CN113388950A - Absorbable high-performance nanofiber woven tendon patch and preparation method thereof - Google Patents
Absorbable high-performance nanofiber woven tendon patch and preparation method thereof Download PDFInfo
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- CN113388950A CN113388950A CN202110659225.8A CN202110659225A CN113388950A CN 113388950 A CN113388950 A CN 113388950A CN 202110659225 A CN202110659225 A CN 202110659225A CN 113388950 A CN113388950 A CN 113388950A
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Abstract
The invention relates to an absorbable high-performance nanofiber woven tendon patch. The tendon patch is formed by weaving warp yarns and weft yarns, wherein the weft yarns form the longitudinal direction of the tendon patch, and the warp yarns form the transverse direction of the tendon patch; the warp yarns are biodegradable nanofiber yarns or biodegradable microfiber yarns, and the weft yarns are biodegradable nanofiber yarns; the biodegradable nanofiber yarn is a pure twisted nanofiber yarn prepared by obtaining twisted primary yarn formed by twisting oriented nanofibers by adopting an electrostatic spinning technology and then carrying out hot drafting treatment on the twisted primary yarn. The invention also provides a preparation method. The tendon patch prepared by the invention can effectively simulate a nanofiber multilevel structure of a natural tendon extracellular matrix, has an ultrahigh specific surface area, can provide more sites for adhesion and growth of cells, has ultrahigh mechanical properties, can provide reliable mechanical support for regeneration of new tissues, and is beneficial to regeneration and reconstruction of injured tendon tissues.
Description
Technical Field
The invention relates to the technical field of artificial tendons and artificial ligaments in sports medicine, in particular to an absorbable high-performance nanofiber woven tendon patch and a preparation method thereof.
Background
With the progress of human civilization and the pursuit of people for health, the outdoor exercises of people are remarkably increased. Due to the continuous occurrence of accidents and the increasingly obvious trend of social aging, more and more sports injuries are caused. Among them, tendon and ligament injuries, of which more than 50% are caused by trauma and degenerative diseases, are very common in the field of clinical medicine, especially in the field of sports medicine. Statistics show that there are approximately over 3000 million tendon injury patients worldwide per year and result in medical expenses in excess of 1560 billion dollars. Due to the low cellular content, low vascularization and very limited innervation of natural tendon tissue, there is essentially no possibility of natural healing, even if healing is accompanied by the formation of fibrotic scar tissue. The bad repair of tendon injury not only affects the quality of daily life, but also interrupts the sporting life of many top athletes. Therefore, the search for a novel method for promoting the physiological regeneration and repair of the tendon has extremely important clinical significance.
At present, three methods of tendon transplantation, autologous tendon transplantation, allogeneic tendon transplantation and tendon prosthesis replacement are mainly used for treating tendon injury. In addition to the scarcity of donors, the use of these grafts presents a number of risks, such as the susceptibility to donor area dysfunction, immune rejection, disease transmission, inadequate repair, failure to achieve functional reconstruction to meet the needs of the body, and the like. Compared with the multiple biological graft technologies such as autologous, allogeneic and xenogeneic technologies, the tissue engineering patch scaffold material provides a new idea for solving the problems.
Tissue engineering is a developing field, and with the development of materials science and biomedicine, the tissue engineering tendon transplantation is expected to become an ideal method for permanently curing tendon injury, so that the development of biological materials capable of being used for muscle repair is urgently needed. Various studies have shown that fiber-based scaffolds produced using a variety of fiber-based manufacturing techniques are considered suitable for replacing anisotropic tissues and promoting their healing, and the specific structure makes it possible to mimic collagen tissue to ensure mechanical support and tissue infiltration during regeneration, which provides a more promising approach for clinical treatment of tendon defects without autologous tendon transplantation. Compared with other fiber-based scaffold preparation technologies, the tendon patch with the textile structure has the mechanical property characteristics of rigidity and flexibility, and has obvious advantages. At present, commercially available tendon patches are processed by adopting a textile technology, such as polyester fiber tendon patches, carbon fiber tendon patches and the like, but the traditional textile tendon patches still have many defects, such as incapability of degradation, strong rejection reaction of organisms and the like. In addition, the yarns used by the existing textile tendon patch are all micron fiber yarns, so that the biological activity is poor, the number of cell adhesion sites is small, and the cell adhesion, growth and functional expression are not facilitated. A large number of researches show that compared with the microfiber, the nanofiber has a remarkably increased specific surface area, can provide more active sites for cell growth, and can effectively regulate and control the behavior of cells through a contact guiding effect. However, the existing nanofiber tendon patch has generally poor mechanical properties and cannot meet the requirements of actual use. How to reconstruct the regenerated tissue similar to the natural tendon in the aspects of histology, biomechanical characteristics and the like to solve the problem of tendon defect is a difficult problem which is aimed at by experts and scholars in the field of sports medicine at home and abroad.
