Responsive orthopedic implant material and method of making and using same

Abstract

The application discloses a responsive orthopedic implant material and a preparation method and application thereof. The responsive orthopedic implant material comprises a first substrate layer comprising an exogenous stimulus thermal effect material; a convex array on one side of the first substrate layer, which is made of raw materials containing a second shape memory material, and the convex array is configured to be oriented along a long range and can be transformed into a columnar array under the thermal effect of the first substrate layer. The responsive orthopedic implant material provided by the application has a long-range oriented microstructure with a cell anti-inflammatory effect in a conventional state, but under external stimulation, the first substrate layer generates a thermal effect, so that the shape memory effect of the convex array acts to be transformed into a columnar array with a bone formation promoting effect, so that through the regulation of external stimulation, the responsive orthopedic implant material can match the needs of different stages in the bone repair process, and achieve the effects of completing bone repair and excellent bone integration.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a responsive orthopedic implant material and a preparation method and application thereof. BACKGROUND

[0002] Bone is an important structural support system of the human body, and bone defect refers to the condition that the structural integrity of the bone is destroyed due to congenital or acquired factors. Bone defects with a size greater than 2-2.5 times the diameter of the bone are difficult to heal by themselves, and autologous transplantation and allogeneic transplantation commonly used in clinical practice are severely limited in their treatment range and clinical effect due to donor shortage and postoperative infection. Constructing microstructures on the surface of the material to improve the osteoinductive activity of the orthopedic implant material and regulate the immune response after implantation of the material is a common means at present, and certain effects have been achieved, but it is still necessary to explore more effective means to achieve complete bone repair and excellent bone integration.

[0003] With the continuous deepening of bone biology research, it has been revealed that the repair of large-sized bone defects is a complex, multi-cellular and time-sequential process. The bone repair process mainly includes an early hematoma and inflammation reaction period, a middle bone callus formation period and a late bone plate plasticity period. The hematoma and inflammation reaction are induced immediately after the implantation of the material, and the migration, proliferation and functional phenotypes of the inflammatory cells such as platelets, macrophages and neutrophils involved therein are the basis for subsequent angiogenesis and new bone formation. In the middle bone callus formation period and the late bone plate plasticity period, the vascular endothelial growth factor secreted by the early inflammatory cells is conducive to the migration and aggregation of a large number of endothelial cells at the defect repair site, and the bone morphogenetic protein is conducive to the aggregation, proliferation and osteogenic differentiation of a large number of bone marrow mesenchymal stem cells at the defect repair site, thereby completing bone repair and achieving bone integration.

[0004] The current bone repair material is more concerned about part of the process in the bone repair process, and on the other hand, the material cannot be programmed to match the different processes of bone repair, resulting in the inability to achieve complete bone repair and excellent bone integration. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a responsive orthopedic implant material and a preparation method and application thereof, which can cope with most of the processes in the bone repair process and achieve complete bone repair and excellent bone integration.

[0006] In a first aspect of the present application, a responsive orthopedic implant material is provided, which comprises:

[0007] a first substrate layer comprising an exogenous stimulus thermal effect material;

[0008] a protrusion array on one side of the first substrate layer is made of a raw material containing a second shape memory material, the protrusion array is configured to be oriented along a long range and is capable of being transformed into a columnar array under the thermal effect of the first substrate layer.

[0009] The beneficial effects of the embodiments of the present application are:

[0010] The responsive orthopedic implant material provided by the present application realizes the timely transformation of the surface microstructure of the bone repair material based on the exogenous stimulus thermal effect material and the shape memory material. Specifically, in the conventional state, the protrusion array is a long-range oriented microstructure with certain anti-inflammatory effect, but under the exogenous stimulus, the first substrate layer generates a thermal effect, so that the shape memory effect of the protrusion array acts to be transformed into a columnar array with a bone formation promoting effect, so that through the regulation of the exogenous stimulus, the responsive orthopedic implant material can match the needs at different stages of the bone repair process, and achieve the effects of completing bone repair and excellent bone integration.

[0011] Among them, the exogenous stimulus thermal effect material refers to a material that can change its temperature under certain external stimulus. Common exogenous stimuli that can produce thermal effects include light, electricity, magnetism, etc. Thus, in some embodiments, the exogenous stimulus thermal effect material includes at least one of a photothermal effect material, a magnetic thermal effect material, and an electric thermal effect material. In some specific embodiments, the photothermal effect material includes at least one of inorganic photothermal effect materials, organic photothermal materials, etc. The inorganic photothermal effect material includes at least one of nano-metal (such as gold, silver, copper, platinum, palladium, germanium), carbon nanotubes, black phosphorus, graphene, etc. The organic photothermal effect material includes at least one of polypyrrole, polyaniline, polyethylene dioxythiophene, polystyrene sulfonate, indocyanine green, porphyrin. In some specific embodiments, the electric thermal effect material includes at least one of carbon black, carbon nanotubes, and graphene. In some specific embodiments, the magnetic thermal effect material includes at least one of Fe2O3, Fe3O4, FeCo, NiFe, CoFeO, NiFeO, MnFeO.

[0012] In some embodiments, the first substrate layer comprises a matrix material and an exogenous stimulus-thermal effect material loaded in the matrix material. In some specific embodiments, the mass percentage of the exogenous stimulus-thermal effect material to the matrix material is no more than 5%, for example, no more than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%. In some specific embodiments, the matrix material is a polymer material. In some specific embodiments, the matrix material is a first shape memory material having a shape memory effect, and the mass percentage of the exogenous stimulus-thermal effect material to the first shape memory material is no more than 5%, for example, no more than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%. In some specific embodiments, the first shape memory material comprises at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, polyhydroxybutyrate.

