CN107875441B - Calcium-lithium silicate system bioactive ceramic scaffold and preparation method and application thereof - Google Patents

Calcium-lithium silicate system bioactive ceramic scaffold and preparation method and application thereof Download PDF

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CN107875441B
CN107875441B CN201711078804.3A CN201711078804A CN107875441B CN 107875441 B CN107875441 B CN 107875441B CN 201711078804 A CN201711078804 A CN 201711078804A CN 107875441 B CN107875441 B CN 107875441B
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吴成铁
陈蕾
邓翠君
常江
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Pinghu High Tech Entrepreneurship Service Center Development Co ltd
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Shanghai Institute of Ceramics of CAS
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Abstract

The invention relates to a novel bioactive ceramic bracket of a calcium-lithium silicate system, a preparation method and application thereof. The calcium-lithium silicate system bioactive ceramic scaffold is a three-dimensional porous scaffold and has a chemical composition of LixCaySizOnWherein x +2y +4z =2 n. The calcium silicate lithium system bioactive ceramic scaffold has good degradability, mechanical property and bioactivity, has good double bioactivity, and can promote osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells and chondrocytes and in vivo bone-cartilage integrated repair.

Description

Calcium-lithium silicate system bioactive ceramic scaffold and preparation method and application thereof
Technical Field
The invention relates to osteochondral defect repair, in particular to a novel calcium-lithium silicate system bioactive ceramic bracket, a preparation method thereof and application thereof in osteochondral defect repair, belonging to the field of biological materials.
Background
Osteochondral injury caused by trauma, osteoarthritis and the like is a very common disease in clinic and is a well-known treatment problem in orthopedics and sports medicine. This is because articular cartilage is a non-vascular and non-neural connective tissue, and has a slow cell metabolism, and once it is damaged, its spontaneous repair and regeneration ability is poor [1 ]. Currently, the main clinical methods for treating cartilage injury include bone marrow stimulation, microarthrosis, autologous/allogeneic osteochondral transplantation, autologous/allogeneic chondrocyte transplantation and the like [2], but these methods have limitations [3 ]. More and more studies have found that damage to cartilage often causes joint dysfunction and involvement of subchondral bone [4,5 ]. Therefore, when repairing cartilage defects, great attention must be paid to the relationship between subchondral bone and articular cartilage, and an attempt must be made to repair cartilage-subchondral bone at the same time.
The tissue engineering strategy provides a feasible method for the integrated repair of cartilage and osteochondral [6, 7 ]. At present, many materials have been applied to the research of in vitro construction of cartilage and osteochondral tissues, such as calcium phosphate ceramics, bioglass, metals, high molecular polymers and the like for use in bone tissue materials, and hyaluronic acid, collagen, chitosan, gelatin, high molecular composite materials and the like for use in cartilage tissue materials. Different materials have merits in composition, microstructure, porosity, surface appearance and mechanical properties [8 ]. However, at present, the tissue engineering scaffold materials are difficult to synergistically promote the growth and regeneration of two tissues (bone and cartilage). The ideal bone and cartilage repairing material is not only suitable for the survival of chondrocytes and can promote the formation of calcified cartilage matrixes, but also the interface has to have the capability of integrating with bone tissues so as to improve the relevant mechanical properties. Therefore, whether a novel bioactive material can be designed or not can release various active ions in the material degradation process to construct an ion microenvironment which can promote differentiation of bones and cartilages, improve the interaction of cells and materials, and further effectively mobilize endogenous stem cells to participate in spontaneous repair of cartilage/bone injury, which may be a new way for solving the problems.
The previous study shows that: bioactive glasses and ceramics of the silicate system have an active activity of stimulating regeneration of hard tissues due to their specific chemical composition, and promote bone regeneration faster than phosphate bioceramics of conventional structure [9,10 ]. This is because Si ions released during the degradation of the material are confirmed to have the function of activating cells and stimulating the gene expression of cells [11 ]. On the other hand, lithium chloride, an earlier drug used for the treatment of bipolar disorder [12], has shown great potential in recent years for the research of arthritis treatment. Lithium chloride was found to inhibit cartilage degradation and cartilage mechanical loss in vitro by the action of the inflammatory factor IL-1 [13,14 ]. Thompson et al found that lithium chloride can also treat osteoarthritis by inhibiting the Hedgehog signaling pathway to avoid excessive chondrocyte hypertrophy, cartilage matrix catabolism [15 ].
