RU2744710C2 - Method of producing nanocomposite joint ligament implant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and, more specifically, to traumatology, and discloses a method for manufacturing a nanocomposite joint ligament with a coating of a composite biocompatible nanomaterial. Method involves application of auxiliary substance on the surface of the workpiece of the ligament joint implant – water-protein dispersion of carbon nanotubes with subsequent formation of nanocomposite coating by exposure to infrared radiation in wavelength range 0.7–1.2 mcm, with power per unit area of 0.1–0.5 W/cm².

EFFECT: method allows improving mechanical properties and degree of biocompatibility of a joint ligament implant and can be used for restoring function of an injured ligament of a knee joint, as well as other joints.

5 cl, 3 dwg, 1 ex

Description

The invention relates to medicine and, in particular, to traumatology. It is intended for the treatment of surgical implants used in the reconstruction of ligaments and tendons with infrared (IR) radiation.

Surgical operation allows the damaged ligaments of the knee to be replaced with artificial implants. Even relatively inert synthetic polymers, being a foreign body in the body, often maintain a chronic inflammatory response and change their physical properties. Consequently, long-term functioning of synthetic implants often becomes impossible. Therefore, to avoid rejection of the implant and reduce the risk of infection after transplantation, a special coating is applied to the surface of the implants.

A known method of populating the implant of the ligament of the knee with fibroblasts, with the impregnation of the implant with an auxiliary substance - collagen [1]. A significant disadvantage of this method of covering the knee ligament implant is the complexity of working with the cellular component, which requires long-term preparation of the material: collection of cells from the patient, cultivation and seeding of the implant with cells.

The known method of fixing the implant of the ligament of the knee joint in the holes of the tibia of a person or an animal, with the transfer of energy to the auxiliary solidifying fusible material, penetrating into the bone tissue [2]. The disadvantage of this method of fixing the knee ligament implant is incomplete physiological compatibility of the proposed fusible material, which may result in the destruction or rejection of the implant.

A known method of processing artificial ligaments of joints and tendons using auxiliary substances - nanofibers, including graphene, its derivatives and carbon nanotubes [3]. The disadvantage of this method of manufacturing and using ligament implants is the lack of functional treatment of the used carbon-containing materials, which can only impart osteinductive properties to the ligament implant material, which determine the stable fixation of the implant in the bone canal, but does not ensure the biological compatibility of the implant with the body tissues.

The closest technical solution to the proposed method of processing artificial ligaments of joints and tendons is the formation of a nanocomposite coating of the knee ligament implant by means of water vaporizing action of laser radiation [4]. The disadvantage of this method of manufacturing and using ligament implants is that it is difficult to control the temperature of local heating of the nanocomposite material and there is a high risk of lowering its functional characteristics. For example, in a nanocomposite material made of a water-protein dispersion of carbon nanotubes, in the case of overheating (temperature over 80 ° C) of the albumin protein, its denaturation begins. Indeed, the laser beam locally, over an area of several tens of square micrometers, heats up the material strongly, which may not affect the recorded integral temperature of the coating. As a result, locally denatured areas are formed on the surface of the implant coating, the presence of which reduces the possibility of cell adhesion and proliferation on the coating surface. This means that the initially high degree of biocompatibility of the nanocomposite coating is significantly reduced. Therefore, for such a joint ligament implant, there is a possibility of an inflammatory reaction or other complications.

Obviously, it is necessary to preserve the effectiveness of the coating of the implant of the ligaments of the joints and to prevent overheating of the nanocomposite coating during the laser treatment.

The objective of the present invention is to increase the degree of biocompatibility of a nanocomposite ligament implant coated with a composite biocompatible nanomaterial and its tensile strength relative to uncoated ligament implants.

The problem is solved by applying a bundle of an auxiliary substance to the surface of the implant blank - a water-protein dispersion of carbon nanotubes (CNTs), and then forming a nanocomposite coating by water-evaporating action of IR radiation in the wavelength range of 0.7-1.2 μm, power per unit area 0.1-0.5 W / cm ² . The concentration of the protein component in the auxiliary substance is in the range from 15 to 30 wt. %. The concentration of carbon nanotubes in the auxiliary substance is in the range from 0.1 to 0.3 wt. %. Albumin and collagen are used as the protein component of the excipient. IR irradiation of the auxiliary substance layer applied to the knee ligament ligament implant blank is carried out while the implant blank is rotated.