[0013] In some embodiments, the first substrate layer comprises a matrix material and an exogenous stimulus-thermal effect material loaded in the matrix material. In some specific embodiments, the mass percentage of the exogenous stimulus-thermal effect material to the matrix material is no more than 5%, for example, no more than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%. In some specific embodiments, the matrix material is a polymer material. In some specific embodiments, the matrix material is a first shape memory material having a shape memory effect, and the mass percentage of the exogenous stimulus-thermal effect material to the first shape memory material is no more than 5%, for example, no more than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%. In some specific embodiments, the first shape memory material comprises at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, polyhydroxybutyrate.

[0014] In some embodiments, the first substrate layer comprises a matrix material and an exogenous stimulus-thermal effect material loaded in the matrix material. In some specific embodiments, the mass percentage of the exogenous stimulus-thermal effect material to the matrix material is no more than 5%, for example, no more than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%. In some specific embodiments, the matrix material is a polymer material. In some specific embodiments, the matrix material is a first shape memory material having a shape memory effect, and the mass percentage of the exogenous stimulus-thermal effect material to the first shape memory material is no more than 5%, for example, no more than 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%. In some specific embodiments, the first shape memory material comprises at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, polyhydroxybutyrate.

[0015] In addition, the long-range oriented protrusion array is converted into a columnar array under the thermal effect of the first substrate layer. Specifically, the shape memory material has an initial shape of a columnar array, the condition of the columnar array is changed under certain conditions, the initial shape is changed, the columnar array is changed into a long-range orientation, and the state of the long-range orientation is fixed and maintained. Under the stimulation of the thermal effect, the long-range oriented protrusion array is restored to the columnar array.

[0016] In the columnar array, the protrusions arranged in an array are in the form of a column perpendicular to the set plane, which can be a cylinder, a prism, but is not limited thereto, and also includes a circular truncated cone, a prism truncated cone, or other columnar or approximately columnar structures known in the art. Each protrusion in the array can be the same or different columnar.

[0017] In some embodiments, the protrusion array can be directly or indirectly compounded on one side of the first substrate layer in any one or several physical or chemical ways known in the art, such as photolithography, 3D printing, etc. In some specific embodiments, the protrusion array is compounded on one side of the first substrate layer through a second substrate layer, wherein the second substrate layer comprises a substrate and a protrusion array, the first substrate layer is located on one side of the substrate, and the protrusion array is located on the other side of the substrate away from the first substrate layer.

[0018] In some embodiments, the substrate is made of raw materials containing a polymer material. In some specific embodiments, the substrate is made of raw materials containing a third shape memory material. In some specific embodiments, the third shape memory material includes at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, and polyhydroxybutyrate.

[0019] In some embodiments, the second substrate layer includes osteogenesis-promoting particles. In some specific embodiments, the substrate and / or the protrusion array includes osteogenesis-promoting particles, i.e., the osteogenesis-promoting particles can be loaded in the substrate and / or the protrusion array. In some specific embodiments, the osteogenesis-promoting particles account for not more than 10% of the mass percentage of the second substrate layer, specifically 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%. In some specific embodiments, the osteogenesis-promoting particles include at least one of hydroxyapatite, silicon dioxide, and silver. In some specific embodiments, the osteogenesis-promoting particles are micron-sized or nanometer-sized osteogenesis-promoting particles.

[0020] In some embodiments, the base and the array of protrusions of the second substrate layer have the same or different compositions. In some specific embodiments, the base and the array of protrusions have the same composition, both including a shape memory material and osteogenic particles. In some specific embodiments, the second substrate layer includes a shape memory material and osteogenic particles loaded in the shape memory material at a mass percentage of no more than 10% of the second substrate layer, the second substrate layer including a base and an array of protrusions on the base.

[0021] In some embodiments, the responsive orthopedic implant material is modified with at least one functional peptide segment, the functional peptide segment including an inflammation regulation functional peptide segment, an angiogenesis promotion functional peptide segment, a bone growth promotion functional peptide segment, an antibacterial peptide, an antioxidant peptide.

[0022] In some embodiments, the inflammation regulation functional peptide segment includes an anti-inflammatory peptide segment. In some specific embodiments, the inflammation regulation functional peptide segment includes a peptide segment having anti-transglutaminase 2 (TGase 2) activity and / or anti-phospholipase A2 (PLA2) activity. In some specific embodiments, the amino acid sequence of the inflammation regulation functional peptide segment includes KVLDGQDP, which can further include 1-10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids, at the N-terminus and / or C-terminus, while ensuring its original inflammation regulation activity. In some specific embodiments, the amino acid sequence of the inflammation regulation functional peptide segment is KVLDGQDP (SEQ ID NO. 3).

[0023] In some embodiments, the angiogenesis promotion functional peptide segment includes an osteopontin (OPN)-derived peptide segment. In some specific embodiments, the angiogenesis promotion functional peptide segment includes an N-terminal osteopontin (OPN) peptide segment. In some specific embodiments, the amino acid sequence of the angiogenesis promotion functional peptide segment includes SVVYGLR, which can further include 1-10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids, at the N-terminus and / or C-terminus, while ensuring its original angiogenesis promotion activity. In some specific embodiments, the amino acid sequence of the angiogenesis promotion functional peptide segment is SVVYGLR (SEQ ID NO. 1).