The lithium calcium silicate system has four ternaryAnd (3) stable phase of the system: li2CaSiO4(LCS)，Li2Ca2Si2O7(LC2S2)，Li2Ca4Si4O13(LC4S4) And Li2Ca3Si6O16(LC3S6)[16]. The previous research mainly focuses on the exploration of the crystal structures of the four stable phases and the synthesis of Eu by the solid-phase reaction method2+Doping with Li2CaSiO4Or Li2Ca2Si2O7For the investigation of luminescent materials [17-19]. The prior solid-phase reaction method has the problems of complicated operation steps, high required reaction temperature and long time. In addition, calcium-lithium silicate system materials have not been studied in the field of biomaterials.
Prior art documents:
[1]Hunziker EB.Articular cartilage repair:basic science and clinicalprogress.A review of the current status and prospects.OsteoarthritisCartilage 2002；10:432-463.
[2]Minas T,Nehrer S.Current concepts in the treatment of articularcartilage defects.Orthopedics 1997；20:525-538.
[3]Dhinsa BS,Adesida AB.Current Clinical Therapies for CartilageRepair,their Limitation and the Role of Stem Cells.Current Stem CellResearch&Therapy 2012；7:143-148.
[4]Minas T,Gomoll AH,Rosenberger R,Royce RO,Bryant T.IncreasedFailure Rate of Autologous Chondrocyte Implantation After Previous TreatmentWith Marrow Stimulation Techniques.Am J Sports Med 2009；37:902-908.
[5]Rudert M.Histological evaluation of osteochondral defects:consideration of animal models with emphasis on the rabbit,experimentalsetup,follow-up and applied methods[J].Cells Tissues Organs,2002；171(4):229-240.
[6]Seo S-J,Mahapatra C,Singh RK,Knowles JC,Kim H-W.Strategies forosteochondral repair: Focus on scaffolds.Journal of tissue engineering 2014；5:1-14.
[7]Martin I,Miot S,Barbero A,Jakob M,Wendt D.Osteochondral tissueengineering.J Biomech 2007；40:750-765.
[8]Nooeaid P,Salih V,Beier JP,Boccaccini AR.Osteochondral tissueengineering:scaffolds,stem cells and applications.J Cell Mol Med 2012；16:2247-2270.
[9]Xu SF,Lin KL,Wang Z,Chang J,Wang L,Lu JX,et al.Reconstruction ofcalvarial defect of rabbits using porous calcium silicate bioactiveceramics.Biomaterials 2008；29:2588-2596.
[10]Hench LL.The story of Bioglass(R).Journal of Materials Science-Materials in Medicine 2006；17:967-978.
[11]Hench LL.Genetic design of bioactive glass.J Eur Ceram Soc 2009；29:1257-65.
[12]Williams RSB,Harwood AJ.Lithium therapy and signaltransduction.Trends Pharmacol Sci 2000；21:61-64.
[13]Minashima T,Zhang Y,Lee Y,Kirsch T.Lithium Protects AgainstCartilage Degradation in Osteoarthritis.Arthritis&Rheumatology 2014；66:1228-1236.
[14]Thompson CL,Yasmin H,Varone A,Wiles A,Poole CA,Knight MM.Lithiumchloride prevents interleukin-1induced cartilage degradation and loss ofmechanical properties.J Orthop Res 2015；33:1552-1559.
[15]Thompson CL,Wiles A,Poole CA,Knight MM.Lithium chloride modulateschondrocyte primary cilia and inhibits Hedgehog signaling.FASEB J 2016；30:716-726.
[16]West AR.Phase Equilibria in the System Li2O-CaO-SiO2J.Am.Ceram.Soc.1978；61:152–155. [17]Villafuertecastrejon ME,Dago A,PomesR.Crystal structure determination of Li2Ca4Si4O13.J Solid State Chem 1994；112:438-440.
[18]Liu J,Sun JY,Shi CS.A new luminescent material:Li2CaSiO4:Eu2+.Mater Lett 2006；60:2830-2833.
[19]Kahlenberg V,Brunello E,Hejny C,Krueger H,Schmidmair D,Tribus M,et al.Li2Ca2Si2O7: Structural,spectroscopic and computational studies on asorosilicate.J Solid State Chem 2015；225:155-167.。
disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a novel bioactive ceramic scaffold of a lithium calcium silicate system, a preparation method thereof and application thereof in osteochondral defect repair.
One aspect of the present disclosure provides a calcium-lithium silicate system bioactive ceramic scaffold, which is a three-dimensional porous scaffold having a chemical composition of LixCaySizOnWherein x +2y +4z is 2 n.