For example, chinese patent publication No. CN 111450316 a specifically discloses an integrated scaffold for simulating mineralization of bone-tendon-bone to non-mineralization gradient structure, the scaffold includes warps and wefts, the warps are nanofiber yarns with zero mineral content, the wefts in the middle of the scaffold are nanofiber yarns with zero mineral content, and the wefts in the middle are sequentially nanofiber yarns with gradually increased mineral content. The integrated bracket for simulating the bone-tendon-bone mineralization-non-mineralization gradient structure constructed by the patent provides an ideal microenvironment for repairing tendon defects, realizes the guidance of tendon tissue regeneration after implantation, promotes tendon-bone healing, and is suitable for tendon-bone injury repair.
And patent publication No. CN 110331486A, a multi-layer structure nanofiber yarn knitted tendon scaffold, and preparation and application thereof. The yarn is of a multi-layer core-spun structure, and during preparation: the core layer yarn passes through a hollow rotary funnel to serve as a receiving device, a positive high-voltage power supply and a negative high-voltage power supply are respectively added to double needles on two sides, the electrospun nanofiber is twisted to the core layer yarn, double-layer continuous nanofiber yarn is prepared, then the double-layer nanofiber yarn serves as a core layer, natural polymer nanofibers are electrospun on the outer layer, and three-layer nanofiber yarn is prepared. And finally knitting the nanofiber yarns with different layers into a three-dimensional (3D) knitted scaffold by a knitting process for tendon repair. The scaffold has the characteristics of long-term enhancement of the mechanical property of regenerated tissues, good biocompatibility, and contribution to repair of tendon injury and recovery of normal functions.
Although the above patents all adopt the nanofiber yarn which is helpful for cell repair and regeneration to prepare the tendon scaffold, the tendon scaffold obtained by adopting the above technical scheme has poor mechanical properties and cannot meet the requirements of practical use, because the above patents only adopt the rotary funnel when specifically preparing the nanofiber yarn, and do not adopt a component which is combined with the rotary funnel to form a split electric field in the opposite surface of the rotary funnel, that is, the electric field does not perform orientation induction on the formed nanofiber, so that the obtained fiber presents a disordered phenomenon. The disorder of the fibers can not effectively stretch the fibers, so that the prepared yarn has poor mechanical property, the strength of the bracket is influenced finally, and the repair of tendon injury and the recovery of normal functions are not facilitated.
In view of this, there is a need for a nanofiber woven tendon patch that can truly simulate extracellular matrix, provide a favorable environment for cell repair and regeneration, and realize bioabsorbability, high bioactivity, high strength, and low immunogenicity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a nanofiber multistage structure capable of effectively simulating natural tendon extracellular matrix (ECM), which has an ultrahigh specific surface area, can provide reliable mechanical support for new tissue regeneration, is beneficial to regeneration and reconstruction of injured tendon tissues, and can be used for preparing an absorbable high-performance nanofiber woven tendon patch which is bioabsorbable, high in bioactivity, high in strength and low in immunogenicity, and a preparation method thereof.
The technical scheme adopted by the invention for realizing the purpose is as follows: an absorbable high-performance nanofiber woven tendon patch, which is woven by warp yarns and weft yarns, wherein the weft yarns form the longitudinal direction of the tendon patch, and the warp yarns form the transverse direction of the tendon patch; the warp yarns are biodegradable micro fiber yarns or biodegradable nano fiber yarns, and the weft yarns are biodegradable nano fiber yarns; the biodegradable nanofiber yarn is a pure twisted nanofiber yarn prepared by twisting oriented nanofibers by using an electrostatic spinning technology to form a twisted primary yarn and then carrying out hot drafting treatment on the twisted primary yarn.
The absorbable high-performance nanofiber woven tendon patch is characterized in that the biodegradable nanofiber yarn is processed by biodegradable natural high polymer, synthetic high polymer or a mixture of multiple high polymers; the natural high polymer is one or more of silk fibroin, gelatin, collagen, hyaluronic acid, chitosan and sodium alginate; the synthetic polymer is a copolymer formed by one or more monomers of poly-L-lactic acid, poly-L-lactide-caprolactone, lactic acid-glycolic acid copolymer, polyglycolic acid, polyhydroxybutyrate, hydroxybutyrate copolyester and polydioxanone.
In the absorbable high-performance nanofiber woven tendon patch, the biodegradable microfiber yarn is one or more of polylactic acid microfiber yarn, chitosan microfiber yarn, collagen microfiber yarn, gelatin microfiber yarn, polycaprolactone microfiber yarn, polylactic acid-glycolic acid copolymer microfiber yarn, silk fibroin microfiber yarn, and poly (p-dioxanone) microfiber yarn.
