A bone organoid scaffold and method of manufacturing the same

Abstract

The application provides a bone-like organ scaffold and a manufacturing method thereof, and relates to the technical field of bone-like organ scaffolds.The scaffold comprises a plurality of functional layers arranged in sequence;each functional layer comprises a plurality of through holes in the transverse direction;the through holes of different functional layers correspond to each other in the longitudinal direction, forming a plurality of first channels penetrating through the scaffold;each functional layer comprises upper and lower parts in the longitudinal direction;the lower part is a solid matrix layer;the upper part is a filling layer;the filling layer comprises a plurality of uniformly arranged solid matrix strips;there are gaps between adjacent solid matrix strips, forming second channels connecting the periphery of the functional layer and the first channels.The scheme can be rapidly produced and manufactured through 3D bioprinting technology, shortening the production cycle of the scaffold;the bone-like organ designed in the scheme, which is coupled with bionic mineralization and new blood vessels, can be implanted in the femoral head necrosis area in the early collapse stage and provide an environment for sufficient mechanical support, blood supply reconstruction and bone repair in combination with marrow core decompression technology.

Description

Technical Field

[0001] This invention relates to the field of bone organ scaffold technology, and in particular to a bone organ scaffold and its manufacturing method. Background Technology

[0002] Osteonecrosis of the femoral head (ONFH), also known as avascular necrosis of the femoral head (ANFH), is a multifactorial progressive osteoarthritis characterized by ischemic necrosis of bone cells, destruction of the trabecular bone microstructure, and decreased mechanical properties of the femoral head, ultimately leading to femoral head collapse and secondary osteoarthritis. ONFH can occur at any age, but it is most common in young and middle-aged adults aged 20-40 and has become a significant public health issue. Although current treatments can improve symptoms and function in ONFH patients to some extent, they cannot effectively restore blood supply to the femoral head and achieve bone regeneration, thus failing to fundamentally stop disease progression. Therefore, how to achieve angiogenesis and new bone formation at the affected site as quickly as possible in the early stages of ONFH to slow the rapid progression of the disease has become a major challenge for the medical community. Summary of the Invention

[0003] The purpose of this invention is to provide a bone organ scaffold and a method for manufacturing the same, so as to solve at least one of the above-mentioned technical problems existing in the prior art.

[0004] In a first aspect, to solve the above-mentioned technical problems, the present invention provides a bone organ scaffold, comprising a plurality of functional layers arranged sequentially; Each functional layer includes several through holes in the horizontal direction; the through holes of different functional layers correspond to each other in the vertical direction, forming several first channels that penetrate the bracket. Each functional layer comprises two parts in the vertical direction: the lower part is a solid matrix layer; the upper part is a filler layer; the filler layer includes multiple uniformly distributed solid matrix strips; gaps are left between adjacent solid matrix strips to form a second channel connecting the periphery of the functional layer with the first channel; the first channel is used to fill with a filler gel containing angiogenesis factors. In this way, after the above-mentioned scaffold is cultured, before the operation, the filling glue is injected into the first channel of the scaffold, and then the scaffold is implanted into the lesion area of ​​femoral head necrosis. Combined with bone grafting, the gap between the scaffold and the cancellous bone particles is filled. Finally, acrylic resin bone cement is used to seal it, so that the scaffold can be tightly integrated with the femoral head and form blood vessels based on the first and second channels, so as to achieve the purpose of providing mechanical support, blood supply reconstruction and bone repair.

[0005] In one feasible implementation, the scaffold is specifically made of GelMA (methacrylamide gelatin), AlgMA (methacrylic acid alginate), and modified nHA (nano-hydroxyapatite); GelMA is used to mimic collagen and provide a photocrosslinking mechanism; AlgMA is used to provide ionic crosslinking sites; nHA is used to form an organic / inorganic hybrid system and mimic the extracellular mechanism of natural bone cells; these substances are extremely safe and reliable for the human body, and due to their high degree of crosslinking, there is a very low probability that they will be lost into the human circulation.

[0006] In one feasible implementation, the filler adhesive is specifically made of (highly cross-linked) polyacrylic acid (pAA), polyethylene glycol diacrylate (PEGDA), and angiogenic factors (such as VEGF-A, i.e., vascular endothelial growth factor-A). These substances are relatively safe for the human body, and through high cross-linking, they have a very low probability of being lost into the human circulation. At the same time, this helps to achieve the slow release effect of angiogenic factors.

[0007] In one feasible implementation, the support is cylindrical in shape to accommodate holes drilled within the lesion area of ​​femoral head necrosis.

[0008] In one feasible implementation, the functional layer has one through-hole, and the through-hole is located in the center of the functional layer, so as to make the radial strength of the stent uniform and the radial blood revascularization path length consistent.

[0009] In one feasible implementation, the solid matrix strip is a straight segment with a square cross-section to meet the requirements of 3D bioprinting and to make the second channel shorter, thereby increasing the rate of blood vessel formation.

[0010] In one feasible implementation, the solid matrix strips of adjacent functional layers are arranged in a circumferentially rotating progressive manner to reduce the stress concentration problem in the axial direction of the support and improve the overall strength of the support.

