Bone-like bioactive polycaprolactone porous scaffold and preparation method thereof

Abstract

The application discloses a kind of bone-like biological activity polycaprolactone porous scaffold and preparation method thereof, belong to biological scaffold field, steps include: 3D printing out initial scaffold with connected hole groove, immerse in pre-gel solution, cultivate to make pre-gel solution gelation, scrape off redundant gel;Wherein, 3D printing raw material is polycaprolactone and alpha-gentio-biose;Pre-gel solution includes thrombin, human umbilical vein endothelial cell acellular matrix and fibrinogen.Initial scaffold connected hole groove as primary hole groove, alpha-gentio-biose is dissolved in pre-gel solution to form secondary hole groove, cultivate process will human umbilical vein endothelial cell acellular matrix be evenly fixed in primary hole groove and secondary hole groove, alpha-gentio-biose is used as sacrificial template, after being dissolved into pre-gel solution and becoming part of gel again, it can provide nutrition for vascular cell proliferation, together with human umbilical vein endothelial cell acellular matrix, promote angiogenesis, more conducive to bone repair.

Description

Bone-inspired bioactive polycaprolactone porous scaffold and its preparation method Technical Field

[0001] This invention relates to a bone-inspired bioactive polycaprolactone porous scaffold and its preparation method, belonging to the field of bioscaffolds. Background Technology

[0002] Autologous bone grafting is the ideal treatment for bone defects caused by various reasons. However, the availability of autologous and allogeneic bone is limited, making it difficult to meet the needs of large-scale bone transplantation. Utilizing biodegradable polymers as bone substitutes has become a popular research direction. Initially, this required seeding cells onto scaffolds, which had limitations including time-consuming cell expansion, reduced cell survival rates after implantation, and potential immune rejection. Later, in-depth research into stem cell biology and materials science revealed that stem cells can respond sensitively to exogenous physicochemical and biological signals, and that biomaterials can be endowed with properties that regulate stem cell biological effects during preparation. Using biomaterials to reconstruct the body's regenerative microenvironment, regulate the behavior of the host's inherent stem cells, and initiate repair effects has led to in situ tissue engineering strategies. However, bone regeneration is highly dependent on rapid and sufficient angiogenesis. The lack of neovascularization after implantation of bioinert scaffolds can lead to secondary hypoxia, resulting in necrosis in the central part of the scaffold or poor osseointegration, manifested as the scaffold being partially or completely encapsulated by fibrous granulation tissue. Therefore, previous in-situ tissue engineering strategies that utilize single factors (growth factors, small molecule compounds, etc.) to promote osteogenic differentiation cannot reproduce the complex microenvironment of bone regeneration. How to promote angiogenesis within bioinert scaffolds remains a challenge in this field. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, the present invention provides a bone-inspired bioactive polycaprolactone porous scaffold and its preparation method, which can induce angiogenesis in polycaprolactone bone substitutes.

[0004] The technical solution adopted by this invention to solve its technical problem is:

[0005] In a first aspect, this application provides a method for preparing a bone-inspired bioactive polycaprolactone porous scaffold, comprising the following steps: fused deposition modeling (FDM) to 3D print an initial scaffold with interconnected pores; immersing the initial scaffold in a pregel solution; cultivating the pregel solution to allow it to gel; and scraping off the gel from the outer surface of the initial scaffold to obtain the bone-inspired bioactive polycaprolactone porous scaffold; wherein the 3D printing raw materials are polycaprolactone and α-gentiobiose; and the pregel solution comprises thrombin, decellularized matrix of human umbilical vein endothelial cells, and fibrinogen.

[0006] The bone-inspired bioactive polycaprolactone porous scaffold prepared by the method described in this application can be 3D printed into various shapes. The connecting pores of the initial scaffold serve as larger primary pores. After the initial scaffold is immersed in the pregel solution, the pregel solution fills the primary pores, and α-gentiobiose gradually dissolves at this time, leaving smaller secondary pores on the initial scaffold. During the incubation process, thrombin converts fibrinogen into fibrin, thereby causing the pregel solution to gel, which relatively uniformly fixes the decellularized matrix of human umbilical vein endothelial cells in the primary and secondary pores. α-gentiobiose and polycaprolactone have similar melting temperatures and can be mixed with polycaprolactone for 3D printing. α-gentiobiose serves as a sacrificial template, dissolving into the pregel solution and becoming part of the gel, and also provides nutrients for cell proliferation. Together with the decellularized matrix of human umbilical vein endothelial cells, it promotes angiogenesis, which is more conducive to bone repair.