The absorbable high-performance nanofiber woven tendon patch has the length of 10-150mm and the width of 0.5-150mm, and the warp and weft yarn densities of the tendon patch include, but are not limited to, 60 yarns/10 cm × 90 yarns/10 cm, 63 yarns/10 cm × 150 yarns/10 cm, 138 yarns/10 cm × 238 yarns/10 cm, and 67 yarns/10 cm × 467 yarns/10 cm.
The absorbable high-performance nanofiber woven tendon patch described above has an appearance including, but not limited to, a ribbon shape, a round cord shape, and a multi-layered laminate shape.
A preparation method of an absorbable high-performance nanofiber woven tendon patch comprises the following steps:
(1) preparing natural high polymer, synthetic high polymer or a plurality of high polymer mixture spinning solutions;
(2) loading the spinning solution obtained in the step (1) into a double liquid storage device, respectively controlling the conveying of the spinning solution by a propelling device to double filament spraying heads, respectively adding positive and negative electric fields to the double filament spraying heads, spraying the nano fibers to a middle metal disc and a hollow metal rod to obtain oriented nano fibers, twisting the oriented nano fibers by the rotation of the metal disc to form a primary yarn, carrying out primary drafting by a primary yarn roller, carrying out hot drafting treatment, and finally receiving by a collecting roller to obtain biodegradable nano fiber yarns;
(3) weaving the biodegradable nanofiber yarn prepared in the step (2) as warp or weft by a weaving process in a weaving technology, or weaving the biodegradable nanofiber yarn prepared in the step (2) as warp and the biodegradable nanofiber yarn prepared in the step (2) as weft to obtain the absorbable high-performance nanofiber woven tendon fabric;
(4) and (4) cutting the absorbable high-performance nanofiber woven tendon fabric prepared in the step (3) according to the required size, and finally preparing the absorbable high-performance nanofiber woven tendon patch.
The preparation method of the absorbable high-performance nanofiber woven tendon patch comprises the step of preparing the spinning solution by mass concentration of 0.1-50%.
In the step (2), the liquid supply speed is 0.1-100ml/h, the voltage applied by the double-nozzle is 1-100Kv, the distance between the two nozzles is 5-100cm, the distance between the metal disc and the hollow metal rod is 1-50cm, and the rotating speed of the metal disc is 50-5000 rpm/min.
In the step (2), a hot drawing device is used for hot drawing, and the drawing temperature is set to be 40-500 ℃.
In the preparation method of the absorbable high-performance nanofiber woven tendon patch, the solvent for preparing the spinning solution in the step (1) includes, but is not limited to, one or more of hexafluoroisopropanol, trifluoroethanol, dichloromethane and trifluoroacetic acid.
The absorbable high-performance nanofiber woven tendon patch has the beneficial effects that: the tendon patch provided by the invention is prepared by combining an electrostatic spinning technology with a hot drawing technology to obtain high-performance (high orientation degree, high crystallinity and high strength) pure nanofiber twisted yarns, and then adopting a weaving machine weaving technology to prepare the high-performance nanofiber woven fabric tendon patch which is bioabsorbable, high in bioactivity, high in strength and low in immunogenicity.
The method has the following advantages and positive effects:
(1) the absorbable high-performance nanofiber woven fabric tendon patch prepared by the method is composed of pure nanofiber twisted yarns and micron fiber yarns or all-pure nanofiber twisted yarns, and breaks through the structural characteristics of the micron yarns of the traditional woven tendon patch; the bundle-shaped structure of the pure nanofiber twisted yarn highly simulates a natural tendon basic structure unit, namely a collagen fiber bundle-shaped structure, and the nanoscale can provide more growth sites for cells, so that cell adhesion is facilitated; the woven structure can provide mechanical properties for tissue regeneration and reconstruction, so the absorbable high-performance nanofiber woven fabric tendon patch is beneficial to regeneration and reconstruction of damaged tendon tissues. Not only solves the outstanding problems of low biological activity, incapability of degradation, strong rejection reaction of organisms and the like of the traditional artificial tendon patch, but also solves the problems that the mechanical property of the current nanofiber tendon patch is generally poor and the actual use requirement cannot be met.
(2) The absorbable high-performance nanofiber woven fabric tendon patch prepared by the invention is adjustable in density according to the warp and weft directions of the yarns, so that the size of the pores of the tendon patch is adjustable; the three-dimensional porous structure is favorable for cell infiltration and three-dimensional growth, and new tissues are formed along with cell adhesion and proliferation, so that the new tissues are integrated with an organism, and the damaged tendon regeneration construction is successful.
