CN113332497A

CN113332497A - Double-sided bracket and preparation method and application thereof

Info

Publication number: CN113332497A
Application number: CN202110485196.8A
Authority: CN
Inventors: 胥伟华; 陈春英; 闫娜
Original assignee: National Center for Nanosccience and Technology China; Institute of Genetics and Developmental Biology of CAS
Current assignee: National Center for Nanosccience and Technology China; Institute of Genetics and Developmental Biology of CAS
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-09-03
Anticipated expiration: 2041-04-30
Also published as: CN113332497B

Abstract

The invention provides a double-sided bracket and a preparation method and application thereof. The double-sided bracket includes: chitosan collagen composite fiber layer and hydroxyapatite modified chitosan collagen composite fiber layer. The scaffold material has a double-layer structure, and the chitosan collagen composite fiber layer is a barrier layer and maintains the space for tissue regeneration; the chitosan collagen composite fiber layer modified by hydroxyapatite is a functional layer and provides active substances for tissue regeneration; and the stem cells are adhered to the functional layer in a combined manner, so that a good microenvironment is provided for tissue regeneration, and a better repairing effect is achieved. In addition, the material has good biocompatibility and the capacity of promoting cell proliferation and differentiation. The double-sided scaffold material is combined with stem cells for tissue regeneration, is beneficial to the retention of exogenous stem cells at an injured part, provides a better tissue regeneration microenvironment for endogenous stem cells and exogenous stem cells, and has guiding significance for the clinical application of tissue regeneration.

Description

Double-sided bracket and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials and stem cells, in particular to a double-sided scaffold and a preparation method and application thereof, and particularly relates to an implanted cell-loaded scaffold material in the field of tissue repair research and a preparation method and application thereof.

Background

Periodontal tissue is composed of the gingiva, the periodontal ligament, the cementum, and the alveolar bone, and is a well-defined tissue, which mainly functions to provide physical and mechanical support for the teeth. Severe periodontitis is an inflammation induced by oral bacterial biofilm, which can cause severe destruction of the soft and hard tissues of the periodontal tissue, thereby impairing the function and beauty of teeth. Although current treatment methods can limit disease progression by controlling aspects of inflammation, complete periodontal regeneration is unpredictable. The ultimate goal of periodontal therapy is to regenerate lost periodontal tissue, including reattachment of the periodontal ligament to the newly formed cementum and alveolar bone. This requires a highly coordinated spatiotemporal healing response, including reattachment of periodontal ligament fibers to the previously contaminated root surface, and bone formation within the periodontal defect. In addition to the challenges presented by the complex structure of the periodontal tissue, periodontal healing is further complicated by the avascular nature of the tooth surface. It is therefore essential to control key wound healing events to achieve periodontal regeneration using various methods of tissue engineering.

The healing principle of periodontal wound is the same as that of other parts of the body. First, a fibrin clot between the flap edge and the root surface is created in the wound surface, maintaining a space for regeneration. After the fibrin clot is broken, the long epithelial cells first attach to the root surface and regeneration fails. Fibrin is a temporary matrix that promotes cell recruitment, regulating the growth and differentiation of stem cells. When the area of injury is large, the fiber clot is prone to collapse, resulting in epithelial cells adhering to the root surface. In the 80's of the 20 th century, guided tissue regeneration with periodontal regeneration promotion by means of a physical barrier membrane, which has functions of space maintenance and selective cell regeneration, was proposed. However, due to the complexity of the oral environment, avascular root surface, microbial membranes and inflammation, the therapeutic effects of GTR techniques show great variability.

At present, the stent materials selected in the repair and reconstruction of periodontal tissues are mainly as follows: acellular tissue matrix, degradable high molecular material and natural high molecular material. The physicochemical properties (porosity, mechanical properties and the like) of the acellular tissue matrix are poor in modifiability; the degradable high molecular material lacks a cell recognition signal, is not beneficial to cell recognition and adhesion, and acidic substances formed during degradation can cause peripheral inflammation; although natural polymer materials have good cell compatibility, the mechanical properties and easy degradation limit the application of the natural polymer materials in periodontal regeneration.