The calcium silicate lithium system bioactive ceramic scaffold has good degradability, mechanical property and bioactivity, has good double bioactivity, can promote osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells and chondrocytes and in vivo bone-cartilage integrated repair, is expected to solve the problems of insufficient cartilage regeneration capacity and the like in bone-cartilage integrated repair, and realizes good repair of large-area cartilage defects.
Preferably, the chemical composition of the calcium-lithium silicate system bioactive ceramic scaffold is Li2Ca2Si2O7、Li2Ca4Si4O13、Li2CaSiO4Or Li2Ca3Si6O16。
In another aspect, the present application provides a method for preparing a bioactive ceramic scaffold of a lithium calcium silicate system, comprising the following steps:
(1) synthesizing calcium silicate lithium system bioactive ceramic powder by a sol-gel method;
(2) preparing a bioactive ceramic support ceramic biscuit by using calcium silicate lithium system bioactive ceramic powder as a raw material through a three-dimensional printing technology; and
(3) and sintering the obtained ceramic biscuit of the bioactive ceramic support to obtain the bioactive ceramic support of the calcium-lithium silicate system.
The invention synthesizes the calcium silicate lithium system bioactive ceramic powder by adopting a sol-gel method for the first time, has the advantages of simple process and easy control of conditions, and further prepares the calcium silicate lithium system bioactive ceramic bracket with controllable macroporous appearance by utilizing a three-dimensional printing technology.
Preferably, step (1) comprises the steps of:
(A) hydrolyzing a silicon source;
(B) adding a lithium source and a calcium source into the hydrolyzed silicon source according to the stoichiometric ratio, and stirring to obtain a clear solution;
(C) aging the obtained clear solution at 40-80 ℃ for 24-48 hours, and drying at 120-150 ℃ for 48-72 hours to obtain dry gel;
(D) and (3) ball-milling and sieving the obtained dry gel, and calcining for 2-6 hours at 800-1050 ℃ to obtain the calcium silicate lithium system bioactive ceramic powder.
Preferably, in the step (a), the silicon source is tetraethoxysilane.
Preferably, the silicon source, water and 1-3 mol.L-1According to a molar ratio of 1: (6-9): (0.06-0.1) and stirring for 0.5-2 hours to completely hydrolyze the silicon source.
Preferably, in the step (B), the lithium source is a lithium salt selected from at least one of lithium nitrate, lithium chloride, and the like; the calcium source is lithium salt, and is selected from at least one of calcium nitrate, calcium chloride and the like; the stirring time is 4-6 hours.
Preferably, the particle size of the calcium-lithium silicate system bioactive ceramic powder is less than or equal to 74 μm.
Preferably, in the step (2), calcium-lithium silicate system bioactive ceramic powder is uniformly mixed with a binder, and a structural model of the bioactive ceramic support ceramic biscuit is constructed by computer-aided design software to perform three-dimensional printing.
Preferably, the binder is sodium alginate and pluronic F127 aqueous solution, the calcium silicate lithium system bioactive ceramic powder: sodium alginate powder: the mass ratio of the pluronic F127 aqueous solution is 1: (0.05-0.10): (0.6-1.2).
Preferably, the particle size of the sodium alginate powder is less than or equal to 74 μm, and the concentration range of the pluronic F127 aqueous solution is 10-30 wt%.
Preferably, in the step (3), the sintering temperature is 850-1100 ℃, and the sintering time is 2-6 hours.
The application also provides application of the calcium-lithium silicate system bioactive ceramic scaffold in preparation of a cartilage-subchondral bone defect integrated repair implant material.
The novel bioactive ceramic scaffold of the calcium silicate lithium system provided by the invention has good degradability, mechanical properties and bioactivity, and animal experiments prove that the novel bioactive ceramic scaffold of the calcium silicate lithium system has a bidirectional biological function of cartilage-subchondral bone integrated repair. Therefore, the novel bioactive ceramic scaffold of the calcium silicate lithium system is a novel bifunctional biological ceramic scaffold, and has bright application prospect in the field of cartilage-subchondral bone defect integrated repair.
