CN105999400A - CS/beta-TCP (calcium silicate/beta-tricalcium phosphate) porous composite material for promoting osteogenesis and vasculogenesis and preparation method thereof - Google Patents

Abstract

The invention discloses a CS/beta-TCP (calcium silicate/beta-tricalcium phosphate) porous composite material for promoting osteogenesis and vasculogenesis and a preparation method thereof. The CS/beta-TCP porous composite material is obtained by mixing the powder of calcium silicate (CS) and tricalcium phosphate (beta-TCP) according to a mass ratio, adding polyvinyl alcohol (PVA) into the powder, uniformly mixing an obtained mixture to be a paste, carrying out 3D (three-dimensional) printing, drying a printed three-dimensional model at a room temperature and then carrying out high-temperature sintering on the dried three-dimensional model, and sections of pore canals are squares of 350 microns multiplied by 350 microns. The CS/beta-TCP porous composite material for promoting the osteogenesis and the vasculogenesis is simple in process, has better biocompatibility and uniformly through pore-canal structures, is beneficial to osteoinduction and promotes the vasculogenesis.

Description

CS/β-TCP porous composite material for promoting osteogenesis and its preparation method

technical field

The present invention relates to a technology in the field of biomedical materials for repairing bone tissue, in particular to a CS/β-TCP (Calcium Silicate/β-Tricalcium Phosphate, calcium silicate/β-tricalcium phosphate) for promoting osteogenesis Calcium) porous composite material and its preparation method.

Background technique

There are tens of millions of patients with bone defect or osteonecrosis caused by trauma, tumor resection, chronic osteomyelitis, and vascular necrosis every year in the world. There is an urgent need for a large number of suitable bone graft materials in clinical practice. At present, the main methods for clinical treatment of bone defects are: autologous bone graft, allogeneic bone graft, and filling with artificial biomaterials. Among them, autologous bone grafting is the current gold standard, but its source is limited, it cannot fill large bone defects, and it will cause secondary injury to the patient, prolonging the operation and recovery time. Allograft bone, as one of the bone repair materials that have been widely used clinically and second to autologous bone, mainly includes allograft bone (cadaver bone) and xenograft bone (bovine bone, pig bone, etc.), although it can be obtained in large quantities, but However, it has certain immunogenicity and is easy to cause infection. In recent years, artificial biomaterials, such as HA, TCP, and PLGA, have been gradually used in clinical practice. However, when repairing bone tissue with insufficient autologous blood supply or severe large-scale bone defects, complications such as bone resorption and osteonecrosis often occur when combined with allogeneic bone or artificial biomaterials. The main reason may be that it is difficult for blood vessels to grow into it, and an integrated active structure with blood supply has not been formed.

At present, in order to ensure good blood supply in the defect area to obtain a better bone repair effect in clinical practice, doctors mainly take autogenous bone with vascular pedicles for treatment, and the bone harvesting sites include iliac crest and fibula. When the amount of autologous bone is not enough, it will be combined with allograft bone or artificial biomaterial to fill the gap, or even treat it as a large segment of scaffold material. Compared with autologous bone, allograft bone transplantation is prone to complications such as infection and rejection, and its osteogenic induction and osteogenic integration are still not ideal, and it does not promote angiogenesis. The common forms of clinically applied artificial inorganic biofilling materials such as TCP and HA are granular to irregular small pieces ranging in volume from 1 cm ³ . HA will not degrade in the body, TCP has no osteoinductive and angiogenic effects, and there is no uniform through-hole structure inside the block, so the effects of osteoconduction and osseointegration are not ideal. In summary, the preparation of an artificial biofilling material with good biocompatibility, uniform through-hole structure, osteogenic induction and angiogenesis through a simple and controllable technology will be beneficial to promote clinical osteogenesis. Treatment of tissue defects.

Contents of the invention

In view of the above-mentioned deficiencies in the prior art, the present invention proposes a CS/β-TCP porous composite material for promoting osteogenesis and its preparation method, and prepares a CS/β-TCP porous composite material with a certain mass ratio by 3D printing technology. Scaffolds, after high-temperature sintering, obtain porous composite materials with good biocompatibility, uniform through-hole structure, osteoinductivity, osteoconductivity, and angiogenesis, and are expected to be used as bone filling materials for bone tissue repair.

The present invention is achieved through the following technical solutions:

The invention relates to a CS/β-TCP porous composite material for promoting osteogenesis, which has a square channel structure.

The pore cross-section of the square pore structure is 350 μm×350 μm, which can significantly promote the ingrowth of blood vessels, bone formation and integration.

The preparation method of the material involved in the present invention is to mix the powders of calcium silicate (CS) and tricalcium phosphate (β-TCP) according to the mass ratio, add polyvinyl alcohol (PVA) and evenly mix them into a paste, and perform 3D printing. The printed three-dimensional scaffolds were dried at room temperature and then sintered at high temperature to obtain CS/β‐TCP porous composites.