[0024] In some embodiments, the bone anabolic functional peptide segment comprises an osteogenic growth peptide (OGP)-derived peptide segment. In some specific embodiments, the bone anabolic functional peptide segment comprises an osteogenic growth peptide (OGP) C-terminal peptide segment. In some specific embodiments, the amino acid sequence of the bone anabolic functional peptide segment comprises YGFGG, which can further comprise 1-10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids at the N-terminus and / or C-terminus, while ensuring its original bone anabolic activity. In some specific embodiments, the amino acid sequence of the bone anabolic functional peptide segment comprises YGFGG (SEQ ID NO. 2).

[0025] In addition, it can be understood that the responsive orthopedic implant material is suitable for use in vivo for bone defects and the like, and thus each different raw material (such as the first shape memory material, the second shape memory material, and the third shape memory material) in the responsive orthopedic implant material preferably has certain biocompatibility and degradability.

[0026] In some embodiments, the polypeptide modified on the bioactive metal layer comprises a plurality of polypeptides, each having the same or different first functional peptide segment, the same or different enzyme cleavage substrate peptide segment, and the same or different second functional peptide segment. For example, different enzyme cleavage substrate peptide segments can be selected according to the strength of the enzyme in different time sequences during bone repair, so as to expose different functional peptide segments, or different first functional peptide segments or different second functional peptide segments in the plurality of polypeptides are combined to expose different functional peptide segments at the same time, so as to have a multi-angle bone repair effect.

[0027] The functional polypeptide chain segment is modified on the surface of the responsive orthopedic implant material to achieve time sequence regulation of macrophage inflammatory phenotype and bone marrow mesenchymal stem cell osteogenic differentiation in different periods of bone repair, to solve the contradiction between contact induction effect in regulating macrophages and bone marrow mesenchymal stem cells in microstructure morphology, so as to promote the time sequence regulation of bone defects and improve the treatment effect.

[0028] In a second aspect, the present application provides a preparation method of a responsive orthopedic implant material, comprising the preparation of a protrusion array, which comprises the following steps:

[0029] The raw material containing the second shape memory material is placed in a template, and the template comprises a concave array matched with the protrusion array, and after drying, a columnar array is obtained:

[0030] The columnar array is oriented to obtain a long-range oriented protrusion array.

[0031] In some embodiments, the preparation of the responsive orthopedic implant material further comprises compounding the protrusion array with the first substrate layer to obtain the responsive orthopedic implant material.

[0032] In some embodiments, the first substrate layer is prepared by mixing a solution of the first shape memory material with a solution of the exogenous stimuli-responsive thermal effect material, and solidifying the mixed solution in a template to obtain the first substrate layer.

[0033] In some embodiments, the solidifying in the template comprises placing the mixed solution in the template, and solidifying at 0-10°C, specifically at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C. In some specific embodiments, the solidifying time is 1-10 days, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days. In some specific embodiments, the drying is performed by vacuum drying. In some specific embodiments, the vacuum drying is performed for 1-3 days, specifically 1, 2, 3 days.

[0034] In some embodiments, the exogenous stimuli-responsive thermal effect material in the solution of the exogenous stimuli-responsive thermal effect material is prepared by a top-down or bottom-up method. In some specific embodiments, the method for preparing the exogenous stimuli-responsive thermal effect material comprises at least one of mechanical ball milling, mechanical exfoliation, liquid phase exfoliation, intercalation exfoliation, vapor deposition, liquid deposition, etc.

[0035] In some embodiments, the responsive orthopedic implant material comprises a second substrate layer, and the second substrate layer comprises a base and an array of protrusions. The second substrate layer is prepared by placing a raw shape memory material in a template, the template comprising an array of recesses matching the array of protrusions, and drying to obtain a base and an array of columns on the base, and orienting the array of columns to obtain a base and a long-range oriented array of protrusions on the base.

[0036] In some embodiments, the columnar array is oriented by hot-pressing. In some specific embodiments, the hot-pressing temperature for the hot-pressing is 40-100℃. It is understood that different hot-pressing temperatures are selected according to the selection of the shape memory material. The specific hot-pressing temperature can be 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, 100℃. In some specific embodiments, the hot-pressing time for the hot-pressing is 1-120min. It is understood that different hot-pressing times are selected according to the selection of the shape memory material. The specific hot-pressing time can be 1min, 2min, 3min, 4min, 5min, 10min, 15min, 20min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 70min, 80min, 90min, 100min, 110min, 120min. In some specific embodiments, the specific steps for the hot-pressing include, after heating for the hot-pressing time, using a polished glass slide to scratch the surface of the columnar array to orient it.

[0037] In some embodiments, the method further comprises modifying a functional peptide segment to the responsive orthopedic implant material. In some specific embodiments, the functional peptide segment is modified to the responsive orthopedic implant material by a click reaction.

[0038] In some specific embodiments, the method of modifying a functional peptide segment to the responsive orthopedic implant material comprises:

[0039] providing a responsive orthopedic implant material, a functional peptide segment, and a linking molecule, the functional peptide segment containing an azido group, and the linking molecule comprising a catechol unit and a DBCO group;

[0040] activating the responsive orthopedic implant material by plasma immersion ion implantation;

[0041] connecting the linking molecule to the responsive orthopedic implant material by chelation of the activated responsive orthopedic implant material and the catechol unit;

[0042] connecting the linking molecule to the functional peptide segment by a click reaction of the DBCO group and the azido group.

[0043] In some specific embodiments, in the plasma immersion ion implantation, the negative radio frequency power is 20%-60%, the sample chamber pressure is 0.2mbar-0.6mbar, and the action time is 3-10min. In some specific embodiments, the plasma gas comprises at least one of oxygen and nitrogen.