[0007] Furthermore, the α-gentiobiose accounts for 3%-8% of the 3D printing raw material, and the polycaprolactone accounts for 92%-97% of the 3D printing raw material. The appropriate ratio of α-gentiobiose is beneficial for the initial scaffold to form sufficient secondary pores after being soaked in the pregel solution, and the remaining initial scaffold is not too loose, and the osmotic pressure of the gel relative to the cells is also avoided.

[0008] Furthermore, the initial support is divided into multiple layers of molding blocks parallel to the horizontal plane. Each layer of the molding block includes multiple parallel wire clusters. Each wire cluster consists of three molten deposited filaments, including two bottom wires and one top wire. The distance between the two bottom wires is the radius of the filament. The top wire is stacked on the bottom wires and located between the two bottom wires, which helps to improve the compression performance of the initial support.

[0009] Furthermore, adjacent wire clusters on the same layer of the molding block have a first gap, and the filaments on the same layer of the molding block are parallel; the wire clusters on two adjacent layers of the molding block form a certain angle, and the first gaps on multiple layers of the molding block constitute the connecting hole groove. Thus, the connecting hole groove can be made by printing the bracket, eliminating the need for mechanical drilling after 3D printing, avoiding the impact of drilling on the internal structure of the initial bracket, and also helping to save 3D printing materials.

[0010] Furthermore, the width of the first gap is 1.5mm-2mm, the filaments on the two adjacent molding blocks form an angle of 25°-40°, and the diameter of the filaments is 0.3mm-0.5mm, which is conducive to forming crisscrossing interconnected grooves, so that the gel can be more uniformly solidified in the initial support.

[0011] Furthermore, the pregel solution also includes vascular fragments, which can utilize the cells in the vascular fragments to accelerate angiogenesis in the porous scaffold.

[0012] Furthermore, the preparation steps of the pregel solution are as follows:

[0013] Prepare a sodium chloride solution of fibrinogen;

[0014] Prepare thrombin-calcium chloride solution;

[0015] Prepare decellularized matrix solution for human umbilical vein endothelial cells;

[0016] The fibrinogen sodium chloride solution, the thrombin calcium chloride solution, and the human umbilical vein endothelial cell decellularization matrix solution are mixed in an ice bath to obtain the pregel solution.

[0017] Furthermore, the preparation steps of the decellularized matrix solution of human umbilical vein endothelial cells are as follows:

[0018] Mix 1 part by volume of 0.5% Triton X-100 solution and 5 parts by volume of 20mM NH4OH solution to prepare a decellularized solution;

[0019] Human umbilical vein endothelial cells were added to the decellularized solution, centrifuged, collected into a pellet, and washed with PBS buffer until the pH was neutral.

[0020] Soak one part of the mass in three parts of 100 U / mL Dnase I solution by volume, soak at 37°C for 45 min, centrifuge to collect the insoluble part and freeze-dry and grind to obtain matrix powder;

[0021] The matrix powder was soaked in 0.1% pepsin-hydrochloric acid solution for 48 hours, then the system was adjusted to neutral with NaOH, and finally diluted with PBS solution to prepare a human umbilical vein endothelial cell decellularized matrix solution containing 10 mg of the matrix powder per milliliter.

[0022] Furthermore, the step of preparing the fibrinogen sodium chloride solution includes: adding fibrinogen powder to physiological saline to prepare a 10 mg / mL fibrinogen sodium chloride solution;

[0023] The step of preparing the thrombin calcium chloride solution includes: preparing thrombin with 1M calcium chloride solution to prepare a 100U / mL thrombin calcium chloride solution;

[0024] The mixing ratio of the fibrinogen sodium chloride solution, the thrombin calcium chloride solution, and the human umbilical vein endothelial cell decellularized matrix solution is 10:1:10.

[0025] In a second aspect, this application provides a bone-inspired bioactive polycaprolactone porous scaffold, which is prepared by the bone-inspired bioactive polycaprolactone porous scaffold preparation method described in the first aspect, and is capable of inducing angiogenesis in the polycaprolactone bone substitute.

[0026] The beneficial effects of this invention are as follows: both α-gentiobiose and polycaprolactone are biodegradable and have similar melting temperatures, allowing them to be mixed and fused together for 3D printing to directly create initial scaffolds of various shapes. The connecting pores of the initial scaffold serve as primary pores. After the initial scaffold is immersed in the pregel solution, the pregel solution fills the primary pores, and α-gentiobiose gradually dissolves, leaving secondary pores smaller than the primary pores on the initial scaffold. During the cultivation process, thrombin causes the pregel solution to gel, uniformly fixing the decellularized matrix of human umbilical vein endothelial cells in the primary and secondary pores. α-gentiobiose serves as a sacrificial template, dissolving into the pregel solution and becoming part of the gel, while also providing nutrients for vascular cell proliferation. Together with the decellularized matrix of human umbilical vein endothelial cells, it promotes angiogenesis, which is more conducive to bone repair.