The essence of the invention is as follows. An auxiliary substance is prepared - a water-protein dispersion of carbon nanotubes. It is used to form a nanocomposite coating on the knee ligament implant. To obtain an excipient, the following steps are carried out:

1. Single-walled CNTs (SWCNTs) or multi-walled CNTs (MWCNTs) are added to distilled water, and the dispersion is stirred in a magnetic stirrer for 60 min, and then dispersed in an ultrasonic (US) disperser at a temperature of ≤ 30 ° C for 50 min to obtain uniform dispersion of black color. The concentration of CNTs is selected in the range of 1–2 wt. %.

2. Bovine serum albumin (BSA) powder at a concentration of 15-30 wt.% Is added to the aqueous CNT dispersion. %. Then bovine collagen (BC) is added to the dispersion at a concentration of 1-2 wt. %. The resulting dispersion in the composition of CNTs, BSA, and BA is carefully moved in a magnetic stirrer for 60 min, then dispersed in an ultrasonic bath at a temperature of ≤ 40 ° C for 60 min.

3. Dispersion BSA / BA / CNT is decanted within 24 hours, filtered and poured into another vessel.

The composition of the nanocomposite based on BSA, BA, and CNT is selected based on the following considerations.

BSA - bovine serum albumin is a biological completely biocompatible material and is widely used in medical practice. Albumin makes up about 60% of blood plasma proteins, its average concentration in plasma is ~ 40 g / l. The transport function of albumin is to transport free fatty acids, cholesterol and other biologically active substances. For albumin, a typical conformation in the secondary or tertiary structure with an insignificant loading of the protein with toxic products. The pattern of a dried drop of a freshly prepared aqueous solution of serum albumin is characterized by the presence of a significant number of radial cracks that form arches along the edge of the drop. The albumin molecule is well studied at the atomic-structural level: it has two modifications (isomers), which are an ellipsoid of revolution and an irregular triangular prism. BSA serves as a biological matrix in the nanocomposite material we use.

BC - bovine collagen makes up more than 30% of the total mass of body proteins and is the main component of connective tissue. Compared to the BSA molecule (~ 69 Da), the collagen molecule has a gigantic molecular weight of 300 kDa. The main advantages of collagen are: high mechanical strength, absence of toxic and carcinogenic properties, weak antigenicity, the ability to form complexes with biologically active substances (heparin, chondroitin sulfate, antibiotics, etc.), the ability to stimulate the regeneration of the body's own tissues, to swelling, resorption, to performing the function of an ion exchanger: collagen ensures membrane semi-permeability, stimulation of reparation, and a hemostatic effect. At the same time, collagen biopolymer possesses the positive qualities of synthetic polymers and implants, for example, high mechanical tensile strength, low elongation, fiber orientation, which determines its use in medical applications.

CNTs - carbon nanotubes have extremely high mechanical, physical, optical and other parameters and unique functional capabilities. In particular, high tensile strength of ~ 50 GPa, elastic modulus about 1 TPa and conductivity, reduced density on the nanotube, ≥20000 See × m ² / kg, as well as an acceptable degree of safety with respect to black is not worse, especially purified and functionalised nanotubes. In nanocomposites, two types of CNTs are widespread: SWCNTs and MWCNTs. Significant high values of mechanical parameters in nanocomposites are realized with MWCNT or SWCNT fillers, but a higher degree of purification is realized in SWCNTs.

The listed positive properties of composite composites make it possible, on their basis, to realize a BSA / BK / CNT nanocomposite material with the required parameters and functional properties and with the possibility of medical use.

A standard artificial binder made of synthetic material, in particular polyethylene terephthalate (PET), serves as the base on which the BSA / BA / CNT dispersion is applied to obtain a coating. In general, the final product, referred to as an "implant", is a PET bundle coated with a BSA / BA / CNT nanocomposite material.

FIG. 1 shows sketches of the system for preliminary cleaning of the bundle (Fig. 1a): 1 - ultrasonic bath, 2 - water in the ultrasonic bath, 3 - vessel in which the bundle is placed, 4 - ethyl alcohol, 5 - bundle; systems for cleaning the bundle (Fig. 1 b): 6 - distilled water; systems for sterilizing the bundle in a UV chamber (Fig. 1 c): 7 - UV chamber.

