CN117205364A - 3D printing biological ink for repairing bone defect, functional bracket and preparation method thereof - Google Patents
3D printing biological ink for repairing bone defect, functional bracket and preparation method thereof Download PDFInfo
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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
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to 3D printing biological ink for repairing bone defects, a functional bracket and a preparation method thereof.
Background
Bone defects caused by trauma, tumor, inflammation and infection have high incidence and large harm, and the repair and reconstruction of the bone defects still remain to be solved in the orthopaedics field and are hot spots for research. Autologous bone grafting is a "gold standard" for treating bone defects, but its clinical application is limited by donor sources and there is a risk of complications such as infection and fracture caused by additional surgical extraction of bone; while allogeneic bone is at risk of eliciting an immune response; artificial bone filler materials generally lack osteoinductive properties. The 3D bioprinted functional scaffold may overcome these limitations and provide personalized benefits to meet the needs of anatomic remodeling and functional repair of bone defects.
However, developing bio-inks with durable osteoinductive activity and rapid vascularization capability is a major challenge for 3D bioprinting scaffolds into clinical transformation. The use of single recombinant growth factors obviously cannot meet the needs of bone defect repair and reconstruction, and the recombinant growth factors are high in price, unstable in physicochemical properties, easy to induce complications such as ectopic ossification and tumor, and further limit the clinical application of the recombinant growth factors. Injectable Platelet Rich Fibrin (iPRF) is the second generation platelet concentrate product following Platelet Rich Plasma (PRP). iPRF is prepared by centrifugation of whole blood of a patient, and after activation, can release various growth factors such as transforming growth factor-beta (TGF-beta), vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), and plays an important role in promoting angiogenesis, osteoblast differentiation of stem cells and regulating immune microenvironment. In addition, their proportions are similar to the physiological proportions in the body, and may better synergistically promote personalized tissue repair. The prior clinical test report shows that PRF can obviously promote soft tissue repair, but the effect of PRF on bone defect repair is not clear.
In the normal fracture healing process, VEGF, which promotes angiogenesis, is released directly at the beginning of the injury and peaks around day 10, while the expression of bone morphogenic protein-2 (BMP-2), which promotes osteogenesis, continues to increase until around day 21. This suggests that a loading system capable of sustained slow release of active agents is needed. iPRF has a more abundant fibrin network than PRP and can act as a slow release growth factor, but this limited slow release effect cannot meet the need for 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 the bone regeneration process. Briefly, the osteogenic differentiation of stem cells requires a rigid matrix, whereas the mechanical rigidity of pure PRF gels is clearly too low. Based on this, it is a promising strategy to construct iPRF-based hydrogel bio-inks with enhanced mechanical strength and sustained release capabilities. The mixture of medical gelatin (Gel) and medical Sodium Alginate (SA) has good biocompatibility and printability, and is widely used in regenerative medicine. After Gel/SA (GS) hydrogel ink is crosslinked and solidified, an intercommunicated uniform pore structure can be formed, and the iPRF-GS composite hydrogel containing iPRF with a certain concentration can further form a stable multi-network structure under the action of thrombin crosslinking agent containing calcium ions. The mechanical strength of the iPRF-GS hydrogel and the capability of continuously and slowly releasing the growth factors are greatly enhanced.
The selection of seed cells is the most fundamental and critical link in bone tissue engineering and 3D bioprinting research. Bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs) are the most widely used two seed cells, and both of these stem cells have multiple differentiation potential and can be differentiated into osteoblasts, chondrocytes, skeletal muscle cells, etc. under different induction conditions. However, the BMSC source is limited, needs to be obtained from a patient by bone marrow puncture, which is easy to cause pain to the patient, in addition, in the previous bone marrow puncture and stem cell enrichment test, the BMSC content in bone marrow is found to be very small, and in order to meet the concentration required by clinical application, the BMSC needs to be amplified in vitro, however, the repeated amplification process cannot ensure that the stem cell characteristics can not be maintained all the time. In contrast, the selection of ADSCs as seed cells has great potential for clinical transformation. The adipose tissue is positioned subcutaneously, can be obtained through minimally invasive puncture, and the amount of adipose tissue required by primary culture is small. ADSC has high controllability, low potential tumorigenic risk, wide sources, simple extraction and preparation mode, small trauma and small ethical dispute, and has great potential for constructing bone repair bio-ink.
