CN113440648A

CN113440648A - BBG/PCL composite porous bone scaffold and preparation method thereof

Info

Publication number: CN113440648A
Application number: CN202110281273.8A
Authority: CN
Inventors: 王俊峰; 韩健
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2021-03-16
Filing date: 2021-03-16
Publication date: 2021-09-28
Anticipated expiration: 2041-03-16
Also published as: CN113440648B

Abstract

The invention discloses a BBG/PCL composite porous bone scaffold and a preparation method thereof, belonging to the technical application field of biomedical materials. The BBG/PCL composite porous bone scaffold provided by the invention is obtained by selectively laser sintering composite porous bone scaffold powder containing BBG powder and PCL powder on the basis of a scaffold model obtained by performing Boolean cross operation on a bone three-dimensional model and an array with a body-centered cubic lattice structure as a basic unit, has excellent pore structure, mechanical property, biodegradability, biocompatibility, protein adsorbability, mineralization capability, in-vivo osteogenesis and angiogenesis promotion effect and the like, and can be used as a bone substitute transplantation material with excellent performance.

Description

BBG/PCL composite porous bone scaffold and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical material application, and particularly relates to a BBG (borate bioactive glass)/PCL (polycaprolactone) composite porous bone scaffold and a preparation method thereof, in particular to a BBG/PCL composite porous bone scaffold prepared by SLS (selective laser sintering) and application thereof in bone defect (especially critical dimension bone defect (CSBD)) repair.

Background

Bone defect repair is one of the most common regenerative surgeries, with over 200 million bone grafts per year worldwide. Aging, trauma, tumor resection, developmental deformity and infection are the main causes of bone defects. Among bone defects, critical-sized bone defects (CSBD) refer to 1/2 with a length exceeding the diameter of a long bone, or to bone loss with a length exceeding 1/5-1/4 of the length of a long bone, and the inherent repair capability of the bone itself cannot achieve effective bridging, so autologous bone graft, allogeneic bone graft and bone replacement graft are mostly used for the treatment. Among them, the main disadvantages of autologous bone grafting are long operation time, large secondary trauma and high infection risk. After allogeneic bone transplantation, the host immune response affects the vascularization and cellularization process of the graft, and complications usually occur in 30-60% of cases. Bone Tissue Engineering (BTE) utilizes a three-dimensional porous bone conduction scaffold to promote bone regeneration, can provide a large number of required donors for CSBD, and is a promising bone replacement transplantation method.

For bone tissue engineering, the scaffold not only has good biocompatibility and sufficient mechanical strength, but also has the characteristics of porous interconnected structure, convenience for cell adhesion, proliferation and tissue growth, controllable geometric dimension, good degradability, convenience for customization and the like. The performances of the scaffold depend on the matrix material and the preparation method thereof to a great extent, and the scaffolds prepared by different matrix materials and different methods have different structural characteristics and can directly influence the biological characteristics of cell adhesion, proliferation, differentiation and the like.

Clinically, due to the fact that CSBD often has irregular and complex three-dimensional geometrical bone defects, a customized support specific to a patient is needed to perfectly fit an anatomical section, but the CSBD is difficult to achieve by adopting traditional methods such as solvent casting, particle leaching, gas foaming and electrostatic spinning. BTEs based on 3D printing technology can fabricate scaffolds with custom geometries and precise pore structures from medical imaging data, while largely overcoming these difficulties, typically require support structures and are not suitable for the fabrication of complex pore structures (e.g., special structures such as three-cycle minimum curves, body-centered cubes, negative poisson's ratio, etc.). At present, a plurality of high molecular materials are used for manufacturing bone tissue engineering scaffolds, such as PCL, which has good thermal elasticity, biocompatibility and solubility in most organic solvents, and has characteristics of high mechanical strength and low melting point (60 ℃), however, due to the defects of high hydrophobicity, lack of cell adhesion sites, low bioactivity and biodegradability of PCL and many other polymers, the wide application of PCL in bone tissue engineering scaffolds is limited.

The Selective Laser Sintering (SLS) technique utilizes CO₂The rapid prototyping technology of selective sintering of polymer or composite powder by laser beam has become an ideal technology for constructing tissue engineering scaffold (such as bone tissue engineering scaffold) because it does not need any supporting structure and is more suitable for the manufacture of complex pore structure. In terms of the selection of the matrix material, a composite material consisting of a polymer or a bioactive material (e.g., Hydroxyapatite (HA), Bioglass (BG), beta tricalcium phosphate (β -TCP), etc.) and PCL is also gradually replaced by a single PCL matrix material, and a bone tissue engineering scaffold having good biocompatibility and biomechanical properties HAs been developed. For example, patent documents CN104441668A (hereinafter referred to as document 1) and guo ling cloud et al (guo ling cloud et al, the study on the construction of HA/PCL bone tissue engineering scaffolds by selective laser sintering technology, journal of oral material apparatus, vol 24, No. 2, 2015, and hereinafter referred to as document 2) both disclose a method for obtaining an HA/PCL bone tissue engineering scaffold having a three-dimensional interconnected pore structure by using a SLS technology and Polycaprolactone (PCL) and Hydroxyapatite (HA) as raw materials, wherein the bone tissue engineering scaffold HAs good biocompatibility. Patent document CN110327496A (hereinafter referred to as document 3) discloses a Sr-GO/PCL composite scaffold obtained by SLS technology and composed of a PCL matrix and Sr-GO composite particles dispersed in the PCL matrix, and the method disclosed in the document utilizes GO as a nucleation matrix and a carrier of Sr nanoparticles to synergistically promote the dispersion of GO and Sr nanoparticles in the PCL matrix, thereby imparting excellent bioactivity, hydrophilicity and mechanical properties to the composite scaffold.

However, although the scaffolds disclosed in the

above documents

1, 2 and 3 have good biocompatibility and mechanical properties (in the

documents

1 and 2, the combination of HA and PCL would rather reduce the mechanical properties of the composite scaffold), the pore structure is simple, which is not favorable for cell adhesion, propagation, differentiation and tissue growth under complex environmental conditions in vivo. On the other hand, the three methods only disclose basic index data in vitro such as porosity, hydrophilicity, mechanical property, biocompatibility and the like of the bone tissue engineering scaffold, and do not disclose key index data such as degradation rate, protein adsorption capacity, osteogenesis and angiogenesis of organisms and the like of the bone tissue engineering scaffold, so that the applicability of the bone tissue engineering scaffold in clinic cannot be determined.

Disclosure of Invention

In view of one or more problems in the prior art, an aspect of the present invention provides a BBG/PCL composite porous bone scaffold, which includes a basic scaffold obtained by selective laser sintering of composite porous bone scaffold powder based on a bone three-dimensional model and a scaffold model obtained by boolean intersection of an array using a body-centered cubic lattice structure as a basic unit, the basic scaffold including end faces at both ends and a circumferential face at the circumference;

the circumferential surface of the basic bracket is distributed with completely interconnected pores, through holes communicated with the end surfaces of the two ends of the basic bracket are distributed in the basic bracket, and the pores and the through holes are communicated with each other;

the body-centered cubic lattice structure comprises four cylinders which are arranged along the diagonal line of an inner body of the body-centered cubic lattice, wherein the proportion relation between the diameter D and the length L of each cylinder is D: L ═ 1: 2-1: 50, preferably D: L ═ 1: 5-1: 10, and further preferably D: L ═ 1: 7-1: 9;

the composite porous bone scaffold powder comprises 5-20 wt% of BBG powder and 80-95 wt% of PCL powder.

The diameter D of each of the four cylinders of the body-centered cubic lattice structure is 0.20 mm-1.00 mm, and the length L is 2 mm-10 mm.

The bone three-dimensional model is selected from a natural bone scanning three-dimensional model or a bone three-dimensional model designed by three-dimensional modeling software.

The porosity of the pores and through holes distributed on the basic support is 50% to 90%, preferably 57% to 70%; the aperture of the pore and the through hole is 0.40 mm-0.90 mm, preferably 0.40 mm-0.70 mm.

The composite porous bone scaffold powder comprises 10-20 wt% of BBG powder and 80-90 wt% of PCL powder, wherein the particle size distribution of the BBG powder is 12-90 mu m, and the particle size distribution of the PCL powder is 28-200 mu m.

