CN115040694B

CN115040694B - Bone regeneration biological composite scaffold with blood vessel and nerve functions and preparation method and application thereof

Info

Publication number: CN115040694B
Application number: CN202210574213.XA
Authority: CN
Inventors: 吴成铁; 秦宸
Original assignee: Shanghai Institute of Ceramics of CAS
Current assignee: Shanghai Institute of Ceramics of CAS
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2023-06-02
Anticipated expiration: 2042-05-25
Also published as: CN115040694A

Abstract

The invention discloses a bone regeneration biological composite scaffold with blood vessel and nerve functions, a preparation method and application thereof. The biological composite scaffold comprises a polymer network formed by solidifying a hydrogel material, and biological ceramic materials and angiogenesis-related cells which are uniformly distributed in the polymer network, wherein the angiogenesis-related cells comprise at least one of vascular endothelial cells, vascular progenitor cells and vascular smooth muscle cells, and the biological ceramic materials contain at least one of silicon, calcium, lithium, magnesium, copper, cobalt, zinc, strontium and manganese.

Description

Bone regeneration biological composite scaffold with blood vessel and nerve functions and preparation method and application thereof

Technical Field

The invention relates to a bone regeneration biological composite scaffold with blood vessel and nerve functions, a preparation method and application thereof, and belongs to the technical field of biological materials.

Background

The current clinical treatment methods do not achieve the goal of complete, efficient and large area tissue repair. The problems of limited bridging of serious tissues, insufficient nutrient transmission and the like faced by massive tissue or organ defects are still the medical problems to be solved. Most tissues and organs of the human body are highly vascularized and innervated, and blood vessels and neural networks are concomitantly distributed in the human body, complement each other, and fully participate in the regeneration process of most human tissues by delivering nutrients, cytokines and neurotransmitters. Therefore, in order to effectively regenerate fully vascularized and innervated tissue, one of the keys is to reconstruct both the blood vessels and the neural network simultaneously. The development of a scaffold capable of promoting vascular-nerve integrated tissue repair in large defects has important practical significance.

Disclosure of Invention

Aiming at the problems, the invention provides a bone regeneration biological composite stent with double functions of blood vessels and nerves, and a preparation method and application thereof. The biological composite scaffold has long-term cell activity, has biological activity of inducing neuroblastosis of nerve cells and directional differentiation of other tissue cells, can realize tissue regeneration of blood vessel-neuronization, and can effectively promote the blood vessel-neuronization repair of tissue defects, thereby being a potential treatment means for treating massive tissue or organ defects.

In a first aspect, the present invention provides a bone regeneration bio-composite scaffold having both vascular and neural functions. The biological composite scaffold comprises a polymer network formed by solidifying a hydrogel material, and biological ceramic materials and angiogenesis-related cells which are uniformly distributed in the polymer network, wherein the angiogenesis-related cells comprise at least one of vascular endothelial cells, vascular progenitor cells and vascular smooth muscle cells, and the biological ceramic materials contain at least one of silicon, calcium, lithium, magnesium, copper, cobalt, zinc, strontium and manganese.

Preferably, the matrix of the hydrogel material is at least one of methacryl gelatin, methacryl hyaluronic acid, gellan gum, sodium alginate, methylcellulose, gelatin and chitosan.

Preferably, the mass concentration of the hydrogel material in the biological composite stent is 5-20%.

Preferably, the mass concentration of the cells related to angiogenesis in the biological composite scaffold is 0.001-1%.

Preferably, the mass of the bioceramic material of the biocomposite stent is less than 10% of the hydrogel material, and preferably 0.001-10%.

Preferably, the biological ceramic material is at least one of calcium silicate, magnesium yellow feldspar, cobalt yellow feldspar, copper tobermorite, lithium magnesium silicate, lithium calcium silicate, strontium silicate, magnesium silicate and manganese silicate.

Preferably, the particle size of the bioceramic material is 10 μm to 200 μm.

In a second aspect, the present invention provides a method for preparing a bone regeneration bio-composite scaffold having both vascular and neural functions as described in any one of the above. The preparation method comprises the following steps: uniformly mixing a biological ceramic material, cells related to angiogenesis and a hydrogel material to form biological ink; setting the temperature of the biological ink to 15-20 ℃, extruding, forming, crosslinking and solidifying the biological ink to obtain the bone regeneration biological composite scaffold with the double functions of blood vessels and nerves.

In a third aspect, the present invention provides the use of a bone regenerating biological composite scaffold having both vascular and neural functions as defined in any one of the above, in tissue repair, organ repair and regeneration.