[0011] Secondly, based on the same inventive concept, this application also provides a method for manufacturing the above-mentioned bone organ scaffold, comprising the following steps: Step 1: Acquire CT images of the patient's femoral head region; after preprocessing the CT images and constructing a femoral head model, extract the femoral head lesion region model.

[0012] Step 2: Generate a scaffold model based on the femoral head lesion area model.

[0013] In one feasible implementation, the specific method for generating the stent model includes: extracting the largest circumscribed circle from the tangential plane of the femoral head lesion area model, and using the diameter of the largest circumscribed circle as the outer diameter of the stent; using the radial length of the femoral head lesion area model as the stent length; dividing the stent length by a preset functional layer thickness and rounding to obtain the number of functional layers; calculating the perforation of the through hole based on a preset through hole diameter; dividing the through hole perforation by the sum of the width of the solid matrix strip and the inner width of the second channel and rounding to obtain the number of solid matrix strips; using the width of the solid matrix strip as the step size for the circumferential rotation of the solid matrix strip to obtain the stent model; thus, personalized stents can be generated according to the size of the femoral head lesion area of ​​different patients.

[0014] In one feasible implementation, the width of the solid matrix strip is 100 micrometers, which enables 3D bioprinting and supports the functional layer without causing collapse.

[0015] In one feasible implementation, the functional layer has a thickness of 200 micrometers, wherein the solid matrix layer has a thickness of 100 micrometers and the filler layer has a thickness of 100 micrometers, which can meet the requirements of 3D bioprinting.

[0016] In one feasible implementation, the specific method for calculating the inner width of the second channel includes: Step a1: Collect blood biochemical indicators from the patient and define the biochemical indicator vector. ;Specific expressions include: ; in, Indicates blood viscosity; Hematocrit indicates the volume of red blood cells; Indicates platelet concentration; Step a2: Construct a microchannel size calculation model, which specifically includes: ; in, Indicates the second channel Width at the location; This indicates the distance measured from the inside (through-hole end); Indicates the inner width of the second channel; Indicates the outer width of the second channel; Indicates the length of the second channel; Step a3: Construct the channel size adjustment coefficient, the specific expression of which includes: ; in, Indicates the channel size adjustment factor; These represent weighting factors, which can be calibrated based on conventional fluid dynamics models and clinical data. For example, , , A negative value indicates that a high platelet concentration may increase the risk of coagulation blockage, requiring an increase in the width of the second channel; This represents the baseline reference value for blood viscosity. This indicates the baseline reference value for hematocrit. This represents the baseline reference value for platelet concentration; Step a4, based on Adjusting the channel width, specific expressions include: ; in, This represents the initial value of the inner width of the second channel; This represents the initial value of the outer width of the second channel; Step a5: Substitute the adjusted width into the microchannel size calculation model to predict the blood inflow rate and vascularization time. The specific expressions include: ; ; in, Indicates the rate of blood inflow; Indicates vascularization time; Indicates blood viscosity, i.e. ; Hematocrit, or hematocrit ; This represents the first calibration coefficient, which can be obtained by fitting experimental data and is used to evaluate the influence of channel average width and blood viscosity on blood inflow velocity. This represents the second calibration coefficient, which can be obtained by fitting experimental data and is used to evaluate the effect of hematocrit on blood inflow velocity. Represents the baseline vascularization time, a constant that can be determined experimentally; This represents the acceleration coefficient, a positive coefficient that can be obtained by fitting experimental data, i.e. Each additional unit can shorten the vascularization time; Step a6, based on and Determine the linear relationship between them, and construct the objective function and constraints. The objective function is used to make Minimize, specifically the following expressions: ; ; in, Indicates the linear scaling factor; The constraints include: ; ; in, This represents the minimum channel width in 3D bioprinting. This indicates the maximum channel width in 3D bioprinting; This represents the minimum blood flow velocity within the physiological range; This indicates the maximum blood flow velocity within the physiological range; Step a7: The optimal inner width of the second channel is obtained by using the golden ratio search method.

[0017] In one feasible implementation, the golden section search method specifically includes: Step a71: Determine the search interval based on the constraints. Among them, the lower limit of the interval equal Upper limit of the interval equal Set the maximum number of iterations; Step a72: Calculate the golden ratio point and The specific formulas include: ; ; in, This represents the golden ratio constant, which can take a value of 1.618. Step a73: For each iteration, calculate and Corresponding objective function value and : like The optimal solution lies in the interval Internal update ; Otherwise, the optimal solution lies in the interval Internal update ; Step a74: Iteratively execute step a72 until the maximum number of iterations is reached.

[0018] Step 3: Extract (primary) mesenchymal stem cells (BMSCs) and vascular endothelial cells (VECs) from the patient and culture them separately.

[0019] In one feasible embodiment, the culture medium for the mesenchymal stem cells comprises: α-MEM medium (α-modified Eagle) supplemented with (10% by volume) fetal bovine serum (FBS), (1% by volume) penicillin / streptomycin, (10-100 nM / L) dexamethasone, (50-200 μM / L) ascorbic acid and (10 mM / L) β-glycerophosphate.

[0020] In one feasible implementation, the culture medium for vascular endothelial cells comprises: a basal culture medium supplemented with (10% by volume) fetal bovine serum, (1% by volume) penicillin / streptomycin, (90 μg / ml) heparin, and (15 μg / ml) endothelial cell growth supplement.