FIG. 2 shows a sketch of a camera for IR irradiation: 8 - a bundle with an applied layer of BSA / BK / CNT dispersion, 9 - an IR illuminator, 10 - an infrared radiation reflector, 11 - a system of fastening and rotation of the bundle, 12 - a common table where they are mounted all parts of the camera.

To obtain an implant, the following procedures were performed:

preliminary cleaning of the binder in an ultrasonic bath in ethanol for 30 min;

cleaning the bundle in an ultrasonic bath in distilled water for 30 minutes;

antibacterial treatment of the binder in an ultraviolet (UV) chamber for 50 minutes;

application of an aqueous dispersion of BSA / BK / CNT by lowering a binder into it or spraying the dispersion using a dispersion spray;

irradiation with infrared radiation layers of dispersion on the binder;

testing the mechanical parameters of the implant, i.e. bonds with nanocomposite BSA / BK / CNT coating.

After cleaning and sterilization, the ends of the binder are fixed in a special holder and the BSA / BA / CNT dispersion is applied to the bond surface on both sides using a dispersion spray, or by completely immersing the binder in the dispersion.

Then the binder is transferred to the chamber for infrared irradiation, where it rotates, and this achieves the obtaining of a layer uniform in the thickness of the nanocomposite coating. In the process of irradiation, the temperature of the layer surface on the binder surface is remotely monitored, and the readings are transmitted to the electronic unit, which controls the rotation of the binder 8. The radiation power of the IR emitter is also controlled 9. The feedback between the temperature control device and the power control device makes it easy to maintain the temperature on the binder surface about 50 ° С with an accuracy of ± 3 ° С. FIG. 2 does not reflect the electronic control unit, the pyrometric temperature recorder, as well as the electrical connections between them and with other parts of the camera for IR irradiation. As a result of the selection of technological parameters, for example: the composition of the aqueous dispersion of BSA / BA / CNT, the speed of rotation of the bundle, the power of the IR emitter and the recorded temperature on the surface of the bundle; the formation of a dried layer of a nanocomposite coating with a thickness of 0.2-1 microns on the bond surface is ensured This thickness is sufficient to increase the strength of the ligament implant without adversely affecting the elasticity of the ligament.

The dried layer of the BSA / BA / CNT nanocomposite coating contains the following components: BSA matrix - ≥ 90 wt. %, BK - ~ 6 wt. %, CNT - ≤ 3 wt. %.

Example.

Distilled water contains the ^OC CNT95TA gel (manufactured by OCSiAl), which is an aqueous dispersion of SWCNTs with a SWCNT concentration of 2.5 wt. %. The gel is mixed with distilled water in a magnetic stirrer for 30 minutes, then this composition is homogenized in an ultrasonic homogenizer for 30 minutes. Taking into account all parts of water (distilled and included in the gel), the resulting composition consists of approximately 0.05 wt. % SWCNT and 99.95 wt. % water. Then BSA powder from AMRESCO and neutral aqueous suspension of BC produced by OOO Makmedi are added. The resulting aqueous dispersion in the composition of BSA, BA, and SWCNT is mixed in a magnetic stirrer and in an ultrasonic bath for 30 minutes, respectively. It should be noted that the processing of the dispersion in an ultrasonic homogenizer and an ultrasonic bath was carried out with temperature control, at which the temperature was not allowed to rise above 30 ° C. The mass composition is selected in such a way that the final aqueous dispersion contains: 15-30 wt. % BSA, 1-2 wt. % BA and 0.2-1.0 wt. % SWCNT, the rest is water.

Knee ligament implant PET strips, with approximate dimensions: length 300 mm, width 10 mm, thickness 1.5 mm, served as test samples, on which layers of composite BSA / BC / SWCNT nanomaterial were applied, and which were then tested for mechanical strength. On both edges of the strip, small cuts are made in the middle with a depth of about 1 mm. Consequently, a constriction is formed at this point, the width of which is 2 mm less relative to the width of the strip in other places, and the sample breaks exactly at this point during testing on a tensile machine. This allows the tensile strength to be determined with an accuracy of a few percent.