The BBG powder is prepared by a traditional melt quenching method, and comprises, or consists essentially of, or consists of, in mass percent: b is₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15% and SrO: 1-8%; or

The BBG powder comprises, or consists essentially of, or consists of the following components in percentage by mass: b is₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15%, SrO: 1-8% and ZnO: 0.5 to 2 percent.

The invention also provides a preparation method of the BBG/PCL composite porous bone scaffold, which comprises the following steps:

1) taking a model obtained by Boolean cross operation of a bone three-dimensional model and an array with a body-centered cubic lattice structure as a basic unit as a support model, deriving the support model into an STL format, and transmitting STL format data information of the support model to a selective laser sintering machine;

2) placing the composite porous bone scaffold powder in a selective laser sintering machine, and processing according to the scaffold model in the step 1) and preset parameters to obtain a BBG/PCL composite porous bone scaffold;

In the above method, the preset parameters include: the preheating temperature is 40-50 ℃, the laser power is 2-15W, the scanning speed is 500-3000 mm/s, the scanning interval is 0.1-0.2 mm, and the thickness of the powder layer is 0.1-0.2 mm.

In the method, the diameter D of each of the four cylinders of the body-centered cubic lattice structure is 0.20mm to 1.00mm, and the length L is 2mm to 10 mm.

In the above method, the bone three-dimensional model is selected from a natural bone scanning three-dimensional model or a bone three-dimensional model designed by three-dimensional modeling software.

In the method, the composite porous bone scaffold powder comprises 10-20 wt% of BBG powder and 80-90 wt% of PCL powder, the particle size distribution of the BBG powder is 12-90 μm, and the particle size distribution of the PCL powder is 28-200 μm. The BBG powder is prepared by a traditional melt quenching method, and comprises, or consists essentially of, or consists of, in mass percent: b is₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15% and SrO: 1-8%; or the BBG powder further comprises ZnO: 0.5 to 2 percent.

Yet another aspect of the present invention also provides a powder composition for bone scaffolds, comprising 5 wt% to 20 wt% of a BBG powder and 80 wt% to 95 wt% of a PCL powder, wherein the BBG powder has a particle size distribution of 12 μm to 90 μm, and the PCL powder has a particle size distribution of 28 μm to 200 μm;

The BBG powder further comprises ZnO: 0.5 to 2 percent.

The application of the BBG powder in improving the adsorption performance of PCL on protein also belongs to the content of the invention, and the BBG/PCL composite porous bone scaffold is prepared by uniformly mixing the BBG powder and the PCL powder and then selectively sintering the mixture by laser, wherein the mass percentage of the BBG powder in the BBG/PCL composite porous bone scaffold is more than 5%.

The BBG/PCL composite porous bone scaffold provided based on the technical scheme is obtained by selectively sintering composite porous bone scaffold powder containing BBG powder and PCL powder by laser on the basis of a natural bone scanning three-dimensional model (such as microcomputer tomography) or a bone three-dimensional model (such as CAD three-dimensional model) designed by three-dimensional modeling software and a scaffold model obtained by Boolean cross operation of an array with a body-centered cubic (BCC) lattice structure as a basic unit, wherein the peripheral surface and the end surfaces of the BBG/PCL composite porous bone scaffold are both of uneven structures, fully interconnected pores are distributed on the peripheral surface, through holes communicated with the end surfaces at two ends of the BBG/PCL composite porous bone scaffold are distributed inside the BBG/PCL composite porous bone scaffold, and the through holes are also communicated with the pores, so that the BBG/PCL composite porous bone scaffold provided by the invention is provided with holes in the three-dimensional space direction, and all the pores are completely interconnected, and compared with the simple pore structure of the bone tissue engineering scaffold disclosed in the documents 1 to 3, the porous structure greatly increases the exposed surface area of the skeleton, and is more favorable for cell adhesion, propagation, differentiation and tissue growth under complex environmental conditions in an organism.

On the other hand, the basic scaffold as the BBG/PCL composite porous bone scaffold is prepared by uniformly mixing BBG and PCL powder through SLS (selective laser sintering), wherein the PCL powder is rough in surface after being subjected to the melting treatment of SLS, the surface area is increased, the exposed surface area of a PCL matrix is further improved due to a complex porous structure, more cell attachment sites and adhesion sites of BBG particles can be provided, attachment, release and contact with bone tissue cells of elements such as B, Na, Ca, Mg, K, Sr and the like in a BBG material uniformly dispersed on the surface of the PCL matrix are facilitated, and propagation, differentiation and tissue growth of the bone tissue cells are further facilitated. In vivo and in vitro experiments prove that compared with a bone scaffold prepared from a single PCL material, the composite porous bone scaffold prepared by introducing a proper amount of BBG material into the PCL material can not only improve the mechanical property, biocompatibility, biodegradability and protein adsorbability of the bone scaffold, but also obviously improve the mineralization capability and bone regeneration capability of the bone scaffold, plays a role in promoting osteogenesis and angiogenesis in organisms, does not generate cytotoxicity to cells, therefore, the powder composition for bone scaffolds comprising BBG and PCL provided by the invention can be used as a raw material of an excellent bone tissue engineering scaffold, and the BBG/PCL composite porous bone scaffold prepared from the powder composition can be used as a bone graft substitute material with excellent performance, the method overcomes the prejudice that the degradation of BBG in the prior art can lead to the fact that the strong alkaline environment is not favorable for cell growth and is not suitable for preparing the bone tissue engineering scaffold.

Drawings

FIG. 1 is a schematic diagram of the structure of a body centered cubic lattice;

FIG. 2 is the results of analysis of the micro-morphology, structure, particle size distribution, elements (B, Ca, Na, K, Sr, Mg elements), etc. of PCL powder and BBG powder, wherein A shows the SEM micro-morphology of BBG powder, B shows the XRD spectrum of BBG powder, C shows the EDS analysis of elements in BBG powder, D shows the particle size distribution curves of BBG powder and PCL powder, and E shows the SEM micro-morphology of PCL powder;

FIG. 3 is a flow chart of SLS preparation of BBG/PCL composite porous bone scaffold;

FIG. 4 is a photograph showing the morphology and characterization of various scaffolds prepared in example 1, wherein a is an SEM image of the surface of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL, b is an SEM image of the surface of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL, C is an SEM image of the surface of 20BBG/PCL scaffold, and d is a partial enlarged view of SEM and EDS-Mapping (elements C, Na, Ca, Mg, K and Sr) at the dotted-frame part in C;

FIG. 5 is a schematic structural view of a BBG/PCL composite porous bone scaffold prepared according to an embodiment of the present invention, wherein A shows a front view and B shows a left view;

FIG. 6 is an SEM image of the surface of 5 scaffolds prepared according to example 1, namely 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40 BBG/PCL;

FIG. 7 is a graph showing the results of performance characterization of various scaffolds prepared in example 1, wherein a is a panel showing the results of detection of compressive strength, b is b panel showing the results of detection of elongation at break, c is a result of detection of porosity, d is a result of measurement of water contact angle, e is a result of measurement of water contact angle of 40BBG/PCL scaffold, and f is a result of detection of protein adsorption performance of various scaffolds;

FIG. 8 is a graph showing the results of measurement of degradability, mineralization ability, biocompatibility and osteogenesis-promoting performance of various scaffolds prepared in example 1, wherein a is a graph showing the change in residual mass of the scaffolds immersed in Tris-HCl, b is a graph showing the change in pH of the scaffolds immersed in Tris-HCl, c is a graph showing the SEM morphology of the scaffolds after soaking in SBF for 1, 3 and 7d, d is a graph showing FITC staining and DAPI staining after culturing h-BMSC cells using the scaffolds for 1 day, e is a graph showing the survival rate of the h-BMSC cells using the scaffolds for 1, 3 and 5 days, and f is a graph showing the activity of alkaline phosphatase (ALP) after culturing the h-BMSC cells using the scaffolds for 3 and 7 days;

FIG. 9 is release profiles of B, Ca and K elements after immersion of various scaffolds prepared in example 1 in Tris-HCl, wherein a represents the release profile of B element, B represents the release profile of Ca element, and c represents the release profile of K element;

FIG. 10 is an SEM image of the surface of the stent after soaking various scaffolds prepared in example 1 in an SBF solution, wherein a is an SEM image of the surface of the stent after soaking 0BBG/PCL in the SBF solution for 5 days, b is an SEM image of the surface of the stent after soaking 20BBG/PCL in the SBF solution for 3 days, and c is an SEM image of the surface of the stent after soaking 40BBG/PCL in the SBF solution for 5 days;