[0027] Other features and advantages of this application will be set forth in the following description and will be apparent in part from the description or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures particularly pointed out in the written description and the accompanying drawings. Attached Figure Description

[0028] Figure 1 is a schematic diagram of an initial support structure provided in an embodiment of this application.

[0029] Figure 2 is a cross-sectional view of each layer of an initial support provided in an embodiment of this application. Detailed Implementation

[0030] With the continuous advancement of orthopedic surgery and sports medicine treatment techniques, clinical methods for healing and treating musculoskeletal diseases such as fractures have been constantly improved. However, fractures and bone diseases caused by various reasons still cannot be repaired or healed, ultimately leading to nonunion and osteonecrosis. The treatment of bone defects caused by sports injuries, traffic accidents, and post-tumor surgery, the rescue of early osteonecrosis, revision of artificial joints, and spinal fusion all rely on the bone repair and regeneration process.

[0031] Research has revealed that bone tissue repair and regeneration is a complex physiological process that cannot be achieved simply by combining cells, biomaterials, and growth factors, and implanting them into the bone defect site. Bone tissue repair and regeneration involves a series of pathological and physiological processes. Angiogenesis occurs after bone injury and hematoma formation and determines the success or failure of bone repair. Angiogenesis refers to the process by which endothelial cells, based on existing blood vessels, receive biological signals released from the injury site, proliferate, migrate, connect, and fuse to form new blood vessels through a "budding" process. These newly formed blood vessels not only deliver oxygen, nutrients, and growth factors to the bone repair and regeneration site but also provide a pathway for the entry of cells involved in repair and osteoblast progenitor cells.

[0032] Although polycaprolactone (PCL) is a high-molecular-weight polymer with good biocompatibility, processability, and excellent biodegradability, PCL scaffolds are generally unable to provide sufficient cell adhesion sites due to their bioinertness, resulting in poor bioactivity.

[0033] To address the above issues, this application provides a method for preparing a bone-inspired bioactive polycaprolactone porous scaffold. The steps include: fused deposition modeling (FDM) to 3D print an initial scaffold with interconnected pores; immersing the initial scaffold in a pregel solution; cultivating the pregel solution to allow it to gel; and scraping off the gel from the outer surface of the initial scaffold to obtain a bone-inspired bioactive polycaprolactone porous scaffold. The 3D printing materials are polycaprolactone and α-gentiobiose; the pregel solution includes thrombin, decellularized matrix of human umbilical vein endothelial cells, and fibrinogen.

[0034] Incubation refers to allowing thrombin to function and convert fibrinogen into fibrin, thereby transforming the pregel solution into a gel. Specifically, incubation involves standing at 37°C for 30 minutes. Pregel solutions that have not been soaked in the initial stent should be stored at 0°C-4°C.

[0035] The preparation steps for the pregel solution are as follows:

[0036] Fibrinogen powder was added to physiological saline to prepare a 10 mg / mL fibrinogen sodium chloride solution.

[0037] Thrombin was prepared using 1M calcium chloride solution to prepare a 100U / mL thrombin calcium chloride solution.

[0038] Prepare decellularized matrix solution for human umbilical vein endothelial cells;

[0039] In an ice bath, fibrinogen sodium chloride solution, thrombin calcium chloride solution, and decellularized matrix solution of human umbilical vein endothelial cells were mixed in a ratio of 10:1:10 to obtain a pregel solution.

[0040] The preparation steps for the decellularized matrix solution of human umbilical vein endothelial cells are as follows:

[0041] Mix 1 part by volume of 0.5% Triton X-100 solution and 5 parts by volume of 20mM NH4OH solution to prepare a decellularized solution;

[0042] Human umbilical vein endothelial cells were added to the decellularized solution, centrifuged, collected into a pellet, and washed with PBS buffer until the pH was neutral.

[0043] Soak one part of the agglomerate in three parts of 100 U / mL Dnase I solution by volume. After soaking at 37°C for 45 min, collect the insoluble part by centrifugation and freeze-dry and grind to obtain matrix powder.