[0021] Step 4: Dissolve gelatin (e.g., 10g) and sodium alginate (e.g., 0.5g) in two separate containers containing deionized water (e.g., 42mL) to obtain two solutions. Mix the two solutions and add a predetermined volume (e.g., 4mL) of methacrylate (MA) to adjust the pH to the range of 8-10 to obtain a mixed solution. Add a predetermined weight (e.g., 8g) of modified nHA powder to the mixed solution to obtain a mixture. Stir the mixture in a water bath with a magnetic stirrer (50°C) for a predetermined time (e.g., 3 hours) and then stop the reaction with deionized water (at room temperature). Dialyze the mixture at a predetermined temperature (e.g., 37°C) and a predetermined time (e.g., 7 days) to remove unreacted small molecules to obtain a bio-ink containing GelMA, AlgMA, and modified nHA.

[0022] In one feasible embodiment, the solid weight ratio of gelatin, sodium alginate, nHA, and methacrylate is as follows: gelatin accounts for 44.444%; sodium alginate accounts for 2.222%; nHA accounts for 35.556%; and methacrylate accounts for 17.778%.

[0023] In one feasible implementation, the modified nHA refers to the RGD peptide being grafted onto the surface of nHA via a PDA coating-Schiff alkalization valence-donating linkage, and the E7 (heptaglutamate) sequence of the VEGF mimic peptide-derived QK peptide being adsorbed onto calcium ions in nHA via electrostatic attraction, thereby achieving the dual functions of scaffold cell adhesion and angiogenesis induction, thereby accelerating the self-assembly of bone organoids and the self-growth of blood vessels.

[0024] In one feasible implementation, the specific method of the modification includes: Step b1: Disperse the nHA dry powder in deionized water according to the preset concentration (e.g., 5 mg / mL); then perform ultrasonic vibration dispersion for the preset time (e.g., 5-10 minutes) to ensure that the powder is fully dispersed and the sample is kept moist, so that the subsequent PDA coating is more uniform. Step b2: At room temperature, prepare a Tris-HCl buffer solution of a predetermined concentration (e.g., 10 mM / L) and adjust its pH to 8.5; dissolve dopamine•HCl (dopamine hydrochloride) in the Tris-HCl buffer solution to a predetermined concentration (e.g., 1–2 mg / mL) to obtain a dopamine solution; suspend the dispersed nHA powder in the dopamine solution for reaction, and gently shake for a predetermined time (e.g., 12–24 hours) to avoid strong shear force. During this process, the coating will gradually darken the color of the solution; then, wash the dopamine solution with (large amounts) deionized water or PBS (phosphate buffered saline) for a predetermined number of times (e.g., 3 times) to remove unreacted dopamine•HCl; recover the PDA-nHA particles (i.e., nano-hydroxyapatite particles coated with PDA) by centrifugation and keep them moist; Step b3: At room temperature, dissolve the RGD peptide at a preset concentration (e.g., 1.5 mg / mL) in Tris buffer (10 mM / L) to obtain an RGD solution (pH 8.5); add PDA-nHA particles to the RGD solution for reaction, and gently shake for a preset time (e.g., 6 hours); then wash the RGD solution with PBS a preset number of times (e.g., 3-5 times); exchange the washing buffer by centrifugation until no free peptides are present in the washing buffer (which can be determined by UV or BCA / protein quantification method). Step b4: Dissolve E7-QK dry powder in deionized water at a preset concentration (e.g., 1 mg / mL) to obtain an E7-QK solution, and dilute it with TBS / PBS buffer (pH 7.4) (e.g., 100 μM / L); suspend the dispersed nHA powder in the E7-QK solution for reaction, and incubate it in the dark with gentle shaking for a preset time (e.g., 1-4 hours); then recover the nHA-E7-QK composite particles by centrifugation or filtration; wash the nHA-E7-QK composite particles with PBS or deionized water a preset number of times (e.g., 3 times) to remove unbound peptides.

[0025] Step 5: Adjust the mesenchymal stem cell concentration to a preset level (e.g., 1.0 × 10⁻⁶). 6 A suspension of cells per ml was mixed with the bio-ink (10 wt% concentration) to obtain cell-loaded 3D printing bio-ink.

[0026] Step 6: Based on the scaffold model, load 3D printing bio-ink into a DLP printer and perform 3D bioprinting according to the preset program to obtain a scaffold. Simultaneously, immerse the scaffold in α-MEM culture medium for further growth and development.

[0027] In one feasible implementation, the α-MEM culture medium in step 6 also includes (10% by volume) fetal bovine serum, (1% by volume) penicillin / streptomycin, (10 nM / L) dexamethasone, (100 μM / L) ascorbic acid and (10 mM / L) β-glycerophosphate.

[0028] Step 7: Place the scaffold in an incubator (37°C, 5% carbon dioxide) for culture. After a preset time (e.g., 7 days), place the scaffold in a culture medium containing vascular endothelial cells for prevascularization treatment to obtain a bone organoid scaffold.

