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CN-122005931-A - 3D printing hydrogel stent for treating bone defect and preparation method thereof

CN122005931ACN 122005931 ACN122005931 ACN 122005931ACN-122005931-A

Abstract

The invention discloses a 3D printing hydrogel bracket for treating bone defect and a preparation method thereof, wherein fish gelatin and fish bone powder are extracted from cold water fish, the fish gelatin and the fish bone powder are mixed to be used as biological ink, vascular endothelial growth factors are loaded in the biological ink, a 3D printing technology is adopted to prepare a methacryloylated fish gelatin bracket doped with the fish bone powder, and a microfluidic technology is adopted to prepare a methacryloylated fish gelatin and alginic acid microsphere with BMP 2. After the microsphere is assembled into the fishbone powder mixed hydrogel bracket, methacrylic acid groups on the surface of the microsphere and in the bracket are polymerized and crosslinked under the action of a light curing agent through ultraviolet irradiation, so that the microsphere and the frame are fixed to form the composite bracket. The invention solves the balance problem between the porosity, the mechanical stability and the active ingredient retention of the bracket, and provides a personalized and multifunctional repairing scheme for the complex bone defect.

Inventors

  • WANG YONGXIANG
  • ZHAO YUANJIN
  • QIU JIAYIN

Assignees

  • 江苏省苏北人民医院

Dates

Publication Date
20260512
Application Date
20260409

Claims (10)

  1. 1. A3D printing hydrogel bracket for treating bone defect is characterized by comprising a fish gelatin bracket prepared from main raw materials of fish methacryloylated gelatin and fish bone powder through 3D printing, and mixed microspheres filled in the bracket and containing the fish methacryloylated gelatin and the methacryloylated alginic acid, wherein the mass ratio of the fish gelatin bracket to the mixed microspheres is (8-15): 1.
  2. 2. The 3D printed hydrogel scaffold for treating bone defects of claim 1, wherein the mass ratio of fish gelatin scaffold to mixed microspheres is 10:1.
  3. 3. The 3D printed hydrogel scaffold for treating bone defects of claim 1, wherein the fish methacryloylated gelatin is fish skin gelatin that has been methacryloylated.
  4. 4. The 3D printed hydrogel scaffold for treating bone defects of claim 1, wherein the fishbone powder is obtained by sieving with a 1000 mesh sieve.
  5. 5. A method of preparing a 3D printed hydrogel scaffold for the treatment of bone defects according to any one of claims 1-4, comprising the steps of: (1) Performing methacryloylation treatment on gelatin derived from fish skin; (2) Collecting fresh cold water fish bones, grinding the fresh cold water fish bones into powder after treatment, and performing decellularization treatment for later use; (3) Preparing biological ink by taking the fish methacryloylated gelatin obtained in the step (1) and the fish bone powder obtained in the step (2) as main raw materials, and printing by a 3D printer; (4) Preparing mixed microspheres by taking fish methacryloyl gelatin and methacryloyl alginic acid as main raw materials, collecting the microspheres by adopting a calcium chloride solution, carrying out illumination crosslinking by using 405nm blue-violet light, and washing the collected microspheres by using water and absolute ethyl alcohol; (5) And (3) assembling the 3D printing bracket prepared in the step (3) and the mixed microsphere prepared in the step (4) at a low temperature in a photo-crosslinking mode, and carrying out illumination by using 405nm blue-violet light, and cleaning to obtain the composite microsphere.
  6. 6. The preparation method according to claim 5, wherein the step (1) comprises completely dissolving the fishskin gelatin in a cell Phosphate Buffer (PBS) solution at a concentration of 10% for 3 hours at 50 ℃, then slowly adding methacrylic acid to the previous solution at a ratio of 5 to 10% with vigorous stirring, reacting for 3 hours at 50 ℃, removing unreacted methacrylic acid by dialysis against pure water, and freeze-drying the resultant reaction product at-40 ℃.
  7. 7. The preparation method according to claim 5, wherein the step (2) comprises collecting fresh cold water fish bone, washing the fish bone with PBS, removing blood vessels, muscles and periosteum on the surface of the fish bone, and washing the fish bone with 10% sodium chloride solution and 3% sodium bicarbonate solution to remove fat residues. The frozen fish bones were ground in liquid nitrogen at-80 ℃ and the powder was sieved using a 1000 mesh sieve, the decellularization treatment comprising the steps of treatment with 1% polyethylene glycol octylphenyl ether (Triton X-100) solution to disrupt cell membranes, followed by subsequent maceration with Tris-HCl buffer containing 0.1% ethylenediamine tetraacetic acid (EDTA) followed by 0.1% Sodium Dodecyl Sulfate (SDS) to eliminate cellular components, and finally freeze-drying the treated fish bone powder at-40 ℃ and storing at-20 ℃ for subsequent use.
  8. 8. The preparation method according to claim 5, wherein the step (3) comprises dissolving the fish methacryloylated gelatin prepared in the step (1) and the fish bone powder (0 to 2%) obtained in the step (2) with deionized water to an optimal value of 1% by mass, and adding 0.5% by mass of a photo-curing agent LAP, 0.1% by mass of a photostabilizer lemon yellow and Vascular Endothelial Growth Factor (VEGF) (2. Mu.g mL 1) Printing with a 3D printer at a thickness of 10 μm for each layer, photocrosslinking for 10s at 405nm for each layer, and washing the support with PBS after printing, and preserving at 4deg.C.
  9. 9. The preparation method according to claim 5, wherein the step (4) comprises assembling a microfluidic device in a concentric circular structure using a capillary glass tube, dissolving 15% of fish methacryloylated gelatin, 5% sodium alginate and 0.5% of lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (LAP) in deionized water as an aqueous phase, using paraffin oil as an oil phase, controlling the flow rate ratio of the aqueous phase to the oil phase to 1:10 with a test syringe pump, preparing microspheres, collecting microspheres with a 5% calcium chloride solution, and crosslinking with 405nm blue violet light for 5 minutes, and washing the collected microspheres with water and absolute ethanol in sequence.
  10. 10. The preparation method according to claim 5, wherein the step (5) comprises assembling the 3D printing support prepared in the step (3) and the mixed microsphere prepared in the step (4) by means of photocrosslinking, sucking the microsphere into the gap of the 3D printing support by using a microinjector, wherein the weight ratio of the 3D printing support to the microsphere is (8-15): 1, irradiating the composite support for 5 minutes by using 405nm blue-violet light, and then washing the uncrosslinked microsphere in the support by using PBS.