In a fourth aspect, the present invention provides a bio-ink for a bone regeneration bio-composite scaffold having both vascular and neural functions. The bio-ink comprises 0.001-10% of bio-ceramic material with the mass concentration of hydrogel material, 5-20% of hydrogel material and 100-500 ten thousand/mL of angiogenesis-related cells, wherein the angiogenesis-related cells comprise at least one of vascular endothelial cells, vascular progenitor cells and vascular smooth muscle cells, and the bio-ceramic material contains at least one of silicon, calcium, lithium, magnesium, copper, cobalt, zinc, strontium and manganese.

Drawings

FIG. 1 is an optical photograph of a composite scaffold of cell-containing (human umbilical vein endothelial cells) organisms prepared according to the invention and having different concentrations of bioceramic (LMS) powder (0%, 1.5%,3%, 4.5%) and fluorescence micrographs after 1, 7, 14, 21 days of culture. Living cells are shown green; dead cells are shown in red.

FIG. 2 is a representation of the angiogenic activity of the bioceramic (LMS) powder (0%, 1.5%,3%, 4.5%) prepared according to the present invention, after 21 days of culture, containing cells (human umbilical vein endothelial cells).

FIG. 3 shows the condition of a new blood vessel after subcutaneous implantation of the biological composite stent prepared by the present invention for two weeks.

FIG. 4 is a representation of neural differentiation after co-culturing PC-12 cells with a biocomposite scaffold.

FIG. 5 is a representation of neural differentiation of Schwann Cells (SCs) co-cultured with a biocomposite scaffold prepared according to the present invention.

FIG. 6 is a representation of osteogenic differentiation of human placental mesenchymal stem cells (hpMSCs) co-cultured with a biocomposite scaffold prepared according to the present invention.

FIG. 7 is a representation of the promotion of bone tissue regeneration after implantation of the biological composite scaffold prepared according to the invention into a rat skull defect.

FIG. 8 is a representation of the promotion of angiogenesis in new bone tissue by the biocomposite scaffolds prepared according to the present invention.

FIG. 9 is a representation of the promotion of nerve regeneration in new bone tissue by the biocomposite scaffold prepared according to the present invention.

Detailed Description

The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof. Unless otherwise specified, each percentage refers to a mass percent.

The invention provides a biological composite scaffold for bone regeneration with double functions of blood vessels and nerves, which can also be called as a biological composite scaffold for promoting the blood vessel-neuronization repair performance or a bone regeneration scaffold with double functions of blood vessels and nerves. The biological composite stent integrates a biological ceramic material with vasogenic activity and angiogenesis related cells and a hydrogel material with biocompatibility. The scaffold has the functional property of inducing vascular-neurogenic tissue repair.

The cells contained in the scaffold are cells related to angiogenesis, can differentiate into blood vessels under the induction of bioactive ceramics, and can induce neural differentiation and tissue cell regeneration through intercellular communication. In some embodiments, the angiogenesis-related cells contained in the scaffold may be at least one of vascular constituent cells such as vascular endothelial cells, endothelial progenitor cells, vascular smooth muscle cells, and the like. The mass concentration of the cells is 0.001-1%, and the activity and differentiation state of the cells can be well maintained at this concentration. If the cell concentration is too high, it may be difficult to obtain a sufficient adhesion space on the surface or inside of the scaffold and sufficient nutrients, resulting in difficulty in maintaining its activity for a long period of time. If the cell concentration is too low, it is difficult to maintain survival due to the low cell density, a scaffold with dense and uniformly distributed cells cannot be prepared, and at the same time, insufficient communication with other cells is caused due to the too low cell concentration.

The hydrogel component in the scaffold can provide support for cells and facilitate the cells to absorb nutrients. In some embodiments, the matrix of the hydrogel material included in the scaffold is a hydrogel material formed by mixing at least one of methacryl gelatin, methacryl hyaluronic acid, gellan gum, sodium alginate, methylcellulose, gelatin, and chitosan with water. For example, the matrix of the hydrogel material is methacryloyl gelatin (GelMA). In some embodiments, the hydrogel material has a mass concentration of 1-20%, preferably 5-20%, more preferably 6%. If the concentration of hydrogel material is too high, the hydrogel may be too hard and the polymer network too dense for proper cell survival. If the concentration of hydrogel material is too low, the polymer network is too sparse to crosslink into a stable hydrogel.