[0029] In one feasible implementation, the culture medium containing vascular endothelial cells in step 7 specifically includes: EBM-2 (Lonza) basal medium supplemented with (2% by volume) fetal bovine serum, (1% by volume) penicillin / streptomycin, (5 ng / mL) human epidermal growth factor (hEGF), (10 ng / mL) human basic fibroblast growth factor (hFGF-β), (20 ng / mL) long-acting human insulin-like growth factor-1 (Long R3 IGF-1), (0.5–10 ng / mL) vascular endothelial growth factor (VEGF-165), (1 μg / mL) ascorbic acid, (0.2 μg / mL) hydrocortisone, and (22.5 μg / mL) heparin.

[0030] In one feasible implementation, the manufacturing method further includes step 8, injecting a filler containing angiogenesis factors into the first channel of the bone organoid scaffold before surgery.

[0031] In one feasible embodiment, the specific preparation method of the filler adhesive includes: Step c1: Prepare a sterile aqueous solution containing pAA; mix PEGDA and pAA thoroughly at a preset mass percentage (e.g., 1:99wt%) to obtain a prepolymer mixture, thereby ensuring that all solutes are fully dispersed under mild conditions; Step c2: Add an aqueous photoinitiator (LAP) to the prepolymer mixture according to a preset mass percentage (e.g., 0.05-0.1wt%). Step c3: Dissolve human recombinant angiogenesis factor VEGF-A165 in sterile PBS (containing 0.1% BSA, bovine serum albumin) at a preset concentration (e.g., 0.5-1 μg / ml) to prepare a stock solution and cool it. Step c4: In an ice bath at a preset temperature (e.g., 4°C), add the cooled mother liquor to the prepolymer mixture that has already had the photoinitiator added, obtain the mixture and mix it (gently) (for about 30 seconds) to avoid the generation of bubbles; Step c5: Fill the mold with the mixture and irradiate it with a 405nm blue LED with a light intensity of about 10–20mW / cm² for 30–120 seconds to obtain a fully cross-linked filler. Step c6: On a shaker at room temperature, repeatedly wash the filling gel with sterile PBS (containing 0.1% BSA) (at least 3 times, for 10-15 minutes each time).

[0032] By adopting the above technical solution, the present invention has the following beneficial effects: This invention provides a bone organoid scaffold and its manufacturing method, which can be rapidly manufactured using 3D bioprinting technology, shortening the scaffold production cycle and allowing for flexible personalized manufacturing. The biomimetic mineralization and neovascularization coupled bone organoids designed in this scheme can be combined with core decompression technology to implant into the femoral head necrosis area in the early stage of collapse, providing sufficient mechanical support and an environment for blood revascularization and bone repair. This scheme adds RGD peptides and VEGF-mimicking peptides to the surface of nano-hydroxyapatite, enabling the scaffold to achieve the dual functions of cell adhesion and angiogenesis induction, thereby accelerating organoid self-assembly and angiogenesis. To facilitate material exchange and angiogenesis, this scheme reserves channels from the periphery to the center, with a continuous gradient in channel width, which promotes vascular infiltration, enhances cell migration and material transport, ensuring the bone organoid grows uniformly in multiple dimensions and achieves rapid vascularization. The organic / inorganic hybrid system constructed in this scheme simulates the extracellular mechanisms of natural bone cells, while the photocrosslinking mechanism of GelMA ensures that the mixed bio-ink can be processed by digital light processing. This solution utilizes a highly cross-linked filler gel to continuously induce blood vessels to grow from the periphery of the stent to the center by slowly releasing pro-angiogenic factors. After implantation, it can accelerate the integration of blood vessels and the stent in the body. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 A frontal sectional view of a bone organ scaffold provided in an embodiment of the present invention; Figure 2 A top sectional view of a bone organ scaffold provided in an embodiment of the present invention; Figure 3 This is an illustration of an installation scenario for a bone organ scaffold provided in an embodiment of the present invention; Figure 4 This is an illustration of a single precession of adjacent functional layers provided in an embodiment of the present invention.

[0035] Figure label: 1-Cancellous bone particles; 2-Scaffold; 3-Filling adhesive; 4-Acrylic resin bone cement; 5-Solid matrix strip; 6-Through hole. Detailed Implementation

[0036] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0038] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0039] The present invention will be further explained below with reference to specific embodiments.

[0040] Example 1: like Figure 1-2 As shown, the bone organ scaffold 2 provided in this embodiment includes multiple functional layers arranged sequentially; Each functional layer includes a through hole 6 in the horizontal direction; the through holes of different functional layers correspond to each other in the vertical direction to form a first channel penetrating the bracket; Each functional layer comprises two parts in the longitudinal direction: the lower part is a solid matrix layer; the upper part is a filling layer; the filling layer includes multiple uniformly distributed solid matrix strips 5; gaps are left between adjacent solid matrix strips 5 to form a second channel connecting the periphery of the functional layer with the first channel; the first channel is used to fill with a filler gel 3 containing angiogenic factors (e.g., VEGF-A). Thus, as Figure 3 As shown, after the above-mentioned scaffold 2 is cultured, before the operation, the filling glue 3 is injected into the first channel of the scaffold 2, and then the scaffold 2 is implanted into the lesion area of ​​femoral head necrosis. The gap between the scaffold and the cancellous bone particles 1 is filled by combined pressure bone grafting. Finally, the gap is sealed with acrylic resin bone cement 4. This allows the scaffold 2 to be tightly integrated with the femoral head and form blood vessels based on the first and second channels, so as to achieve the purpose of providing mechanical support, blood supply reconstruction and bone repair.