(3) The yarns used in the invention are all made of biodegradable polymers, and can be gradually degraded along with the regeneration process of tendon tissues after being transplanted in vivo; the degradation product is non-toxic and harmless, and can be discharged out of the body along with the metabolism of the human body, thereby improving the possibility of repairing the tendon injury on the premise of not causing secondary damage to the body and reducing the disease infection.
(4) The absorbable high-performance nanofiber woven fabric tendon patch prepared by the invention can be added with anti-inflammatory and angiogenesis promoting drugs or growth factors in the spinning process according to clinical requirements, so that the inflammatory reaction is reduced for tendon regeneration, the nutrition supply is improved, the tendon repair is promoted, and the body movement function is recovered.
The tendon patch can be used for repairing tendon injury and ligament injury, has the advantages of bioabsorption, high bioactivity, high strength, low immunogenicity, simple preparation method, suitability for industrial production and wide popularization and application value.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the biodegradable nanofiber yarn of the present invention;
fig. 2 is an SEM picture of a woven fabric tendon patch composed of Silk Fibroin (SF)/poly-l-lactic acid (PLLA) nanofiber yarns with different ratios in the longitudinal direction and PLLA microfiber yarns in the transverse direction;
fig. 3 is an X-ray diffraction spectrum of a woven fabric tendon patch formed by Silk Fibroin (SF)/poly-l-lactic acid (PLLA) nanofiber yarns with different ratios in the longitudinal direction and PLLA microfiber yarns in the transverse direction;
fig. 4 shows the tensile mechanical properties of a woven fabric tendon patch formed by Silk Fibroin (SF)/poly-l-lactic acid (PLLA) nanofiber yarns with different ratios in the longitudinal direction and PLLA microfiber yarns in the transverse direction;
FIG. 5 is SEM image of cell adhesion of human Achilles tendon cells growing for 7 days on a woven fabric tendon patch made of Silk Fibroin (SF)/poly L-lactic acid (PLLA) nanofiber yarns with different ratios in the longitudinal direction and PLLA micrometer fiber yarns in the transverse direction;
FIG. 6 is an immunofluorescence staining pattern of human Achilles tendon cells growing for 7 days on a woven fabric tendon patch composed of Silk Fibroin (SF)/poly L-lactic acid (PLLA) nanofiber yarns with different ratios in the longitudinal direction and PLLA micrometer fiber yarns in the transverse direction;
fig. 7 is a graph showing the proliferation status of human achilles tendon cells growing on a woven fabric tendon patch formed by Silk Fibroin (SF)/poly-L-lactic acid (PLLA) nanofiber yarns with different ratios in the longitudinal direction and PLLA microfiber yarns in the transverse direction for 1 day, 3 days, and 7 days, respectively;
FIG. 8 is a physical diagram and SEM photograph of a full PLLA nanofiber yarn woven fabric tendon patch with adjustable pore size, wherein (a) the physical diagram is shown, and (b) the SEM photograph is shown;
fig. 9 is a physical diagram and an SEM photograph of the absorbable high-performance nanofiber tendon patch prepared in the present invention, which are respectively prepared in a style 1: biodegradable nanofiber yarn-biodegradable microfiber yarn machine fabric tendon patch; style 2: biodegradable nanofiber yarn-biodegradable nanofiber yarn machine fabric tendon patch, wherein (a) the physical picture, (b) the SEM picture;
FIG. 10 is a schematic diagram showing the simulation of the electric field formed by the device after applying positive and negative voltages;
FIG. 11 is a diagram of a metal disc and a hollow metal rod inducing fiber orientation during the preparation of nanofiber yarn according to the present invention;
FIG. 12 is a schematic diagram of the nanofiber yarn formed by the rotation of the metal disc during the preparation of the nanofiber yarn according to the present invention.
Detailed Description
The invention is further explained in detail with reference to the drawings and the specific embodiments;
example 1
An absorbable high-performance nanofiber woven tendon patch is formed by weaving warp yarns and weft yarns, wherein the weft yarn direction forms the longitudinal direction of the tendon patch, and the warp yarn direction forms the transverse direction of the tendon patch; the warp yarns are biodegradable microfiber yarns or biodegradable nanofiber yarns, and the weft yarns are biodegradable nanofiber yarns; the biodegradable nanofiber yarn is a pure twisted nanofiber yarn prepared by twisting oriented nanofibers obtained by an electrostatic spinning technique and then subjecting the twisted primary yarn to a hot-drawing process, as shown in fig. 1. In this embodiment, the biodegradable nanofiber yarn is a pure twisted nanofiber yarn obtained by electrospinning high polymer poly-L-lactic acid (PLLA), and then the obtained pure twisted nanofiber yarn is used as weft yarn, and the biodegradable PLLA microfiber yarn is used as warp yarn, and the tendon patch with a strip-shaped appearance is formed by a weaving process. Wherein the density of warp and weft yarns of the tendon patch is 60 yarns/10 cm multiplied by 90 yarns/10 cm.