The bioactive ceramic powder in the bracket can release bioactive ions such as silicon, calcium, lithium, magnesium, copper, cobalt, strontium, manganese and the like, promote vascular related cells in the bracket to form blood vessels, promote directional differentiation of various other tissue cells, and efficiently promote vascular-neurogenic regeneration of massive tissue defects. In some technical schemes, the bioactive ceramics contained in the bracket have more than or equal to three constituent elements, and comprise at least one element which is favorable for vascularization, such as silicon, calcium, lithium, magnesium, copper, cobalt, zinc, strontium, manganese and the like. The bioactive ceramic powder comprises at least one of calcium silicate, magnesium yellow feldspar, cobalt yellow feldspar, copper tobermorite, lithium magnesium silicate, lithium calcium silicate, strontium silicate, magnesium silicate, manganese silicate and other bioceramics. Lithium magnesium silicate bioceramic powder (LMS) is selected for use in embodiments of the present invention. The mass of the biological ceramic material of the biological composite stent is less than 10 percent of that of the hydrogel material (matrix), preferably 0.001-10 percent. If the content of the ceramic powder is too high, a large amount of active ions such as Li, mg and Si ions are released, and toxic and side effects are generated on cells.

The composite scaffold can induce the neurogenic differentiation of the nerve-related cells. The cells related to the nerve capable of being induced comprise one or more of neural stem cells, oligodendrocytes, schwann cells, PC-12 cells, neurons and the like with neurogenic characteristics. The composite scaffold can also induce osteogenic differentiation of bone-related cells. The bone related cells capable of being induced include one or more cells with osteogenic differentiation properties, such as bone marrow mesenchymal stem cells, placenta mesenchymal stem cells, embryonic stem cells, osteoprogenitor cells, osteoblasts, etc. In conclusion, the composite scaffold has the function of promoting the repair of the vascularized tissue, and can be used for treating massive tissue defects.

The prior art adopts nerve-related cells and bone-related cells to prepare the active scaffold with upper/lower layer structure and multiple cells for printing and neurogenic bone regeneration, and the cells are distributed in a bionic design mode to obtain the bone regeneration scaffold with the neurogenic function. The invention adopts another completely different mode to directly print the biological ink containing the biological ceramic material, the hydrogel material and the angiogenesis related cells into the bracket, and utilizes the cooperation of the angiogenesis related cells and the biological ceramic material to obtain the bone regeneration bracket with double functions of blood vessels and nerves. This is the first time the present invention has been proposed and implemented.

In addition, although chinese patent CN112426569a mentions vascular endothelial cells, it still uses the arrangement of fibroblasts and vascular endothelial cells to simulate the bionic properties of skin tissue, and there is no way to obtain a bone regeneration scaffold with dual functions of blood vessels and nerves. In addition, the bone regeneration stent with the blood vessel and nerve functions plays a role in slowly releasing the angiogenesis-related cells loaded by the stent, the cells in the stent maintain high activity for a long time in the culture process of 21 days, and can grow out of the stent gradually, so that the bone regeneration stent has dynamics, which cannot be realized in Chinese patent CN 112426569A.

The method for preparing the bone regeneration scaffold having both vascular and neural functions according to the present invention will be described. The scaffold is manufactured by a 3D printing method, and specifically comprises the steps of preparing bio-ink containing bioactive ceramics and cells related to angiogenesis, and printing the scaffold with specific size based on the bio-ink.

The bio-ink comprises 0.001-10% of a bio-ceramic material with a mass concentration of a hydrogel material (matrix), 5-20% of a hydrogel material and angiogenesis-related cells with a density of 100-500 ten thousand/mL, wherein the angiogenesis-related cells comprise at least one of vascular endothelial cells, vascular progenitor cells and vascular smooth muscle cells, and the bio-ceramic material contains at least one of silicon, calcium, lithium, magnesium, copper, cobalt, zinc, strontium and manganese. The concentration of the cells is 100-500 ten thousand/mL, and the activity and differentiation state of the cells can be well maintained at the concentration. If the cell concentration is too high, it may be difficult to obtain a sufficient adhesion space on the surface or inside of the scaffold and sufficient nutrients, resulting in difficulty in maintaining its activity for a long period of time. If the cell concentration is too low, it is difficult to maintain survival due to the low cell density, a scaffold with dense and uniformly distributed cells cannot be prepared, and at the same time, insufficient communication with other cells is caused due to the too low cell concentration. For example, the concentration of cells is 200 ten thousand/mL.