FIG. 11 is a Ca/P deposition SEM scan image of the stent surface after soaking 20BBG/PCL prepared in example 1 in SBF solution for 1, 3 and 7 days;

FIG. 12 is a schematic view of the structure of a cell culture system;

fig. 13 is a schematic diagram of a rabbit radius defect bone repair process and a bone repair characterization result, in which, a shows the schematic diagram of the rabbit radius defect bone repair process, b shows a micro-CT reconstructed three-dimensional model image of a rabbit radius defect site, c and d show the new bone volume and bone volume/tissue volume at 6 weeks and 12 weeks after stent implantation, respectively, and e to j show the relative quantitative results of bone formation related gene analysis of the new tissue at 6 weeks and 12 weeks after stent implantation;

FIG. 14 is a photograph of a site of repair of a rabbit radius defect bone 6 weeks and 12 weeks after implantation of a stent, wherein a shows 6 weeks after implantation of a stent and b shows 12 weeks after implantation of a stent;

FIG. 15 is a histological image of H & E and M-T staining of a repair site of rabbit radius defect bone 12 weeks after stent implantation, wherein a shows an H & E staining image and b shows an M-T staining image;

FIG. 16 is a histological image of H & E and M-T staining of a repair site of rabbit radius defect bone 6 weeks after stent implantation, wherein a shows an H & E staining image and b shows an M-T staining image;

FIG. 17 is an M-T stained histological image of a radius defect bone repair site of a rabbit at 12 weeks after implantation of a 20BBG/PCL scaffold;

FIG. 18 is an M-T stained histological image of a repair site of rabbit radius defect bone 12 weeks after implantation of scaffolds (0BBG/PCL and 20BBG/PCL) and a control group without scaffolds;

FIG. 19 is an H & E stained image of the stent site after 6 weeks and 12 weeks of the implanted stents (0BBG/PCL and 20BBG/PCL) and the control group of non-implanted stents, in which a shows the H & E stained image of the repair site of each group after 12 weeks, b shows a partially enlarged image of a middle dark frame, c shows the analysis result of the area of the residual pores of the stent after 6 weeks and 12 weeks of the implanted stents (0BBG/PCL and 20BBG/PCL), d shows a partially enlarged image of a middle light frame, and E shows the analysis result of the VEGF-related genes generated in the blood vessels of the new tissues after 6 weeks and 12 weeks of the implanted stents (0BBG/PCL and 20BBG/PCL) and the control group of non-implanted stents.

Detailed Description

Aiming at the defects that the pore structure of the bone tissue engineering scaffold in the prior art is simple and is not beneficial to the adhesion, propagation, differentiation and tissue growth of cells under complex environmental conditions in organisms, and the clinical applicability of the existing bone tissue engineering scaffold cannot be clarified due to the lack of in vivo application data, the invention provides the BBG/PCL composite porous bone scaffold with a complex porous structure design on the basis of a Selective Laser Sintering (SLS) technology, which takes uniform mixed powder of BBG and PCL as raw materials, a natural bone scanning three-dimensional model or a bone three-dimensional model designed by three-dimensional modeling software and a scaffold model obtained by Boolean crossing operation of an array with a body-centered cubic lattice structure as a basic unit, and performs in vitro and in vivo experiments by using the BBG/PCL composite porous bone scaffold.

As used herein, the term "Borate Bioactive Glass (BBG)" is a boron-based bioactive glass, which is different from conventional silicate or phosphate bioactive glass containing elements of Si, P, etc., wherein the Si element, which cannot be degraded by biological metabolism, is not contained at all, and the P element is not contained, and mainly contains elements of B, Ca, K, Na, Mg, Sr, etc. BBG materials have been demonstrated to have good biocompatibility and bioactivity, and have been a major advance in the field of soft tissue repair. However, since BBG degrades faster in body fluids, the pH of the surrounding tissue rises rapidly in a short time (>10) Too high pH can generate obvious toxic action on cells, and BBG can release high-concentration B element in the process of rapid degradation, and can also cause obvious cytotoxic action, so that the BBG material cannot be directly used as a soft tissue repair material or other medical engineering materials (such as bone tissue engineering scaffold materials). Although patent document CN109985272A discloses that BBG can be modified by phosphate buffer treatment, and patent document CN108275883A discloses that AlO can be introduced into boron-phosphorus bioactive glass₄The structural method slows down the degradation of BBG in organisms, thereby reducing the toxic action on the organisms and being well used as a soft tissue repair material. However, due to the inherently brittle, less tough nature of BBG materials, even when phosphate modified BBG is used or AlO is introduced into phosphate modified BBG₄Neither structure can alter these properties inherent to the BBG material itself,so that the modified BBG material can not be directly used for preparing bone tissue engineering scaffolds with certain structural toughness, necessary hole structure design and mechanical properties.

However, the inventors surprisingly found that when a proper amount of BBG is introduced into PCL, the BBG material with inherent characteristics of brittle texture and poor toughness does not cause the reduction of mechanical properties of the composite material as when HA is introduced (the BBG material HAs the characteristics of brittle texture and poor mechanical properties) but maintains the mechanical properties of the composite material to a certain extent, and can even obviously improve the mechanical properties and structural toughness of the composite material and also obviously improve the porosity and biocompatibility (hydrophilicity) of the composite material. On the other hand, after BBG is introduced into the PCL polymer, the degradation of the BBG can be slowed down by the PCL polymer, so that the rapid increase of the pH value caused by the rapid degradation of the BBG in body fluid is avoided, and the weak alkaline environment in the body fluid caused by the slow degradation of the BBG under the control action of the PCL polymer is avoided. And because BBG is slowly degraded under the control action of PCL, the controllable slow release of various elements in BBG can be directly influenced, high-concentration elements such as B, Ca, K, Na, Mg, Sr and the like in body fluid can not be caused, and the elements are continuously and slowly released for a long time, so that the absorption and utilization of cells and tissues are facilitated. On the other hand, the inventor also surprisingly finds that the introduction of a proper amount of BBG into PCL can also significantly improve the adsorption capacity of the composite material (composite porous bone scaffold) on proteins, which is more beneficial to the attachment, migration and growth of cells on the surface of the composite bone scaffold. Therefore, the BBG material and the PCL material can be compounded to prepare the bone tissue engineering scaffold, wherein the BBG and the PCL play a synergistic and complementary role to obviously maintain or improve the mechanical property, the structural toughness, the biocompatibility, the mineralization capacity, the protein adsorption property and the like of the composite material, the potential cytotoxic effect is not generated, the biodegradability of the composite material is obviously improved, and in-vivo experiments prove that compared with a pure PCL scaffold, the BBG/PCL composite porous bone scaffold provided by the invention can obviously promote bone regeneration and angiogenesis and can be used as an excellent bone substitute transplanting material.

As used herein, the term "body-centered cubic lattice" is a structure comprising four cylinders arranged along the internal body diagonals of the body-centered cubic lattice, as shown in fig. 1, which shows a schematic structural view of the body-centered cubic lattice. In some embodiments, the ratio of the diameter D to the length L of each cylinder of the body-centered cubic lattice is preferably D: L ═ 1:2 to 1:50, preferably D: L ═ 1:5 to 1:10, and more preferably D: L ═ 1:7 to 1: 9. In some embodiments, each cylinder of the body-centered cubic lattice has a diameter D of 0.20mm to 1.00mm and a length L of 2mm to 10mm, but the diameter D and the length L of each cylinder of the body-centered cubic lattice are not limited thereto and will vary adaptively according to the actual size of the three-dimensional model of the bone.

As used herein, the term "pore diameter" refers to the maximum width of a pore or through-hole in the cross-sectional direction.

As used herein, the term "wt%" refers to a mass percent content.

The invention is further illustrated by the following examples. It should be understood that the specific examples are intended to be illustrative of the invention and are not intended to limit the scope of the invention.

The methods used in the following examples are conventional methods unless otherwise specified, and specifically, the methods disclosed in documents 1 to 3 can be referred to.

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

In the following description, a BBG/PCL composite porous bone scaffold is prepared by taking a CSBD model of rabbit radius as an example, but the method of the present invention is not limited to the preparation of a BBG/PCL composite porous bone scaffold of a CSBD model of rabbit radius, and can also be used to prepare a BBG/PCL composite porous bone scaffold of any three-dimensional model of bone, wherein the three-dimensional model of bone can be a natural bone scanning three-dimensional model or a three-dimensional model of bone designed by three-dimensional modeling software.