Drawings
FIG. 1.Li2Ca4Si4O13XRD pattern of the bioactive ceramic powder. It is shown that pure phase Li can be successfully prepared by sol-gel method2Ca4Si4O13Bioactive ceramic powder;
FIG. 2.Li2Ca4Si4O13And (3) analyzing the morphology and XRD of the bioactive ceramic bracket. (A)3D printing Li2Ca4Si4O13Biological activity ceramic support digital photo, (B)3D printing Li2Ca4Si4O13Optical photo of bioactive ceramic scaffold, (C, D)3D printing Li2Ca4Si4O13SEM photograph of bioactive ceramic scaffold. (E-H)3D printing Li2Ca4Si4O13SEM photographs and XRD analysis of bioactive ceramic scaffolds soaked in simulated body fluids for 14 days. Shown therein, 3D printing of prepared Li2Ca4Si4O13Bioactive ceramic scaffoldHas controllable aperture with high communication. Li2Ca4Si4O13The bioactive ceramic scaffold has the capacity of inducing the mineralization of the bone-like hydroxyapatite;
FIG. 3 (A) Li of different pore diameters2Ca4Si4O13Digital photograph of bioactive ceramic support, (B) Li with different pore diameters2Ca4Si4O13Micro-CT picture of bioactive ceramic support, (C) Li2Ca4Si4O13Porosity and mechanical strength of bioactive ceramic scaffold, (D) Li2Ca4Si4O13Mechanical curve of bioactive ceramic scaffolds. The figure shows that Li can be effectively regulated and controlled by regulating and controlling the pore size (170-400 mu m) of the bracket2Ca4Si4O13The porosity (37-61%) and the mechanical strength (15-40 MPa) of the bioactive ceramic scaffold. Li2Ca4Si4O13The bioactive ceramic bracket has the characteristics of high connectivity and high strength;
FIG. 4 (A) Li2Ca4Si4O13The pH value of the bioactive ceramic bracket soaked in Tris-HCl changes, (B) Li2Ca4Si4O13The quality change of the bioactive ceramic bracket soaked in Tris-HCl at different time points, (C-F) Li2Ca4Si4O13And (3) soaking the bioactive ceramic support in Tris-HCl for different time points according to the change curve of the accumulative release amount of Ca, Si, Li and P elements. Li2Ca4Si4O13The bioactive ceramic bracket is soaked in Tris-HCl, and Li is added along with the increase of the soaking time2Ca4Si4O13The bioactive ceramic bracket keeps continuous ion release and mass loss, and has good degradability;
FIG. 5 shows the gene expression profiles of chondrocytes and mesenchymal stem cells (rBMSC), (A-D) the expression of the cartilage-specific genes COL II, Aggrecan, N-cadh, SOX9, and (E-I) the expression of the osteogenic differentiation-critical genes RUNX2, OCN, OPN, BMP2, COL I. In the figure, CTR represents a blank control, left column in a double columnThe child represents β -TCP, the right hand column represents Li2Ca4Si4O13A bioactive ceramic. Shown in the figure, Li2Ca4Si4O13The bioactive ceramic leaching liquor can obviously promote the expression of cartilage specific genes and osteogenic differentiation key genes in a certain concentration range;
FIG. 6 is a graph showing the characterization of alkaline phosphatase (ALP) activity and mineralization, (A) quantification of ALP, (B) ALP staining, (C) alizarin red quantification, (D) alizarin red staining, wherein the left column in the two-column in the graph shows β -TCP, and the right column shows Li2Ca4Si4O13A bioactive ceramic. Shown in the figure, Li2Ca4Si4O13The bioactive ceramic leaching liquor can remarkably improve ALP activity of bone marrow mesenchymal stem cells and effectively promote formation of calcium nodules;
FIG. 7 bone marrow mesenchymal stem cells and chondrocytes in Li2Ca4Si4O13The bioactive ceramic bracket is adhered. (A) Bone marrow mesenchymal stem cells are adhered to the scaffold, and (B) chondrocytes are adhered to the scaffold. Shown in the figure, Li2Ca4Si4O13The bioactive ceramic scaffold remarkably promotes the adhesion of chondrocytes and mesenchymal stem cells;
FIG. 8.Li2Ca4Si4O13The bioactive ceramic scaffold is implanted into rabbit body with repairing effect for 8 weeks and 12 weeks, A1-C4Material implantation for 8 weeks (A)1-4) Blank control group, (B)1-4) TCP group, (C)1-4)Li2Ca4Si4O13Group (d); d1-F4Material implantation for 12 weeks, (D)1-4) Blank control group, (E)1-4) TCP group, (F)1-4)Li2Ca4Si4O13And (4) grouping. Shown in the figure, Li2Ca4Si4O13Compared with a blank control group and a TCP group, the bioactive ceramic scaffold group remarkably promotes cartilage-subchondral bone repair;
FIG. 9.Li2Ca2Si2O7XRD pattern of bioactive ceramic powderSpectra. It is shown that pure phase Li can be successfully prepared by sol-gel method2Ca2Si2O7Bioactive ceramic powder;