Preparing the biological ink. The bio-ceramic material, the angiogenesis-related cells and the hydrogel material are uniformly mixed to form the bio-ink. In some technical schemes, biological ceramic material powder, angiogenesis-related cells and biocompatible hydrogel materials are mixed at 25-37 ℃ to prepare uniform aqueous solution, and then the aqueous solution is rapidly cooled at 4 ℃ to form the biological ink for printing.

Setting the temperature of the biological ink to 15-20 ℃, extruding, forming, crosslinking and solidifying the biological ink to obtain the bone regeneration bracket with the double functions of blood vessels and nerves. In some technical schemes, the temperature of the biological ink is set to be 15-20 ℃, the biological ink is extruded on a platform at 4 ℃ according to a designed shape according to a program, and then the biological ink is irradiated and crosslinked for 10-300 seconds by using a light source with the wavelength of 400-450 nm.

As an example, the method for preparing a bone regeneration scaffold having both vascular and neural functions includes the steps of:

material preparation: the lyophilized GelMA xerogel was first dissolved in Phosphate Buffered Saline (PBS), heated and stirred at 60℃for 30 minutes until complete dissolution, stopped heating, and centrifuged at 1000rpm for 2 minutes to remove the upper layer of foam. The homogeneous liquid obtained by centrifugation was filtered through a 0.22 μm filter and sterilized, and the sterile GelMA solution was incubated at 37 ℃ for use. Taking lithium magnesium silicate ceramic (LMS) powder, sterilizing by ultraviolet irradiation for 1 hour, adding the powder into GelMA solution at 37 ℃, and uniformly mixing to obtain the LMS-GelMA composite ink. The concentration of GelMA may be 1-20wt%, preferably 6wt%; the concentration of LMS powder can be 0-10% of GelMA (gel) mass.

Cell preparation: preparing at least one of vascular endothelial cells, endothelial progenitor cells and vascular smooth muscle cells, and dispersing the vascular endothelial cells, the endothelial progenitor cells and the vascular smooth muscle cells in the LMS-GelMA composite ink to prepare the biological ink containing cells.

The printing process comprises the following steps: printing on a cooled glass plate by adopting a cooling platform heat preservation and extrusion type printing method, wherein the temperature of the glass plate is set to be 4 ℃ and the temperature of a charging barrel containing biological ink is set to be 18 ℃. The bio-ink is extruded for printing according to a set program using a pneumatic extrusion needle. After printing, the bone regeneration stent with double functions of blood vessels and nerves is obtained by crosslinking by using 405nm blue light irradiation. In some embodiments, the extrusion pressure of the bio-ink is set at 30-80Kpa.

The invention firstly uses the method to prepare the bone regeneration bracket with blood vessel and nerve functions, constructs a tissue engineering construct with blood vessel-nerve repair function, realizes the physiological functions of long-time high activity survival and dynamic growth of cells, has good adhesion, proliferation and differentiation performances, can show good vascularization performance and neuronization promoting performance after long-time culture in vitro and in vivo, can realize the tissue repair of blood vessel-nerve, and has the potential of efficiently treating massive tissue defects.

The invention provides application of the bone regeneration scaffold with blood vessel and nerve functions in aspects of tissue repair, organ repair and regeneration, including application in aspects of culturing vascular tissue engineering constructs, blood vessel repair and regeneration, nerve tissue repair and regeneration, promoting tissue cell directional differentiation, promoting massive bone tissue repair, promoting blood vessel-nerve tissue repair and the like.

The invention also provides an in vitro culture method of the bone regeneration bracket with the double functions of blood vessels and nerves. In one embodiment of the present invention, after the preparation of a bone regeneration scaffold having both vascular and neural functions on a glass plate according to the above-described preparation method, the scaffold was transferred to a 12-well culture plate, 2mL of cell culture medium was added to the well in which the scaffold was placed, the cover of the culture plate was covered, and the culture plate was placed at 37℃with 5% CO ₂ The culture was performed in the cell culture chamber of (2), and the liquid-changing operation was performed every 1 day during the subsequent culture.

The present invention will be further illustrated by the following examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.