[0044] In some embodiments, the chelation of the responsive orthopedic implant material with the catechol unit comprises incubating the activated responsive orthopedic implant material with a solution of the linking molecule for 12-24 hours. In some specific embodiments, the temperature for incubating the responsive orthopedic implant material with the solution of the linking molecule is 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C.

[0045] In some embodiments, the solvent in the solution of the linking molecule is water or a buffer, such as a phosphate buffer, e.g., a PBS buffer, etc. It can be appreciated that the amount of the linking molecule introduced to the responsive orthopedic implant material through the chelation can be reasonably adjusted by controlling the concentration of the solution of the linking molecule and the incubation time, etc.

[0046] In some embodiments, the click reaction of the DBCO group with the azido group comprises incubating the responsive orthopedic implant material with the solution (dispersion) of the functional peptide segment for 12-24 hours. In some specific embodiments, the temperature for incubating the responsive orthopedic implant material with the solution of the functional peptide segment is 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C. In some specific embodiments, the solvent in the solution of the functional peptide segment is water or a buffer, such as a phosphate buffer, e.g., a PBS buffer, etc. It can be appreciated that the amount of the functional peptide segment introduced to the responsive orthopedic implant material through the click reaction can be reasonably adjusted by controlling the concentration of the solution of the functional peptide segment and the incubation time, etc.

[0047] In some embodiments, the linking molecule comprises 2-10 catechol units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 catechol units. In some embodiments, the linking molecule comprises a main chain and a side chain, at least one end group of the main chain is a DBCO group, and the side chain comprises catechol units. In some embodiments, the linking molecule comprises a repeating unit of , and the number of the repeating unit is 2 or more, such as 2-10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 repeating units.

[0048] In some embodiments, the linking molecule comprises a repeating unit of at one end and a DBCO group at the other end.

[0049] In some embodiments, the linking molecule has the following structural formula:

[0050] wherein n is a positive integer of 2 or more, and x is 1-5.

[0051] In some embodiments, the structure of the connecting molecule is

[0052]

[0053] In a third aspect, the present application provides a medical product, which comprises the responsive orthopedic implant material as described above, or the responsive orthopedic implant material prepared by the method as described above, or the substrate formed with the bone repair layer by the method as described above.

[0054] In some embodiments, the medical product comprises an implantable medical device.

[0055] In a fourth aspect, the present application provides the use of the responsive orthopedic implant material as described above in the preparation of a medical product.

[0056] The time sequence active regulation of the surface microtopography of the responsive orthopedic implant material provided by the present application responds to the effector cells in different repair stages, and then matches each stage of bone tissue repair, promotes bone repair and bone integration; in the early stage of implantation, the acute inflammatory reaction of the body is inhibited to promote osteogenesis; in the middle and late stages of implantation, under the condition of applying an external field such as near-infrared light or a magnetic field, the transition of the convex array surface microtopography from the long-range oriented morphology for regulating inflammation to the microcolumn array morphology for promoting osteogenesis can be actively controlled through the thermal effect, which effectively promotes bone regeneration; the surface modification of the material with the functional polypeptide segment effectively regulates the inflammatory response and promotes osteogenic differentiation. In addition, the technologies involved in the present application have the remarkable advantages of being simple, efficient, and highly reproducible, which guarantees the subsequent batch and industrial production.

[0057] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is a schematic diagram of the preparation method in the embodiments of the present application.

[0059] Figure 2 A and B in are scanning electron microscope photos of the plane and cross section of M-H8 in the embodiments of the present application, respectively.

[0060] Figure 3 is the static water contact angle result of the P, P / H, H8, M-H8-O2, and M-H8+Y samples in the embodiments of the present application, and the embedded photo is the water droplet photo when the corresponding group of samples is tested.

[0061] Figure 4 is the temperature rise curve of the first substrate layer containing 0.05% BP, 0.1% BP, and 0.15% BP and PBS under 808 nm near-infrared laser irradiation in the embodiments of the present application.

[0062] Figure 5 are scanning electron microscope photos of macrophages directly cultured on each group of samples for 1 day in the embodiments of the present application.

[0063] Figure 6 are immunofluorescence confocal images of macrophages directly cultured on each group of samples for 1 day in the embodiments of the present application.

[0064] Figure 7 are CD163 marker immunofluorescence quantitative results of macrophages directly cultured on each group of samples for 1 day in the embodiments of the present application.

[0065] Figure 8 are alkaline phosphatase staining conditions of human bone marrow mesenchymal stem cells directly cultured on different groups of samples for 7 days and 14 days in the embodiments of the present application.

[0066] Figure 9 are extracellular matrix mineralization quantitative detection conditions of human bone marrow mesenchymal stem cells directly cultured on different groups of samples for 14 days and 21 days in the embodiments of the present application.

[0067] Figure 10 are alkaline phosphatase staining conditions of human bone marrow mesenchymal stem cells cultured in different macrophage conditioned media for 7 days in the embodiments of the present application.

[0068] Figure 11 are micro-CT skull three-dimensional reconstruction maps of skull defect SD rats implanted with bone repair materials for 4 weeks and 8 weeks in the embodiments of the present application.

[0069] Figure 12 are micro-CT skull osteogenesis-related parameter results of skull defect SD rats implanted with bone repair materials for 4 weeks and 8 weeks in the embodiments of the present application. DETAILED DESCRIPTION

[0070] The concept and the technical effects of the present application will be described in detail below in combination with embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0071] The embodiments of the present application will be described in detail below. The described embodiments are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0072] In the description of the present application, the meaning of several is more than one, the meaning of multiple is more than two, greater than, less than, more than, etc. are understood as not including the number, above, below, etc. are understood as including the number, the meaning of about is within the number ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. If there is a description to the first, second, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or implicitly indicating the sequence of indicated technical features.

