3D printing biological ink, functional scaffold for repairing bone defects and preparation method thereof

Abstract

This invention provides a 3D-printed bio-ink, a functional scaffold, and a method for preparing the same for bone defect repair. The 3D-printed bio-ink comprises patient-derived injectable platelet-rich fibrin (iPRF), autologous adipose-derived stem cells (ADSC), gelatin, and sodium alginate. This invention uses polycaprolactone (PCL) and hydroxyapatite (HA) as the load-bearing structural materials of the scaffold, and employs autologous iPRF solution and ADSC as the main active ingredients of the bio-ink. This results in good bioactivity while avoiding immune rejection. Furthermore, the prepared 3D bioprinted bone defect repair scaffold exhibits high mechanical strength and a strong ability to release growth factors, which is beneficial for inducing angiogenesis and osteogenic differentiation of stem cells, making it suitable for large-scale application.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a 3D-printed bio-ink, a functional scaffold, and a method for preparing the same for bone defect repair. Background Technology

[0002] Bone defects caused by trauma, tumors, inflammation, and infection have a high incidence and pose significant risks. Their repair and reconstruction remain urgent problems and research hotspots in orthopedics. Autologous bone grafting is the "gold standard" for treating bone defects, but its clinical application is limited by donor availability and carries risks of complications such as infection and fracture due to additional bone harvesting. Allogeneic bone, on the other hand, carries the risk of inducing immune responses; and artificial bone filling materials generally lack osteogenic induction properties. 3D bioprinted functional scaffolds can overcome these limitations and provide personalized benefits to meet the needs of anatomical remodeling and functional repair of bone defects.

[0003] However, developing bio-inks with durable osteogenic induction activity and rapid vascularization capabilities is a major challenge for the clinical translation of 3D bioprinted scaffolds. Using single recombinant growth factors is clearly insufficient for bone defect repair and reconstruction; moreover, these recombinant growth factors are expensive, chemically unstable, and prone to inducing complications such as heterotopic ossification and tumors, further limiting their clinical application. Injectable platelet-rich fibrin (iPRF) is a second-generation platelet concentrate following platelet-rich plasma (PRP). iPRF is prepared by centrifuging whole blood from patients and, upon activation, releases multiple growth factors, such as transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF), which play important roles in promoting angiogenesis, osteogenic differentiation of stem cells, and regulating the immune microenvironment. Furthermore, their proportions are similar to the physiological proportions in vivo, allowing for better synergistic promotion of personalized tissue repair. Previous clinical trials have reported that PRF can significantly promote soft tissue repair, but the role of PRF in bone defect repair remains unclear.

[0004] During normal fracture healing, VEGF, which promotes angiogenesis, is released directly at the onset of injury and peaks around day 10, while the expression of bone morphogenetic protein-2 (BMP-2), which promotes osteogenic growth, continues to increase until around day 21. This indicates a need for a loading system capable of sustained, slow release of active factors. iPRF has a richer fibrin network than PRP, which can facilitate the slow release of growth factors, but this limited slow release effect cannot meet the needs of bone regeneration. On the other hand, in addition to the sustained induction of growth factors, the mechanical microenvironment of stem cells is also considered a key regulator in bone regeneration. Simply put, osteogenic differentiation of stem cells requires a rigid matrix, and the mechanical rigidity of pure PRF gel is clearly too low. Based on this, constructing iPRF-based hydrogel bioinks with enhanced mechanical strength and sustained-release capabilities is a promising strategy. A mixture of medical gelatin (Gel) and medical sodium alginate (SA) is widely used in regenerative medicine due to its good biocompatibility and printability. After cross-linking and curing, gel / SA(GS) hydrogel inks can form interconnected and uniform porous structures. iPRF-GS composite hydrogels containing a certain concentration of iPRF can further form stable multi-network structures under the action of thrombin cross-linking agents containing calcium ions. The mechanical strength and sustained growth factor release capacity of iPRF-GS hydrogels are significantly enhanced.

[0005] The selection of seed cells is the most fundamental and crucial step in bone tissue engineering and 3D bioprinting research. Bone marrow mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs) are the two most widely used seed cells. Both types of stem cells possess multiple differentiation potentials and can differentiate into osteoblasts, chondrocytes, skeletal muscle cells, etc., under different induction conditions. However, the source of BMSCs is limited, requiring autologous extraction via bone marrow aspiration, which can cause patient discomfort. Furthermore, our previous bone marrow aspiration and stem cell enrichment experiments revealed that the content of BMSCs in bone marrow is extremely low. To meet the concentration required for clinical applications, in vitro expansion is necessary, but repeated expansion processes cannot ensure that their stem cell characteristics can be maintained. In contrast, selecting ADSCs as seed cells has great potential for clinical translation. Adipose tissue is mostly located subcutaneously and can be obtained through minimally invasive puncture, requiring a small amount of adipose tissue for primary culture. ADSCs offer high controllability, low potential tumorigenicity, wide availability, simple extraction and preparation methods with minimal trauma and ethical controversy, making them a promising candidate for constructing bone repair bio-inks.