Example 1

The invention relates to preparation of a bone regeneration biological composite scaffold with blood vessel and nerve functions, which comprises the following steps:

LMS-containing inks were prepared by incorporating LMS particles into GelMA hydrogels. First, gelMA (6 wt.%) and lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (LAP) (0.3 wt.%) in a lyophilized state were dissolved in Phosphate Buffered Saline (PBS) at 58 ℃ for 30 minutes. The obtained GelMA solution was then filtered through a 0.22 μm filter to be sterilized. Then adding LMS bioceramic particles sterilized by ultraviolet into GelMA solution (the addition amounts are 0%,1.5%,3%,4.5%, 6% of GelMA respectively), thereby preparingA biological ink containing LMS. Will have a 1 x 10 ⁷ A cell suspension (100. Mu.L) of Human Umbilical Vein Endothelial Cells (HUVECs) was added to 5mL of the hydrogel ink and mixed uniformly to form a HUVECs-loaded bio-ink.

The scaffold was printed using a biological 3D printer. The whole printing process is carried out in an ultra clean bench. The HUVECs loaded bio-ink was transferred to a stainless steel cartridge and left to cool at 4 ℃ for 20 minutes. Subsequently, an extrusion needle (inside diameter of needle: 170 μm) was mounted on the cartridge and mounted on a bioprinter for printing. The cartridge temperature was set at 18 ℃ and the print platform was set at 4 ℃ to also maintain the shape of the scaffold before the bio-ink was crosslinked. After printing, the scaffold was crosslinked for 60s with light of 405nm wavelength. Finally, the scaffolds were transferred to a culture plate and medium was replenished at 37℃with a volume fraction of 5% CO ₂ Is cultured in a humidified incubator.

Example 2

Application of bone regeneration biological composite scaffold with blood vessel and nerve functions in-vitro culture of vascular tissue engineering construct

Cell-containing composite scaffolds with gradient LMS content GelMA,1.5LMS-GelMA,3LMS-GelMA,4.5LMS-GelMA (0%, 1.5%,3%,4.5% for LMS content, respectively) were prepared as described in example 1. And at 37℃with a volume fraction of 5% CO ₂ The culture was continued for 21 days in a humidified incubator, and the scaffolds were stained with a live/dead cell stain, in which live cells were stained green and dead cells were stained red, at 1, 7, 14, 21 days of culture. As shown in fig. 1, the cell viability was high in each set of scaffolds, and after 21 days of culture, living cells proliferated and adhered to the surface of the covered scaffolds.

To characterize its vascularization function in vitro, the expression of the cell vascular specific protein CD31 in the scaffold was characterized by immunofluorescent staining methods, in the following manner: first, the stent is immersed in a 4% paraformaldehyde solution by mass for fixation for at least 30 minutes. The scaffolds were then permeabilized with a mass fraction of 0.5% Triton-X solution and blocked with a mass fraction of 5% BSA solution for 30 min at room temperature. Thereafter, the scaffolds were incubated in primary antibody working fluid (CD 31: abcam, ab 28364) and at 4℃overnight. The next day, the scaffolds were washed 3 times with PBS for 5 min each, then incubated with the corresponding secondary antibody working solution for 1h at 37 ℃, after which the scaffolds were washed 3 times with PBS. Next, cytoskeleton and nuclei of cells in the scaffold were stained with Alex Fluor 488 conjugated phalloidin dye and diamidinophenyl indole Dye (DAPI), respectively. Finally, a laser confocal microscope was applied to capture photographs of the scaffold. As shown in fig. 2, each group of scaffolds formed a dense endothelial cell network on the scaffolds after 21 days of culture, the intercellular vascular specific protein network was also quite clear, and particularly in the scaffolds containing LMS, the CD31 protein network was quite dense, demonstrating that the composite scaffolds formed vascularized in vitro tissues after long-term culture.

Example 3

Application of the bone regeneration biological composite scaffold with blood vessel and nerve functions in the field of blood vessel repair and regeneration

To explore the in vivo vascularization capacity of bioprinted angiogenic anterior scaffolds, four sets of scaffolds were prepared as described in example 1. The four groups of brackets are respectively: gelMA (LMS and cell free scaffold), 3LMS-GelMA (cell free, LMS containing scaffold), HUVECs-GelMA (cell containing, LMS free scaffold), and HUVECs-3LMS-GelMA (LMS and cell containing scaffold) were then implanted subcutaneously in nude mice. The specific operation mode is as follows: SPF-grade male BALB/c-nude mice were used 6-8 weeks old for subcutaneous implantation. Each set of printed scaffolds (6 mm long, 3mm wide, 2mm thick) was cultured in vitro for 3 days and then implanted. Implantation is performed in a pathogen free (SPF) environment. First, anesthesia was performed by injecting chloral hydrate into nude mice through the abdominal cavity. After anesthesia, a subcutaneous cavity was created and placed in the stent, open on both sides of their back, and then the incision was sutured. Finally, the nude mice were kept in cages for 2 to 4 weeks under SPF, and then scaffolds were taken and fixed in 4% paraformaldehyde solution by mass fraction. As shown in fig. 3, the cell-free scaffold did not have a significant morphological change after implantation, whereas the cell-loaded scaffold was completely covered by surrounding tissue. At the same time, some blood vessels can be observed on/in the scaffold, where more blood vessels can be observed in the scaffold containing cells. It can be seen that in the HUVECs-3LMS-GelMA stent, the growing blood vessels extend directly into the interior of the stent through the printed holes, indicating that they are able to induce more vascular ingrowth. The bone regeneration biological composite scaffold with the blood vessel and nerve functions has wide application prospect in the field of blood vessel repair.