[0073] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the exemplary description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.

[0074] Embodiment 1

[0075] The present embodiment provides a responsive orthopedic implant material, referring to Figure 1 , taking degradable high molecular orthopedic implant material polylactic acid as a representative of orthopedic implant material, nano-hydroxyapatite particles as pro-osteogenic nanoparticles, anti-inflammatory short peptide modified with azide (N3-KVLDGQDP) and pro-osteogenic short peptide modified with azide (N3-YGFGG) as functional polypeptide segments, applying extracorporeal near-infrared light, and the thermal effect material is black phosphorus nanosheet. The specific preparation process includes the following steps:

[0076] (1) Dissolve polylactic acid in chloroform to prepare a polylactic acid solution with a mass concentration of 0.125 g / mL. Dissolve hydroxyapatite in absolute ethanol to prepare a hydroxyapatite solution with a mass concentration of 0.2 g / mL. Mix the polylactic acid solution and the hydroxyapatite solution in a volume ratio of 500:3 to prepare a mixed solution of polylactic acid containing 1.2 mg / mL of hydroxyapatite. Then pour the mixed solution into a template with a micropore array, place it in a 4℃ refrigerator for one day, then transfer it to a vacuum drying oven for three days. Take the material out of the template to obtain a second substrate layer with a columnar array, denoted as H8.

[0077] (2) The bulk black phosphorus was mixed with N-methyl pyrrolidone (NMP) solvent to prepare a mixture of 1 mg / mL. The mixture was ground to obtain a coarse dispersion liquid. The coarse dispersion liquid was ultrasonicated at 300 W power in a 5 °C constant temperature water bath for 10 h. The supernatant was centrifuged at 4000 rpm for 15 min. The supernatant was centrifuged again at 7000 rpm for 15 min. The supernatant was centrifuged at 10000 rpm for 15 min. The precipitate was directly dispersed in NMP to obtain a black phosphorus nanosheet solution of 2.0 mg / mL. The polylactic acid solution of 0.125 g / mL was mixed with the black phosphorus nanosheet solution of 2.0 mg / mL at a mass ratio of black phosphorus nanosheets to polylactic acid of 0.05%, 0.1%, and 0.15%, respectively. The mixture was poured into a template with a smooth surface. The template was placed in a 4 °C refrigerator for three days and then moved to a vacuum drying oven for one day. The polylactic acid loaded with black phosphorus nanosheets was obtained as a first substrate layer. The samples were recorded as 0.05% BP, 0.1% BP, and 0.15% BP, respectively.

[0078] (3) The 0.1% BP and H8 were bonded together after the interface was brushed with an appropriate amount of chloroform. Finally, a shape memory double-layer material with a columnar array was obtained. The sample was recorded as M-H8.

[0079] (4) The shape memory double-layer material with a columnar array M-H8 was placed in a 55 °C oven for a period of time. A polished glass slide was used to scratch the surface of the second substrate layer with a columnar array in the length direction. The originally regular columnar array was converted into a long-range oriented structure array extending in the column height direction. The sample was recorded as M-O.

[0080] (5) The M-O was treated by gas plasma immersion ion implantation technology. Oxygen was selected as the gas source. The specific treatment process was as follows: the radio frequency power was 25%, the sample chamber pressure was 0.4 mbar, and the action time was 5 minutes. Then, the sample was immersed in a 0.1 mg / mL DBCO solution at 4 °C for 24 hours. The sample was washed with pure water for two times. The sample with a surface modified with DBCO was obtained. The sample with a surface modified with DBCO was immersed in a mixed solution of 0.1 mg / mL anti-inflammatory polypeptide segment with a tail modified with an azide group at 4 °C for 24 hours. The obtained sample was recorded as M-O+K. The sample with a surface modified with DBCO was immersed in a mixed solution of 0.1 mg / mL pro-osteogenic polypeptide segment with a tail modified with an azide group and 0.1 mg / mL anti-inflammatory polypeptide segment with a tail modified with an azide group at 4 °C for 24 hours. The obtained sample was recorded as M-O+K+Y.

[0081] The final sample M-O+K+Y includes a first substrate layer and a second substrate layer, the first substrate layer includes polylactic acid shape memory material and black phosphorus nanosheets loaded therein, and the second substrate layer includes a hydroxyapatite-loaded polylactic acid shape memory material base and a long-range oriented protrusion array on the base. In addition, the first substrate layer and the second substrate layer are also modified with anti-inflammatory polypeptide segments and osteogenic polypeptide segments.

[0082] In addition, in step (1), a mixed solution of 1.2 mg / mL hydroxyapatite polylactic acid is poured into a smooth-surfaced template, and after being placed in a 4°C refrigerator for one day, it is transferred to a vacuum drying box for three days. The material is taken out of the template to obtain a second substrate layer of hydroxyapatite-loaded polylactic acid shape memory material formed by a smooth surface, denoted as P / H.

[0083] After 0.1% BP and P / H are adhered together by brushing an appropriate amount of chloroform at the interface, a smooth-surfaced shape memory double-layer material is finally obtained, denoted as M-F.

[0084] In addition, in step (1), polylactic acid is dissolved in chloroform to prepare a polylactic acid solution with a mass concentration of 0.125 g / mL, which is poured into a smooth-surfaced template and placed in a 4°C refrigerator for one day, and then transferred to a vacuum drying box for three days. The material is taken out of the template to obtain a second substrate layer of polylactic acid shape memory material formed by a smooth surface, denoted as P.