Therefore, multifunctional scaffold materials for mesenchymal stem cell therapy in combination with corresponding tissues require further research.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a double-sided stent and a preparation method and application thereof, in particular to an implanted cell-carrying stent material in the field of tissue repair research and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a double-sided stent comprising: chitosan collagen composite fiber layer and hydroxyapatite modified chitosan collagen composite fiber layer.

In the invention, the double-sided bracket comprises a chitosan collagen composite fiber layer and a chitosan collagen composite fiber layer modified by hydroxyapatite, wherein the chitosan collagen composite fiber layer is an unmodified chitosan collagen composite fiber layer and is a compact layer (compact layer), the smaller aperture of the compact layer hinders the invasion of epithelial cells, and a certain time is provided for the functional layer to mineralize on the surface of a tooth root; the chitosan collagen composite fiber layer modified by the hydroxyapatite is a functional layer (functional layer), the larger aperture of the functional layer is beneficial to the contact between cells and the hydroxyapatite, and the local calcium and phosphorus ion concentration is improved to promote osteogenic differentiation. When the double-sided scaffold is used for inoculating stem cells, the cell forms of the cells in the functional layer and the compact layer are good, which shows that the two layers have good biocompatibility; and the cells have better spreading shape, proliferation and migration capacity on the scaffold material.

Preferably, the chitosan collagen composite fiber layer is prepared by electrostatic spinning of chitosan collagen spinning solution containing RGD (Arg-Gly-Asp).

Preferably, the chitosan collagen spinning solution containing RGD comprises, by mass: 1-3% of chitosan, 1-15% of collagen, 0.25-0.75% of polyoxyethylene and the balance of solvent.

The chitosan content is 1-3%, such as 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, etc., based on 100% of the chitosan collagen spinning solution containing RGD.

The collagen content is 1-15%, such as 1%, 2%, 4%, 6%, 8%, 9%, 10%, 12%, 13%, 14%, 15%, etc., based on 100% by mass of the chitosan collagen spinning solution containing RGD.

The polyoxyethylene content is 0.25-0.75% by mass of the chitosan collagen spinning solution containing RGD, such as 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.75% and the like.

Preferably, the concentration of RGD in the RGD-containing chitosan collagen spinning solution is 0.02-1mg/mL, such as 0.02mg/mL, 0.05mg/mL, 0.08mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, and the like.

Preferably, the chitosan has a molecular weight of 10⁴-10⁵Da, e.g. 1X 10⁴Da、2.5×10⁴Da、4×10⁴Da、5×10⁴Da、6×10⁴Da、7×10⁴Da、8×10⁴Da、9×10⁴Da、1×10⁵Da, etc.

Preferably, the collagen is type i collagen, preferably type i collagen of bovine tendon.

Preferably, the polyethylene oxide has a molecular weight of 10⁴-10⁵Da, e.g.10⁴Da、2.5×10⁴Da、4×10⁴Da、5×10⁴Da、6×10⁴Da、7×10⁴Da、8×10⁴Da、9×10⁴Da、10⁵Da, etc.

Preferably, the solvent is an aqueous acetic acid solution, preferably 85-95 wt% (e.g., 85 wt%, 86 wt%, 87 wt%, 88 wt%, 90 wt%, 92 wt%, 94 wt%, 95 wt%, etc.) aqueous acetic acid.

Preferably, the chitosan collagen composite fiber layer modified by hydroxyapatite comprises the chitosan collagen composite fiber layer and hydroxyapatite deposited between the chitosan collagen composite fiber layers.

Preferably, the mass of the hydroxyapatite accounts for 5-20% of the total mass of the hydroxyapatite-modified chitosan collagen composite fiber layer, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 14%, 16%, 18%, 20%, etc.

Preferably, the fiber diameter of the double-sided scaffold is 0.5-1.5 μm, such as 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, and the like.

Preferably, the pore size of the chitosan collagen composite fiber layer is 10 μm or less, for example, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, etc.

Preferably, the pore size of the hydroxyapatite-modified chitosan collagen composite fiber layer is 20-30 μm, such as 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, and the like. Wherein the pore size is expressed in terms of the diameter of the pores.