Example 4

Application of the bone regeneration biological composite scaffold with blood vessel and nerve functions in nerve tissue repair and regeneration

In order to explore the ability of the bone regeneration biological composite scaffold with blood vessel and nerve functions to promote neurogenesis, a transwell method is adopted to indirectly co-culture the PC-12 cells and the Schwann cells related to the nerves with the scaffold. The preparation method of the biocomposite scaffold is as described in example 1. These cells were co-cultured with three sets of scaffolds, 3LMS-GelMA (LMS scaffold without cells), HUVECs-GelMA (scaffold loaded with HUVECs without LMS) and HUVECs-3LMS-GelMA (scaffold containing LMS and loaded with HUVECs), respectively, and their differentiation was then explored, with untreated hpMSCs cultured in normal medium as control.

First, the stimulation of the neuronal axons by the cell-containing composite scaffolds was investigated using PC-12 cells (hypodifferentiation) with neural differentiation properties as model cells. The growth of the PC-12 axon was analyzed by immunofluorescence staining. The specific operation mode is as follows: first, the stent is immersed in a 4% paraformaldehyde solution by mass for fixation for at least 30 minutes. The scaffolds were then permeabilized with a mass fraction of 0.5% Triton-X solution and blocked with a mass fraction of 5% BSA solution for 30 min at room temperature. Thereafter, the scaffolds were incubated in primary antibody working solution (α/β -Tubulin, cell Signaling, 2148) and overnight at 4 ℃. The next day, the scaffolds were washed 3 times with PBS for 5 min each, then incubated with the corresponding secondary antibody working solution for 1h at 37 ℃, after which the scaffolds were washed 3 times with PBS. Next, nuclei of cells in the scaffolds were stained using DAPI stain. Finally, a laser confocal microscope was applied to capture photographs of the scaffold. The images in FIG. 4 show that each set of scaffolds promoted neurite elongation in PC-12 cells compared to the control. We found that the HUVECs-3LMS-GelMA scaffolds containing LMA loaded with HUVECs had significant stimulatory effects on neurite elongation.

In addition, another cell, schwann Cells (SCs), which plays an important role in nerve regeneration, was also co-cultured with each set of scaffolds in the same manner. The expression of specific protein S100 associated with neurogenesis in SCs was detected by immunofluorescence in the same manner. According to the results of FIG. 5, the biocomposite scaffold (HUVECs-3 LMS-GelMA scaffold containing both LMS and vascular-related cells) was able to significantly up-regulate S100 in SCs. In conclusion, the biological composite scaffold greatly enhances the nerve differentiation activity of PC-12 cells and SCs, and shows that the scaffold has the function of promoting nerve repair.

Example 5

Application of bone regeneration biological composite scaffold with blood vessel and nerve functions in promoting directional differentiation of other tissue cells

In order to explore the application of the biological composite scaffold in promoting the differentiation of other tissue cells, mesenchymal stem cells are used as model cells, and the stimulation effect of the biological composite scaffold on the osteogenic differentiation of the mesenchymal stem cells is further studied. Mesenchymal stem cells derived from placenta (hpMSCs) were co-cultured indirectly with scaffolds using the transwell method, and the scaffolds were prepared as described in example 1. hpMS were co-cultured with three sets of scaffolds, 3LMS-GelMA (LMS scaffold without cells), HUVECs-GelMA (scaffold loaded with HUVECs without LMS) and HUVECs-3LMS-GelMA (scaffold containing LMS and loaded with HUVECs), respectively, and their osteogenic differentiation was explored, with untreated hpMS cultured in normal medium as control. As shown in FIG. 6, each group of scaffolds up-regulated the expression of osteogenic related proteins in hpMSCs, further indicating that HUVECs-3LMS-GelMA scaffolds have good ability to induce osteogenic differentiation. In conclusion, the biocomposite scaffolds significantly enhance osteoblast differentiation of mesenchymal stem cells, demonstrating that they have the function of promoting directional differentiation of other tissue cells.