FIG. 10.Li2Ca2Si2O7And (3) analyzing the morphology and XRD of the bioactive ceramic bracket. (A-B)3D printing Li2Ca2Si2O7Bioactive ceramic support digital photo, (C)3D printing Li2Ca2Si2O7Optical photo of bioactive ceramic scaffold, (D, E)3D printing Li2Ca2Si2O7SEM photograph of bioactive ceramic scaffold. (F, G)3D printing Li2Ca2Si2O7SEM photographs of bioactive ceramic scaffolds after 14 days immersion in simulated body fluids. (H)3D printing Li2Ca2Si2O7XRD analysis before and after the bioactive ceramic scaffold is soaked in simulated body fluid for 14 days. Shown therein, 3D printing of prepared Li2Ca2Si2O7The bioactive ceramic scaffold has highly interconnected controllable pore sizes. Li2Ca2Si2O7The bioactive ceramic scaffold has the capacity of inducing the mineralization of the bone-like hydroxyapatite;
FIG. 11.Li2Ca2Si2O7The bioactive ceramic scaffold is implanted into rabbit body for 12 weeks to achieve repairing effect, A1-C4Micro-CT results 12 weeks after material implantation, (A)1-4) Blank control group, (B)1-4) TCP group, (C)1-4)Li2Ca2Si2O7And (4) grouping. Shown in the figure, Li2Ca2Si2O7The bioactive ceramic scaffold group significantly promoted cartilage-subchondral bone repair compared to the placebo and TCP groups.
Detailed Description
The present invention is further described below in conjunction with the following embodiments and the accompanying drawings, it being understood that the drawings and the following embodiments are illustrative of the invention only and are not limiting.
The inventors have considered Si alone4+、Li+Ion is respectively coupled to the fine particlesThe osteogenic differentiation of cells and the maintenance of cartilage phenotype, the lithium silicate ceramic scaffold was considered for the study of bone-cartilage integrated repair.
One embodiment of the invention provides a calcium-lithium silicate system bioactive ceramic scaffold which is a three-dimensional porous scaffold and has the chemical composition of LixCaySizOnWherein x +2y +4z is 2 n. For example, the chemical composition may be Li2Ca2Si2O7，Li2Ca4Si4O13，Li2CaSiO4Or Li2Ca3Si6O16。
The calcium-lithium silicate system bioactive ceramic bracket has high connectivity. The pore diameter can be 170-400 μm, and the porosity can be 37-61%. The porosity and the mechanical strength can be regulated and controlled by regulating the pore size. The calcium-lithium silicate system bioactive ceramic scaffold has good mechanical properties, for example, the compressive strength of the scaffold can be 15-40 MPa.
In one embodiment of the invention, the calcium silicate lithium system bioactive ceramic powder is synthesized by a sol-gel method, and the calcium silicate lithium system bioactive ceramic scaffold with controllable macroporous morphology is further prepared by a three-dimensional printing technology.
The preparation of the bioactive ceramic scaffold of the lithium calcium silicate system is specifically described below.
Firstly, synthesizing calcium-lithium silicate system bioactive ceramic powder, the chemical composition of which is LixCaySizOn。
The synthesis raw materials comprise a silicon source, a lithium source and a calcium source. The silicon source may be a silicate, for example selected from ethyl orthosilicate. The lithium source may be a lithium salt, preferably a water-soluble lithium salt, for example selected from lithium nitrate, lithium chloride and the like. The calcium source may be a calcium salt, preferably a water soluble calcium salt, for example selected from calcium nitrate, calcium chloride and the like.
The silicon source is hydrolyzed, preferably completely hydrolyzed. In one example, the silicon source is mixed with an acid and stirred to hydrolyze the silicon source. The acid may be nitric acid, hydrochloric acid, etc. For example, a silicon source, water, 1 to 3 mol.L-1Nitric acid ofThe molar ratio is 1: (6-9): (0.06-0.1) and stirring for 0.5-2 hours to completely hydrolyze the silicon source.
Adding a lithium source and a calcium source into the hydrolyzed silicon source according to a stoichiometric ratio. After addition, stirring may be carried out for 4-6 hours until a clear solution is obtained.
The resulting clear solution is heat treated to obtain a xerogel. Before the heat treatment, an aging treatment can be carried out to ensure that the clear solution forms a uniform wet gel network. In an example, the aging is carried out at 40-80 ℃ for 24-48 hours, and the heat treatment is carried out at 120-150 ℃ for 48-72 hours.
And ball-milling and sieving the xerogel to obtain a precursor. The ball milling time can be 4-8 h. The sieving can be 100-300 mesh sieving.