[0041] Furthermore, the specific materials of the scaffold 2 include GelMA (methacrylamide gelatin), AlgMA (methacrylic acid alginate), and modified nHA (nano-hydroxyapatite); GelMA is used to mimic collagen and provide a photocrosslinking mechanism; AlgMA is used to provide ionic crosslinking sites; nHA is used to form an organic / inorganic hybrid system and mimic the extracellular mechanism of natural bone cells; these substances are extremely safe and reliable for the human body, and due to the high degree of crosslinking, there is a very low probability that they will be lost into the human circulation.

[0042] Furthermore, the support 2 is cylindrical in shape to accommodate holes drilled within the lesion area of ​​femoral head necrosis.

[0043] Furthermore, the functional layer has one through-hole, and the through-hole is located in the center of the functional layer, so as to ensure that the radial strength of the stent is uniform and the radial blood revascularization path length remains consistent.

[0044] Furthermore, the solid matrix strip 5 is a straight line segment with a square cross-section to meet the requirements of 3D bioprinting and to make the second channel shorter, thereby increasing the rate of blood vessel formation.

[0045] Furthermore, such as Figure 4 As shown, the solid matrix strips 5 of adjacent functional layers are arranged in a circumferentially rotating manner to reduce the stress concentration problem in the axial direction of the support and improve the overall strength of the support 2.

[0046] Example 2: This embodiment provides a method for manufacturing the above-mentioned bone organ scaffold, including the following steps: Step 1: Acquire CT images of the patient's femoral head region; after preprocessing the CT images and constructing a femoral head model, extract the femoral head lesion region model.

[0047] Step 2: Generate a scaffold model based on the femoral head lesion area model.

[0048] Furthermore, the specific method for generating the stent model includes: extracting the largest circumscribed circle from the tangential plane of the femoral head lesion area model, and using the diameter of the largest circumscribed circle as the outer diameter of the stent; using the radial length of the femoral head lesion area model as the stent length; dividing the stent length by a preset functional layer thickness and rounding to obtain the number of functional layers; calculating the perforation of the perforation based on a preset perforation diameter; dividing the perforation of the perforation by the sum of the width of the solid matrix strip and the inner width of the second channel and rounding to obtain the number of solid matrix strips; using the width of the solid matrix strip as the step size for the circumferential rotation of the solid matrix strip to obtain the stent model; in this way, personalized stents can be generated according to the size of the femoral head lesion area of ​​different patients.

[0049] Furthermore, the width of the solid matrix strip is 100 micrometers (and the height is also 100 micrometers), which enables 3D bioprinting and supports the functional layer to prevent collapse.

[0050] Furthermore, the functional layer has a thickness of 200 micrometers, wherein the solid matrix layer has a thickness of 100 micrometers and the filler layer has a thickness of 100 micrometers, which can meet the requirements of 3D bioprinting.

[0051] Furthermore, the specific calculation method for the inner width of the second channel includes: Step a1: Collect blood biochemical indicators from the patient and define the biochemical indicator vector. ;Specific expressions include: ; in, Indicates blood viscosity; Hematocrit indicates the volume of red blood cells; Indicates platelet concentration; Step a2: Construct a microchannel size calculation model, which specifically includes: ; in, Indicates the second channel Width at the location; This indicates the distance measured from the inside (through-hole end); Indicates the inner width of the second channel; Indicates the outer width of the second channel; Indicates the length of the second channel; Step a3: Construct the channel size adjustment coefficient, the specific expression of which includes: ; in, Indicates the channel size adjustment factor; These represent weighting factors, which can be calibrated based on conventional fluid dynamics models and clinical data. For example, , , A negative value indicates that a high platelet concentration may increase the risk of coagulation blockage, requiring an increase in the width of the second channel; This represents the baseline reference value for blood viscosity. This indicates the baseline reference value for hematocrit. This represents the baseline reference value for platelet concentration; Step a4, based on Adjusting the channel width, specific expressions include: ; in, This represents the initial value of the inner width of the second channel; This represents the initial value of the outer width of the second channel; Step a5: Substitute the adjusted width into the microchannel size calculation model to predict the blood inflow rate and vascularization time. The specific expressions include: ; ; in, Indicates the rate of blood inflow; Indicates vascularization time; Indicates blood viscosity, i.e. ; Hematocrit, or hematocrit ; This represents the first calibration coefficient, which can be obtained by fitting experimental data and is used to evaluate the influence of channel average width and blood viscosity on blood inflow velocity. This represents the second calibration coefficient, which can be obtained by fitting experimental data and is used to evaluate the effect of hematocrit on blood inflow velocity. Represents the baseline vascularization time, a constant that can be determined experimentally; This represents the acceleration coefficient, a positive coefficient that can be obtained by fitting experimental data, i.e. Each additional unit can shorten the vascularization time; Step a6, based on and Determine the linear relationship between them, and construct the objective function and constraints. The objective function is used to make Minimize, specifically the following expressions: ; ; in, Indicates the linear scaling factor; The constraints include: ; ; in, This represents the minimum channel width in 3D bioprinting. This indicates the maximum channel width in 3D bioprinting; This represents the minimum blood flow velocity within the physiological range; This indicates the maximum blood flow velocity within the physiological range; Step a7: The optimal inner width of the second channel is obtained by using the golden ratio search method.