[0006] Polycaprolactone (PCL) and hydroxyapatite (HA) are commonly used main scaffold materials for bioprinted bone repair scaffolds. They have good biocompatibility and osteointegration properties, and both are biodegradable and absorbable. Composite materials of the two have stronger mechanical properties and excellent osteoinductive properties.

[0007] Based on the above analysis, this invention combines iPRF, gelatin, sodium alginate and ADSC in appropriate proportions to construct a novel bone repair bio-ink, and then uses PCL / HA material to print layer by layer to construct a bone defect repair scaffold. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention provides a 3D printed bio-ink for bone defect repair, a functional scaffold, and a method for preparing the same.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A first aspect of the present invention is to provide a 3D printing bio-ink for bone defect repair, comprising injectable platelet-rich fibrin (iPRF), adipose-derived stem cells (ADSC), gelatin, and sodium alginate.

[0011] Furthermore, the aforementioned 3D printing bio-ink comprises components of the following concentrations: 2–8% (w / v) medical-grade gelatin, 0.05–2% (w / v) medical-grade sodium alginate, 2–15% (v / v) iPRF solution, and 0.5–2.0 × 10⁻⁶ ppm. 7 / ml adipose-derived stem cells, preferably comprising the following components at the following concentrations: 5% (w / v) medical gelatin, 1% (w / v) medical sodium alginate, 10% (v / v) iPRF solution and 1.5 × 10 7 / ml adipose-derived stem cells.

[0012] Furthermore, the iPRF solution described above is a blood extract from the patient's own blood, an allogeneic blood, or a xenogeneic blood; preferably, it is a blood extract from the patient's own blood.

[0013] Further, the preparation method of the above-mentioned iPRF solution is as follows: take 10-30 ml of fresh venous blood, centrifuge, and aspirate the upper light yellow liquid as the iPRF solution; preferably, take 10-30 ml of fresh venous blood, add sodium citrate solution, centrifuge, and aspirate the upper light yellow liquid as the iPRF solution; more preferably, take 10-30 ml of fresh venous blood, add 0.5-2.5 ml of 2.5% sodium citrate solution, centrifuge, and aspirate the upper light yellow liquid as the iPRF solution; even more preferably, the volume of 2.5% sodium citrate solution is 1 / 15 of the volume of fresh venous blood, which can maintain the non-coagulating state of the bio-ink for about 2 hours and does not affect the stability of the gel component in the final stent.

[0014] Furthermore, the aforementioned adipose-derived stem cells are derived from autologous umbilical abdominal fat, obtained by digesting and culturing adipose tissue to the third generation.

[0015] The second aspect of the present invention is to provide a method for preparing the above-mentioned 3D printing bio-ink, wherein a mixed solution of medical gelatin and medical sodium alginate is first prepared, then iPRF solution is added, and finally adipose-derived stem cells are resuspended in the above mixed solution at a certain concentration to obtain the 3D printing bio-ink.

[0016] A third aspect of the present invention is to provide a method for preparing a functional scaffold for bone defect repair, comprising the following steps:

[0017] Step 1: Obtain the above-mentioned 3D printing bio-ink;

[0018] Step 2: Hydroxyapatite and polycaprolactone are melt-mixed in a certain mass ratio to obtain the support load-bearing structure material;

[0019] Step 3: Perform a CT scan on the patient's bone defect area, and then use 3D reconstruction software to create a 3D model of the bone defect area.

[0020] Step four involves loading the scaffold load-bearing structural material and 3D printing bio-ink into different cartridges of the 3D bioprinter, and then printing layer by layer to construct a functional scaffold for bone defect repair.

[0021] Furthermore, the above preparation method also includes: immersing the bone defect repair scaffold in a crosslinking agent for crosslinking; preferably, the crosslinking agent is a thrombin crosslinking agent with a concentration of 2% (w / v) CaCl2, and the crosslinking time is 10 to 50 minutes, more preferably 30 minutes.

[0022] Furthermore, the mass ratio of hydroxyapatite to polycaprolactone is 1:9 to 3:7, preferably 1:4.

[0023] Furthermore, in the aforementioned bone defect repair scaffold, the thickness of the scaffold load-bearing structural material layer is 500–700 μm, and the thickness of the bio-ink layer is 400–500 μm, with the two layers being alternately stacked and printed.