The formulation of the BBG bioactive glass used in the following examples is not particularly limited, but the following component systems are preferred in mass percent: b is₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15% and SrO: 1-8% (component system 1); or B₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15%, SrO: 1-8% and ZnO: 0.5-2% (component system 2). The component system 1 is an initial boron glass component disclosed in patent document CN108164135A, which has been proved to have good bioactivity and biocompatibility, and the component system 2 is formed by adding ZnO on the basis of the component system 1, so as to further provide Zn element for BBG bioactive glass, which is more beneficial to promoting osteogenic repair. It was verified that both the above component system 1 and the component system 2 can achieve the effects of the present invention. The BBG bioactive glass used in the following examples was prepared by melt quenching (melt-quenching) to obtain a bulk glass, which was ground and sieved to obtain the BBG bioactive glass.

Example 1: preparation of BBG/PCL composite porous bone scaffold

Experiment 1.1 preparation of composite porous bone scaffold powder

(1) The BBG bioactive glass used in the experiment has the formula (all in mass percent): b is₂O₃：52％、MgO：5％、CaO：20％、Na₂O：6％、K₂O: 12%, SrO: 4% and ZnO: 1 percent. The formula is used as a raw material, blocky glass (the melting temperature is 1000-1350 ℃, the heat preservation time is 0.5-4 hours) is prepared by a traditional melting quenching method, BBG powder is obtained by sieving after the blocky glass is fully ground, and the BBG powder has microscopic morphology, structure, particle size distribution and elements (B, Ca)Na, K, Sr, Mg elements) and the like. As shown in fig. 2, the micro-morphology (a), XRD spectrum (B), EDS elemental analysis result (C) and particle size distribution curve (D) of the BBG powder are shown. The BBG powder prepared is of pure amorphous and amorphous structures, namely an XRD spectrogram has two wide diffusion scattering peaks of 20-40 degrees 2 theta and 40-50 degrees 2 theta; the particle size distribution is 12-90 μm, and the average particle size is about 40 μm; the results of EDS elemental analysis also showed that the BBG powder obtained contained B, Ca, Na, K, Sr, Mg, and completely no Si or P.

(2) The PCL powder used in the experiment was purchased from solvay corporation, and the microscopic morphology, particle size distribution, and the like of the PCL powder were analyzed. The results are shown in FIG. 2 (E) and (D), wherein (E) is the micro-morphology of the PCL powder and (D) is the particle size distribution curve of the PCL powder, and it can be seen that the particle size distribution of the PCL powder is 28 μm to 200 μm and the average particle size is about 90 μm.

(3) Mixing BBG powder and PCL powder at a certain ratio, wherein the BBG accounts for 0%, 5%, 10%, 20% and 40% of the mixed powder by mass respectively. After sufficient shaking and stirring, BBG/PCL mixed powder with different BBG powder contents is obtained respectively, namely the composite porous bone scaffold powder, and is used in the following experiments.

Experiment 1.2 preparation of BBG/PCL composite porous bone scaffold

The experiment takes a CSBD model of rabbit radius as an example, a BBG/PCL composite porous bone scaffold similar to the CSBD model of natural rabbit radius is prepared, and the method specifically includes the following steps in combination with the flow diagram shown in fig. 3.

(1) As shown in a of fig. 3, a 34mm long radius bone was cut along the longitudinal axis of the entire rabbit radius bone, and a micro computer tomography (micro-CT) scan was performed on the cut radius bone with a spatial resolution of 9 μm to obtain a scanned three-dimensional model image of the rabbit radius defect.

(2) The three-dimensional model image obtained by scanning in the step (1) is introduced into a Mimics 20.0 software to reconstruct a three-dimensional model of a rabbit radius defect (for example, a natural bone may be a hollow structure with a medullary cavity (the diameter of the hollow structure may be designed as required), or a solid structure without the medullary cavity.

(3) An array with a body centered cubic lattice (BCC) structure (shown as C in fig. 3) as the basic unit was constructed in the mics 20.0 software, where each BCC comprises four cylinders arranged along the diagonal of the body centered cubic lattice with a diameter D and a length L designed to be 0.5mm and 4mm, respectively. The diameters D and the lengths L of four cylinders which are selected and arranged in the body-centered cubic lattice inner body diagonal line can also be respectively designed to be 0.2mm, 10 mm; or D is 0.3mm, L is 9 mm; or D is 0.4mm, L is 2 mm; or D is 0.5mm, L is 5 mm; or D is 0.8mm, L is 8 mm; or D is 1.0mm, L is 8mm, etc.

(4) And (3) performing Boolean crossing operation on the CSBD model reconstructed in the step (2) and the array which is established in the step (3) and takes the body-centered cubic lattice structure as the basic unit to reserve an intersecting part, obtaining a customized micropore support model as shown in D in figure 3, exporting the micropore support model into an STL format, and transmitting STL format data information of the micropore support model to a commercial Selective Laser Sintering (SLS) machine.

(5) As shown in E of fig. 3, 5 kinds of composite porous bone scaffold powders prepared in experiment 1 were respectively placed in a selective laser sintering machine, and sintering was performed according to STL format data information of the microporous scaffold model of step (4) according to preset parameters (preheating temperature may be 40 to 50 ℃, laser power may be 2 to 15W, scanning speed may be 500 to 3000mm/s, scanning interval may be 0.1 to 0.2mm, thickness of the powder layer may be 0.1 to 0.2 mm; parameters set in this example are specifically preheating temperature 45 ℃, laser power 13W, scanning speed 1000mm/s, scanning interval 0.1mm, thickness of the powder layer 0.15mm), unsintered composite porous bone scaffold powders attached to the surface or trapped in the pores may be removed using a wind turbine, 5 kinds of BBG/PCL composite porous bone scaffolds (length is 34mm each, the width and the height are both 6.5mm and 6.5mm), as shown in F in FIG. 3, the BBG content is respectively named as 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL (which are collectively called rabbit radius model bracket). For different research purposes, two additional scaffolds with the same unit structure as the rabbit radius model scaffold but different three-dimensional geometries (cubic scaffold for porosity determination (10 mm in length, width and height, respectively), cuboid scaffold for compression experiment (40 mm in length, 20mm in width and 10mm in height) and a solid disc for protein adsorption experiment (6 mm in diameter and 1mm in height) were prepared at the same BBG content, and the morphology of each BBG/PCL scaffold is shown as a in fig. 4.

As shown in fig. 5, a micro-CT reconstruction structure diagram of 20BBG/PCL prepared by the experiment is shown by taking a 20BBG/PCL rabbit radius model scaffold as an example, wherein a is a main view of 20BBG/PCL, B is a left view of 20BBG/PCL, it can be seen that the 20BBG/PCL obtained by selectively laser sintering composite porous bone scaffold powder based on a bone three-dimensional model and a scaffold model obtained by boolean intersection operation with an array having a body-centered cubic lattice structure as a basic unit according to the present invention comprises end surfaces 2 at both ends and a circumferential surface 1 at the circumference, both the circumferential surface 1 and the end surfaces 2 of the scaffold are rugged structures (which can provide more attachment sites for cells), and a plurality of pores 3 completely interconnected are substantially uniformly distributed on the scaffold, a plurality of through holes 4 (4 shown in the figure) are distributed inside and communicated with the end surfaces 2 at both ends of the 20BBG/PCL scaffold, and all the through holes 4 and all the pores 2 are also communicated with each other, and the complex pore structure also greatly increases the exposed area of the surface of the stent, thereby being more beneficial to the attachment of cells. Therefore, the BBG/PCL composite porous bone scaffold provided by the invention is provided with holes in the three-dimensional space direction, all the holes are completely interconnected, and the surface is of an uneven complex porous three-dimensional structure, which is difficult to realize by the traditional template method, the freeze drying method, the gas foaming method and the common 3D printing technology (such as FDM and DIW).

The following example 2 will characterize the BBG/PCL composite porous bone scaffold provided by the present invention in detail by a specific experiment.