The CS/β-TCP mass ratio is 1-10%.

Preferably, the CS/β-TCP mass ratio is 1-5%.

The mass ratio of the polyvinyl alcohol to CS/β-TCP mixed powder is 6%.

The present invention relates to a porous bulk scaffold material, which is made based on the above-mentioned CS/β-TCP composite material.

technical effect

Compared with the prior art, the present invention has been verified by experiments that the CS/β‐TCP porous composite material with a mass ratio of 5% has good biocompatibility and a uniformly penetrating 350 μm × 350 μm square channel structure, with a large specific surface area and a It is beneficial to the adhesion and proliferation of cells, the ingrowth of blood vessels, and the formation and integration of bone; the presence of silicon ions in the porous composite material makes it osteoinductive and has the effect of promoting angiogenesis, which is expected to reduce bone resorption and osteogenesis in clinical practice. The incidence of complications such as necrosis.

Description of drawings

Fig. 1 is the schematic diagram of preparation method of CS/β-TCP porous composite material of the present invention;

Fig. 2 is the result figure of HBMSCs cell proliferation experiment;

Figure 3 is a graph showing the results of HUVECs cell proliferation experiments;

Figure 4 is a scanning electron micrograph of the growth state of HBMSCs and HUVECs on the porous composite material;

In the figure: (a) is HBMSCs on β-TCP, (b) is HBMSCs on 5% CS/β-TCP, (c) is HUVECs on β-TCP, (d) is 5% CS/β-TCP % of HUVECs on CS/β‐TCP;

Fig. 5 is a graph of alkaline phosphatase activity detection result;

In the figure: (a) is the β-TCP group, (b) is the 5% CS/β-TCP group;

Figure 6 is a graph showing the results of tube formation of endothelial cells in vitro;

In the figure: (a) is the β-TCP group, (b) is the 5% CS/β-TCP group;

Figure 7 is the results of subcutaneous masson detection of osteogenesis in nude mice;

In the figure: (a) is the β-TCP group, (b) is the 5% CS/β-TCP group;

Figure 8 is a diagram showing the effect of subcutaneous masson detection of blood vessels in nude mice.

detailed description

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

Example 1

This embodiment relates to a block scaffold material for promoting osteogenesis, which is made of CS/β‐TCP composite material, and the cross section of the channel is a square of 350 μm×350 μm.

As shown in Figure 1, this embodiment includes the following steps:

Step 1. Weigh 5g of calcium silicate powder and β‐tricalcium phosphate powder according to the mass ratio of calcium silicate accounting for 0%, 5%, and 10% of the total powder, and sieve and mix them in two 50mL centrifuge tubes , add 0.3g PVA one by one and mix evenly to form a paste, and put it into the barrel of the 3D bio-plotter printer.

The calcium silicate powder is self-made in the laboratory. Stir and mix the sodium silicate and calcium nitrate at a molar ratio of 1:1 overnight, then filter the suspension, wash the precipitate with deionized water for 3 times, and wash with absolute ethanol for 2 After the first time, dry overnight under vacuum; calcined at 800°C for 2 hours, and pulverized with a pulverizer for 15 minutes to obtain calcium silicate powder.

The purity of the β-TCP powder is greater than 98%.

The sieve mesh is 500 mesh.

The mass ratio of the PVA and CS/β-TCP mixed powder is 6%.

Step 2. Use the computer-aided software CAD to model the 3D model, save the material parameters in STL format, and import the 3D bio‐plotter to print the 3D structural model layer by layer.

The size of the three-dimensional model is 1 cm×1 cm×0.5 cm, and the channel structure is a square of 500 μm×500 μm.

The pressure of the layer-by-layer printing is 300-450kPa, the extrusion speed is 6mm/s, and the printing needle is 21G.

Step 3. After drying the printed three-dimensional structure model at room temperature for 24 hours, perform high-temperature sintering to obtain the sintered CS/β-TCP porous composite material.

The temperature of the high-temperature sintering is 1200° C., and the time is 2 hours.

The sintered CS/β-TCP porous composite material has a shrinkage rate of 20% to 30%, the final size is 0.7cm×0.7cm×0.35cm, and the channel cross-sectional size is 350μm×350μm.

Step 4. Perform experimental detection on the obtained CS/β-TCP porous composite material.

The experimental tests include: CCK-8 cell proliferation test, scanning electron microscope test of cell growth state, ALP (alkaline phosphatase, alkaline phosphatase) activity test, gel laying test and animal in vivo test.