A preparation method of an absorbable high-performance nanofiber woven tendon patch comprises the following steps:
(1) preparing a spinning solution: weighing PLLA with the mass of 1.0g by using an electronic balance, dissolving the PLLA in 10mL of hexafluoroisopropanol, and stirring overnight on a magnetic stirrer at normal temperature to prepare PLLA spinning solution with the mass concentration of 10%;
(2) preparing biodegradable nanofiber yarns: loading the PLLA spinning solution with the mass concentration percentage of 10% obtained in the step (1) into a double liquid storage device, respectively controlling the PLLA spinning solution to be conveyed to double spinning nozzles by a propelling device, wherein the liquid supply speed is 0.8mL/h, respectively providing a high-voltage driving force of +/-12 kV for the spinning nozzles by using a high-voltage electrostatic generator, spraying nano fibers to a metal disc and a hollow metal rod to obtain oriented nano fibers, twisting the oriented nano fibers by rotating the metal disc to form primary yarns, collecting the primary yarns by a primary yarn roller, further conveying the primary yarns to a hot drawing device for hot drawing, and finally receiving the primary yarns by a collecting roller to obtain biodegradable nano fiber yarns; wherein the distance between the two nozzles is controlled to be 20cm, the distance between the metal disc and the hollow metal rod is controlled to be 10cm, the rotating speed of the metal disc is 250rpm, and the temperature of the hot drawing device is controlled to be 80 ℃; if the distance between the double-wire-spraying heads is too far, the electrostatic attraction effect is weakened, and well-ordered fibers cannot be effectively formed;
(3) and (3) weaving the biodegradable nanofiber yarn prepared in the step (2) as weft yarn and the biodegradable microfiber yarn as warp yarn by using a weaving process in a weaving technology to obtain the biodegradable nanofiber yarn-biodegradable microfiber yarn machine fabric tendon patch.
Example 2
The same points as those in example 1 are not repeated, except that in this example, the biodegradable nanofiber yarn is a pure twisted nanofiber yarn obtained by electrospinning a mixture of high polymer Silk Fibroin (SF) and poly-l-lactic acid (PLLA), and then the tendon patch with a strip-shaped appearance is formed by weaving warp yarns and weft yarns. Wherein the density of warp and weft yarns of the tendon patch is 63 yarns/10 cm multiplied by 150 yarns/10 cm.
When preparing a spinning solution, weighing 0.2g of SF and 0.8g of PLLA by using an electronic balance, dissolving the SF and the PLLA in 10mL of hexafluoroisopropanol, and stirring overnight on a magnetic stirrer at normal temperature to prepare an SF/PLLA blended spinning solution with the mass concentration percentage of 10%; loading the prepared SF/PLLA spinning solution with the mass concentration of 10% into a double liquid storage device to obtain biodegradable nanofiber yarns; and finally, interweaving the biodegradable nanofiber yarns as weft yarns and the biodegradable PLLA (polylactic acid) micron fiber yarns as warp yarns on a loom according to a plain weave by using a weaving process in a weaving and weaving technology, wherein the warp yarn density is 63 yarns/10 cm, and the weft yarn density is 150 yarns/10 cm, so as to prepare the biodegradable nanofiber yarn-biodegradable micron fiber yarn machine fabric tendon patch capable of absorbing high performance.
Example 3
The same points as those in example 2 will not be repeated, except that in this example, the tendon patch has warp and weft yarn density of 138 pieces/10 cm × 238 pieces/10 cm.
When preparing a spinning solution, weighing 0.35g of SF and 0.65g of PLLA by using an electronic balance, dissolving the SF and the PLLA in 10mL of hexafluoroisopropanol, and stirring the mixture overnight on a magnetic stirrer at normal temperature to prepare an SF/PLLA blended spinning solution with the mass concentration percentage of 10%; loading the prepared SF/PLLA spinning solution with the mass concentration of 10% into a double liquid storage device to obtain biodegradable nanofiber yarns; and finally, interweaving the biodegradable nanofiber yarns as weft yarns and the biodegradable PLLA (polylactic acid) microfiber yarns as warp yarns on a loom according to a plain weave by using a weaving process in a weaving and weaving technology, wherein the warp yarn density is 138 pieces/10 cm, and the weft yarn density is 238 pieces/10 cm, so as to prepare the biodegradable nanofiber yarn-biodegradable microfiber yarn machine fabric tendon patch which can absorb high performance.