And calcining the obtained precursor to obtain the calcium-lithium silicate system bioactive ceramic powder. The calcination temperature can be 800-1050 ℃. The calcination time may be 2 to 6 hours. And after calcination, the material can be naturally cooled. The obtained calcium-lithium silicate-based bioactive ceramic powder is known to be a pure phase by X-ray diffraction (XRD) (see, for example, fig. 1 and 9).
And carrying out three-dimensional printing on the obtained calcium-lithium silicate system bioactive ceramic powder to obtain a bioactive ceramic support ceramic biscuit. In one example, calcium-lithium silicate system bioactive ceramic powder is uniformly mixed with a binder, and a printing program is designed by software to perform three-dimensional printing. The binder can be sodium alginate and pluronic F127 aqueous solution. The mass ratio of the calcium silicate lithium system bioactive ceramic powder, the sodium alginate and the pluronic F127 aqueous solution can be 1: (0.05-0.10): (0.6-1.2). The particle size of the calcium-lithium silicate system bioactive ceramic powder can be less than or equal to 74 mu m. The powder with the granularity can easily pass through the printing needle head, so that the needle head is not easy to block. The particle size of sodium alginate may be less than or equal to 74 μm. The sodium alginate with the granularity range is easy to be uniformly mixed with the ceramic powder, so that the slurry is better in uniformity. The concentration range of the F127 aqueous solution can be 10-30 wt%.
Sintering the obtained ceramic biscuit of the bioactive ceramic support to obtain the calcium silicate lithium system bioactive ceramic support. The sintering temperature can be 850-1100 ℃. The sintering time can be 2-6 hours.
The means such as Scanning Electron Microscope (SEM), Micro-CT and the like show that the bioactive ceramic scaffold of the lithium calcium silicate system has the characteristics of highly communicated controllable pore diameter, high connectivity and high strength (for example, see figures 2, 3 and 10). For example, the porosity (37-61%) and the mechanical strength (15-40 MPa) of the bioactive ceramic scaffold of the calcium-lithium silicate system can be effectively controlled by controlling the pore size (170-400 μm) of the bioactive ceramic scaffold of the calcium-lithium silicate system. It is known that the calcium-silicate-lithium system bioactive ceramic scaffold has the ability to induce mineralization of bone-like hydroxyapatite (see, for example, (E-H) in fig. 2, and (F-H) in fig. 10). In addition, the bioactive ceramic scaffold of the lithium calcium silicate system of the present invention can maintain sustained ion release and mass loss, and has good degradability (see, for example, fig. 4).
Research on physical and chemical properties of bioactive ceramic bracket of calcium-lithium silicate system
The calcium-lithium silicate system bioactive ceramic scaffold (6 parallel samples) was tested for compressive strength. The results show that the bioactive ceramic scaffold of the lithium calcium silicate system has good mechanical properties (for example, see fig. 3).
Soaking calcium silicate lithium system bioactive ceramic scaffold (3 parallel samples) in Tris-HCl solution (V)Tris-HCl/Mscaffold200ml/g), soaking in a thermostat at 37 ℃ for 35 days, and observing the degradation of the stent. The results indicate that the calcium-lithium silicate system bioactive ceramic scaffold has good degradation properties (see, e.g., fig. 4).
Soaking calcium-lithium silicate system bioactive ceramic scaffold (3 parallel samples) in SBF solution (V)SBF/Mscaffold200ml/g), soaking in a 37 ℃ incubator for 14 days, and observing the mineralization of the stent. The results show that the calcium-lithium silicate system bioactive ceramic scaffold has good biomineralization performance (for example, see (E-H) in FIG. 2 and (F-H) in FIG. 10).
Bidirectional biological performance research of calcium silicate lithium system bioactive ceramic
Calcium-lithium silicate system bioactive ceramic in-vitro osteogenesis induction promoting property.
The rabbit bone marrow mesenchymal stem cells are cultured by using different calcium silicate lithium system bioactive ceramic leaching solutions, the adhesion and proliferation of materials to the bone marrow mesenchymal stem cells and the expression of genes and proteins related to osteogenesis are researched, the early osteogenesis performance is quantitatively and qualitatively analyzed by using an alkaline phosphatase kit, and the mineralization at the final stage of osteogenesis is evaluated by using an alizarin red staining method. Research results show that the ion products released by the bioactive ceramic of the lithium calcium silicate system remarkably promote the adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (for example, see (A-D) in figure 5, and (A) in figures 6 and 7), and prove that the material has osteogenic promotion.
