and angiogenesis in vitro, thereby promoting tissue regeneration.

[0058] Fig. 9 A is a simulation diagram of animal experiments. Fig. 9 B shows the skin wound healing of rats in the blank group, GelMA group and iPRF-MA group at different time points after surgery. Fig. 9 C is the wound healing simulation diagram of the blank group, GelMA group and iPRF-MA group. Fig. 9D is the wound healing rate of the blank group, GelMA group and iPRF-MA group. A full-thickness wound model of diabetic rats with a diameter of 5 mm was created, and GelMA and iPRF-MA hydrogels were applied topically to the wounds, while the blank control group was treated with normal saline. The wounds were systematically monitored and photographed at 0, 3, 6, 9 and 12 days after surgery, and the remaining wound area was calculated using ImageJ software. The results showed that diabetic wounds treated with iPRF-MA hydrogel showed the fastest healing process at all time points, and no serious infection was found in the iPRF-MA group during the healing period, while the GelMA group and the control group showed signs of infection 3 and 6 days after surgery. After treatment with iPRF-MA hydrogel, the healing of diabetic wounds made significant progress, and a reduction of more than 50% in wound area was observed on the 6th day after surgery. By the 21st day, the wounds in the iPRF-MA group showed a state close to healing, and the newly formed wounds were completely covered with epithelial tissue. It can be seen that the iPRF-MA hydrogel of the present invention has a certain therapeutic effect on wounds.

[0059] Fig.10 The H&E staining and Masson staining images of skin tissue in a rat full-thickness wound model using iPRF-MA. As shown in the figure, the iPRF-MA group showed abundant newly formed granulation tissue and intact epithelium on the wound surface, indicating active healing and regeneration. In contrast, the control group and the GelMA group only showed scattered newly formed tissue. Only a small amount of collagen fibers appeared in the control group, and they were loose and disordered. The iPRF-MA group showed more collagen deposition, and the collagen fibers in the skin tissue were denser, thicker, and better arranged. It can be seen that the iPRF-MA hydrogel prepared by this method has a significant therapeutic effect on full-thickness wounds in rats.

[0060] Treatment of tissue defects caused by factors such as trauma, tumors, and infections is a key clinical problem. As one of the main means of reconstruction and repair of defects, tissue engineering has become a promising repair method by implanting a scaffold that promotes tissue healing to assist the repair and recovery of defective tissues. APC is increasingly being selected to treat tissue defects because it has an optimal ratio of endogenous growth factors and is completely derived from the patient himself without any side effects. However, due to problems such as poor mechanical properties, too fast release of growth factors, and high sensitivity of clinical technology, its further application in clinical practice is limited. The present invention adopts an extremely simple preparation technology to synthesize a personalized iPRF-MA hydrogel scaffold. While improving its mechanical properties, it is endowed with the characteristics of light-controlled gelation, so that it can fill irregular tissue defects in an injectable manner, and then form a hydrogel with good mechanical properties in situ in the tissue by light, which greatly facilitates clinical use. At the same time, through the encapsulation effect of the hydrogel and the affinity of the GelMA component in the hydrogel for the growth factor, the problem of explosive release of growth factors in the early stage of traditional APC is overcome, and the sustained release of growth factors is achieved, so that it can play a role in the process of tissue regeneration for a long time. The growth factors in iPRF give the hydrogel strong biological activity, promote cell migration, adhesion and proliferation, induce cell positioning, adhesion and colonization while providing a microenvironment suitable for cell growth, and guide cell proliferation and differentiation. In addition, the various growth factors in the hydrogel can greatly promote vascularization and achieve rapid regeneration of tissues. The iPRF-MA hydrogel constructed by the present invention has shown ideal mechanical properties in relevant characterizations, and has been proven to play a key role in the process of tissue defect repair in in vitro and in vivo experiments, and has greatly promoted the expression of angiogenesis-related genes. Therefore, it can be applied to clinical treatments related to tissue defect repair.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a photocurable autologous platelet concentrate hydrogel, characterized in that: A method for preparing a photocurable autologous platelet concentrate hydrogel comprises the following steps: Step 1, preparation of GelMA; Step 2, preparation of iPRF; Step 3: Preparation of iPRF-MA hydrogel: Step 3.1, weigh 500 mg of GelMA hydrogel precursor and place it in 4 ml of PBS, place it in a 55°C water bath, and wait for it to fully dissolve to obtain a GelMA solution with a mass fraction of 10%; weigh 1 mg of LAP photoinitiator and add it to the above solution, and stir for 5 minutes; Step 3.2: Pour the prepared liquid into a centrifuge tube, then add 1 ml of iPRF, use a syringe to fully mix the two components, and irradiate with 405 nm ultraviolet light for 30 seconds to cross-link them to obtain iPRF-MA hydrogel.

2. The method for preparing the photocurable autologous platelet concentrate hydrogel according to claim 1, characterized in that: The step 1 comprises the following specific steps: Step 1.1, 5 g of type A pig skin gelatin was added to 50 ml of diphosphate buffered saline in portions, and the system was placed in a 50° C. water bath and continuously stirred at 400 rpm until completely dissolved, thereby obtaining a GelMA hydrogel precursor solution with a concentration of 10%; Step 1.2, the GelMA hydrogel precursor solution was stirred at 400 rpm in a 50°C environment, and 1.5-3 ml of methacrylic acid was added dropwise at a rate of 0.5 ml / min. After the reaction was continued for 3 hours, NaOH solution or powder was added until the pH was adjusted to 7.2; Step 1.3, pour the GelMA hydrogel precursor solution into 300 ml of mild PBS solution at 50°C, transfer the liquid to a 14000KD dialysis membrane after mixing evenly, and keep it dialyzed in a 50°C distilled water bath for 3-7 days; during this period, replace the distilled water every 2 hours for the first two days, and reduce the frequency of water change to 3-4 times / day in the later period until equilibrium is reached; Step 1.4: After dialysis, the GelMA hydrogel precursor solution was filtered using a filter with a pore diameter of 0.22 μm; Step 1.5: The GelMA hydrogel precursor solution after filtration was aspirated out in batches and transferred to a large dish for freeze drying for 2 days to finally obtain a white porous foam-like GelMA hydrogel precursor.

3. The method for preparing the photocurable autologous platelet concentrate hydrogel according to claim 2, characterized in that: The step 2 comprises the following specific steps: Step 2.1, take 5 ml of whole blood and put it into a plastic blood collection tube without anticoagulant; Step 2.2: Centrifuge at 300 g for 3 minutes at room temperature and collect the upper light yellow liquid, which is iPRF.

4. An application of a photocurable autologous platelet concentrate hydrogel, iPRF-MA hydrogel prepared by the method for preparing a photocurable autologous platelet concentrate hydrogel according to any one of claims 1 to 3, characterized in that: iPRF-MA hydrogel is used as a scaffold to treat wound healing and tissue regeneration after defect formation.

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