[0024] A fourth aspect of the present invention is to provide a bone defect repair scaffold prepared by the above-described preparation method.

[0025] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0026] This invention uses polycaprolactone and hydroxyapatite as scaffold load-bearing structural materials, and employs autologous iPRF solution and ADSC as the main active ingredients of bio-ink. It has good bioactivity while avoiding immune rejection. Moreover, the prepared 3D bioprinted bone defect repair scaffold has high mechanical strength and strong ability to release growth factors, which is conducive to inducing angiogenesis and osteogenic differentiation of stem cells, and is suitable for large-scale promotion and use. Attached Figure Description

[0027] Figure 1 This is a front view of a 3D bioprinted bone defect repair scaffold according to an embodiment of the present invention;

[0028] Figure 2 This is a top view of a 3D bioprinted bone defect repair scaffold in one embodiment of the present invention;

[0029] Figure 3 This is the compressive stress-strain curve of a 3D bioprinted bone defect repair scaffold in one embodiment of the present invention.

[0030] Figure 4 The results of the release kinetics detection of key growth factors in a 3D bioprinted bone defect repair scaffold according to an embodiment of the present invention are shown; Figure A: TGF-β, Figure B: VEGF, Figure C: PDGF. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings to enable a better understanding of the invention. However, the following embodiments do not limit the scope of the invention.

[0032] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0033] Example 1

[0034] This embodiment provides a functional scaffold for bone defect repair, and the specific fabrication process is as follows:

[0035] 1. Preparation of autologous adipose-derived stem cells from the patient: Extracted adipose tissue is used to extract human adipose-derived stem cells using collagenase I digestion combined with tissue block culture. The specific steps are as follows:

[0036] 1) Under sterile conditions, approximately 1.5g of fat was extracted from the patient's umbilical abdomen via minimally invasive puncture and rinsed with sterile saline until no blood was visible.

[0037] 2) Add an equal volume of 0.1% type I collagenase to the adipose tissue and digest for 20 min in a cell culture incubator. Stop digestion with DMEM medium containing 10% fetal bovine serum, then centrifuge at 1000 rpm for 10 min and discard the supernatant. Resuspend the incompletely digested tissue fragments and the underlying cell clusters in DMEM complete medium and seed them into culture dishes. When the cells reach approximately 90% confluence, passage them and collect third-generation adipose-derived stem cells for constructing bioactive ink.

[0038] 2. Preparation of patient-derived injectable platelet-rich fibrin:

[0039] Under aseptic conditions, approximately 28 ml of venous blood was drawn from the patient. Then, 2 ml of 2.5% sodium citrate solution was added and gently mixed (the volume percentage of sodium citrate solution was 1 / 15). The mixture was centrifuged at 700 rpm for 3 minutes. After centrifugation, the liquid in the tube separated into two layers. The upper, light yellow liquid was collected to obtain the injectable platelet-rich fibrinogen solution.

[0040] 3. Preparation of the composite bio-ink: First, a mixed solution of medical-grade gelatin and medical-grade sodium alginate was prepared. Then, iPRF solution was added. Finally, adipose-derived stem cells were resuspended in the above mixed solution to construct the bio-ink. In this bio-ink, the concentration of medical-grade gelatin was 5% (w / v), the concentration of medical-grade sodium alginate was 1% (w / v), the concentration of iPRF solution was 10% (v / v), and the concentration of adipose-derived stem cells was 1.5 × 10⁻⁶. 7 / ml.

[0041] 4. Preparation of load-bearing structural materials for the support frame:

[0042] Hydroxyapatite and polycaprolactone were melt-mixed at a mass ratio of 1:4 to obtain the support load-bearing structural material.

[0043] In existing bone repair materials, hydroxyapatite and polycaprolactone are typically mixed in a mass ratio between 1:9 and 3:7. However, in practice, we found that melting at a 3:7 mass ratio resulted in some hydroxyapatite powder precipitation. Therefore, through actual melt mixing and printing tests, we optimized the material ratio to a 1:4 mass ratio. This ensures that no hydroxyapatite powder precipitation occurs while maximizing the utilization of hydroxyapatite's osteoinductive properties.

[0044] 5. Parameters for designing the 3D bioprinted bone repair scaffold: CT scans are performed on the patient's bone defect area, and then 3D modeling is performed using 3D reconstruction software to obtain a three-dimensional model of the bone defect area. Based on the actual clinical situation, the required shape, size, and thickness of the scaffold's main load-bearing layer and bio-ink layer are determined (in this embodiment, the PCL / HA scaffold main layer thickness is 600μm, and the iPRF / Gel / SA / ADSC bio-ink layer thickness is 450μm).