Preferably, the double-sided scaffold further comprises fibrinogen and thrombin. The function of adding the fibrin is to further enhance the mechanical property of the prepared scaffold material and obtain smaller pore diameter, thereby further promoting wound repair. The thrombin acts to promote the degree of cross-linking of the scaffold material fibre network.

Preferably, the fibrin content of the double-sided scaffold is 1-20%, such as 1%, 2%, 4%, 6%, 8%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.

Preferably, the content of thrombin in the double-sided scaffold is 10-30U/cm²E.g. 10U/cm²、12U/cm²、15U/cm²、18U/cm²、20U/cm²、22U/cm²、25U/cm²、30U/cm²And the like.

In a second aspect, the present invention provides a method for preparing a double-sided stent as described in the first aspect, the method comprising the steps of:

(1) preparing a chitosan collagen composite fiber layer through electrostatic spinning;

(2) soaking the chitosan collagen composite fiber layer in hydroxyapatite deposition liquid to obtain a chitosan collagen composite fiber layer modified by hydroxyapatite;

(3) and bonding the chitosan collagen composite fiber layer with the hydroxyapatite-modified chitosan collagen composite fiber layer to obtain the double-sided scaffold.

Preferably, in the step (1), the specific preparation method of the chitosan collagen composite fiber layer comprises the following steps: mixing chitosan, collagen, polyoxyethylene, RGD and a solvent to obtain a spinning solution, preparing the chitosan collagen composite fiber by an electrostatic spinning technology, washing and drying to obtain the chitosan collagen composite fiber layer.

Preferably, the parameters of the electrospinning technique are: a20-23 gauge needle (e.g., 20 gauge, 21 gauge, 22 gauge, 23 gauge), an extrusion rate of 0.1-1cm/min (e.g., 0.1cm/min, 0.2cm/min, 0.4cm/min, 0.6cm/min, 0.8cm/min, 1cm/min, etc.), a low pressure of-5-0 kV (e.g., -5kV, -4kV, -3kV, -2kV, -1kV, 0kV, etc.), and a high pressure of 15-25kV (e.g., 15kV, 16kV, 18kV, 20kV, 22kV, 24kV, 25kV, etc.) are used.

In the present invention, spinning is performed with the parameters of the electrospinning technique described above, and a fiber diameter of appropriate thickness can be spun to obtain an appropriate pore diameter of the dense layer upon drying.

Preferably, after the soaking, the soaking is further performed with washing, which is performed with ethanol at least 3 times (e.g., 3 times, 4 times, 5 times, 6 times, etc.), and then with water at least 3 times (e.g., 3 times, 4 times, 5 times, 6 times, etc.).

Preferably, the drying is freeze drying, the temperature of the drying is-90 to-70 ℃, such as-90 ℃, 85 ℃, 82 ℃, 80 ℃, 78 ℃, 75 ℃, 70 ℃ and the like, and the time of the drying is 12 to 24h, such as 12h, 14h, 16h, 18h, 20h, 22h, 24h and the like.

Preferably, in step (2), the concentration of the hydroxyapatite deposit solution is 0.1-10mg/mL, such as 0.1mg/mL, 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, etc.

Preferably, in step (2), the soaking is performed by shaking on a shaking table, the shaking frequency is 70-90rpm, such as 70rpm, 75rpm, 80rpm, 85rpm, 90rpm, and the like, and the shaking time is 20-60min, such as 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, and the like.

Preferably, in step (2), after the soaking, washing is further performed, the washing uses ethanol with a volume concentration of 40-60% (e.g., 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, etc.), and the number of times of washing is at least 3 times, e.g., 3 times, 4 times, 5 times, 6 times, etc.

Preferably, in the step (3), the specific steps of bonding are as follows: and after the chitosan collagen composite fiber layer and the chitosan collagen composite fiber layer modified by the hydroxyapatite are jointed, adding a fibrinogen solution and thrombin from one side of the chitosan collagen composite fiber layer for reaction to obtain the double-sided bracket.

In the present invention, the reason why the fibrinogen solution and thrombin are added to the unmodified chitosan collagen composite fiber layer side, i.e., the dense layer, is selected as follows: the pore diameter of the compact layer can be improved, thereby achieving the purpose of reducing the pore diameter.