Example 4
The same points as those in example 2 are not repeated, except that in this example, the tendon patch has a warp and weft yarn density of 67 pieces/10 cm × 467 pieces/10 cm.
When preparing a spinning solution, weighing 0.5g of SF and 0.5g of PLLA by using an electronic balance, dissolving the SF and the PLLA in 10mL of hexafluoroisopropanol, and stirring overnight on a magnetic stirrer at normal temperature to prepare an SF/PLLA blended spinning solution with the mass concentration percentage of 10%; loading the prepared SF/PLLA spinning solution with the mass concentration of 10% into a double liquid storage device to obtain biodegradable nanofiber yarns; and finally, interweaving the biodegradable nanofiber yarns as weft yarns and the biodegradable PLLA (polylactic acid) micron fiber yarns as warp yarns on a loom according to a plain weave by using a weaving process in a weaving and weaving technology, wherein the warp yarn density is 67 pieces/10 cm, and the weft yarn density is 467 pieces/10 cm, so as to prepare the biodegradable nanofiber yarn-biodegradable micron fiber yarn machine fabric tendon patch which can absorb high performance. In actual use, the required size is cut at will for use.
Different ratios of SF/PLLA nanofiber yarn-PLLA microfiber yarn woven tendon patches made from examples 1-4 are shown in fig. 2. The tendon patch is of a three-dimensional woven structure, so that infiltration and three-dimensional growth of cells are facilitated, the nanofiber can provide more adhesion sites for the cells, the high length-diameter ratio of the nanofiber yarn simulates a tendon structural unit-collagen fiber bundle, and the three-dimensional braided structure provides mechanical support.
The different ratios of SF/PLLA nanofiber yarn-PLLA microfiber yarn woven tendon patches prepared in the above examples 1-4 were analyzed for crystallization by a wide-angle X-ray diffractometer, respectively, with a scanning range of 2 θ being 5 ° to 60 ° and a scanning speed of 5 °/min. The XRD pattern of the tendon patch is shown in fig. 3, and the intensity of the diffraction peak of the tendon patch decreases gradually near 16.4 ° when the SF content increases.
Then, the mechanical properties of the tendon patch in the longitudinal direction were analyzed by a uniaxial tensile tester for the SF/PLLA nanofiber yarn-PLLA microfiber yarn woven tendon patch prepared in the above examples 1-4 with different ratios, the clamping distance was 20mm, and the tensile speed was 120 mm/min. The load-elongation curve of the tendon patch is shown in fig. 4, and shows a trend that the breaking load gradually decreases with the increase of the SF content, and has the same rule as the XRD data analysis.
The SF/PLLA nanofiber yarn-PLLA microfiber yarn woven tendon patches prepared in examples 1-4 with different ratios were cut into round samples with a radius of about 0.35mm, placed in a biological safety cabinet for UV sterilization for 2h, soaked in alcohol at 4 ℃ for 2h, and soaked in culture solution overnight. Cell density per sample was 1X 105The human achilles tendon cells were planted on the samples, respectively, and cultured in a cell incubator for 7 d. On day 7, fixing the cells on the sample by glutaraldehyde to obtain a cell-sample community, dehydrating with gradient alcohol, drying, spraying gold for 90s, and observing the adhesion condition of the cells by a scanning electron microscope. As shown in fig. 5, the SEM picture of human achilles tendon cell adhesion shows that the cells can be uniformly adhered and grown on the tendon patch, and the cells are more adhered on the nanofiber yarn than the microfiber yarn; the porous structure of the three-dimensional woven structure indicates that cells are able to grow inward.
On the 7 th day, fixing cells on a sample by using paraformaldehyde, dyeing the cell-sample community by using far infrared DNA dye and phalloidin after penetrating and sealing, and observing the adhesion and three-dimensional growth state of the cells on the sample under a laser confocal microscope. An immunofluorescence photograph of human achilles tendon cells on a sample is shown in fig. 6, wherein the cell nucleus is larger and is circular or oval, which indicates that the sample is nontoxic and is beneficial to cell growth; the cytoskeleton grows along the direction of the nano fibers, almost the whole sample is covered, and the growth state of the cells is good.
MTT was used to test the proliferation of the SF/PLLA nanofiber woven tendon patch cell-sample community at different ratios in examples 1-4, and the cellular absorbance values were measured at day 1, day 3, and day 7, respectively, as shown in FIG. 7. The results showed that the OD values of the cells of each group increased gradually with time, and on the third day, the OD values of the 35:65 and 50:50 groups were more significant than those of the other two groups (. P < 0.05), and on day 7, the difference in significance was further increased (. P < 0.001); and OD values for 35:65 and 50:50 groups were significantly higher than 20:80 groups at day 7 (P < 0.05). The prepared tendon patch is free from cytotoxicity, cells can gradually proliferate on a sample along with time, and the sample has good biocompatibility. In addition, the addition of the natural polymer SF promotes the proliferation of cells.