Promotion of chondrogenic differentiation in vitro of bioactive ceramic bodies of the calcium-lithium silicate system
Different bioactive ceramic leaching liquids of calcium silicate lithium systems are used for culturing rabbit chondrocytes, and the adhesion and proliferation of the materials to the chondrocytes and the expression of genes and proteins specific to the cartilage are researched. Research results show that ion products released by the bioactive ceramic of the lithium calcium silicate system remarkably promote the adhesion and proliferation of chondrocytes and the expression of specific genes and proteins thereof (for example, see (E-H) in figure 5 and (B) in figure 7), and prove that the material has the biological activities of forming cartilage and protecting chondrocytes.
Effect of calcium silicate lithium system bioactive ceramic scaffold on cartilage-subchondral bone integrated repair
The invention proves that the calcium-lithium silicate system bioactive ceramic scaffold has the cartilage-subchondral bone integrated repair efficiency for the first time. Micro-CT results show that the new cartilage and subchondral bone formed at the defect position by implanting the calcium silicate lithium system bioactive ceramic scaffold are remarkably increased compared with a blank group control and a pure TCP group. Histochemical staining analysis shows that after the material is implanted for 8 weeks, a small amount of mixture of new bone and fibrous tissue is formed around the bone defect of the blank control group and the pure TCP group, and a certain amount of new cartilage and subchondral bone is formed around the bone defect and in the center of the bracket of the calcium-lithium silicate system bioactive ceramic bracket group. After 12 weeks of implantation, the new cartilage and subchondral bone of the calcium-lithium-silicate bioactive ceramic scaffold group completely covered the defect, while the blank control group and the pure TCP group had defects, and the resultant had a certain amount of fibrous tissue (see, for example, fig. 8 and 11). The results show that the calcium-lithium silicate system bioactive ceramic scaffold has excellent in-vivo cartilage-subchondral bone integrated repair performance.
The calcium-lithium silicate system bioactive ceramic scaffold can be used as a novel bioactive implant material.
The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Comparative example 1
Tricalcium phosphate (β -TCP) scaffolds were prepared using three-dimensional printing techniques as controls: the method is characterized in that commercially available beta-TCP powder is used as a raw material, and the beta-TCP powder, sodium alginate and F-127 are mixed according to a mass ratio of 1: 0.06: 0.8, printing and molding, and calcining at 1100 ℃ for 3 hours to obtain the beta-TCP support.
Example 1
Preparation of Li by sol-gel method2Ca4Si4O13Bioactive ceramic scaffold
(1) At room temperature, ethyl orthosilicate, deionized water and 2 mol.L-1According to a molar ratio of 1: 8: 0.08 mixing and stirring for 0.5h to completely hydrolyze. According to the stoichiometric ratio Li: ca: si is 2: 4: and 4, sequentially adding lithium nitrate and calcium nitrate, and stirring for 5 hours to obtain a clear solution. Then sealing the prepared clear solution in an oven at 60 ℃ for aging for 24 hours and drying at 120 ℃ for 72 hours to obtain xerogel; ball-milling the xerogel for 6h, sieving, calcining at 940 ℃ for 3 h, and naturally cooling to obtain Li2Ca4Si4O13The XRD pattern of the bioactive ceramic powder is shown inFIG. 1, which is known to be a pure phase;
(2) the obtained Li2Ca4Si4O13Bioactive ceramic powder: sodium alginate: f127 is mixed according to the mass ratio of 1: 0.06: 0.8, and three-dimensional printing is carried out by utilizing a software design printing program, and the printing support is sintered for 2 hours at 960 ℃ to obtain Li2Ca4Si4O13A bioactive ceramic scaffold;
(3) for Li2Ca4Si4O13The bioactive ceramic scaffold is used for researching phase composition, macroporous structure on the surface of the scaffold, microscopic morphology, mechanical property, degradation property, osteogenesis, chondrogenesis property and in vivo repair effect, the research method is as described above, and the results are shown in figures 2-8.