[0052] Furthermore, the golden section search method specifically includes: Step a71: Determine the search interval based on the constraints. Among them, the lower limit of the interval equal Upper limit of the interval equal Set the maximum number of iterations; Step a72: Calculate the golden ratio point and The specific formulas include: ; ; in, This represents the golden ratio constant, which can take a value of 1.618. Step a73: For each iteration, calculate and Corresponding objective function value and : like The optimal solution lies in the interval Internal update ; Otherwise, the optimal solution lies in the interval Internal update ; Step a74: Iteratively execute step a72 until the maximum number of iterations is reached.

[0053] Furthermore, the inner width of the second channel can be 50 micrometers; the outer (surface) width of the second channel can be 500 micrometers; the length of the solid matrix strip can be 8000 micrometers; and the number of solid matrix strips in each filling layer can be 112; thus, it is suitable for most femoral head necrosis area surgeries.

[0054] Step 3: Extract (primary) mesenchymal stem cells (BMSCs) and vascular endothelial cells (VECs) from the patient and culture them separately.

[0055] Furthermore, the culture medium for the mesenchymal stem cells comprises: α-MEM medium (α-modified Eagle) supplemented with (10% by volume) fetal bovine serum (FBS), (1% by volume) penicillin / streptomycin, (10 nM / L) dexamethasone, (100 μM / L) ascorbic acid and (10 mM / L) β-glycerophosphate.

[0056] Furthermore, the culture medium for vascular endothelial cells consists of a basal medium supplemented with (10% by volume) fetal bovine serum, (1% by volume) penicillin / streptomycin, (90 μg / ml) heparin, and (15 μg / ml) endothelial cell growth supplement.

[0057] Step 4: Dissolve gelatin (e.g., 10g) and sodium alginate (e.g., 0.5g) in two separate containers containing deionized water (e.g., 42mL) to obtain two solutions. Mix the two solutions and add a predetermined volume (e.g., 4mL) of methacrylate (MA) to adjust the pH to the range of 8-10 to obtain a mixed solution. Add a predetermined weight (e.g., 8g) of modified nHA powder to the mixed solution to obtain a mixture. Stir the mixture in a water bath with a magnetic stirrer (50°C) for a predetermined time (e.g., 3 hours) and then stop the reaction with deionized water (at room temperature). Dialyze the mixture at a predetermined temperature (e.g., 37°C) and a predetermined time (e.g., 7 days) to remove unreacted small molecules to obtain a bio-ink containing GelMA, AlgMA, and modified nHA.

[0058] Furthermore, the solid weight ratio of gelatin, sodium alginate, nHA and methacrylate is as follows: gelatin accounts for 44.444%; sodium alginate accounts for 2.222%; nHA accounts for 35.556%; and methacrylate accounts for 17.778%.

[0059] Furthermore, the modified nHA refers to the RGD peptide being grafted onto the surface of nHA via a PDA coating-Schiff alkalization valence-donating linkage, and the E7 (heptaglutamate) sequence of the VEGF mimic peptide-derived QK peptide being adsorbed onto calcium ions in nHA via electrostatic attraction, thereby achieving the dual functions of scaffold cell adhesion and angiogenesis induction, and thus accelerating the self-assembly of bone organoids and the self-growth of blood vessels.

[0060] Furthermore, the specific methods of the modification include: Step b1: Disperse the nHA dry powder in deionized water according to the preset concentration (e.g., 5 mg / mL); then perform ultrasonic vibration dispersion for the preset time (e.g., 5-10 minutes) to ensure that the powder is fully dispersed and the sample is kept moist, so that the subsequent PDA coating is more uniform. Step b2: At room temperature, prepare a Tris-HCl buffer solution of a predetermined concentration (e.g., 10 mM / L) and adjust its pH to 8.5; dissolve dopamine•HCl (dopamine hydrochloride) in the Tris-HCl buffer solution to a predetermined concentration (e.g., 1–2 mg / mL) to obtain a dopamine solution; suspend the dispersed nHA powder in the dopamine solution for reaction, and gently shake for a predetermined time (e.g., 12–24 hours) to avoid strong shear force. During this process, the coating will gradually darken the color of the solution; then, wash the dopamine solution with (large amounts) deionized water or PBS (phosphate buffered saline) for a predetermined number of times (e.g., 3 times) to remove unreacted dopamine•HCl; recover the PDA-nHA particles (i.e., nano-hydroxyapatite particles coated with PDA) by centrifugation and keep them moist; Step b3: At room temperature, dissolve the RGD peptide at a preset concentration (e.g., 1.5 mg / mL) in Tris buffer (10 mM / L) to obtain an RGD solution (pH 8.5); add PDA-nHA particles to the RGD solution for reaction, and gently shake for a preset time (e.g., 6 hours); then wash the RGD solution with PBS a preset number of times (e.g., 3-5 times); exchange the washing buffer by centrifugation until no free peptides are present in the washing buffer (which can be determined by UV or BCA / protein quantification method). Step b4: Dissolve E7-QK dry powder in deionized water at a preset concentration (e.g., 1 mg / mL) to obtain an E7-QK solution, and dilute it with TBS / PBS buffer (pH 7.4) (e.g., 100 μM / L); suspend the dispersed nHA powder in the E7-QK solution for reaction, and incubate it in the dark with gentle shaking for a preset time (e.g., 1-4 hours); then recover the nHA-E7-QK composite particles by centrifugation or filtration; wash the nHA-E7-QK composite particles with PBS or deionized water a preset number of times (e.g., 3 times) to remove unbound peptides.