Example 5
The same as example 1 is not repeated, but in this example, the prepared PLLA nanofiber yarns are used as warp yarns and weft yarns, and the tendon patches with the both warp and weft directions of the PLLA nanofiber yarns are prepared by weaving the PLLA nanofiber yarns on a weaving machine according to the warp and weft yarn densities of 60/10 cm × 90/10 cm, 63/10 cm × 150/10 cm, 138/10 cm × 238/10 cm, 67/10 cm × 467/10 cm.
In the full PLLA nanofiber yarn woven tendon patch with adjustable pore size prepared in this embodiment, as shown in fig. 8, the pore size of the tendon patch can be adjusted by adjusting the yarn density, so that three-dimensional tendon patches with different pore sizes are prepared to meet different clinical requirements.
Fig. 9 is a physical diagram and an SEM photograph of the absorbable high-performance nanofiber tendon patch prepared in the present invention, which are respectively prepared in a style 1: biodegradable nanofiber yarn-biodegradable microfiber yarn machine fabric tendon patch; style 2: biodegradable nanofiber yarn-biodegradable nanofiber yarn machine fabric tendon patch, wherein (a) a physical drawing and (b) SEM pictures are included.
The principle of the invention for preparing the oriented and stretched nano-fiber is as follows: the two nozzles are oppositely arranged and respectively charged with opposite high-voltage static electricity, and then a high-voltage electrostatic field is formed in the whole device area. As can be seen from the device electric field simulation fig. 10 and fig. 11 and 12, due to the combined action of the metal disk and the hollow metal rod, the electric field lines are split when approaching the metal disk and the hollow metal rod, and are respectively directed to the metal disk and the hollow metal rod. The nano fibers sprayed from the double spray heads are oppositely charged, fly to the area formed by the metal disc and the hollow metal rod under the action of electrostatic attraction, and are respectively pulled to the metal disc and the hollow metal rod under the action of an electric field force generated by the split electric field lines close to the metal disc and the hollow metal rod, so that the orientation and the stretching of the nano fibers are realized, the nano fibers are changed into bundles in a well-ordered sequence, and a foundation is laid for the subsequent hot drawing treatment. However, if only the metal disc is used, a split electric field cannot be formed, which causes the fibers to be in a disordered state, and the fibers cannot be effectively stretched, resulting in poor strength of the obtained twisted nanofibers. That is, the invention firstly orients and stretches the nano-fiber, then carries out hot drawing, then obtains the high-strength pure nano-fiber twisted yarn, and then utilizes the pure nano-fiber twisted yarn to weave the tendon patch. As is known, human collagen fibers are fiber bundles with the diameter of 0.5-20 mu m and distributed in bundles, while the tendon patch is woven by pure nanofiber yarns with bundle-shaped structures, so that the tendon patch can highly simulate the natural tendon basic structural unit, namely the collagen fiber bundle-shaped structures, and on one hand, the nano-scale can provide more growth sites for cells and is beneficial to cell adhesion; on the other hand, the woven structure can provide mechanical properties for tissue regeneration and reconstruction, so the absorbable high-performance nanofiber woven fabric tendon patch is beneficial to regeneration and reconstruction of damaged tendon tissues. Therefore, the tendon patch prepared by the invention not only can provide a favorable environment for cell repair and regeneration, but also solves the technical problems that the existing nanofiber tendon patch has generally poor mechanical properties and cannot meet the actual use requirements.
The tendon patch prepared by the invention is prepared by firstly obtaining high-performance (high orientation degree, high crystallinity and high strength) pure nanofiber twisted yarns through an electrostatic spinning technology combined with a hot drawing process, then preparing the high-performance nanofiber woven fabric tendon patch capable of being bioabsorbed, high in bioactivity, high in strength and low in immunocompetence by adopting a weaving process of a weaving machine, breaking through the structure of micron fiber yarns and composite yarns of the traditional woven artificial tendon patch, effectively simulating the nanofiber multilevel structure of a natural tendon extracellular matrix (ECM), having an ultrahigh specific surface area, providing more sites for the adhesion and growth of cells, providing reliable mechanical support for the regeneration of new tissues, being beneficial to the regeneration and reconstruction of damaged tendon tissues, and thoroughly solving the outstanding problems of low bioactivity, non-degradability, strong rejection reaction and the like of the traditional artificial tendon patch, is worthy of being widely popularized and applied.
The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. All equivalent changes or modifications made according to the spirit of the present disclosure should be covered within the scope of the present disclosure.