[0085] In addition, step (4) is omitted, and M-H8 is directly treated by the gas plasma immersion ion implantation technology in step 5. Oxygen is used as the gas source, the radio frequency power is 25%, the sample chamber pressure is 0.4 mbar, and the action time is 5 minutes to obtain M-H8-O2. Then M-H8-O2 is immersed in a 0.1 mg / mL DBCO solution at 4°C for 24 hours, washed with pure water twice, and then immersed in a 0.1 mg / mL osteogenic polypeptide segment with an azide group at the tail end for 24 hours at 4°C to obtain M-H8+Y.

[0086] Figure 2 The scanning electron microscope photos of the plane and cross section of M-H8. As can be seen from the figure, after being combined with the first substrate layer, the satellite hairs of the columnar array on the second substrate layer are not affected, indicating that the responsive orthopedic implant material with a columnar array morphology on the surface is successfully prepared by the steps in the above method.

[0087] Example 2

[0088] The static contact angles of the P, P / H, H8, M-H8-O2, M-H8+Y samples prepared in Example 1 were tested respectively using a contact angle tester to characterize the hydrophilic or hydrophobic properties of the surfaces of the samples. The specific steps are as follows:

[0089] The clean samples were fixed on the sample stage of the contact angle tester, and 20 μL of ultrapure water droplets were added to the surface of the sample by a syringe using the droplet method to measure the contact angle of the droplet and the material interface.

[0090] The results are shown in Table 1. Figure 3 The P sample did not load hydroxyapatite, and the surface had no micro-morphology, and the contact angle was 91.04±0.36°; the P / H group loaded hydroxyapatite nanoparticles, but the surface still had no micro-morphology, and the contact angle was 96.91±0.61°; the H8 group loaded hydroxyapatite nanoparticles, and the surface had a columnar array of micro-morphology, and the contact angle further increased to 115.90±2.11°; the M-H8-O2 group was treated with oxygen plasma on the surface of the H8 group, and the contact angle decreased to 65.36°±1.91°; and the M-H8+Y group was grafted with a bone-promoting polypeptide segment, and the contact angle increased to 82.27±3.78°.

[0091] Overall, the contact angles of the surfaces of the samples in each group showed a trend of H8>P / H>P>M-H8+Y>M-H8-O2, indicating that the gas plasma immersion ion implantation technology can significantly improve the hydrophilicity of the surface of the material.

[0092] Example 3

[0093] The 0.05% BP, 0.1% BP, and 0.15% BP in Example 1 were cut into circular samples with a diameter of 1 cm, and were respectively immersed in 250 μL of phosphate buffer solution. The samples were irradiated with 808 nm near-infrared laser, the distance between the near-infrared light source and the sample was 10 cm, and a thermal imager was used to record the temperature change of the sample during near-infrared irradiation. The results are shown in Table 2. Figure 4 The temperature of the 0.1% BP sample reached 55°C after 10 seconds of laser irradiation, and the temperature was between 55°C and 60°C in the interval of 10-25 seconds. The above results show that after applying an external field of near-infrared light, the 0.1% BP sample as the first substrate layer can be heated to the phase transition temperature of the shape memory material in the second substrate layer in a short time, and after reaching the phase transition temperature, the heating is slow, reducing the possibility of damage to the surrounding tissue of the implanted site due to rapid heating.

[0094] Example 4

[0095] The mouse macrophage-like cell line RAW264.7 cells were inoculated and cultured on the surfaces of the samples in each group (P, M-F, M-O, M-O+K) in Example 1 (the samples with a protrusion array are the surfaces with a protrusion array) for 1 day, fixed with a 2.5% glutaraldehyde solution at 4°C for 2 hours, then dehydrated with a gradient ethanol solution, and finally the morphology of the proliferated macrophages on the material interfaces of each group was observed by a scanning electron microscope (SEM) to evaluate the effect of the material on regulating inflammation.

[0096] The results are shown in Figure 5 The macrophages grown on the M-O and M-O+K groups of materials with a long-range oriented protrusion array were more elongated and showed obvious M2-type macrophage-like morphology compared with the macrophages grown on the P and M-F groups of materials with smooth surfaces. The above results show that the prepared sample with a long-range oriented protrusion array has the ability to inhibit inflammatory response in the early stage after implantation, can inhibit the polarization of macrophages to M1 type, and promote the polarization to M2 type.

[0097] Example 5

[0098] The mouse macrophage-like cell line RAW264.7 cells were inoculated and cultured on the surfaces of the samples in each group (P, M-F, M-O, M-O+K) in Example 1 (the samples with a protrusion array are the surfaces with a protrusion array) for 1 day, and the culture medium was aspirated and the cells were washed with PBS for 3 times. Subsequently, the cells were incubated in 4% paraformaldehyde at room temperature for 30 minutes, then 0.1% Triton X-100 was added for incubation for 10 minutes, and the blocking solution was added for incubation of the cells for 10 minutes. Then, the CD163 (a marker of M2-type macrophages) antibody was used as a primary antibody for overnight incubation. The FITC-labeled secondary antibody was added to the samples with the CD163 antibody, and the cells were incubated for 1 hour in the dark. Finally, DAPI working solution was added for incubation of the cells for 4.5 minutes, and then the cells were washed with PBS for 3 times, and observed and photographed by a confocal fluorescence microscope.