Polycaprolactone (PCL) and Hydroxyapatite (HA) are used as main body stent materials commonly used for bioprinting bone repair stents, have good biocompatibility and osseointegration effect, and can be degraded and absorbed, and the composite material of the Polycaprolactone (PCL) and the Hydroxyapatite (HA) HAs stronger mechanical property and excellent osteoinductive property.
Based on the analysis, the invention combines iPRF, gelatin, sodium alginate and ADSC according to a proper proportion to construct a novel bone repair biological ink, and prints the novel bone repair biological ink with PCL/HA material layer by layer through a double-channel spray head to construct a bone defect repair functional bracket.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides 3D printing biological ink for repairing bone defects, a functional bracket and a preparation method thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a first aspect of the present invention is to provide a 3D printed bio-ink for bone defect repair comprising Injectable Platelet Rich Fibrin (iPRF), adipose-derived stem cells (ADSC), gelatin and sodium alginate.
Further, the 3D printing bio-ink comprises the following components in concentration: 2-8% (w/v) medical gelatin, 0.05-2% (w/v) medical sodium alginate, 2-15% (v/v) iPRF solution and 0.5-2.0X10 7 Preferably, the adipose-derived stem cells comprise the following components in the following concentrations: 5% (w/v) medical gelatin, 1% (w/v) medical sodium alginate, 10% (v/v) iPRF solution, and 1.5X10 7 /ml adipose-derived stem cells.
Further, the iPRF solution is a blood extract from autologous, allogeneic or xenogeneic sources; autologous blood extracts are preferred.
Further, the preparation method of the iPRF solution comprises the following steps: taking 10-30 ml of fresh venous blood, centrifuging, and sucking the pale yellow liquid on the upper layer to obtain an iPRF solution; preferably, 10-30 ml of fresh venous blood is taken, sodium citrate solution is added, and the mixture is centrifuged, and the upper pale yellow liquid is taken as iPRF solution; more preferably, 10-30 ml of fresh venous blood is taken, 0.5-2.5 ml of sodium citrate solution with the concentration of 2.5% is added, and the mixture is centrifuged, and the upper pale yellow liquid is taken to be the iPRF solution; more preferably, the volume of the 2.5% sodium citrate solution is 1/15 of the volume of fresh venous blood, and the non-coagulated state of the bio-ink can be maintained for about 2 hours without affecting the stability of the gel component in the final scaffold.
Further, the adipose-derived stem cells are derived from autologous umbilical abdominal fat, and are obtained by subjecting adipose tissue to digestion and culture until the third generation.
The second aspect of the present invention provides a method for preparing the 3D printing bio-ink, first preparing a mixed solution of medical gelatin and medical sodium alginate, then adding an iPRF solution, and finally re-suspending adipose-derived stem cells in the mixed solution at a certain concentration to obtain the 3D printing bio-ink.
A third aspect of the present invention provides a method for preparing a bone defect repair functional scaffold, comprising the steps of:
step one, acquiring the 3D printing biological ink;
step two, melting and mixing hydroxyapatite and polycaprolactone according to a certain mass ratio to obtain a support bearing structure material;
thirdly, CT scanning is carried out on the bone defect part of the patient, and then three-dimensional modeling is carried out through three-dimensional reconstruction software, so that a three-dimensional model of the bone defect part is obtained;
and step four, filling the support bearing structural material and 3D printing biological ink into different charging barrels of a 3D biological printer, and then printing layer by layer to construct the bone defect repair functional support.
Further, the preparation method further comprises the following steps: immersing the bone defect repair function bracket into a cross-linking agent for cross-linking; preferably, the crossingThe coupling agent is CaCl with the concentration of 2% (w/v) 2 The thrombin crosslinking agent of (2) is crosslinked for 10 to 50 minutes, more preferably 30 minutes.
Further, the mass ratio of the hydroxyapatite to the polycaprolactone is 1:9 to 3:7, preferably 1:4.
further, in the bone defect repair functional stent, the thickness of the supporting structure material layer of the stent is 500-700 mu m, the thickness of the biological ink layer is 400-500 mu m, and the supporting structure material layer and the biological ink layer are alternately laminated and printed.
A fourth aspect of the present invention provides a bone defect repair functional scaffold prepared by the above preparation method.
Compared with the prior art, the invention has the following technical effects:
the invention takes polycaprolactone and hydroxyapatite 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.