[0061] Step 5: Adjust the mesenchymal stem cell concentration to a preset level (e.g., 1.0 × 10⁻⁶). 6 A suspension of cells per ml was mixed with the bio-ink (10 wt% concentration) to obtain cell-loaded 3D printing bio-ink.

[0062] Step 6: Based on the scaffold model, load 3D printing bio-ink into a DLP printer and perform 3D bioprinting according to the preset program to obtain a scaffold. Simultaneously, immerse the scaffold in α-MEM culture medium for further growth and development.

[0063] Furthermore, the α-MEM culture medium in step 6 also includes (10% by volume) fetal bovine serum, (1% by volume) penicillin / streptomycin, (10 nM / L) dexamethasone, (100 μM / L) ascorbic acid and (10 mM / L) β-glycerophosphate.

[0064] Step 7: Place the scaffold in an incubator (37°C, 5% carbon dioxide) for culture. After a preset time (e.g., 7 days), place the scaffold in a culture medium containing vascular endothelial cells for prevascularization treatment to obtain a bone organoid scaffold.

[0065] Furthermore, the culture medium containing vascular endothelial cells in step 7 specifically includes: EBM-2 (Lonza) basal medium supplemented with (2% by volume) fetal bovine serum, (1% by volume) penicillin / streptomycin, (5 ng / mL) human epidermal growth factor (hEGF), (10 ng / mL) human basic fibroblast growth factor (hFGF-β), (20 ng / mL) long-acting human insulin-like growth factor-1 (Long R3IGF-1), (0.5–10 ng / mL) vascular endothelial growth factor (VEGF-165), (1 μg / mL) ascorbic acid, (0.2 μg / mL) hydrocortisone, and (22.5 μg / mL) heparin.

[0066] Furthermore, the manufacturing method also includes step 8, injecting a filler containing angiogenesis factors into the first channel of the bone organoid scaffold before surgery.

[0067] Furthermore, the specific materials of the filler include polyethylene glycol diacrylate (PEGDA), polyacrylic acid (pAA), and angiogenic factors (such as VEGF-A). These substances are relatively safe for the human body, and through high cross-linking, they have a very low probability of being lost into the human circulation. At the same time, they help to achieve the slow release effect of angiogenic factors.

[0068] Furthermore, the specific preparation method of the filler adhesive includes: Step c1: Prepare a sterile aqueous solution containing pAA; mix PEGDA and pAA thoroughly at a preset mass percentage (e.g., 1:99wt%) to obtain a prepolymer mixture, thereby ensuring that all solutes are fully dispersed under mild conditions; Step c2: Add an aqueous photoinitiator (LAP) to the prepolymer mixture according to a preset mass percentage (e.g., 0.05-0.1wt%). Step c3: Dissolve human recombinant angiogenesis factor VEGF-A165 in sterile PBS (containing 0.1% BSA, bovine serum albumin) at a preset concentration (e.g., 0.5-1 μg / ml) to prepare a stock solution and cool it. Step c4: In an ice bath at a preset temperature (e.g., 4°C), add the cooled mother liquor to the prepolymer mixture that has already had the photoinitiator added, obtain the mixture and mix it (gently) (for about 30 seconds) to avoid the generation of bubbles; Step c5: Fill the mold with the mixture and irradiate it with a 405nm blue LED with a light intensity of about 10–20mW / cm² for 30–120 seconds to obtain a fully cross-linked filler. Step c6: On a shaker at room temperature, repeatedly wash the filling gel with sterile PBS (containing 0.1% BSA) (at least 3 times, for 10-15 minutes each time).

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bone organoid scaffold, characterized in that, It includes multiple functional layers arranged sequentially; Each functional layer includes several through holes in the horizontal direction; the through holes of different functional layers correspond to each other in the vertical direction, forming several first channels that penetrate the bracket. Each functional layer comprises two parts in the vertical direction: the lower part is a solid matrix layer; the upper part is a filler layer; the filler layer includes multiple uniformly distributed solid matrix strips; gaps are left between adjacent solid matrix strips to form a second channel connecting the periphery of the functional layer with the first channel; the first channel is used to fill with a filler adhesive containing angiogenesis factors; the solid matrix strips of adjacent functional layers are arranged in a circumferentially rotating progressive manner. The specific materials of the scaffold include GelMA, AlgMA, and modified nHA; The specific materials of the filler include polyacrylic acid, polyethylene glycol diacrylate, and angiogenesis-promoting factors; The angiogenesis-promoting factor is VEGF-A.