The CCK-8 cell proliferation experiment refers to: HBMSCs (Human bone marrow mesenchymal stem cells, human bone marrow mesenchymal stem cells) and HUVECs (human umbilical cord endothelial cells) were respectively mixed with β-TCP, 5% CS /β‐TCP and 10% CS/β‐TCP porous composites were cultured and tested for CCK‐8.

As shown in Figure 2 and Figure 3, the prepared material has good biocompatibility. The experimental results observed for HBMSCs and HUVECs were similar, and with the growth of culture time, the three groups of cells all showed an upward trend. There was no significant difference in cell viability among the groups when CCK‐8 was detected on day 1, but the biocompatibility of 5% CS/β‐TCP group was higher than that of 10% CS when tested on days 3 and 5. The /β-TCP group has good biocompatibility, so 5% CS/β-TCP is the preferred group.

The scanning electron microscope detection of the cell growth state refers to: inoculate HBMSCs and HUVECs on the porous composite material of β-TCP and CS/β-TCP with a mass ratio of 5%, respectively, and perform scanning electron microscope observation after culturing for 3 days.

As shown in Figure 4, the material structure of 5% CS/β-TCP was uniform, and the internal pore structure was a square of 350 μm × 350 μm, and HBMSCs and HUVECs proliferated significantly on the 5% CS/β-TCP porous composite, Cell spreading posture is good.

The ALP activity detection refers to: inoculate HBMSCs in a 24-well plate, place β‐TCP and 5% SC/β‐TCP in corresponding Transwell chambers, add osteogenic induction solution for 7 days of induction ALP activity detection.

Since HBMSCs can differentiate into osteoblasts and secrete alkaline phosphatase (ALP) under the induction of osteogenic induction medium, the high expression of ALP indicates better osteogenesis in vitro.

As shown in Figure 5, the 5% CS/β-TCP group has higher ALP activity expression than the porous composite material of the β-TCP group, that is, it has better osteogenic induction.

The gel-spreading experiment refers to: spread 250 μl of matrigel in a 24-well plate, after the matrigel is solidified, inoculate endothelial cells on the matrigel, place a transwell chamber in the corresponding well, place a transwell chamber in the transwell chamber Inject the corresponding β-TCP or 5% CS/β-TCP porous composite material, and observe the tube formation of endothelial cells on Matrigel after culturing in a 37°C incubator for 2 hours.

As shown in Fig. 6, the tube-forming effect of endothelial cells in the 5% CS/β-TCP group was better than that in the β-TCP group.

The animal experiment in vivo refers to implanting β-TCP and 5% CS/β-TCP porous composite material subcutaneously on the back of nude mice, and taking samples for masson analysis after 8 weeks.

Since the subcutaneous environment on the back of nude mice is a relatively ischemic environment, and stem cells are scarce, it can better simulate the environment of poor blood supply and less cells in the center of large bone defects in the human body, as well as the environment of bone defects that are not rich in blood supply. It is a classic model of ectopic osteogenesis.

As shown in Figure 7 and Figure 8, the 5% CS/β‐TCP group had better angiogenesis than the β‐TCP group, and the blood vessels grew into the porous composite material; the 5% CS/β‐TCP channel had Better bone formation effect, with significant difference.

Claims (7)

1. A CS/β-TCP porous composite material for promoting osteogenesis, characterized in that it has a square channel structure. 2. The porous composite material according to claim 1, characterized in that, the cross section of the square pore structure is 350 μm×350 μm. 3. A preparation method for producing the porous composite material according to claim 1, characterized in that, by mixing the powders of calcium silicate and tricalcium phosphate by mass ratio, adding polyvinyl alcohol and uniformly mixing into paste, carrying out 3D Printing, the printed three-dimensional model is dried at room temperature and then sintered at high temperature to obtain CS/β-TCP porous composite material. 4. The preparation method according to claim 2, characterized in that, the mass ratio of CS/β-TCP is 1-10%. 5. preparation method according to claim 2 is characterized in that, the mass ratio of described polyvinyl alcohol and CS/β-TCP mixed powder is 6%. 6. The method according to claim 3, characterized in that, specifically comprising the following steps: Step 1. Weigh 5g of calcium silicate powder and β‐tricalcium phosphate powder according to the mass ratio of calcium silicate to the total powder mass of 0% to 10%, sieve and mix them in two 50mL centrifuge tubes, and add them one by one Mix 0.3g of PVA evenly into a paste, and put it into the barrel of the 3Dbio-plotter printer; Step 2, use computer-aided software CAD to carry out 3D model modeling, save material parameters in STL format, import 3D bio-plotter to print 3D structural model layer by layer; Step 3. The printed three-dimensional structure model was dried at room temperature for 24 hours and then sintered at high temperature to obtain the sintered CS/β-TCP porous composite material. 7. A block-shaped porous support, characterized in that it is made of the CS/β-TCP porous composite material according to any one of the preceding claims.