Preferably, the fibrinogen solution has a concentration of 1-9mg/mL, such as 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, etc., and the solvent is physiological saline.

Preferably, the thrombin concentration is 5-50U/mL, such as 5U/mL, 10U/mL, 15U/mL, 20U/mL, 25U/mL, 30U/mL, 35U/mL, 40U/mL, 45U/mL, 50U/mL, and the like.

Preferably, the reaction temperature is 30-40 ℃, such as 30 ℃, 32 ℃, 34 ℃, 36 ℃, 38 ℃, 40 ℃ and the like, and the reaction time is 20-30h, such as 20h, 22h, 24h, 26h, 28h, 30h and the like.

Preferably, the reaction is performed by shaking in a constant temperature shaker at a frequency of 70-90rpm, such as 70rpm, 75rpm, 80rpm, 85rpm, 90rpm, etc.

In a third aspect, the present invention provides the use of a double-sided scaffold according to the first aspect in the preparation of a tissue regeneration assisting material.

In the invention, the double-sided bracket has the function of promoting osteogenesis, and meanwhile, the paracrine function of stem cells can construct a good regeneration microenvironment, so that the double-sided bracket has certain clinical application in the aspect of promoting tissue regeneration.

Preferably, the tissue is periodontal tissue.

The scaffold material has good capability of promoting odontogenesis and osteogenesis, and promotes better tissue repair. Wherein, the bracket material can obstruct epithelial cell from attaching to the root surface and avoid incomplete regeneration of periodontal.

In a fourth aspect, the present invention provides a tissue regeneration assisting material comprising stem cells and the double-sided scaffold of the first aspect.

Preferably, the stem cells are dental stem cells.

Preferably, the number of passages of the stem cells is less than 5, e.g., 4, 3, 2, etc.

Preferably, the load of the stem cells is 10⁷-10⁹Individual cell/cm²E.g. 10⁷Individual cell/cm²、5×10⁷Individual cell/cm²、10⁸Individual cell/cm²、5×10⁸Individual cell/cm²、10⁹Individual cell/cm²And the like.

In a fifth aspect, the present invention provides a method for preparing the tissue regeneration assisting material according to the fourth aspect, wherein the method for preparing the tissue regeneration assisting material comprises:

(a) wetting a double-sided stent as described in the first aspect;

(b) and inoculating the stem cells to one side of the chitosan collagen composite fiber layer of the double-sided bracket, and culturing to obtain the tissue regeneration auxiliary material.

Preferably, in step (a), the wetting is with PBS buffer.

Preferably, in step (a), the temperature of the wetting is 35-40 ℃, such as 35 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ and the like, and the time of the wetting is 2-4h, such as 2h, 2.5h, 3h, 3.5h, 4h and the like.

Preferably, in step (b), the stem cells are inoculated in an amount of 10⁷-10⁹Individual cell/cm²E.g. 10⁷Individual cell/cm²、5×10⁷Individual cell/cm²、10⁸Individual cell/cm²、5×10⁸Individual cell/cm²、10⁹Individual cell/cm²And the like.

Preferably, in step (b), the temperature of the culture is 35-40 ℃, such as 35 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ and the like, and the time of the culture is 2-3 days, such as 2 days, 2.5 days, 3 days and the like.

Compared with the prior art, the invention has the following beneficial effects:

(1) the scaffold material has a double-layer structure, and the chitosan collagen composite fiber layer is a barrier layer and maintains the space for tissue regeneration; the chitosan collagen composite fiber layer modified by hydroxyapatite is a functional layer and provides active substances for tissue regeneration; the barrier layer is combined with stem cells to be adhered to the functional layer, so that a good microenvironment is provided for tissue regeneration, and a good repairing effect is achieved; the scaffold has good biocompatibility and the capacity of promoting cell proliferation and differentiation, and the cells have good spreading form, proliferation and migration capacity on the scaffold material;

(2) the preparation method of the bracket material is simple, mild in addition, low in cost and easy to store; organic reagents are not used in the preparation process, so that the harm of organic reagent residues to tissues is avoided;

(3) the double-sided bracket has good capacity of promoting odontogenesis and osteogenesis, promotes better repair of tissues, can block epithelial cells from attaching to the root surface, and avoids incomplete regeneration of periodontal; the double-sided scaffold combined with the stem cells is used for tissue regeneration, is beneficial to the retention of exogenous stem cells at an injured part, provides a better tissue regeneration microenvironment for endogenous stem cells and exogenous stem cells, and has guiding significance for the clinical application of tissue regeneration.