2. A method for manufacturing the stent as described in claim 1, characterized in that, include: Step 1: Acquire CT images of the patient's femoral head region; After preprocessing the CT images and constructing a femoral head model, the femoral head lesion area model is extracted; Step 2: Generate a scaffold model based on the femoral head lesion area model; Step 3: Extract mesenchymal stem cells and vascular endothelial cells from the patient and culture them separately; Step 4: Dissolve gelatin and sodium alginate in two containers containing deionized water at a predetermined weight ratio to obtain two solutions; mix the two solutions and add a predetermined volume of methacrylate to adjust the pH to the range of 8-10 to obtain a mixed solution; add a predetermined weight of modified nHA powder to the mixed solution to obtain a mixture; pass the mixture through a water bath magnetic stirrer for a predetermined time, and then stop the reaction by passing deionized water; dialyze the mixture at a predetermined temperature and time to remove unreacted small molecules to obtain a bio-ink containing GelMA, AlgMA and modified nHA; Step 5: Adjust the mesenchymal stem cells to a suspension of a preset concentration and mix them with the bio-ink to obtain 3D printing bio-ink loaded with cells; Step 6: Based on the scaffold model, load 3D printing bio-ink into a DLP printer and perform 3D bioprinting according to the preset program to obtain a scaffold. Simultaneously, immerse the scaffold in α-MEM culture medium. Step 7: Place the scaffold in an incubator for culture. After a preset time, place the scaffold in a culture medium containing vascular endothelial cells for prevascularization treatment to obtain a bone organoid scaffold.

3. The manufacturing method according to claim 2, characterized in that, The specific calculation method for the inner width of the second channel includes: Step a1: Collect blood biochemical indicators from the patient and define the biochemical indicator vector. ;Specific expressions include: ； in, Indicates blood viscosity; Hematocrit indicates the volume of red blood cells; Indicates platelet concentration; Step a2: Construct a microchannel size calculation model, which specifically includes: ； in, Indicates the second channel Width at the location; Indicates the distance measured from the inside; Indicates the inner width of the second channel; Indicates the outer width of the second channel; Indicates the length of the second channel; Step a3: Construct the channel size adjustment coefficient, the specific expression of which includes: ； in, Indicates the channel size adjustment factor; These represent the weighting factors; This represents the baseline reference value for blood viscosity. This indicates the baseline reference value for hematocrit. This represents the baseline reference value for platelet concentration; Step a4, based on Adjusting the channel width, specific expressions include: ； in, This represents the initial value of the inner width of the second channel; This represents the initial value of the outer width of the second channel; Step a5: Substitute the adjusted width into the microchannel size calculation model to predict the blood inflow rate and vascularization time. The specific expressions include: ； ； in, Indicates the rate of blood inflow; Indicates vascularization time; Indicates blood viscosity, i.e. ; Hematocrit, or erythrocyte volume index, is a measure of the number of red blood cells. ; Indicates the first calibration factor; Indicates the second calibration factor; Indicates the baseline vascularization time; Indicates the acceleration coefficient; Step a6, based on and Determine the linear relationship between them, and construct the objective function and constraints. The objective function is used to make Minimize, specifically the following expressions: ； ； in, Indicates the linear scaling factor; The constraints include: ； ； in, This represents the minimum channel width in 3D bioprinting. This indicates the maximum channel width in 3D bioprinting; This represents the minimum blood flow velocity within the physiological range; This indicates the maximum blood flow velocity within the physiological range; Step a7: The optimal inner width of the second channel is obtained by using the golden ratio search method.

4. The manufacturing method according to claim 2, characterized in that, The solid weight ratio of gelatin, sodium alginate, nHA and methacrylate is as follows: gelatin accounts for 44.444%; sodium alginate accounts for 2.222%; nHA accounts for 35.556%; and methacrylate accounts for 17.778%.

5. The manufacturing method according to claim 2, characterized in that, The modified nHA refers to the nHA surface on which RGD peptides are grafted via a PDA coating-Schiff alkalization valence-donating linkage, and the E7 sequence of the VEGF-mimicking peptide-derived QK peptide is adsorbed onto the nHA by electrostatic attraction.

6. The manufacturing method according to claim 5, characterized in that, The specific methods of the modification include: Step b1: Disperse nHA dry powder in deionized water according to the preset concentration; then perform ultrasonic vibration dispersion for the preset time. Step b2: At room temperature, prepare a Tris-HCl buffer solution of a predetermined concentration and adjust its pH to 8.5; dissolve dopamine•HCl in the Tris-HCl buffer solution to a predetermined concentration to obtain a dopamine solution; suspend the dispersed nHA powder in the dopamine solution for reaction and gently shake for a predetermined time; then wash the dopamine solution with deionized water or PBS for a predetermined number of times; recover the PDA-nHA particles by centrifugation and keep them moist. Step b3: At room temperature, dissolve the RGD peptide at a preset concentration in Tris buffer to obtain an RGD solution; add PDA-nHA particles to the RGD solution for reaction and gently shake for a preset time; then wash the RGD solution with PBS for a preset number of times; exchange the washing buffer by centrifugation until no free peptides are present in the washing buffer. Step b4: Dissolve E7-QK dry powder in deionized water according to the preset concentration to obtain E7-QK solution, and dilute it with TBS / PBS buffer; suspend the dispersed nHA powder in the E7-QK solution for reaction, and incubate gently with shaking in the dark for a preset time; then recover the nHA-E7-QK composite particles by centrifugation or filtration; wash the nHA-E7-QK composite particles with PBS or deionized water for a preset number of times to remove unbound peptides.

Images