Example 6

Application of bone regeneration biological composite scaffold with blood vessel and nerve functions in promoting repair of massive bone defects

LMS-containing inks were prepared by incorporating LMS particles into GelMA hydrogels. First, gelMA (6 wt.%) and lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (LAP) (0.3 wt.%) in a lyophilized state were dissolved in Phosphate Buffered Saline (PBS) at 58 ℃ for 30 minutes. The obtained GelMA solution was then filtered through a 0.22 μm filter to be sterilized. Then, the LMS bioceramic particles sterilized by ultraviolet are added into GelMA solution (the addition amount is 3% of the mass of GelMA), thereby preparing the LMS-containing bio-ink. Will have a 1 x 10 ⁷ A cell suspension (100. Mu.L) of Rat Umbilical Vein Endothelial Cells (RUVECs) was added to 5mL of the hydrogel ink and mixed uniformly to form a RUVECs-loaded bio-ink.

The scaffold was printed using a biological 3D printer. The whole printing process is carried out in an ultra clean bench. The RUVECs-loaded bio-ink was transferred to a stainless steel cartridge and left to cool at 4 ℃ for 20 minutes. Subsequently, an extrusion needle (inside diameter of needle: 170 μm) was mounted on the cartridge and mounted on a bioprinter for printing. The cartridge temperature was set at 18 ℃ and the print platform was set at 4 ℃ to also maintain the shape of the scaffold before the bio-ink was crosslinked. After printing, the scaffold was crosslinked for 60s with light of 405nm wavelength. Finally, the scaffolds were transferred to a culture plate and medium was replenished at 37℃with a volume fraction of 5% CO ₂ Is cultured in a humidified incubator. After 3 days of culture, the scaffolds were perforated with a 5mm diameter punch to prepare 5mm diameter and 2mm thick samples to be implanted.

Skull defect repair experiments were performed using 8 week old male SD rats. Two defects of 5mm diameter were drilled in the left and right sides of the sagittal suture of the rat skull, respectively. The prepared biocomposite scaffold samples (RUVECs-3 LMS-GelMA (scaffolds containing both LMS and RUVECs)) were then implanted, in contrast to the remaining three groups: blank (no scaffold implanted at defect), 3LMS-GelMA (scaffold containing LMS without cells), RUVECs-GelMA (scaffold loaded with RUVECs without LMS). After 8 weeks of implantation, the whole skull of the rat was drawn and then fixed in 4% paraformaldehyde solution.

After the materials are taken, the regeneration condition of the new bone tissue is evaluated by a Micro-computer tomography (Micro-CT) reconstruction method. The specific operation is as follows: all the skull obtained was scanned layer by a Micro-CT instrument (SKYSCAN 1172, bruker, germany). After scanning, the defective bone volume/total volume (BV/TV) values, bone small Liang Shu (tb.n) and Bone Mineralization Density (BMD) were analyzed by CT-analyzer software (CTAn, bruker). During the analysis, the threshold value of all samples was set to 70-255, and the parameter remained unchanged. Finally, CT-Volume (CTVol, bruker) software is applied to carry out 3D reconstruction on the image. The newly formed bone tissue within the skull defect is shown in fig. 7 in coronal and sagittal sections (green). In the group implanted with scaffolds, new bone tissue was formed within the defect, with the RUVECs-3LMS-GelMA scaffold having optimal bone repair capability.

Example 7

Application of bone regeneration biological composite scaffold with blood vessel and nerve functions in promoting vascular-neurogenic tissue repair