[0099] Figure 6 and Figure 7 The results show that the macrophages grown on the surfaces of the M-O and M-O+K groups of materials with a long-range oriented protrusion array have higher CD163 expression than the macrophages grown on the surfaces of the P and M-F groups of materials with smooth surfaces without micro-topography. This shows that the long-range oriented protrusion array has the effect of promoting the polarization of macrophages to M2 type and inhibiting inflammatory response, and the CD163 expression of the M-O+K group is higher than that of the M-O group, indicating that the anti-inflammatory peptide synergistically promotes the polarization of macrophages to M2 type.

[0100] Example 6

[0101] Human bone marrow mesenchymal stem cells (BMSCs) were seeded and cultured on the surfaces of each group of samples (P, M-F, M-H8, M-H8+Y) in Example 1 (the surfaces of samples with raised arrays were the surfaces with raised arrays), and the BMSCs on the surfaces of each group of samples were induced to differentiate into osteoblasts using bone induction medium. After 7 days and 14 days of culture, the BMSCs were fixed with 4% paraformaldehyde and stained with an alkaline phosphatase (ALP) qualitative kit for semi-quantitative study of their ALP expression. After 14 days and 21 days of culture, the mineralized calcium nodes of the cells were stained with alizarin red, and the cells of each group were fixed with 75% ethanol for 1 hour. Subsequently, the cells were stained with an alizarin red solution for 30 minutes, the samples were washed with deionized water to remove excess dye, and when the deionized water after washing the samples was not red, the alizarin red bound to the mineralized calcium nodes on the samples was eluted with 10% cetylpyridinium chloride, and the absorbance value of the eluate at 570 nm was measured using a microplate reader to analyze the mineralization level of the extracellular matrix (ECM) on different samples. The results are shown in Figure 8 and Figure 9 It is shown that among the above four groups of samples, the ALP expression and extracellular matrix mineralization of the BMSCs cultured on the surface of the M-H8+Y group of samples are the best, indicating that under the dual action of the columnar array microtopography and the osteogenic polypeptide chain segment, the osteogenic differentiation of the BMSCs is effectively promoted.

[0102] Example 7

[0103] Mouse macrophage-like cell line RAW264.7 cells were seeded and cultured on the surfaces of each group of samples (P, M-F, M-O, M-O+K) in Example 1 (the surfaces of samples with raised arrays were the surfaces with raised arrays) for 3 days, and the cell culture supernatant was collected every day. The collected medium was mixed with fresh medium at a ratio of 1:1 to prepare a conditioned medium without RAW264.7 cells. Human bone marrow mesenchymal stem cells (BMSCs) were seeded in a 48-well plate and cultured for 7 days using the conditioned medium, and then the BMSCs were fixed with 4% paraformaldehyde and stained with an alkaline phosphatase (ALP) qualitative kit for semi-quantitative study of their ALP expression. The results are shown in Figure 10 As can be seen from the figure, the ALP expression of the BMSCs of the four groups of P, M-F, M-O, and M-O+K increased in turn. In combination with Example 5, it is shown that long-range oriented microtopography can promote the timely and effective conversion of macrophages from the M1 phenotype to the M2 phenotype, and M2 macrophages can secrete osteoblast factors (such as TGF-β) to promote the osteogenesis of BMSCs.

[0104] Example 8

[0105] Sprague Dawle (SD) rats (8 weeks, male) were anesthetized and a 5mm diameter defect was made on the top of the skull using a 5mm diameter drill bit. The skull fragments were carefully removed without affecting the dura mater, and the bone repair materials (P, M-F, M-O+K+Y) in Examples 2-4 were placed on the surface of the bone defect after careful hemostasis. Finally, the wound was sutured and the mice were monitored appropriately. At 1 week after the operation, the implanted site was irradiated with an 808nm near-infrared laser to heat the material to above 55 degrees Celsius for 10 seconds. At 4 and 8 weeks after the operation, the mice were sacrificed and the skulls were removed for analysis of bone regeneration using a micro-CT. The samples were reconstructed in three dimensions using software provided by the instrument manufacturer, and the results are shown in Figure 11 , to obtain parameters such as bone volume fraction (BV / TV), trabecular bone crossbeam number (Tb.N), and trabecular bone separation degree (Tb.Sp), and the results are shown in Figure 12 .

[0106] BV / TV represents the proportion of bone tissue in the scanned area, and a higher value indicates that there is relatively more bone tissue in the scanned area; Tb.Sp represents the average interval distance between trabecular bones, reflecting the arrangement density of the trabecular bones, and a smaller value indicates that the interval between the trabecular bones is narrower, indicating a higher trabecular bone density; Tb.N represents the number of trabecular bones per unit length, and this parameter provides information about the arrangement density of the trabecular bones, and a larger value indicates a larger number of trabecular bones and a higher density. From Figure 11 and Figure 12 it can be seen that at 4 and 8 weeks, the bone repair material in the M-O+K+Y group has the best effect on repairing the bone defect.

[0107] Example 9

[0108] This example provides a responsive orthopedic implant material M-O+K+Y+S, which differs from M-O+K+Y in Example 1 in that the connected polypeptide segments include the anti-inflammatory short peptide KVLDGQDP, the pro-osteogenic short peptide YGFGG, and the pro-angiogenic short peptide SVVYGLR.

[0109] Example 10

[0110] This example provides a responsive orthopedic implant material M-O+K+Y+G, which differs from M-O+K+Y in Example 1 in that the connected polypeptide segments include the anti-inflammatory short peptide KVLDGQDP, the pro-osteogenic short peptide YGFGG, and the anti-oxidative short peptide GGC. The responsive orthopedic implant material provided in this example has certain anti-oxidative ability while promoting angiogenesis and osteogenesis, effectively solving the problem of poor bone repair effect caused by oxidative stress.