Example 2 characterization of BBG/PCL composite porous bone scaffolds

2.1 morphological characteristics of BBG/PCL composite porous bone scaffold

The morphological characteristics of the 5 scaffolds were characterized by SEM scan imaging and EDS-Mapping elemental analysis of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL (based on rabbit radius model scaffold). SEM images of the surfaces of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL, as shown in b in FIG. 4, and an enlarged view of the SEM images of the 5 scaffolds, respectively, is shown in FIG. 6; as shown in FIG. 4, c, an SEM image of a 20BBG/PCL scaffold is shown; as shown in d in FIG. 4, a partial enlarged view of SEM and EDS-Mapping (elements of C, Na, Ca, Mg, K and Sr) at the dotted frame portion in C in FIG. 4 is shown.

From the SEM image of the 20BBG/PCL scaffold shown in c of fig. 4 and the micro-CT reconstruction structure diagram of the 20BBG/PCL shown in fig. 5, it can be seen that the 20BBG/PCL scaffold has PCL powders bonded to each other as a skeleton, the BBG powders are uniformly dispersed on the surface of the PCL skeleton, and a micro-pore structure (including pores and through holes) is present between the PCL powders bonded to each other, and the pore size of the pores in the pore structure is about 600 to 700 μm. The pore sizes of the holes scattered on other 3 kinds of bone scaffolds containing BBG were also tested, wherein the pore size of the holes scattered on the surface of 5BBG/PCL scaffold was about 400-500 μm, the pore size of the holes scattered on the surface of 10BBG/PCL scaffold was about 500-600 μm, and the pore size of the holes scattered on the surface of 40BBG/PCL scaffold was about 900-1000 μm, it can be seen that the pore size of the holes in the pore structure increases with the increase of the content of BBG, which may be due to the different material properties of the composite porous bone scaffold powders with different BBG contents, i.e. the different flow properties under the selective laser sintering condition, resulting in the different pore sizes of the holes on the surface of the bone scaffolds prepared from the composite porous bone scaffold powders with different BBG contents. However, the 40BBG/PCL stent surface still has a large number of non-design pores, which may be the main reason for the reduced mechanical strength of the 40BBG/PCL stent (detailed below). Generally, the pore diameter of more than 150 μm is a suitable choice for a bone regeneration scaffold, which provides necessary conditions for the growth of cells and blood vessels in the scaffold in the bone defect repair process, so that the BBG/PCL composite porous bone scaffold prepared by the invention meets the requirements on the pore diameter (400-700 μm) of the surface pore structure.

As is clear from the EDS-Mapping elemental analysis result of the 20BBG/PCL scaffold shown in d in FIG. 4, the surface of the 20BBG/PCL scaffold contains Na, Ca, Mg, K, Sr and other elements which are only present in BBG, so that the region where these elements are present is the BBG-present region. Similarly, BBG particles were also visible on all other BBG/PCL scaffolds and were uniformly dispersed in the PCL matrix.

2.2 mechanical Properties of BBG/PCL composite porous bone scaffold

The mechanical properties of 5 scaffolds were characterized by testing the compressive strength and elongation at break of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL (based on cuboid scaffold samples). The results are shown in fig. 7, where panel a shows the compressive strength results for 5 stents and panel b shows the elongation at break results for 5 stents. In the figure, n is 3, error bars represent mean ± SD, indicating significant differences compared to 0BBG/PCL, P <0.05, P <0.01, P < 0.001.

As can be seen from panel a in FIG. 7, as the BBG content in the composite scaffold increases, the compressive strength of the scaffold increases first and then decreases, compared with the pure PCL sample (0.82 + -0.012 MPa), the compressive strength of 5BBG/PCL reaches a maximum of 1.01 + -0.015 MPa, which is improved by about 18.6%, the compressive strength of 10BBG/PCL group decreases to 0.97 + -0.019 MPa, the compressive strength of 20BBG/PCL (0.86 + -0.019 MPa) is substantially the same as that of 0BBG/PCL scaffold, and the compressive strength of 40BBG/PCL decreases to 0.44 + -0.035 MPa (which may be related to the existence of a large amount of non-designed pores on the surface of 40BBG/PCL scaffold, as shown in FIG. 6). Obviously, although the BBG is a material having inherent characteristics of being brittle and poor in toughness like HA, when a proper amount (for example, less than 20% by mass of the composite system) of BBG is incorporated into PCL, the BBG material does not cause a reduction in mechanical properties (for example, compressive strength) of the composite material like HA, but maintains the mechanical properties of the composite material to a certain extent, and even obviously improves the mechanical properties of the composite material.

As can be seen from panel b of fig. 7, the elongation at break of the test specimen steadily increased from 23.2 ± 0.929% to 39.8 ± 2.183% as the BBG content in the composite scaffold increased, which means that the composite scaffold had stronger ductility. It is clear that although BBG is a material with inherent properties of being more brittle and less tough, its incorporation into PCL can, in turn, significantly increase the ductility, i.e., structural toughness, of the composite.

2.3 porosity of BBG/PCL composite porous bone scaffold

Porosity assays were performed on 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL (based on cubic scaffold samples). The results are shown in fig. 7, panel c, where n is 3, error bars represent mean ± SD, indicating significant differences compared to 0BBG/PCL, P <0.05, P <0.01, P < 0.001.

From the results shown in panel c of FIG. 7, it can be seen that the porosities of the 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL scaffolds were 56.44. + -. 0.64%, 57.94. + -. 0.59%, 63.56. + -. 0.81%, 68.45. + -. 1.01% and 74.85. + -. 0.98%, respectively. It is clear that the porosity of the composite scaffold increases with the percentage by mass of BBG in the scaffold (with a porosity of about 57% to 70% in the range of 5% to 20% BBG). The porosity of the scaffold necessary for bone regeneration needs to be at least 50%, so the BBG/PCL composite porous bone scaffold provided by the invention meets the requirement of bone regeneration, and the interconnected pores in the scaffold enable cells to rapidly migrate from the surface of the scaffold to the interior of the scaffold after implantation and promote angiogenesis and nutrient permeation in the regeneration process.

2.4 hydrophilicity of BBG/PCL composite porous bone scaffold

Water contact angle measurements were performed on 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL (based on cubic scaffold samples) to characterize the hydrophilicity of the 5 scaffolds. The results are shown in fig. 7, panel d, where n is 3, error bars represent mean ± SD, indicating significant differences compared to 0BBG/PCL, P <0.05, P <0.01, P < 0.001.

As shown by the panel d in FIG. 7, it can be seen that the surface of the pure PCL scaffold (i.e. 0BBG/PCL) has strong hydrophobicity, and the contact angle with water is 97.6 + -0.76 degrees, which limits the biological application to a certain extent. The water contact angle of the 5BBG/PCL bracket is reduced to 95.2 +/-0.76 degrees. When the BBG mass fraction increased to 10%, the water contact angle further decreased to 88.2 ± 0.29 °, transitioning from hydrophobic to hydrophilic. The water contact angle of the 20BBG/PCL bracket is changed more obviously and is reduced to 63.2 +/-0.57 degrees. The 40BBG/PCL scaffold surface was completely wet and the water contact angle could not be measured because the water drop was present on the surface for too short a time (about 0.20s) (as shown in e-bar in fig. 7). Obviously, the proper introduction of BBG in PCL has a positive effect on improving the hydrophilicity of the PCL scaffold, and since the cell adhesion of the scaffold is closely related to the hydrophilicity of the scaffold surface, the more hydrophilic the scaffold surface is, the more favorable the cell adhesion is, therefore, the proper introduction of BBG in PCL can obviously improve the adhesion of the composite scaffold to cells.

2.5 protein adsorption of BBG/PCL composite porous bone scaffold

Protein (green fluorescent protein GFP) adsorption experiments were performed on 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL (based on solid disc samples) to characterize the protein adsorption performance of the 5 scaffolds. The f panels in FIG. 7 show fluorescence images of the scaffolds after 24h incubation with GFP solution.

As shown in figure 7, panel f, the BBG-added scaffolds absorbed protein significantly better than the pure PCL scaffold compared to the blank control (PBS instead of GFP). Specifically, as the BBG content increases, more and more areas are displayed in the fluorescence image brightens, and the brightness increases. It is inconceivable that the 40BBG/PCL group was over-exposed at the same shooting parameters as the other groups, which means that a large amount of GFP was attached to its surface. The blank control group had no change in dark state between the scaffolds. Indicating that the adsorption capacity of the scaffold for proteins increases with increasing BBG content, which may be caused by the irreversible interaction of electrostatic forces between the amine groups of the proteins and the negatively charged bioactive glass components on the scaffold.