Drawings

Fig. 1 is a scanning electron microscope image of the functional layer of the double-sided support provided in example 1 at a magnification of 1.0K.

FIG. 2 is a scanning electron micrograph of the dense layer of the double-sided stent provided in example 1 at a magnification of 1.0K.

Fig. 3 is a scanning electron microscope image of the functional layer of the double-sided support provided in example 1 at a magnification of 5.0K.

FIG. 4 is a scanning electron micrograph of the dense layer of the double-sided stent provided in example 1 at a magnification of 5.0K.

Fig. 5 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 2.

Fig. 6 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 3.

Fig. 7 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 4.

FIG. 8 is a fluorescence image of cell adhesion after stem cell seeding of the double-sided scaffolds provided in example 5.

Fig. 9 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 6.

Fig. 10 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 7.

Fig. 11 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 8.

Fig. 12 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 9.

Fig. 13 is a scanning electron microscope image of the hydroxyapatite-modified chitosan collagen composite fiber provided in example 10.

Fig. 14 is a scanning electron microscope image of the hydroxyapatite-modified chitosan collagen composite fiber provided in example 11.

FIG. 15 is a scanning electron micrograph of fibers of a double-sided scaffold provided in example 12.

FIG. 16 is a scanning electron micrograph of fibers of a double-sided scaffold provided in example 13.

FIG. 17 is an electron micrograph of the morphology of cells on the functional layer of the double-sided scaffold provided in example 1.

FIG. 18 is an electron micrograph of the morphology of cells on the dense layer of the double-sided scaffold provided in example 1.

FIG. 19 is a two-dimensional fluorescence plot of the morphology of cells on the functional layer of the double-sided scaffold provided in example 1.

FIG. 20 is a two-dimensional fluorescence plot of the morphology of cells on the dense layer of the double-sided scaffold provided in example 1.

FIG. 21 is a 3D fluorescence plot of the morphology of cells on the functional layer of the double-sided scaffold provided in example 1.

FIG. 22 is a 3D fluorescence image of the morphology of cells on the dense layer of the double-sided scaffold provided in example 1.

FIG. 23 is a fiber diameter distribution plot for the double-sided stents provided in examples 1-3.

FIG. 24 is a histogram of fiber diameters before and after washing of the double-sided stent provided in examples 1-3.

Fig. 25 is an ir spectrum of the raw material and the double-sided stent provided in example 1.

FIG. 26 is a DSC chart of the raw materials and the double-sided stent provided in example 1.

Fig. 27 is a graph showing proliferation of dental pulp mesenchymal stem cells on the double-sided scaffold provided in example 1, cultured under osteogenic differentiation conditions.

Fig. 28A is an electron micrograph of the functional layer side of the double-sided scaffold provided in example 1 after 28 days and a scanning electron micrograph (70 μm) of the mesenchymal stem cells of dental pulp.

Fig. 28B is an electron micrograph of the functional layer side of the double-sided scaffold provided in example 1 after 28 days and a scanning electron micrograph (10 μm) of the mesenchymal stem cells of dental pulp.

Fig. 28C is an electron micrograph of the functional layer side of the double-sided scaffold provided in example 1 after 28 days and a fluorescence image of live and dead staining (AM, 70 μm) of the dental pulp mesenchymal stem cells.

Fig. 28D is an electron micrograph of the functional layer side of the double-sided scaffold provided in example 1 after 28 days and a fluorescence image of live and dead staining (PI, 70 μm) of the dental pulp mesenchymal stem cells.

FIG. 29 is a graph showing alkaline phosphatase activity of stem cells cultured on 2D planar and double-sided scaffold in osteogenic differentiation media.

FIG. 30A is a graph of a toxicity test of a degradation solution of a stent material on human gingival epithelial cells.

Fig. 30B is a graph of an experiment on the toxicity of a degraded solution of a scaffold material to human-derived dental pulp mesenchymal stem cells.