[0111] Example 11

[0112] The embodiment provides a responsive orthopedic implant material, which is different from the embodiment 1 in that the osteogenesis particles in the second substrate layer are nano-silver.

[0113] Embodiment 12

[0114] The embodiment provides a responsive orthopedic implant material, which is different from the embodiment 1 in that the exogenous stimulus heat effect material in the first substrate layer is magnetic iron oxide particles. The responsive orthopedic implant material provided by the embodiment changes the micro-morphology of the convex array under the corresponding action based on the magnetic induction heat effect, thereby playing a time-sequential repair role.

[0115] Embodiments 9-12 are tested by referring to embodiments 2-8, and the corresponding results are similar to those of the embodiment 1, which will not be described herein again.

[0116] From the above results, it can be seen that the time-sequential active regulation of the surface micro-morphology of the responsive orthopedic implant material provided by the application responds to the effector cells in different repair stages, and then matches each stage of bone tissue repair, promotes bone repair and bone integration; in the early implantation stage, acute inflammatory reaction of the body is inhibited to promote osteogenesis; in the middle and late implantation stages, under the condition of applying an external field such as near-infrared light or a magnetic field, the surface micro-morphology of the convex array can be actively controlled to change from the long-range oriented morphology for regulating inflammation to the micro-column array morphology for promoting osteogenesis, thereby effectively promoting bone regeneration; and the surface modification of the material with corresponding functional polypeptide segments can effectively regulate inflammation and promote osteogenic differentiation or promote angiogenesis, antioxidant and other functions. In addition, the technologies involved in the application have the advantages of simplicity, high efficiency and high repeatability, which guarantee the subsequent batch and industrial production.

[0117] The above combines the embodiments to make a detailed description of the application, but the application is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary skilled persons in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Responsive orthopedic implant material, characterized in that, Comprising: a first substrate layer, the first substrate layer comprising a first shape memory material and an exogenous stimulus-thermal effect material loaded in the first shape memory material, the mass percentage of the exogenous stimulus-thermal effect material in the first shape memory material being no more than 5%, the first shape memory material comprising at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, polyhydroxybutyrate, the exogenous stimulus-thermal effect material comprising at least one of a photothermal effect material, a magnetocaloric effect material, an electrocaloric effect material; a second substrate layer, the second substrate layer comprising a base and a protrusion array, the first substrate layer being located on one side of the base, the protrusion array being located on the other side of the base away from the first substrate layer, the protrusion array being made of a raw material containing a second shape memory material, the protrusion array being configured to be oriented along a long-range and being capable of being transformed into a columnar array under the thermal effect of the first substrate layer, the second shape memory material comprising at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, polyhydroxybutyrate, the base being made of a raw material containing a third shape memory material, the third shape memory material comprising at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyethylene glycol, polyvinyl alcohol, polypyrrolidone, polydopamine, polyvinyl butyral, polyhydroxybutyrate; the second substrate layer comprising osteogenesis-promoting particles, the responsive orthopedic implant material being modified with at least one functional peptide segment, the functional peptide segment comprising an inflammation regulation functional peptide segment, an angiogenesis-promoting functional peptide segment, an osteogenesis-promoting functional peptide segment, an antibacterial peptide, an antioxidant peptide.

2. The responsive orthopedic implant material of claim 1, wherein, The photothermal effect material comprises at least one of an inorganic photothermal effect material and an organic photothermal effect material.

3. The responsive orthopedic implant material of claim 2, wherein, The inorganic photothermal effect material comprises at least one of a nano-metal, a carbon nanotube, black phosphorus, graphene.

4. The responsive orthopedic implant material of claim 2, wherein, The organic photothermal effect material comprises at least one of polypyrrole, polyaniline, polyethylene dioxythiophene, polystyrene sulfonate, indocyanine green, porphyrin.

5. The responsive orthopedic implant material of claim 1, wherein, The magnetocaloric effect material comprises at least one of Fe2O3, Fe3O4, FeCo, NiFe, CoFeO, NiFeO, MnFeO.

6. The responsive orthopedic implant material of claim 1, wherein, The electrocaloric effect material comprises at least one of carbon black, carbon nanotube, graphene.

7. The responsive orthopedic implant material of claim 1, wherein, The osteogenesis-promoting particles comprise at least one of hydroxyapatite, silicon dioxide.

8. The responsive orthopedic implant material of claim 1, wherein, The amino acid sequence of the inflammation regulation functional peptide segment comprises KVLDGQDP.

9. The responsive orthopedic implant material of claim 1, wherein, The amino acid sequence of the angiogenesis-promoting functional peptide segment comprises SVVYGLR.

10. The responsive orthopedic implant material of claim 1, wherein, The amino acid sequence of the osteogenesis-promoting functional peptide segment comprises YGFGG.

11. The method of producing a responsive orthopedic implant material according to any one of claims 1 to 10, characterized in that, The preparation of the protrusion array comprises the following steps: putting a raw material containing a second shape memory material into a template, the template comprising a concave array matching the protrusion array, and drying to obtain a columnar array; orienting the columnar array to obtain a long-range oriented protrusion array.

12. A medical article characterized by, The responsive orthopedic implant material of any one of claims 1 to 10, or the responsive orthopedic implant material prepared by the method of claim 11.

13. Use of the responsive orthopedic implant material of any one of claims 1 to 10 in the manufacture of a medical product.

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