CN117205364A - A 3D printing bioink, functional scaffold and preparation method for bone defect repair - Google Patents

Abstract

The invention provides 3D printing biological ink for repairing bone defects, a functional bracket and a preparation method thereof. The 3D printing biological ink comprises patient autologous Injectable Platelet Rich Fibrin (iPRF), autologous Adipose Derived Stem Cells (ADSCs), gelatin and sodium alginate. The invention takes Polycaprolactone (PCL) and Hydroxyapatite (HA) as support bearing structural materials, adopts autologous iPRF solution and ADSC as main active components of the biological ink, HAs good biological activity, avoids immune rejection reaction, and the prepared 3D biological printing bone defect repairing functional support HAs high mechanical strength and strong capability of slowly releasing growth factors, is favorable for inducing angiogenesis and osteogenic differentiation of stem cells, and can be suitable for large-scale popularization and use.

Description

A 3D printing bioink, functional scaffold and preparation for bone defect repair method

Technical field

The invention relates to the technical field of biomedical engineering, and specifically to a 3D printing bioink for bone defect repair, a functional scaffold and a preparation method thereof.

Background technique

Bone defects caused by trauma, tumors, inflammation and infection have a high incidence and great harm, and their repair and reconstruction are still urgent problems and research hotspots in the field of orthopedics. Autologous bone transplantation is the "gold standard" for the treatment of bone defects, but its clinical application is limited by the source of donors, and there is a risk of complications such as infection and fracture caused by additional surgical bone removal; allogeneic bone has the risk of inducing immune reactions. ;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.

However, the development of bioinks with durable osteogenic induction activity and rapid vascularization ability is a major challenge for the clinical translation of 3D bioprinted scaffolds. The use of a single recombinant growth factor obviously cannot meet the needs of bone defect repair and reconstruction. Moreover, these recombinant growth factors are expensive, have unstable physical and chemical properties, and can easily induce complications such as ectopic ossification and tumors, further limiting their clinical application. Injectable platelet-rich fibrin (iPRF) is a second-generation platelet concentrate product after platelet-rich plasma (PRP). iPRF is prepared by centrifugation of patients' whole blood. After activation, it can release a variety of growth factors, such as transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and promote angiogenesis. , stem cells play an important role in osteogenic differentiation and regulating the immune microenvironment. In addition, their proportions are similar to the physiological proportions in the body and can better synergistically promote personalized tissue repair. Previous clinical trial reports have shown that PRF can significantly promote soft tissue repair, but the effect of PRF on bone defect repair is still unclear.

During normal fracture healing, VEGF, which promotes angiogenesis, is released directly at the beginning of injury and reaches a peak around day 10, while the expression of bone morphogenetic protein-2 (BMP-2), which promotes osteogenesis, continues to increase. Until around day 21. This suggests the need for a loading system capable of sustained and slow release of active factors. iPRF has a richer fibrin network than PRP and can slowly release 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 to be a key regulatory factor in the bone regeneration process. Simply put, osteogenic differentiation of stem cells requires a rigid matrix, and the mechanical rigidity of pure PRF gel is obviously too low. Based on this, constructing iPRF-based hydrogel bioinks with enhanced mechanical strength and sustained release capability is a promising strategy. The mixture of medical gelatin (Gel) and medical sodium alginate (SA) is widely used in regenerative medicine due to its good biocompatibility and printability. Gel/SA(GS) hydrogel ink can form an interconnected and uniform pore structure after cross-linking and solidification. The iPRF-GS composite hydrogel containing a certain concentration of iPRF can be formed under the action of thrombin cross-linking agent containing calcium ions. Further form a stable multi-network structure. The mechanical strength and sustained release of growth factors of iPRF-GS hydrogel have been greatly enhanced.

The selection of seed cells is the most basic and critical link in bone tissue engineering and 3D bioprinting research. Bone marrow mesenchymal stem cells (BMSC) derived from bone marrow and adipose-derived stem cells (ADSC) derived from fat are the two most widely used seed cells. Both types of stem cells have multiple differentiation potentials and can be differentiated under different induction conditions. Differentiate into osteoblasts, chondrocytes, skeletal muscle cells, etc. However, the source of BMSC is limited and needs to be obtained from the patient's own body through bone marrow puncture, which can easily cause pain to the patient. In addition, we found in previous bone marrow puncture and stem cell enrichment experiments that the content of BMSC in the bone marrow is very small. In order to meet the requirements for clinical application The concentration needs to be expanded in vitro, but repeated expansion processes cannot ensure that its stem cell characteristics can always be maintained. In contrast, selecting ADSC as seed cells has great potential for clinical transformation. Adipose tissue is mostly located under the skin and can be obtained through minimally invasive puncture. The amount of adipose tissue required for primary culture is small. ADSC is highly controllable, has low potential tumorigenic risk, is widely sourced, has a simple extraction and preparation method, is less invasive, and has less ethical controversy. It has great potential to be used to construct bone repair bioinks.