Example 2
Preparation of Li by sol-gel method2Ca2Si2O7Bioactive ceramic scaffold
(1) At room temperature, ethyl orthosilicate, deionized water and 3 mol.L-1According to a molar ratio of 1: 9: 0.06 mixing and stirring for 1h to completely hydrolyze. According to the stoichiometric ratio Li: ca: si is 2: 2: and 2, sequentially adding lithium nitrate and calcium nitrate, and stirring for 5 hours to obtain a clear solution. Then sealing the prepared clear solution in a 50 ℃ oven for aging for 36 hours and drying at 120 ℃ for 60 hours to obtain xerogel; ball milling the xerogel for 5h, sieving, calcining at 850 ℃ for 2 h, and naturally cooling to obtain Li2Ca2Si2O7The XRD spectrum of the bioactive ceramic powder is shown in figure 9, and the bioactive ceramic powder is a pure phase;
(2) the obtained Li2Ca2Si2O7Bioactive ceramic powder: sodium alginate: f127 is mixed according to the mass ratio of 1: 0.08: 1.0, and three-dimensionally printing by using a software-designed printing program, and sintering the printing support at 870 ℃ for 3 hours to obtain Li2Ca2Si2O7A bioactive ceramic scaffold;
(3) for Li2Ca2Si2O7Performing morphology and XRD (X-ray diffraction) division on the bioactive ceramic bracketThe results are shown in FIG. 10. For Li2Ca2Si2O7The bioactive ceramic scaffold was studied for osteogenic, chondrogenic and in vivo repair effects, as described above, and the results are shown in fig. 11.

Claims (9)

1. The calcium silicate lithium system bioactive ceramic scaffold is characterized by being a three-dimensional porous scaffold and having a chemical composition of LixCaySizOnWherein x +2y +4z =2 n; the calcium silicate lithium system bioactive ceramic support has a highly-communicated controllable pore size which is 170-400 mu m; the porosity of the calcium silicate lithium system bioactive ceramic support is 37-61%, and the mechanical strength is 15-40 MPa.
2. The calcium-lithium silicate system bioactive ceramic scaffold as claimed in claim 1, wherein the chemical composition of said calcium-lithium silicate system bioactive ceramic scaffold is Li2Ca2Si2O7、Li2Ca4Si4O13、Li2CaSiO4Or Li2Ca3Si6O16。
3. A method for preparing a calcium-lithium silicate system bioactive ceramic scaffold according to claim 1 or 2, comprising the steps of:
(1) synthesizing calcium silicate lithium system bioactive ceramic powder by a sol-gel method;
(2) preparing a bioactive ceramic support ceramic biscuit by using calcium silicate lithium system bioactive ceramic powder as a raw material through a three-dimensional printing technology; and
(3) sintering the obtained ceramic biscuit of the bioactive ceramic support to obtain the bioactive ceramic support of the calcium-lithium silicate system;
the step (1) comprises the following steps:
(A) hydrolyzing a silicon source: mixing silicon source, water, 1-3 mol.L-1According to a molar ratio of 1: (6-9): (0.06-0.1), and stirring for 0.5-2 hours to completely hydrolyze the silicon source;
(B) adding a lithium source and a calcium source into the hydrolyzed silicon source according to a stoichiometric ratio, and stirring to obtain a clear solution;
(C) aging the obtained clear solution at 40-80 ℃ for 24-48 hours, and drying at 120-150 ℃ for 48-72 hours to obtain dry gel;
(D) and (3) ball-milling and sieving the obtained dry gel, and calcining for 2-6 hours at 800-1050 ℃ to obtain the calcium silicate lithium system bioactive ceramic powder.
4. The method according to claim 3, wherein in the step (A), the silicon source is tetraethoxysilane.
5. The method according to claim 3, wherein in the step (B), the lithium source is a lithium salt selected from at least one of lithium nitrate and lithium chloride; the calcium source is calcium salt selected from at least one of calcium nitrate and calcium chloride; the stirring time is 4-6 hours.
6. The method according to claim 3, wherein the particle size of the calcium-lithium silicate-based bioactive ceramic powder is less than or equal to 74 μm.
7. The preparation method according to claim 3, wherein in the step (2), the calcium-lithium silicate system bioactive ceramic powder is uniformly mixed with the binder, and a structural model of the bioactive ceramic support ceramic biscuit is constructed by using computer aided design software to perform three-dimensional printing;
the adhesive is sodium alginate and pluronic F127 water solution, the calcium silicate lithium system bioactive ceramic powder is as follows: sodium alginate: the mass ratio of the pluronic F127 aqueous solution is 1: (0.05-0.10): (0.6 to 1.2);
the particle size of the sodium alginate is less than or equal to 74 mu m, and the concentration range of the pluronic F127 aqueous solution is 10-30 wt%.
8. The method according to claim 3, wherein in the step (3), the sintering temperature is 850 to 1100 ℃ and the sintering time is 2 to 6 hours.
9. Use of the calcium-lithium silicate system bioactive ceramic scaffold as claimed in claim 1 or 2 in the preparation of cartilage-subchondral bone defect integrated repair implant material.
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