After cleaning and sterilization, the ends of the bond are fixed in a special holder and the BSA / BA / CNT dispersion is applied to the bond surface on both sides using a dispersion spray. After the end of the process of applying the dispersion, the binder is transferred to the chamber for IR irradiation (see Fig. 2). Irradiation is performed here in the following mode: the speed of rotation of the bundle - 0.5 rev / s; specific power of irradiation - 0.5 W / cm ² , irradiation time 60 s. In this mode, a layer of BSA / BA / SWCNT nanocomposite material with a thickness of 0.5 μm is formed on the bond surface by heating it to approximately 50 ° C. This temperature regime is automatically controlled and maintained by an electronic control unit. After the completion of IR irradiation, the sample was dried under room conditions for 60 min, and then its mechanical characteristics were measured in a tensile testing machine. The stress was defined as the ratio of the loaded force to the cross-sectional area at the narrowed area of the sample, and the relative tension as the relative change in the length of the sample.

Note that the uncoated PET sample of the knee joint ligament was considered the control, but the test sample was coated. In this case, the total mass of the layers was estimated as ≤3 mg, and the mass of SWCNTs in it was ≤10 μg. In the body, the implant coating can be resorbed for a long time (more than a month), therefore, the content of free SWCNT particles in the body does not exceed the total background content of carbon-containing substances that are contained in the city air and which a person breathes every day.

FIG. 3 shows graphs showing the dependence of the tensile stress on the relative tension. The graphs 1 and 2 presented here correspond to the samples of bonds without a coating and with a coating layer, respectively. It can be seen that in the latter case, the tensile strength (~ 20%) and the modulus of elasticity (~ 30%) noticeably increase.

Let us note some important properties of the proposed coating method:

a coating layer of a composite biocompatible nanomaterial in the composition of a matrix of biological materials (albumin, collagen) and a filler of carbon nanotubes is formed on the surface of a synthetic implant ligaments by applying an aqueous dispersion on the surface and the action of infrared radiation in the wavelength range of 0.7-1.2 μm and low power per unit surface area 0.1-0.5 W / cm ² ;

low power of infrared radiation does not overheat the layer of composite biocompatible nanomaterial (temperature ≤50 ° C), as a result of which its high degree of biocompatibility is not disturbed or reduced;

a small amount (≤ 3 wt%) of single-walled carbon nanotubes in the nanomaterial can significantly increase the tensile strength (≥20%) and elastic modulus (≥30%) of the synthetic ligament implant and practically does not reduce the high degree of biocompatibility of the nanomaterial matrix.

Thus, the task has been completed. A method of manufacturing a joint ligament implant with a nanocomposite coating is proposed, which makes it possible to increase its mechanical properties and the degree of biocompatibility. This method is simple and affordable and promising for restoring the function of the damaged ligament of the knee joint, as well as other joints.

Sources of information

1. French patent 2.651.99.

2. RF patent 2567603.

3. US patent 8142501.

4. RF patent 2632114 - prototype.

Claims (5)

1. A method of manufacturing a knee ligament ligament implant, including applying a nanocomposite coating based on an auxiliary substance - a water-protein dispersion of carbon nanotubes on the surface of an implant blank, characterized in that a nanocomposite coating of a knee ligament implant is formed on the basis of a water-protein dispersion of carbon nanotubes by means of a water-vaporizing exposure to infrared radiation (IR) in the wavelength range of 0.7-1.2 microns and a power per unit surface area of 0.1-0.5 W / cm ² . 2. A method of manufacturing a knee ligament implant according to claim 1, characterized in that the concentration of the protein component in the water-protein dispersion of carbon nanotubes is in the range from 15 to 30 wt. %. 3. A method of manufacturing a knee ligament implant according to claim 1, characterized in that the concentration of carbon nanotubes in the water-protein dispersion of carbon nanotubes is in the range from 0.1 to 0.3 wt. %. 4. A method of manufacturing a knee ligament implant according to claim 1, characterized in that bovine serum albumin and bovine collagen are used as the protein component of the water-protein dispersion of carbon nanotubes. 5. A method of manufacturing a knee ligament implant according to claim 1, characterized in that infrared irradiation of a layer of a water-protein dispersion of carbon nanotubes applied to a knee ligament implant blank is performed while the implant blank rotates.

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