Since the attachment, migration and growth of cells are affected by proteins absorbed from the surrounding environment to the surface of the scaffold, and most of the proteins interacting with the scaffold in vivo are hydrophilic extracellular proteins (such as GFP), the increase of BBG content in the composite scaffold not only improves the hydrophilicity of the scaffold, but also enables more proteins to be adsorbed on the surface of the scaffold, thereby being more beneficial to the adhesion, migration and growth of cells.

2.6 biodegradability of BBG/PCL composite porous bone scaffold

To characterize the biodegradability of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL, these 5 scaffolds (cubic scaffold samples) were soaked in Tris-HCl solution at pH 7.4 for 28 days, the scaffold weight and the change in B, Ca, K ion concentration in the soaking solution were monitored and the change in pH of the solution within 24h of soaking was monitored. The results are shown in fig. 8, panels a and b, and fig. 9.

As shown in a in FIG. 8, it can be seen that the residual mass fractions of 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL scaffolds after 28d soaking were 98.55 + -0.84%, 97.52 + -0.21%, 95.63 + -0.20%, 88.89 + -0.51% and 58.22 + -0.19%, respectively. And with the prolonging of the soaking time, the weight loss of all the scaffolds is increased, and the degradation rate is slowed down. Under the same conditions, the degradation rate of the bracket with high BBG concentration is higher, particularly the degradation speed of the 40BBG/PCL bracket is obviously higher than that of other brackets, the quality of the bracket is reduced by 27.82% in the first 3 days, and the possible reason is that the bracket collapses in a short time due to the excessively high degradation speed of the 40BBG/PCL bracket. As shown in fig. 9, it can be seen that more ions are released from the scaffold with the loss of scaffold mass, wherein the release tendency of ions B (a in fig. 9), Ca (B in fig. 9) and K (c in fig. 9) is very consistent with the weight loss tendency, and especially the 40BBG/PCL scaffold shows the release of high concentration of ions B, Ca and K. These elements can be detected after 28 days of soaking, which shows that the BBG/PCL scaffold has excellent drug controlled release capability.

As shown in panel b of fig. 8, it can be seen that the pH value of the soaking solution gradually increases with the passage of time, and the higher the BBG content in the scaffold, the higher the pH value of the soaking solution, especially the pH value of the solution soaking 40BBG/PCL reaches 8 at 4h, and even can reach 10 with the increase of time, and shows stronger alkalinity, and has obvious cytotoxicity to the adjacent cell tissues; however, the composite scaffold with BBG content not higher than 20% has weak alkaline pH value in 24 hours, which indicates that the BBG degradation speed is slow, and the release speed of B, Ca and K ions is slow (as shown in fig. 9), so that the pH value of the solution rises slowly.

The above results indicate that there is an obvious positive correlation between the pH value of the soaking solution and the degradation quality loss of the scaffold, and the too strong alkalinity of 40BBG/PCL may cause the scaffold to collapse in a short time (as shown in a panel in fig. 8), so that PCL cannot control the degradation of BBG, which results in too fast BBG degradation rate, and release high-concentration ions of B, Ca, K, etc., which finally results in the fast increase of the pH value of the solution. In the composite scaffold with BBG content not higher than 20%, the composite scaffold does not collapse, and can obviously delay the degradation rate of BBG and the release rate of B, Ca, K and other ions, so that the solution soaking the composite scaffold maintains a weak alkaline environment for a long time, and proves that the weak alkaline environment does not cause toxic effect on adjacent tissues and cells (detailed below), and shows that the degradation of the composite scaffold is favorable, as shown in a figure 8, compared with a pure PCL scaffold, the residual mass fraction of the composite scaffold with BBG content not higher than 20% is obviously reduced, but the composite scaffold does not degrade too fast like a 40BBG/PCL scaffold, the matching relation between the scaffold degradation rate and the bone tissue repair rate can be realized, the strong alkaline environment with toxic effect on cell tissues can not be caused, and the controllable degradation of the composite scaffold and the controllable degradation of B in BBG can be realized by adjusting the BBG content in the composite scaffold, Ca. Controlled release of the K plasma.

2.7 mineralization Capacity of BBG/PCL composite porous bone scaffold

To characterize the mineralization capacity of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL, these 5 scaffolds (cubic scaffold samples) were soaked in simulated body fluid (SBF solution) for 7 days and the apatite formation capacity of the 5 scaffold surfaces was monitored. The results are shown in fig. 8, c, 10 and 11, wherein in fig. 8, c is the SEM topography (magnification: x 4000) of the scaffolds after 5 scaffolds are soaked in SBF solution for 1, 3 and 7 days (1D, 3D and 7D); FIG. 10 is SEM images of stent surfaces of different stents after soaking in SBF solution, wherein a shows SEM images of stent surfaces of 0BBG/PCL after soaking in SBF solution for 5 days and drying in air, b shows SEM images of stent surfaces of 20BBG/PCL after soaking in SBF solution for 3 days and drying in air, and c shows SEM images of stent surfaces of 40BBG/PCL after soaking in SBF solution for 5 days and drying in air; FIG. 11 is an SEM image of Ca/P deposition (based on EDS elemental analysis) of 20BBG/PCL after 1, 3, and 7 days soaking in SBF solution.

From the SEM images shown in fig. 8, panel c and fig. 10, it can be seen that all the scaffold surfaces formed mineralization and that the mineral particles on the scaffold became larger and thicker with increasing soaking time and BBG content. From the analysis results shown in fig. 11, it is understood that the mineral surface formed on the surface of the scaffold contains both P and Ca elements, but the composition of the phosphate ore precipitate changes with the change in the soaking time. For the 20BBG/PCL scaffold, the mineral calcium-phosphorus ratios formed after SBF soaking for 1, 3 and 7 days were 1.33, 1.61 and 1.88, respectively. The hydrophilic surface is favorable for forming hydroxyapatite, and after BBG is added into PCL, the hydrophilicity of the composite scaffold is improved, so that the formation of the hydroxyapatite on the surface of the scaffold is further favorable, and the mineralization capability of the composite scaffold is more prominent along with the improvement of the content of the BBG.

2.8 in vitro biocompatibility, cell proliferation and osteogenic differentiation of human bone marrow Stem cells (hBMSC) of BBG/PCL composite porous bone scaffolds

To characterize the in vitro biocompatibility, cell proliferation capacity and osteogenic differentiation capacity of hBMSC of 5 scaffolds, 0BBG/PCL, 5BBG/PCL, 10BBG/PCL, 20BBG/PCL and 40BBG/PCL, these 5 scaffolds (cuboid scaffold samples) were placed in the co-culture system shown in FIG. 12. In this co-culture system, scaffolds with different BBG content were evaluated for their effect on hbmscs growth and differentiation using a plug-in culture dish purchased from millipore, with a mesh separating the cells from the scaffold, the scaffold placed above the mesh, and the cells seeded at the bottom of the culture dish. The results are shown in the panel d, e and f in FIG. 8, wherein the panel d in FIG. 8 shows the culture of hBMSC cells using different scaffolds (FITC for cytoskeleton: (B) (II))

phallodin), nuclei stained with DAPI) confocal microscope images after 1 day; FIG. 8, panel e shows the survival rate of hBMSC cells cultured on different scaffolds after 1, 3 and 5 days (using CCK-8 kit for analysis, absorbance value at 450nm is shownThe higher the absorbance value, the higher the cell viability (qualitative characterization)); FIG. 8 is a graph showing the activity of hBMSC alkaline phosphatase (ALP) after culturing hBMSC cells with different scaffolds for 3 and 7 days. In panels e and f of fig. 8, n is 3, error bars represent mean ± SD, and P represents a significant difference from 0BBG/PCL<0.05，**P<0.01，***P<0.001。

From the results shown in fig. 8, d, it can be seen that hbmscs cultured using all scaffolds were attached and extended at the bottom of the dish, and most of the seed cells were elongated (flattened) to form pseudopodia filiformis. In three composite scaffolds, namely 5BBG/PCL, 10BBG/PCL and 20BBG/PCL, the number of hBMSCs in the same visual field range is correspondingly increased along with the increase of the content of BBG, and is obviously more than 0BBG/PCL, the number of cell pseudo-feet is also increased, the interaction and communication among cells are facilitated, and the excellent biocompatibility is shown. The number of cells in the 40BBG/PCL group is obviously reduced compared with that of the other three groups of composite scaffolds, which is probably because the excessive BBG in the 40BBG/PCL scaffold causes a strong alkaline microenvironment and is not beneficial to the growth of the cells.