Polycaprolactone (PCL) and hydroxyapatite (HA) are commonly used main scaffold materials for bioprinted bone repair scaffolds. They have good biocompatibility and osseointegration, and both can be degraded and absorbed. The composite of the two The material has stronger mechanical properties and excellent osteoinductive properties.

Based on the above analysis, the present invention combines iPRF, gelatin, sodium alginate and ADSC in appropriate proportions to construct a new type of bone repair bioink, and prints it with PCL/HA material layer by layer through dual-channel nozzles to construct a functional scaffold for bone defect repair. .

Contents of the invention

In order to overcome the deficiencies in the prior art, the present invention provides a 3D printing bioink for bone defect repair, a functional scaffold and a preparation method thereof.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The first aspect of the present invention is to provide a 3D printing bioink for bone defect repair, which includes injectable platelet-rich fibrin (iPRF), adipose-derived stem cells (ADSC), gelatin and sodium alginate.

Further, the above-mentioned 3D printing bio-ink includes components at the following concentrations: 2-8% (w/v) medical gelatin, 0.05-2% (w/v) medical sodium alginate, 2-15% (v/v) The iPRF solution and 0.5-2.0×10 ⁷ /ml adipose-derived stem cells preferably include 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.

Further, the above-mentioned iPRF solution is an autologous, allogeneic or xenogeneic blood extract; preferably, an autologous blood extract is obtained.

Further, the preparation method of the above-mentioned iPRF solution is: take 10-30ml of fresh venous blood, centrifuge, and absorb the upper light yellow liquid to obtain the iPRF solution; preferably, take 10-30ml of fresh venous blood, add sodium citrate solution, and centrifuge. , absorb the light yellow liquid in the upper layer to become the iPRF solution; more preferably, take 10-30ml of fresh venous blood, add 0.5-2.5ml of sodium citrate solution with a concentration of 2.5%, centrifuge, and absorb the light yellow liquid in the upper layer to become the iPRF solution ;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-coagulation state of the bio-ink for about 2 hours without affecting the stability of the gel component in the final stent.

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

The second aspect of the present invention is to provide a method for preparing the above-mentioned 3D printing bioink. First, prepare a mixed solution of medical gelatin and medical sodium alginate, then add the iPRF solution, and finally resuspend the adipose-derived stem cells in the above mixed solution at a certain concentration. , the 3D printing bioink was obtained.

The third aspect of the present invention provides a method for preparing a functional scaffold for bone defect repair, which includes the following steps:

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

Step 2: Melt and mix hydroxyapatite and polycaprolactone according to a certain mass ratio to obtain a stent load-bearing structural material;

Step three: perform a CT scan of the patient's bone defect site, and then perform 3D modeling through 3D reconstruction software to obtain a three-dimensional three-dimensional model of the bone defect site;

Step 4: Load the load-bearing structural materials of the scaffold and 3D printing bio-ink into different barrels of the 3D bioprinter, and then print them layer by layer to build a functional scaffold for bone defect repair.

Further, the above preparation method also includes: immersing the bone defect repair functional scaffold in a cross-linking agent for cross-linking; preferably, the cross-linking agent is a thrombin cross-linking agent with a concentration of 2% (w/v) _CaCl2 , and the cross-linking agent The time is 10 to 50 minutes, and more preferably 30 minutes.

Further, the mass ratio of hydroxyapatite and polycaprolactone is 1:9 to 3:7, preferably 1:4.

Further, in the above-mentioned functional scaffold for bone defect repair, the thickness of the load-bearing structural material layer of the scaffold is 500-700 μm, and the thickness of the bio-ink layer is 400-500 μm, and the two are alternately stacked and printed.

The fourth aspect of the present invention is to provide a functional scaffold for bone defect repair prepared by the above preparation method.

The present invention adopts the above technical solution and has the following technical effects compared with the existing technology:

The present invention uses polycaprolactone and hydroxyapatite as the load-bearing structural materials of the scaffold, and uses autologous iPRF solution and ADSC as the main active ingredients of the bio-ink. It has good biological activity while avoiding immune rejection, and is prepared The 3D bioprinted functional scaffold for bone defect repair has high mechanical strength and strong ability to slowly release growth factors, which is beneficial to inducing angiogenesis and osteogenic differentiation of stem cells, and can be suitable for large-scale promotion and use.