Drawings
FIG. 1 is a front view of a 3D bioprinted bone defect repair functional stent in accordance with one embodiment of the present invention;
FIG. 2 is a top view of a 3D bioprinted bone defect repair functional stent according to one embodiment of the present invention;
FIG. 3 is a compressive stress strain curve of a 3D bioprinted bone defect repair functional stent in accordance with one embodiment of the present invention;
FIG. 4 shows the results of the kinetics of release of key growth factors from a 3D bioprinted bone defect repair functional stent according to one embodiment of the present invention; graph a: TGF- β, panel B: VEGF, panel C: PDGF.
Detailed Description
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
The embodiment provides a bone defect repair function bracket, which is prepared by the following steps:
1. preparation of autologous adipose-derived stem cells of patients: taking the extracted adipose tissue, and extracting the human adipose-derived stem cells by adopting a collagenase I digestion combined tissue block culture method, wherein the method comprises the following specific steps of:
1) Extracting about 1.5g of abdomen fat of the patient by minimally invasive puncture under aseptic condition, and flushing with sterile physiological saline until no blood color exists;
2) Adding 0.1% type I collagenase with the same volume as adipose tissue, digesting for 20min in a cell culture box, stopping digestion with DMEM medium containing 10% fetal bovine serum, centrifuging at 1000r/min for 10min, and discarding the supernatant. Undigested complete tissue mass and underlying cell mass were resuspended in DMEM complete medium and inoculated into petri dishes for culture. And after the cell fusion reaches about 90%, carrying out passage, and collecting third-generation adipose-derived stem cells for constructing the bioactive ink.
2. Preparation of autologous injectable platelet rich fibrin:
about 28ml of venous blood of a patient is extracted under the whole aseptic operation, 2ml of 2.5% sodium citrate solution is added and gently mixed (the volume ratio of the sodium citrate solution is 1/15), and the mixture is centrifuged at 700r/min for 3min. After centrifugation, the liquid in the tube is divided into an upper layer and a lower layer, and the pale yellow liquid on the upper layer is sucked to obtain the injectable platelet-rich fibrin solution.
3. Preparation of composite biological ink: firstly, preparing a mixed solution of medical gelatin and medical sodium alginate, then adding an iPRF solution, and finally, re-suspending adipose-derived stem cells in the mixed solution to construct biological ink, wherein in the biological ink, the concentration of the medical gelatin is 5% (w/v), the concentration of the medical sodium alginate is 1% (w/v), the concentration of the iPRF solution is 10% (v/v), and the concentration of the adipose-derived stem cells is 1.5×10 7 /ml。
4. Preparation of a support bearing structure material:
and (3) melting and mixing the hydroxyapatite and the polycaprolactone according to a mass ratio of 1:4 to obtain the support bearing structure material.
In the existing bone repair materials, hydroxyapatite and polycaprolactone are generally used as 1:9 to 3: the mass ratio between 7 was uniform, however, we found in practice that according to 3:7 mass ratio, and part of hydroxyapatite powder is separated out. Therefore, the material proportion is optimized through actual melt mixing and printing tests, and the preparation is carried out according to the mass ratio of 1:4, so that the precipitation of partial hydroxyapatite powder can be avoided, and the osteoinductive capacity of the hydroxyapatite is utilized to the greatest extent.
5. Parameters of a 3D bioprinted bone repair function scaffold were designed: CT scanning is carried out on the bone defect part of a patient, then three-dimensional modeling is carried out through three-dimensional reconstruction software, and a three-dimensional model of the bone defect part is obtained, so that the shape, the size, the thickness and other parameters of a support main body bearing layer, a biological ink layer thickness and the like of a required 3D biological printing functional support are determined according to clinical actual conditions (in the embodiment, the PCL/HA support main body layer thickness is 600 mu m, and the iPRF/Gel/SA/ADSC biological ink layer thickness is 450 mu m).
6. Respectively filling PCL/HA material into a first feed cylinder of a 3D biological printer, filling iPRF/Gel/SA/ADSC biological ink into a second feed cylinder of the biological printer, then printing layer by layer to construct a bone defect repair function bracket, and finally immersing the bone defect repair function bracket into a biological ink containing CaCl with the concentration of 2% (w/v) 2 The thrombin crosslinking agent is crosslinked for about 30 minutes, and the obtained 3D bioprinted bone defect repair functional stent is shown in figures 1-2.
Example 2
In this example, performance of the bone defect repair stent provided in example 1 was tested, and specific experimental procedures and results were as follows:
1. compressive Strength test
And detecting a stress-strain curve of the 3D bioprinting bone repair function bracket by using a universal mechanical testing machine, and obtaining the compressive strength and the compressive modulus value of the bracket according to stress-strain curve data. As shown in FIG. 3, the compressive strength of the biofunctional scaffold was 8.56.+ -. 0.38MPa, and the compressive modulus was 135.26.+ -. 3.89MPa.