Description

3D printing hydrogel stent for treating bone defect and preparation method thereof Technical Field The invention relates to the technical field of human tissue engineering, in particular to a 3D printing hydrogel bracket for treating bone defects and a preparation method thereof. Background Bone is one of the largest organ systems of the human body, and the weight of the bone accounts for 15% -20% of the total weight of the human body, so that mechanical support is provided for exercise. Bone defects are common refractory diseases in clinical orthopaedics, and are mostly induced by various factors such as tumors, wounds, infection and the like. Due to the importance of bone organs on normal physiological movement of human bodies, bone defects not only seriously damage the movement function of patients and reduce the quality of life, but also bring heavy medical resource consumption and socioeconomic burden, and become the current difficult problem to be solved urgently in orthopedics. Despite the potential for self-healing, bone injuries of critical size are difficult to repair by themselves, requiring bone fillers to promote bridging of the bone fragments. At present, various repair strategies have been explored and applied in the clinical and scientific fields, wherein a repair scheme based on biological materials has good biocompatibility and tissue affinity, and is a core research direction in the field of bone repair and is widely applied. The biological material is mainly divided into two major types of collagen materials and natural bone-derived materials, and certain repair effects are achieved in clinical application, wherein the collagen materials comprise animal-derived gelatin, plant collagen extracts and the like, and the natural bone-derived materials comprise human bone grafts and animal-derived bone tissue derivatives such as cattle, pigs and the like. However, both types of materials have inherent defects, which significantly limit their clinical application prospects. On the one hand, two types of materials are widely subjected to safety risks and ethical disputes, animal-derived materials possibly carry species-specific pathogenic microorganisms, the risk of cross infection is increased, and human-derived materials face double limitations of donor shortage and ethical considerations. On the other hand, the materials generally lack reasonable structural design in the preparation and application processes, are difficult to accurately match the porous structure and mechanical supporting performance of natural bone tissues, and cannot provide ideal microenvironment for bone cell adhesion, proliferation and differentiation. The defects limit the clinical application value of the existing biological materials together, and develop a novel bone repair material with biological safety, excellent structural characteristics and biological activity, thereby having important academic research significance and urgent clinical transformation requirements. The prior art currently has the following limitations and disadvantages: (1) The existing bone repair material has double defects of hidden biosafety hazards (such as cross infection) and insufficient mechanical properties, and a single material is difficult to simultaneously meet the structural requirement of bone cell growth and the supply of osteogenic activity. (2) The growth factor release of the traditional bone repair scaffold lacks time sequence and cannot match the natural bone repair process of early angiogenesis and later osteogenic differentiation. (3) The structural design of the existing composite scaffold and the microenvironment adaptability of the bone defect are insufficient, and the comprehensive requirements of cell adhesion, nutrition exchange and active factor retention are difficult to be met. Disclosure of Invention Aiming at the problems in the prior art, the invention provides a 3D printing hydrogel bracket for treating bone defects and a preparation method thereof, so as to solve or at least alleviate part or all of the technical problems in the prior art. In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme: the invention provides a 3D printing hydrogel bracket for treating bone defects, which comprises a fish gelatin bracket prepared by 3D printing of main raw materials of fish methacryloylated gelatin and fish bone powder, and mixed microspheres filled in the bracket and containing the fish methacryloylated gelatin and the methacryloylated alginic acid, wherein the mass ratio of the fish gelatin bracket to the mixed microspheres is (8-15): 1. As a preferred embodiment, the mass ratio of the fish gelatin scaffold to the mixed microspheres is 10:1. In a preferred embodiment, the fish methacryloylated gelatin is obtained by subjecting fish skin gelatin to methacryloylation treatment. In a preferred embodiment, the fishbone powder is obtained by sieving with a