[0044] The matrix powder was soaked in 0.1% pepsin-hydrochloric acid solution for 48 hours, then the system was adjusted to neutral with NaOH, and finally diluted with PBS solution to prepare a human umbilical vein endothelial cell decellularized matrix solution containing 10 mg of matrix powder per milliliter.

[0045] Existing technologies have demonstrated that decellularized matrix of human umbilical vein endothelial cells promotes angiogenesis. [1] and promote osteogenic differentiation [2] This application focuses more on how to apply decellularized human umbilical vein endothelial cell matrix to FDM (Fused Deposition Modeling) 3D printing of polycaprolactone, and provides optimized scaffold composition and structure. The resulting bone-inspired bioactive polycaprolactone porous scaffold can be 3D printed into various shapes. The connecting pores of the initial scaffold serve as larger primary pores. After the initial scaffold is immersed in the pregel solution, the pregel solution fills the primary pores, and α-gentiobiose gradually dissolves at this time, leaving tiny secondary pores on the initial scaffold. During the incubation process, thrombin converts fibrinogen into fibrin, thereby causing the pregel solution to gel, which more uniformly fixes the decellularized human umbilical vein endothelial cell matrix in the primary and secondary pores. The tiny secondary pores are more conducive to cell adhesion. α-Gentianobiose has a similar melting temperature to polycaprolactone and can be mixed with polycaprolactone for 3D printing. α-Gentianobiose serves as a sacrificial template, dissolving into the pregel solution and becoming part of the gel. It also provides nutrients for cell proliferation and promotes angiogenesis together with the decellularized matrix of human umbilical vein endothelial cells, which is more conducive to bone repair.

[0046] The higher the amount of α-gentiobiose used, the more secondary pores remain after dissolving in the pregel solution, resulting in stronger connectivity. However, this reduces the mechanical properties of the scaffold. Since the bone-like scaffold is ultimately implanted into the bone defect, excessive α-gentiobiose, after dissolving in the pregel solution and solidifying, creates a gel with too much sugar, leading to excessive osmotic pressure relative to bone marrow stem cells, which is detrimental to stem cell differentiation. Preferably, α-gentiobiose accounts for 3%-8% of the 3D printing raw materials, and polycaprolactone accounts for 92%-97%. An appropriate ratio of α-gentiobiose helps the initial scaffold form sufficient secondary pores after being soaked in the pregel solution, while preventing the remaining initial scaffold from becoming too loose, and also provides an appropriate amount of nutrition for cell proliferation.

[0047] Referring to Figures 1 and 2, the initial scaffold is composed of multi-layered molding blocks parallel to the horizontal plane. Each molding block includes multiple parallel filament clusters, and each filament cluster consists of three molten deposited filaments, including two bottom filaments and one top filament. The distance between the two bottom filaments is the radius of the filament, and the top filament is stacked on top of the bottom filaments and located between the two bottom filaments. On the one hand, this allows the initial scaffold to have more compressible space in the vertical direction, which can improve the compressibility of the biological scaffold. On the other hand, these compressible spaces can also be filled with pregel solution, allowing the gel to be distributed more widely and branched more within the scaffold, which is conducive to inducing the formation of a new blood vessel network and is more conducive to bone repair.

[0048] As shown in Figure 1, adjacent wire clusters on the same layer of the molding block have a first gap, and the wires on the same layer of the molding block are parallel; the wire clusters on two adjacent layers of the molding block form a certain angle, and the first gaps on multiple layers of the molding block form a connecting groove. The left side of Figure 2 is a side view of Figure 1, with a total of 6 layers of molding blocks. The 6 layers are disassembled on the right side of Figure 2 to more clearly show the structure. The 6 layers of the molding block, from bottom to top, include layers A, B, C, D, E, and F. The wires on layer B form a 30° angle with the wires on layer A; the wires on layer C form a 30° angle with the wires on layer B, and a 60° angle with the wires on layer A; the wires on layer D form a 30° angle with the wires on layer C, a 60° angle with the wires on layer B, a 90° angle with the wires on layer A, and so on. In layer D of Figure 2, with one end of the thread facing the observer, a top thread can be seen stacked on top of two bottom threads. Of course, in other embodiments, the molding block may have other numbers of layers, not necessarily six; the specific number of layers depends on the required bone thickness to be repaired.

[0049] Specifically, the width of the first gap is 1.5mm-2mm, the filaments on the adjacent two molding blocks form an angle of 25°-40°, and the diameter of the filaments is 0.3mm-0.5mm, which is conducive to forming crisscrossing interconnected grooves, so that the gel can be more uniformly solidified in the initial scaffold.