FIG. 31 is a HE and immunohistochemical map of ex-situ mineralization of stem cell double-sided scaffold material under the skin of nude mice.

FIG. 32 is a graph showing HE staining of heart, liver, spleen, lung and kidney after subcutaneous implantation in nude mice.

FIG. 33 is a drawing of periodontal CT and HE sections of a rat with periodontal defect repaired with a stem cell double-sided scaffold material.

FIG. 34 is a CT view of periodontal pockets after repair of periodontal defects in a mini-pig with a stem cell double-sided scaffold material.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

The embodiment provides a double-sided bracket, which is prepared by the following preparation method:

(1) preparing a chitosan collagen spinning solution containing RGD: mixing 3 wt% chitosan (M)_W3.1×10⁵～3.75×10⁵Da), 3 wt% collagen (M)_W 1.2×10⁶Da), 0.50 wt% of polyEthylene oxide (M)_w9.5×10⁴Da), 0.02mg/mL RGD (Arg-Gly-Asp) and 90 wt% acetic acid water solution are mixed to obtain spinning solution;

adding the spinning solution into a 10mL needle tube, performing electrostatic spinning at an extrusion rate of 0.5cm/min by using a No. 23 needle under the conditions of low pressure of-2 kV and high pressure of 20kV, and receiving the composite fiber obtained by spinning by using an aluminum foil; washing with ethanol for three times and washing with deionized water for three times; finally drying for 18h at-80 ℃ to obtain a chitosan collagen composite fiber layer;

(2) soaking 2g of chitosan collagen composite fiber layer in 5mL of 1mg/mL hydroxyapatite deposition solution at room temperature on a shaking table for oscillation at the frequency of 80rpm for 40 min; washing the mixture for three times by sequentially adopting 50 v% ethanol water solution; finally drying for 18h at-80 ℃ to obtain a chitosan collagen composite fiber layer modified by hydroxyapatite;

(3) taking 2g, 10mm²The chitosan collagen composite fiber layer and 2g and 10mm²Laminating the chitosan collagen composite fiber layer modified by the hydroxyapatite; adding 9mg/mL fibrinogen solution and 20U/mL thrombin from one side of the chitosan collagen composite fiber layer; and finally, placing the material in a constant-temperature shaking table for full reaction at the temperature of 35 ℃ for 24h, oscillating at the frequency of 80rpm, and drying at the temperature of-80 ℃ for 18h to obtain the double-sided support.

Test example 1

Characterization of double-sided scaffold Material

Observing the functional layer and the compact layer of the stent prepared in the example 1 by adopting an electron microscope (manufacturer: Hitachi, model: SU 8220);

fig. 1 to 4 are scanning electron microscope images of the functional layer and the dense layer of the double-sided scaffold provided in example 1 under different magnifications, and as shown in fig. 1 to 4, the electron microscope images of the double-sided scaffold material can see that the functional layer (i.e., hydroxyapatite-modified chitosan collagen composite fiber layer) and the dense layer (i.e., unmodified chitosan collagen composite fiber layer) have different pore sizes. The functional layer has larger aperture, which is beneficial to the contact between cells and hydroxyapatite, and improves the local calcium and phosphorus ion concentration to promote osteogenic differentiation; the smaller pore diameter of the compact layer hinders the invasion of epithelial cells and provides a certain time for the functional layer to mineralize on the surface of the tooth root.

Example 2

This example provides a double-sided stent, which is different from example 1 only in that, in step (1), the content of polyethylene oxide in the spinning solution is 0.25 wt%, and the contents of other components and the preparation steps are the same as in example 1.

Fig. 5 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 2, as shown in fig. 5, the PEO content is small, the fiber diameter is low, but the uniformity of the fiber is good.

Example 3

This example provides a double-sided stent, which is different from example 1 only in that, in step (1), the content of polyethylene oxide in the spinning solution is 0.75 wt%, and the contents of other components and the preparation steps are the same as in example 1.

Fig. 6 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 3, as shown in fig. 6, the PEO content is higher, the fiber diameter is higher, but the uniformity of the fiber is reduced.