Description of drawings

Figure 1 is a front view of a 3D bioprinted bone defect repair functional scaffold in one embodiment of the present invention;

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

Figure 3 is a compressive stress strain curve of a 3D bioprinted functional scaffold for bone defect repair in an embodiment of the present invention;

Figure 4 shows the release kinetics detection results of key growth factors in the 3D bioprinted functional scaffold for bone defect repair in one embodiment of the present invention; Figure A: TGF-β, Figure B: VEGF, Figure C: PDGF.

Detailed ways

The present invention will be described in detail and concretely below through specific embodiments and drawings, so as to better understand the present invention. However, the following embodiments do not limit the scope of the present invention.

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

Example 1

This embodiment provides a functional scaffold for bone defect repair. The specific preparation process is as follows:

1. Preparation of patient's autologous adipose-derived stem cells: Take the extracted adipose tissue and use collagenase I digestion combined with tissue block culture to extract human adipose-derived stem cells. The specific steps are as follows:

1) Extract about 1.5g of the patient’s umbilical and abdominal fat through minimally invasive puncture under sterile conditions, and rinse with sterile saline until there is no blood color;

2) Add 0.1% type I collagenase in the same volume as the adipose tissue, digest in a cell culture incubator for 20 minutes, terminate the digestion with 10% fetal bovine serum DMEM medium, and then centrifuge at 1000 r/min for 10 minutes, and discard the supernatant. Resuspend the incompletely digested tissue pieces and underlying cell clusters in DMEM complete medium, then inoculate them into a culture dish for culture. When the cell fusion reaches about 90%, the cells are passaged, and the third generation adipose-derived stem cells are collected to construct bioactive ink.

2. Prepare the patient’s autologous injectable platelet-rich fibrin:

Extract about 28 ml of the patient's venous blood under aseptic operation, then add 2 ml of 2.5% sodium citrate solution and mix gently (the volume ratio of the sodium citrate solution is 1/15), and centrifuge at 700 r/min for 3 minutes. . After centrifugation, the liquid in the tube is divided into upper and lower layers. The upper layer of light yellow liquid is sucked to obtain an injectable platelet-rich fibrin solution.

3. Preparation of composite bioink: First prepare a mixed solution of medical gelatin and medical sodium alginate, then add the iPRF solution, and finally resuspend the adipose-derived stem cells in the above mixed solution to construct a bioink. In the bioink , the concentration of medical gelatin is 5% (w/v), the concentration of medical sodium alginate is 1% (w/v), the concentration of iPRF solution is 10% (v/v), and the concentration of adipose-derived stem cells is 1.5 ×10 ⁷ /ml.

4. Preparation of load-bearing structural materials for the bracket:

Hydroxyapatite and polycaprolactone are melted and mixed at a mass ratio of 1:4 to obtain a stent load-bearing structural material.

Among existing bone repair materials, hydroxyapatite and polycaprolactone are usually mixed at a mass ratio between 1:9 and 3:7. However, we found in actual operations that they are melted at a mass ratio of 3:7. Some hydroxyapatite powder will precipitate. Therefore, the material ratio is optimized through actual melt mixing and printing tests, and is prepared according to a mass ratio of 1:4. At this time, it can be ensured that no part of the hydroxyapatite powder will precipitate, and the hydroxyapatite can be utilized to the maximum extent. osteoinductive ability.

5. Design the parameters of the 3D bioprinted bone repair functional scaffold: conduct a CT scan of the patient's bone defect site, and then perform 3D modeling through 3D reconstruction software to obtain a three-dimensional three-dimensional model of the bone defect site, thereby determining the needs based on the actual clinical situation The shape, size, and thickness of the stent body load-bearing layer and bio-ink layer of the 3D bioprinted functional scaffold (in this example, the PCL/HA stent body layer thickness is 600 μm, and the iPRF/Gel/SA/ADSC bio-ink layer layer thickness Thick 450μm).

6. Load the PCL/HA material into the barrel one of the 3D bioprinter, and load the iPRF/Gel/SA/ADSC bio-ink into the barrel two of the bioprinter. Then print it layer by layer to build a functional scaffold for bone defect repair, and finally immerse it. Cross-linking is carried out in a thrombin cross-linking agent containing 2% (w/v) CaCl ₂ for about 30 minutes, and the resulting 3D bioprinted bone defect repair functional scaffold is shown in Figure 1-2.