Claims (10)
1. An absorbable high-performance nanofiber woven tendon patch, which is woven by warps and wefts and is characterized in that: the weft yarns form the longitudinal direction of the tendon patch, and the warp yarns form the transverse direction of the tendon patch; the warp yarns are biodegradable micro fiber yarns or biodegradable nano fiber yarns, and the weft yarns are biodegradable nano fiber yarns; the biodegradable nanofiber yarn is a pure twisted nanofiber yarn prepared by twisting oriented nanofibers by using an electrostatic spinning technology to form a twisted primary yarn and then carrying out hot drafting treatment on the twisted primary yarn.
2. The absorbable high performance nanofiber woven tendon patch of claim 1, which is characterized by: the biodegradable nanofiber yarn is processed by biodegradable natural high polymer, synthetic high polymer or mixture of a plurality of high polymers; the natural high polymer is one or more of silk fibroin, gelatin, collagen, hyaluronic acid, chitosan and sodium alginate; the synthetic polymer is a copolymer formed by one or more monomers of poly-L-lactic acid, poly-L-lactide-caprolactone, lactic acid-glycolic acid copolymer, polyglycolic acid, polyhydroxybutyrate, hydroxybutyrate copolyester and polydioxanone.
3. The absorbable high performance nanofiber woven tendon patch of claim 1, which is characterized by: the biodegradable micron fiber yarn is one or more of polylactic acid micron fiber yarn, chitosan micron fiber yarn, collagen micron fiber yarn, gelatin micron fiber yarn, polycaprolactone micron fiber yarn, polylactic acid-glycolic acid copolymer micron fiber yarn, silk fibroin micron fiber yarn and poly (p-dioxanone) micron fiber yarn.
4. The absorbable high performance nanofiber woven tendon patch of claim 1, which is characterized by: the tendon patch has a length of 10-150mm and a width of 0.5-150mm, and the warp and weft yarn densities of the tendon patch include, but are not limited to, 60/10 cm × 90/10 cm, 63/10 cm × 150/10 cm, 138/10 cm × 238/10 cm, 67/10 cm × 467/10 cm.
5. The absorbable high performance nanofiber woven tendon patch of claim 1, which is characterized by: the tendon patch appearance includes, but is not limited to, a ribbon shape, a round cord shape, a multi-layered laminate shape.
6. A preparation method of an absorbable high-performance nanofiber woven tendon patch is characterized by comprising the following steps:
(1) preparing natural high polymer, synthetic high polymer or a plurality of high polymer mixture spinning solutions;
(2) loading the spinning solution obtained in the step (1) into a double liquid storage device, respectively controlling the conveying of the spinning solution by a propelling device to double filament spraying heads, respectively adding positive and negative electric fields to the double filament spraying heads, spraying the nano fibers to a middle metal disc and a hollow metal rod to obtain oriented nano fibers, twisting the oriented nano fibers by the rotation of the metal disc to form a primary yarn, carrying out primary drafting by a primary yarn roller, carrying out hot drafting treatment, and finally receiving by a collecting roller to obtain biodegradable nano fiber yarns;
(3) weaving the biodegradable nanofiber yarn prepared in the step (2) as warp or weft by a weaving process in a weaving technology, or weaving the biodegradable nanofiber yarn prepared in the step (2) as warp and the biodegradable nanofiber yarn prepared in the step (2) as weft to obtain the absorbable high-performance nanofiber woven tendon fabric;
(4) and (4) cutting the absorbable high-performance nanofiber woven tendon fabric prepared in the step (3) according to the required size, and finally preparing the absorbable high-performance nanofiber woven tendon patch.
7. The method for preparing the absorbable high-performance nanofiber woven tendon patch as claimed in claim 6, is characterized in that: the mass concentration percentage of the spinning solution is 0.1-50%.
8. The method for preparing the absorbable high-performance nanofiber woven tendon patch as claimed in claim 6, is characterized in that: in the step (2), the liquid supply speed is 0.1-100ml/h, the voltage applied by the double-spray-head is 1-100Kv, the distance between the two spray heads is 5-100cm, the distance between the metal disc and the hollow metal rod is 1-50cm, and the rotating speed of the metal disc is 50-5000 rpm/min.
9. The method for preparing the absorbable high-performance nanofiber woven tendon patch as claimed in claim 6, is characterized in that: in the step (2), a hot drawing device is adopted for hot drawing treatment, and the drawing temperature is set to be 40-500 ℃.
10. The method for preparing the absorbable high-performance nanofiber woven tendon patch as claimed in claim 6, is characterized in that: the solvent for preparing the spinning solution in the step (1) includes, but is not limited to, one or more of hexafluoroisopropanol, trifluoroethanol, dichloromethane and trifluoroacetic acid.
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