[0045] 6. Load PCL / HA material into barrel one of the 3D bioprinter, and iPRF / Gel / SA / ADSC bio-ink into barrel two. Then, print layer by layer to construct a bone defect repair scaffold. Finally, immerse it in a thrombin crosslinking agent containing 2% (w / v) CaCl2 for approximately 30 minutes for crosslinking. The resulting 3D bioprinted bone defect repair scaffold is shown below. Figure 1-2 As shown.

[0046] Example 2

[0047] This embodiment tests the performance of the bone defect repair scaffold provided in Example 1. The specific experimental steps and results are as follows:

[0048] 1. Compressive strength test

[0049] The stress-strain curves of the 3D bioprinted bone repair scaffold were tested using a universal mechanical testing machine, and the compressive strength and compressive modulus of the scaffold were then obtained based on the stress-strain curve data. For example... Figure 3 As shown, the compressive strength of the bio-functional scaffold is 8.56±0.38MPa, and the compressive modulus is 135.26±3.89MPa.

[0050] 2. Release kinetics test

[0051] The 3D bioprinted bone defect repair scaffold was immersed in a 50ml centrifuge tube containing 10ml of PBS and gently shaken on a shaker in a 37℃ incubator. At predetermined time points (1d, 3d, 7d, 14d, 21d), all the PBS solution in the centrifuge tube was aspirated, and the tube was centrifuged at 3000 rpm for approximately 10 minutes. The supernatant was collected and stored at -80℃ for later use. Simultaneously, 10ml of fresh PBS solution was added to the centrifuge tube for collection at the next time point. Before detecting growth factor concentrations in the sustained-release solutions collected at each time point, the solutions were transferred from -80℃ to 4℃ for approximately 2 hours to equilibrate before testing. The sustained-release samples collected at each time point were tested according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kits for platelet transforming factor-β (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor-BB (PDGF-BB). Figure 4 As shown, the biofunctional scaffold constructed in this embodiment can continuously release growth factors (TGF-β, VEGF, and PDGF) for more than 3 weeks.

[0052] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for preparing a functional scaffold for bone defect repair, characterized in that, Includes the following steps: Step 1: Obtain 3D printing bio-ink, which comprises the following components at the following concentrations: 2%~15% (v / v) injectable platelet-rich fibrin iPRF solution, 0.5×10 7 cells / mL ~2.0×10 7 Adipose-derived stem cells (ADSC) per mL, 2%–8% (w / v) medical gelatin, and 0.05%–2% (w / v) medical sodium alginate; Step 2: Hydroxyapatite and polycaprolactone are melt-mixed in a certain mass ratio to obtain the support load-bearing structure material; Step 3: Perform a CT scan on the patient's bone defect area, and then use 3D reconstruction software to create a 3D model of the bone defect area. Step 4: Load the scaffold load-bearing structural material and 3D printing bio-ink into different barrels of the 3D bioprinter, and then print layer by layer to build a bone defect repair scaffold. It also includes: immersing the bone defect repair scaffold in a cross-linking agent for cross-linking; the cross-linking agent is a thrombin cross-linking agent with a concentration of 2% (w / v) CaCl2, and the cross-linking time is 10~50 minutes.

2. The preparation method according to claim 1, characterized in that, The 3D printing bio-ink comprises the following components at the following concentrations: 5% (w / v) medical-grade gelatin, 1% (w / v) medical-grade sodium alginate, 10% (v / v) iPRF solution, and 1.5 × 10⁻⁶ ppm. 7 Adipose-derived stem cells per mL.

3. The preparation method according to claim 1, characterized in that, The iPRF solution is a blood extract from the patient's own blood, an allogeneic blood, or a xenogeneic blood.

4. The preparation method according to claim 1, characterized in that, The iPRF solution is prepared as follows: Take 10mL~30mL of fresh venous blood, add a 2.5% sodium citrate solution, the volume of which is 1 / 15 of the volume of the fresh venous blood, centrifuge, and collect the upper light yellow liquid as the iPRF solution.

5. The preparation method according to claim 1, characterized in that, The adipose-derived stem cells are derived from autologous umbilical abdominal fat, obtained by digesting and culturing adipose tissue to the third generation.

6. The preparation method according to claim 1, characterized in that, The preparation method of the 3D printing bio-ink is as follows: First, a mixed solution of medical gelatin and medical sodium alginate is prepared, then iPRF solution is added, and finally adipose-derived stem cells are resuspended in the mixed solution at a certain concentration to obtain the 3D printing bio-ink.

7. The preparation method according to claim 1, characterized in that, The mass ratio of hydroxyapatite to polycaprolactone is 1:9 to 3:

7.

8. A bone defect repair scaffold prepared by the preparation method according to any one of claims 1-7.

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