2. Release kinetics test
The 3D bioprinted bone defect repair function scaffold was immersed in a 50ml centrifuge tube containing 10ml PBS and placed on a shaker in a 37℃incubator with gentle shaking. All PBS solution in the centrifuge tube was aspirated at predetermined time points (1 d, 3d, 7d, 14d, 21 d), and then centrifuged at 3000r/min for about 10min, and after supernatant was extracted, stored in a refrigerator at-80℃for use. At the same time, 10ml of fresh PBS solution was added again to the centrifuge tube, and the collection operation was performed at the next time point. Before the growth factor concentration of the slow-release liquid collected in each time period is detected, the slow-release liquid is transferred from a refrigerator at-80 ℃ to a refrigerator at 4 ℃ for balancing for about 2 hours for standby detection. The samples of the sustained release solutions collected at each time point were subjected to experimental procedures according to the instructions of the platelet transforming factor (TGF-. Beta.), vascular Endothelial Growth Factor (VEGF), and platelet derived growth factor (PDGF-BB) ELISA kit. As shown in FIG. 4, the biofunctional scaffolds constructed in the examples can continuously release growth factors (TGF-. Beta., VEGF and PDGF) for more than 3 weeks.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (10)
1. A 3D printing biological ink for repairing bone defects, which is characterized by comprising iPRF, ADSC, gelatin and sodium alginate.
2. The 3D printing bio-ink of claim 1 comprising the following concentrations of components: 2-8% (w/v) medical gelatin, 0.05-2% (w/v) medical sodium alginate, 2-15% (v/v) iPRF solution and 0.5-2.0X10 7 Fat/mlAdipose-derived stem cells preferably comprise the following concentrations of components: 5% (w/v) medical gelatin, 1% (w/v) medical sodium alginate, 10% (v/v) iPRF solution, and 1.5X10 7 /ml adipose-derived stem cells.
3. The 3D printed bio-ink of claim 2 wherein said iPRF solution is a blood extract from autologous, allogeneic or xenogeneic sources; autologous blood extracts are preferred.
4. The 3D printing bio-ink of claim 2 wherein the method of preparing the iPRF solution is: taking 10-30 ml of fresh venous blood, centrifuging, and sucking the pale yellow liquid on the upper layer to obtain an iPRF solution; preferably, 10-30 ml of fresh venous blood is taken, sodium citrate solution is added, and the mixture is centrifuged, and the upper pale yellow liquid is taken as iPRF solution; more preferably, 10-30 ml of fresh venous blood is taken, 0.5-2.5 ml of sodium citrate solution with the concentration of 2.5% is added, and the mixture is centrifuged, and the upper pale yellow liquid is taken to be 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 bio-ink according to claim 2 wherein said adipose derived stem cells are derived from autologous umbilical abdominal fat and are obtained by digestion culture of adipose tissue to the third generation.
6. The method for preparing 3D printing bio-ink according to any one of claims 1 to 5, wherein the 3D printing bio-ink is prepared by first preparing a mixed solution of medical gelatin and medical sodium alginate, then adding an iPRF solution, and finally re-suspending adipose-derived stem cells in the mixed solution at a certain concentration.
7. A method for preparing a bone defect repair functional bracket, which is characterized by comprising the following steps:
step one, obtaining the 3D printing bio-ink according to any one of claims 1-5;
step two, melting and mixing hydroxyapatite and polycaprolactone according to a certain mass ratio to obtain a support bearing structure material;
thirdly, CT scanning is carried out on the bone defect part of the patient, and then three-dimensional modeling is carried out through three-dimensional reconstruction software, so that a three-dimensional model of the bone defect part is obtained;
and step four, filling the support bearing structural material and 3D printing biological ink into different charging barrels of a 3D biological printer, and then printing layer by layer to construct the bone defect repair functional support.
8. The method of manufacturing according to claim 7, further comprising: immersing the bone defect repair function bracket into a cross-linking agent for cross-linking; preferably, the cross-linking agent is CaCl at a concentration of 2% (w/v) 2 The thrombin crosslinking agent of (2) is crosslinked for 10 to 50 minutes, more preferably 30 minutes.
9. The preparation method according to claim 7, wherein the mass ratio of the hydroxyapatite to the polycaprolactone is 1:9 to 3:7, preferably 1:4.
10. a bone defect repair function stent prepared by the preparation method according to any one of claims 7 to 9.
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