As can be seen from the results shown in panel e of fig. 8, the o.d. value (at 450 nm) representing the number of cells increased significantly with the increase of the culture time, indicating that all scaffolds promoted the proliferation of hbmscs. In the three composite scaffolds of 5BBG/PCL, 10BBG/PCL and 20BBG/PCL, the proliferation capacity of hBMSC cells is enhanced along with the increase of BBG content, and the cell proliferation rate of the 20BBG/PCL group is higher than that of the other groups (P <0.01) under all conditions, which is consistent with the results shown in the d-panel of FIG. 8. The cell proliferation activity of the 40BBG/PCL scaffold is only lower than that of the 0BBG/PCL scaffold, and potential cytotoxicity is suggested, which is probably because excessive BBG in the 40BBG/PCL scaffold causes a strong alkaline microenvironment and is not beneficial to cell growth.

As can be seen from the results shown in panel f of fig. 8, the alkaline phosphatase activity of each group of hbmscs increased with the increase of the culture time, indicating that all scaffolds promoted differentiation of hbmscs and expression of osteoblast phenotype. Except for the 40BBG/PCL scaffold, the ALP activity is in positive correlation with the BBG content in the scaffold, and the ALP activity of the 20BBG/PCL group is obviously higher than that of other groups (P <0.01), which shows that the osteogenic differentiation of hBMSC cells is obviously up-regulated.

Example 3: in vivo effect of BBG/PCL composite porous bone scaffold

This example demonstrates the in vivo effects (including osteogenesis, in vivo degradation, and angiogenesis) of the BBG/PCL composite porous bone scaffold provided by the present invention, using the 20BBG/PCL scaffold prepared in example 1 (rabbit radius model scaffold) as an example, and using the 0BBG/PCL scaffold (rabbit radius model scaffold) as a negative control.

3.1 in vivo osteogenesis function of BBG/PCL composite porous bone scaffold

Specifically, as shown in a panel in fig. 13, a 1.8cm defect is generated by performing an operation on a rabbit forelimb radius, then a 20BBG/PCL scaffold matched with the size and shape of the defect is implanted into the defect part, a 0BBG/PCL scaffold is used as a negative control group, an untreated rabbit radius CSBD (the rabbit forelimb radius has a 1.8cm defect) is used as a blank control group, then the repair state of the rabbit radius defect bone of each group is monitored, and the volume of new bone after the operation and the bone formation related gene analysis of new bone tissue are quantitatively analyzed. There was no evidence of inflammation or infection in all animals throughout the post-operative period, suggesting that all stents had good biocompatibility and safety in vivo. The results are shown in fig. 13 b to j and fig. 14, wherein b in fig. 13 shows micro-CT images of the rabbit forelimb radial defect sites at 6 and 12 weeks of the implantation of 0BBG/PCL, 20BBG/PCL stent and the non-implanted group; in FIG. 13, panels c and d show NBV (new bone volume, mm) at 6 weeks and 12 weeks after surgery for each group, respectively³) And BV/TV (bone volume/tissue volume,%); in FIG. 13, panels e to j show the relative expression levels of the bone formation-related genes OPN (osteopontin-expressing gene), OCN (osteocalcin-expressing gene), BMP-2 (bone morphogenetic protein-expressing gene), ALP, RUNX2 and COL-1 (collagen-1-expressing gene) in the neogenetic tissues at 6 weeks and 12 weeks after the operation, respectively (RT-PCR method, normalization of expression by quantitative GAPDH). FIG. 14 shows photographs of rabbit forelimb radial defect sites at week 6 (a) and week 12 (b) of the implanted group with 0BBG/PCL, 20BBG/PCL scaffolds and the non-implanted group.

As shown by the results in FIG. 14, it can be seen that the repair at the site of the radius defect of the forelimb of each group of rabbits occurred to different degrees with the increase of time. micro-CT reconstruction three-dimensional models are carried out on the front limb radius defect parts of all groups of rabbits, and as shown in the b-frame image in figure 13, three images in different directions show that under the condition of no support guide (namely a blank control group), only irregular and spine-shaped new bones appear at two ends of an operation gap, the defect is too large, self-repair cannot be carried out, and a continuous radius is formed. The negative control group and the 20BBG/PCL bracket group are implanted with the bracket, the mechanical properties of the bracket are enough to maintain the shape of the bracket in the bone repair process, and occupy necessary space for the stable growth of bone tissues in a defect area, but the 0BBG/PCL bracket is obviously improved relative to a blank control group, can provide a certain osteogenesis guiding effect, but the new bone grows slowly, and still has a larger defect part 12 weeks after the operation. And for the 20BBG/PCL scaffold, compared with a negative control group, the growth of new bones is obviously promoted, the newly formed bones are well fused with adjacent native bones, no bone dysplasia phenomenon occurs, and the defect part is basically repaired 12 weeks after the operation.

As shown in panels c and d of FIG. 13, it can be seen that NBV and BV/TV increase in each group with increasing implant time. 6 weeks after implantation, 20BBG/PCL scaffold groups NBV and BV/TV (46.14 mm)³27.12%) was significantly higher than the 0BBG/PCL scaffold group (28.62 mm)³16.88%) and blank control (27.88 mm)³16.44%). BV and BV/TV of 20BBG/PCL scaffold group 12 weeks after implantation (78.83 mm)³45.36%) was also significantly higher than the 0BBG/PCL scaffold group (50.96 mm)³29.32%) and untreated group (40.14 mm)³23.10%), therefore, the composite scaffold for introducing BBG into PCL provided by the invention has more excellent in vivo bone defect repair effect compared with a pure PCL scaffold.

As shown in the results from panel e to panel j in fig. 13, it can be seen that the expression level of all osteogenic-related genes in the 20BBG/PCL scaffold group is significantly increased compared to the blank control group and the 0BBG/PCL control group, which is consistent with the results that the BBG/PCL composite scaffold can promote osteogenic differentiation of hbmscs and osteoblast phenotype expression in vitro experiments.

In this experiment, H & E staining and M-T staining were also performed on bone defect sites after implantation of 0BBG/PCL and 20BBG/PCL scaffolds for 6 weeks and 12 weeks, and the results are shown in FIG. 15, FIG. 16, FIG. 17 and FIG. 18, in which FIG. 15 shows H & E staining histological images (a frames) and M-T staining histological images (b frames) of bone defect sites after implantation of 0BBG/PCL and 20BBG/PCL scaffolds for 12 weeks; FIG. 16 shows H & E stained histological images (a frames) and M-T stained histological images (b frames) of bone defect portions 6 weeks after implantation of 0BBG/PCL and 20BBG/PCL scaffolds; FIG. 17 shows a histological image of M-T staining of a bone defect 12 weeks after implantation of a 20BBG/PCL scaffold; FIG. 18 shows M-T stained histological images of bone defects 12 weeks after implantation of 0BBG/PCL, 20BBG/PCL scaffolds and non-implanted groups. In fig. 15 and 16, MB denotes an aged bone; NB represents new bone; BV represents a blood vessel; SM denotes the scaffold material.

From the results shown in FIGS. 15 and 16, it can be seen that the new bone areas of the two groups (0BBG/PCL and 20BBG/PCL) gradually increased with the passage of time. The high power microscope shows that no matter the pure PCL bracket or the 20BBG/PCL bracket, new bone tissues appear in the defect area, and a large amount of bone tissues similar to a bone trabecular structure appear in the repair area. From the results shown in FIG. 18, it is evident that there are more mature new bone tissue areas, less hole areas, and increased secretion of collagen on the 20BBG/PCL scaffold compared to the 0BBG/PCL scaffold. Furthermore, from the results shown in FIG. 17, it can be seen that a continuous marrow cavity is observed between the 20BBG/PCL scaffold and the defective end, which is more favorable for cell proliferation, migration and tissue growth. Therefore, the 20BBG/PCL scaffold has excellent bone forming ability and high bone regeneration efficiency compared with the pure PCL scaffold without BBG.