[0050] Preferably, the pregel solution also includes vascular fragments, which can utilize the vascular endothelial cells in the vascular fragments to accelerate angiogenesis in the porous scaffold, wherein the vascular fragments can be taken from autologous arteries.

[0051] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0052] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

[0053] References:

[0054] [1] Carvalho MS, Silva JC, Cabral JMS, et al. Cultured cell -derived extracelfular matrices to enhance the osteogenic differentiation andangiogenic properties of human mesenchymalstem / stromal cells[J]. Journal of Tissue Engineering and Regenerative Medicine, 2019, 13(9);1544-1558.

[0055] [2] Kang Y, Kim S, Bishop J, et a1. The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and β-TCP scaffold[]. Biomaterials, 2012, 33(29):6998-7007.

Claims

1. A method for preparing a bone-inspired bioactive polycaprolactone porous scaffold, characterized in that, The steps include: A preliminary scaffold with interconnected pores was 3D printed by fused deposition modeling. The preliminary scaffold was then immersed in a pregel solution and cultured to allow the pregel solution to gel. The gel on the outer surface of the preliminary scaffold was scraped off to obtain the bone-inspired bioactive polycaprolactone porous scaffold. The 3D printing raw materials were polycaprolactone and α-gentiobiose. The pregel solution included thrombin, decellularized matrix of human umbilical vein endothelial cells, and fibrinogen.

2. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 1, characterized in that, The α-gentiobiose accounts for 3%-8% of the 3D printing raw material, and the polycaprolactone accounts for 92%-97% of the 3D printing raw material.

3. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 1, characterized in that, The initial support is divided into multiple layers of molding blocks parallel to the horizontal plane. Each layer of the molding block includes multiple parallel thread clusters. Each thread cluster consists of three molten deposited filaments, including two bottom lines and one top line. The distance between the two bottom lines is the radius of the filament. The top line is stacked on the bottom lines and located between the two bottom lines.

4. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 3, characterized in that, The adjacent wire clusters on the same layer of the molding block have a first gap, and the wires on the same layer of the molding block are parallel; the wire clusters on two adjacent layers of the molding block form a certain angle, and the first gaps on multiple layers of the molding block constitute the connecting hole groove.

5. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 4, characterized in that, The width of the first gap is 1.5mm-2mm, the filaments on the two adjacent molding blocks form an angle of 25°-40°, and the diameter of the filaments is 0.3mm-0.5mm.

6. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 1, characterized in that, The pregel solution also includes vascular fragments.

7. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 1, characterized in that, The preparation steps of the pregel solution are as follows: prepare fibrinogen sodium chloride solution; prepare thrombin calcium chloride solution; prepare human umbilical vein endothelial cell decellularized matrix solution; mix the fibrinogen sodium chloride solution, the thrombin calcium chloride solution and the human umbilical vein endothelial cell decellularized matrix solution in an ice bath to obtain the pregel solution.

8. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 7, characterized in that, The preparation steps of the decellularized matrix solution for human umbilical vein endothelial cells are as follows: Mix 1 part of 0.5% Triton X-100 solution and 5 parts of 20mM NH4OH solution by volume to prepare the decellularized solution; Human umbilical vein endothelial cells were added to the decellularized solution, centrifuged, and collected into a clump. The clump was washed with PBS buffer until the pH was neutral. One part of the clump was soaked in three parts of 100 U / mL Dnase I solution at 37°C for 45 min. The insoluble portion was collected by centrifugation and lyophilized to obtain matrix powder. The matrix powder was soaked in 0.1% pepsin-hydrochloric acid solution for 48 h. The system was then adjusted to neutral with NaOH. Finally, it was diluted with PBS solution to prepare a human umbilical vein endothelial cell decellularized matrix solution containing 10 mg of the matrix powder per milliliter.

9. The method for preparing a bone-like bioactive polycaprolactone porous scaffold according to claim 8, characterized in that, The step of preparing the fibrinogen sodium chloride solution includes: adding fibrinogen powder to physiological saline to prepare a 10 mg / mL fibrinogen sodium chloride solution; the step of preparing the thrombin calcium chloride solution includes: preparing thrombin with 1M calcium chloride solution to prepare a 100 U / mL thrombin calcium chloride solution; the mixing ratio of the fibrinogen sodium chloride solution, the thrombin calcium chloride solution, and the human umbilical vein endothelial cell decellularized matrix solution is 10:1:

10.

10. A bone-inspired bioactive polycaprolactone porous scaffold, characterized in that, It is prepared by the method for preparing bone-like bioactive polycaprolactone porous scaffold according to any one of claims 1 to 9.

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