Example 2

This example performs performance testing on the functional scaffold for bone defect repair provided in Example 1. The specific experimental steps and results are as follows:

1. Compressive strength testing test

A universal mechanical testing machine was used to detect the stress-strain curve of the 3D bioprinted bone repair functional scaffold, and then the compressive strength and compressive modulus values of the scaffold were obtained based on the stress-strain curve data. As shown in Figure 3, the compressive strength of the biofunctional scaffold is 8.56±0.38MPa, and the compressive modulus is 135.26±3.89MPa.

2. Release kinetics detection test

Dip the 3D bioprinted bone defect repair functional scaffold into a 50ml centrifuge tube containing 10ml PBS, and place it on a shaker in a 37°C incubator to shake gently. At predetermined time points (1d, 3d, 7d, 14d, 21d), absorb all the PBS solution in the centrifuge tube, then centrifuge at 3000r/min for about 10min, extract the supernatant, and store it in a -80°C refrigerator for later use. At the same time, add 10 ml of fresh PBS solution to the centrifuge tube again, and wait for the collection operation at the next time point. Before testing the growth factor concentration of the sustained-release solution collected at each time period, first transfer the sustained-release solution from the -80°C refrigerator to the 4°C refrigerator to equilibrate for about 2 hours and then set aside for testing. The sustained-release fluid samples collected at each time point were subjected to experimental operations according to the instructions of the platelet transforming factor (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF-BB) enzyme-linked immunoassay kits. As shown in Figure 4, the biofunctional scaffold constructed in the embodiment can sustain the sustained release of growth factors (TGF-β, VEGF and PDGF) for more than 3 weeks.

The specific embodiments of the present invention have been described in detail above, but they are only used as 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 included in the scope of the present invention.

Claims (10)

1. A 3D printing bioink for bone defect repair, characterized by including iPRF, ADSC, gelatin and sodium alginate. 2. The 3D printing bio-ink according to claim 1, characterized in that it includes components at the following concentrations: 2-8% (w/v) medical gelatin, 0.05-2% (w/v) medical sodium alginate , 2-15% (v/v) iPRF solution and 0.5-2.0×10 ⁷ /ml adipose-derived stem cells, preferably including the following concentrations of components: 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. 3. The 3D printing bioink according to claim 2, wherein the iPRF solution is an autologous, allogeneic or xenogeneic blood extract; preferably, an autologous blood extract. 4. The 3D printing bioink according to claim 2, characterized in that the preparation method of the iPRF solution is: take 10-30ml of fresh venous blood, centrifuge, and absorb the upper light yellow liquid to obtain the iPRF solution; preferably, Take 10-30ml of fresh venous blood, add sodium citrate solution, centrifuge, and suck the upper light yellow liquid to become the iPRF solution; more preferably, take 10-30ml of fresh venous blood, add sodium citrate solution with a concentration of 2.5% 0.5 to 2.5 ml, centrifuge, and absorb the upper light yellow liquid to obtain the iPRF solution; more preferably, the volume of the 2.5% sodium citrate solution is 1/15 of the volume of fresh venous blood. 5. The 3D printing bioink according to claim 2, characterized in that the adipose-derived stem cells are derived from autologous umbilical abdominal fat and are obtained by digesting and culturing adipose tissue to the third generation. 6. The preparation method of 3D printing bioink according to any one of claims 1 to 5, characterized in that first, a mixed solution of medical gelatin and medical sodium alginate is prepared, then the iPRF solution is added, and finally the adipose-derived stem cells are added. Resuspend in the mixed solution at a certain concentration to obtain the 3D printing bioink. 7. A method for preparing a functional scaffold for bone defect repair, which is characterized by comprising the following steps: Step 1, obtain the 3D printing bio-ink as described in any one of claims 1-5; Step 2: Melt and mix hydroxyapatite and polycaprolactone according to a certain mass ratio to obtain a stent load-bearing structural material; Step three: perform a CT scan of the patient's bone defect site, and then perform 3D modeling through 3D reconstruction software to obtain a three-dimensional three-dimensional model of the bone defect site; Step 4: Load the load-bearing structural materials of the scaffold and 3D printing bio-ink into different barrels of the 3D bioprinter, and then print them layer by layer to build a functional scaffold for bone defect repair. 8. The preparation method according to claim 7, further comprising: immersing the bone defect repair functional scaffold in a cross-linking agent for cross-linking; preferably, the cross-linking agent is CaCl with a concentration of 2% (w/v) For the thrombin cross-linking agent ₂ , the cross-linking time is 10 to 50 minutes, more preferably 30 minutes. 9. The preparation method according to claim 7, characterized in that the mass ratio of hydroxyapatite and polycaprolactone is 1:9 to 3:7, preferably 1:4. 10. The functional scaffold for bone defect repair prepared by the preparation method according to any one of claims 7-9.