3.2 in vivo degradation and angiogenesis action of BBG/PCL composite porous bone scaffold

In the experiment, H & E staining is carried out on bone defect parts after 6 weeks and 12 weeks of implantation of 0BBG/PCL and 20BBG/PCL stents, the diameters of residual holes of the two groups of stents at 6 weeks and 12 weeks after operation are quantitatively analyzed, H & E staining is carried out on blood vessels on the stents after 6 weeks and 12 weeks of implantation of the 0BBG/PCL and 20BBG/PCL stents, and VEGF related gene analysis (qRT-PCR quantitative analysis) is carried out on angiogenesis of new tissues. The results are shown in FIG. 19, in which a pieces represent the size of the remaining pores of the stent reflected by H & E staining images of each group of defect sites at 12 weeks after the operation; panel b shows an H & E stained image of the 0BBG/PCL and 20BBG/PCL scaffold residual well regions (solid line box in panel a in FIG. 19); panel c shows the results of quantitative analysis of the residual pore area of two groups of scaffolds (0BBG/PCL and 20BBG/PCL) at 6 weeks and 12 weeks after operation; d pieces of H & E staining images (broken line frame in a of FIG. 19) of blood vessels representative of the bone defect sites 0BBG/PCL and 20BBG/PCL stents at 12 weeks after the operation; panel e shows the results of VEGF related gene analysis of neogenetic tissue angiogenesis on both the scaffolds at 6 and 12 weeks post-surgery. In fig. 19, the arrows indicate the vessels on the stent.

From the results shown in the panels a and b in fig. 19 and the result shown in the panel a in fig. 8, because the degradation performance of the composite scaffold can be improved by adding the BBG material into the PCL, new bone tissue can grow from the edge of the scaffold to the center along with the continuous degradation of the scaffold, and after more scaffolds are degraded, more spaces are left to form new bone tissue filling defect parts, so that the size of the scaffold hole is reduced, and the dynamic balance between tissue filling and scaffold degradation is further realized. And because the degradation rate of the pure PCL scaffold is low, the mismatch between the degradation of the scaffold and the growth of new bone tissues can be caused, and the repair process of CSBD can be blocked to a certain extent, so that the size of the scaffold hole is large. From the statistical results shown in panel c of FIG. 19, it can also be seen that although the residual pore areas of both sets of scaffolds (0BBG/PCL and 20BBG/PCL) decreased with the passage of time, under the same conditions, for example, after 6 weeks of implantation, the residual pore area of 20BBG/PCL scaffold (0.084 mm)²) Obviously less than the residual hole area of 0BBG/PCL bracket (0.783 mm)²Almost 9 times that of the 20BBG/PCL stent group); after 12 weeks of implantation, the residual pore area of the 20BBG/PCL stent decreased to 0.025mm²Still significantly less than 0.424mm of the residual pore area of the 0BBG/PCL stent²The result is consistent with the in vitro experiment result, namely BBG added into PCL can obviously improve the degradation rate of the composite scaffold, and realize dynamic balance between the filling of the regenerated tissue and the degradation of the scaffold.

From the results shown in panel d of FIG. 19, it can be seen that at 12 weeks after surgery, both 0BBG/PCL and 20BBG/PCL scaffolds showed abundant angiogenesis, but the 20BBG/PCL scaffold had more abundant angiogenesis relative to 0BBG/PCL, and these vessels can provide sufficient blood circulation and nutrients for the cells attached to the scaffold, thereby promoting proliferation and differentiation of the cells, and finally ensuring new bone formation. From the qRT-PCR quantitative analysis results of the angiogenic genes in the new tissues shown in panel e of FIG. 19, it can be seen that the angiogenic genes in the same group increase with time, and after the stent implantation for 6 weeks and 12 weeks, the 20BBG/PCL stent group is significantly higher than 0BBG/PCL and the blank control group, indicating that the addition of BBG to PCL is more beneficial to angiogenesis in vivo and shows better osteogenic repair effect.

In summary of the results of the examples, after introducing a suitable amount (e.g. 5 wt% to 20 wt%, more preferably 10 wt% to 20 wt%) of BBG into PCL and preparing the BBG/PCL composite porous bone scaffold by SLS method, comparable or more excellent structural performance can be obtained compared to pure PCL scaffold, mainly shown in that: (1) the pore structure is more complex and the porosity is obviously improved, thereby being more beneficial to the adhesion, migration, propagation and tissue growth of cells; (2) after the BBG material with the inherent characteristics of more brittle texture and poorer mechanical property is introduced into the PCL material, the mechanical property (compressive strength and structural toughness) of the composite bracket is not deteriorated, but the mechanical property of the composite bracket is equivalent to or obviously superior to that of a pure PCL bracket; (3) the nano-composite material has more excellent hydrophilic property, biocompatibility and mineralization capability, and is more beneficial to cell adhesion; (4) the protein adsorbent has excellent protein adsorption performance, and is more beneficial to the adhesion, migration and growth of cells; (5) the composite scaffold has improved biodegradability of the composite scaffold, slowed degradation rate of BBG and controllable release of various elements in BBG, after BBG materials are introduced into PCL polymer, the PCL polymer has the function of controlling BBG degradation, so that BBG degradation and release of various elements (such as B, Ca, K and the like) in BBG can be delayed, only weak alkaline environment adjacent to body fluid can be caused, the weak alkaline environment can not generate toxicity to adjacent cells and tissues, and the degradation of the composite scaffold can be promoted, so that the matching relation between the scaffold degradation rate and the bone tissue repair rate is realized; (6) the cell proliferation and the osteogenic differentiation of hBMSC are more facilitated; (7) has more excellent in vivo osteogenesis, improved in vivo biodegradation performance and angiogenesis promoting effect. Therefore, the powder composition for bone scaffolds comprising BBG powder and PCL powder provided by the invention can be used as an excellent raw material for preparing bone tissue engineering scaffolds, and the BBG/PCL composite porous bone scaffold prepared by SLS can be used as an excellent bone substitute graft material.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A BBG/PCL composite porous bone scaffold comprises a basic scaffold obtained by selectively sintering composite porous bone scaffold powder by laser on the basis of a bone three-dimensional model and a scaffold model obtained by Boolean cross operation of an array with a body-centered cubic lattice structure as a basic unit, wherein the basic scaffold comprises end faces at two ends and a circumferential face at the circumference;

2. The composite porous bone scaffold of claim 1, each of the four cylinders of the body centered cubic lattice structure having a diameter D of 0.20mm to 1.00mm and a length L of 2mm to 10 mm.

3. The composite porous bone scaffold of claim 1 or 2, said three-dimensional model of bone being selected from a natural bone scanning three-dimensional model or a three-dimensional model of bone designed by three-dimensional modeling software.

4. The composite porous bone scaffold according to claim 1 or 2, wherein the porosity of the pores and through-holes spread over the primary scaffold is between 50% and 90%, preferably between 57% and 70%; the aperture of the pore and the through hole is 0.40 mm-0.90 mm, preferably 0.40 mm-0.70 mm.

5. The composite porous bone scaffold according to claim 1 or 2 comprising 10-20 wt% of BBG powder having a particle size distribution of 12-90 μm and 80-90 wt% of PCL powder having a particle size distribution of 28-200 μm.

6. The composite porous bone scaffold according to claim 1 or 2, wherein the BBG powder is prepared by a conventional melt quenching method and comprises the following components in percentage by mass: b is₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15% and SrO: 1-8%; or

The BBG powder further comprises ZnO: 0.5 to 2 percent.

7. A preparation method of a BBG/PCL composite porous bone scaffold comprises the following steps:

8. The method of claim 7, wherein the predetermined parameters include: the preheating temperature is 40-50 ℃, the laser power is 2-15W, the scanning speed is 500-3000 mm/s, the scanning interval is 0.1-0.2 mm, and the thickness of the powder layer is 0.1-0.2 mm.

9. A powder composition for a bone scaffold comprising 5-20 wt% of BBG powder and 80-95 wt% of PCL powder, wherein the BBG powder has a particle size distribution of 12-90 μm and the PCL powder has a particle size distribution of 28-200 μm.

10. The powder composition for bone scaffolds according to claim 9, wherein the BBG powder is prepared by a conventional melt quenching method, and comprises the following components in percentage by mass: b is₂O₃：45～60％、MgO：2～8％、CaO：15～25％、Na₂O：3～10％、K₂O: 10-15% and SrO: 1-8%; or

The BBG powder further comprises ZnO: 0.5 to 2 percent.

The application of BBG in improving the adsorption performance of PCL on protein is to uniformly mix BBG powder and PCL powder and then prepare the BBG/PCL composite porous bone scaffold through selective laser sintering, wherein the BBG powder accounts for more than 5% of the BBG/PCL composite porous bone scaffold by mass.