In order to evaluate the application of the bone regeneration biological composite scaffold with the blood vessel and nerve functions in promoting the repair of blood vessel-nerve tissue, the skull defect is further used as a model, and the regeneration condition of blood vessel and nerve in the bone tissue of the new bone of the scaffold is explored. Preparation of scaffolds RUVECs-3LMS-GelMA biocomposite scaffolds containing LMS ceramic and rat umbilical vein endothelial cells were prepared as described in example 6. Subsequently, 8 week old male SD rats were used for skull defect repair experiments. Two defects of 5mm diameter were drilled in the left and right sides of the sagittal suture of the rat skull, respectively. The prepared biocomposite scaffold samples (RUVECs-3 LMS-GelMA (scaffolds containing both LMS and RUVECs)) were then implanted, in contrast to the remaining three groups: blank (no scaffold implanted at defect), 3LMS-GelMA (scaffold containing LMS without cells), RUVECs-GelMA (scaffold loaded with RUVECs without LMS). After 8 weeks of implantation, the whole skull of the rat was drawn and then fixed in 4% by mass paraformaldehyde solution. After the material is obtained, the obtained sample is subjected to tissue section staining to evaluate the generation of blood vessels and nerves in newly formed bone tissue. Specific proteins associated with vascularization and nerve fibers were stained. The specific operation mode is as follows: all the samples were dehydrated by alcohol gradient and embedded in paraffin. The obtained wax block was then sliced to prepare a slice having a thickness of 8. Mu.m, and immunofluorescent staining was performed. First, the prepared sections were boiled in modified citrate antigen retrieval solution (P0083, beyotime, china) for 30 minutes to retrieve the antigen. Next, the sections were incubated overnight at 4 ℃ in primary antibody working solution (CD 31, abcam, ab28364; neuroframe NF-H, neuromics, CH 22104), then they were incubated with secondary antibody working solution for 1 hour at room temperature and washed with water for 5 minutes. Finally, a capping piece (BL 701A, biosharp, china) containing DAPI dye was dropped on the slice to complete the capping. Finally, the sections were observed using a confocal laser scanning microscope (TCS SP8, leica, germany).

As shown in fig. 8, CD31 protein closely related to angiogenesis was most expressed in the biocomposite scaffolds RUVECs-3LMS-GelMA, indicating that the scaffolds significantly promote vascularization of new bone (green). Subsequently, nerve fibers in new bones were labeled by NF-H. As can be seen from FIG. 9, more nerve fibers grew into new bone in RUVECs-3LMS-GelMA scaffolds (green, arrowed position) than scaffolds without RUVECs or LMS. The results show that the biological composite scaffold can obviously promote the innervation in the process of forming new bones.

In conclusion, the bone regeneration biological composite scaffold with the blood vessel and nerve functions can remarkably enhance bone regeneration and promote vascularization and nerve regeneration in new bone tissues.

Claims

1. The application of the bone regeneration biological composite scaffold in preparing the bone regeneration biological composite scaffold with blood vessel and nerve functions is characterized in that the biological composite scaffold comprises a polymer network formed by solidifying a hydrogel material, and biological ceramic materials and blood vessel formation related cells which are uniformly distributed in the polymer network, wherein the blood vessel formation related cells comprise at least one of blood vessel endothelial cells, blood vessel progenitor cells and blood vessel smooth muscle cells, the constituent elements of biological activity ceramic contained in the biological composite scaffold comprise silicon, lithium and magnesium, the biological ceramic material is lithium magnesium silicate, and the mass of the biological ceramic material is 1.5-10% of that of the hydrogel material.

2. The use according to claim 1, wherein the matrix of the hydrogel material is at least one of methacryl gelatin, methacryl hyaluronic acid, gellan gum, sodium alginate, methylcellulose, gelatin, chitosan.

3. The use according to claim 1, characterized in that the mass concentration of hydrogel material in the biocomposite scaffold is 5-20%.

4. The use according to claim 1, wherein the mass concentration of angiogenesis-related cells in the biocomposite scaffold is 0.001-1%.

5. The use according to claim 1, characterized in that the bio-ceramic material has a particle size of 10 μm-200 μm.

6. The use according to any one of claims 1 to 5, wherein the preparation method comprises: uniformly mixing a biological ceramic material, cells related to angiogenesis and a hydrogel material to form biological ink; setting the temperature of the biological ink to 15-20 ℃, extruding, forming, crosslinking and solidifying the biological ink to obtain the bone regeneration biological composite scaffold with the double functions of blood vessels and nerves.

7. The application of the bio-ink in preparing the bone regeneration bio-composite scaffold with the functions of blood vessels and nerves is characterized in that the bio-ink comprises 1.5-10% of bio-ceramic material with the mass concentration of hydrogel material, 5-20% of hydrogel material with the mass concentration of hydrogel material and 100-500 ten thousand/mL of angiogenesis-related cells, wherein the angiogenesis-related cells comprise at least one of vascular endothelial cells, vascular progenitor cells and vascular smooth muscle cells, the constituent elements of bioactive ceramic contained in the bio-ceramic material comprise silicon, lithium and magnesium, and the bio-ceramic material is lithium magnesium silicate.