Example 4

This example provides a double-sided stent, which is different from example 1 only in that the content of chitosan in the spinning solution is 1 wt% in step (1), and the contents of other components and the preparation steps are the same as those of example 1. The fiber diameter may be reduced after changing the chitosan content.

Fig. 7 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 4, and as shown in fig. 7, the fiber diameter of the prepared double-sided scaffold is reduced after adjusting the concentration of chitosan.

Example 5

This example provides a double-sided stent, which is different from example 1 only in that, in step (1), the concentration of RGD in the spinning solution is 0.5mg/mL, and the contents of other components and the preparation steps are the same as those of example 1.

Fig. 8 is a fluorescence graph of cell adhesion after stem cells are seeded on the double-sided scaffold provided in example 5, and as shown in fig. 8, the diameter of the fiber of the prepared double-sided scaffold does not change with the change of the RGD concentration, but the adhesion degree of cells is affected, and the cell adhesion amount is increased.

Example 6

This example provides a double-sided stent, which is different from example 1 only in that the spinning solution contains collagen in an amount of 1 wt% in step (1), and the other components are contained in the same amounts and the preparation steps as in example 1.

Fig. 9 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 6, and as shown in fig. 9, after adjusting the concentration of collagen, the diameter of the prepared double-sided scaffold is reduced.

Example 7

This example provides a double-sided stent, which is different from example 1 only in that the concentration of the aqueous acetic acid solution in the spinning solution in step (1) is 50 wt%, and the contents of other components and the preparation steps are the same as those of example 1.

Fig. 10 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 7, and as shown in fig. 10, the spinning solution is slowly volatilized due to the high water content, and a string-like phenomenon may occur.

Example 8

This example provides a double-sided stent, which differs from example 1 only in that in step (1), the electrospinning parameters are: no. 18 needle, extrusion rate of 0.05cm/min, low pressure of-6 kV, high pressure of 10kV, other component contents and preparation steps were the same as example 1.

Fig. 11 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 8, and as shown in fig. 11, the diameter of the fiber becomes thicker with the decrease of voltage, and a beading may occur.

Example 9

This example provides a double-sided stent, which differs from example 1 only in that in step (1), the electrospinning parameters are: no. 25 needle, extrusion rate of 2cm/min, low pressure of 1kV, high pressure of 30kV, other component contents and preparation steps are the same as example 1.

Fig. 12 is a scanning electron microscope image of the chitosan collagen composite fiber provided in example 9, as shown in fig. 12, increasing voltage increases extrusion rate, and the stability of fiber preparation decreases, but the spinning efficiency increases.

Example 10

This example provides a double-sided stent, which differs from example 1 only in that in step (2), the concentration of the hydroxyapatite deposit solution is 0.1mg/mL, and the contents of other components and the preparation steps are the same as those of example 1.

Fig. 13 is a scanning electron microscope image of the chitosan collagen composite fiber modified by hydroxyapatite according to example 10, as shown in fig. 13, the deposition of hydroxyapatite nanoparticles on the surface of the fiber is closely related to the concentration of the deposition solution, and when the concentration of the deposition solution is 0.1mg/mL, the number ratio of the nanoparticles on the surface of the fiber is reduced.

Example 11

This example provides a double-sided stent, which differs from example 1 only in that in step (2), the concentration of the hydroxyapatite deposit solution is 10mg/mL, and the contents of other components and the preparation steps are the same as those of example 1.

Fig. 14 is a scanning electron microscope image of the hydroxyapatite-modified chitosan collagen composite fiber provided in example 11, and as shown in fig. 14, when the concentration of the deposition solution is 10mg/mL, the density of hydroxyapatite nanoparticles on the surface of the fiber is increased, and the fiber is almost coated with the hydroxyapatite nanoparticles, but some nanoparticles are aggregated.

Example 12

This example provides a double-sided scaffold, which differs from example 1 only in that in step (3), the fibrinogen solution concentration is 1mg/mL, and the other component contents and preparation steps are the same as example 1.

FIG. 15 is a scanning electron microscope image of the fibers of the double-sided scaffold provided in example 12, as shown in FIG. 15, the degree of crosslinking of the fiber surface is low and the pore size is large.

Example 13

This example provides a double-sided scaffold, which differs from example 1 only in that in step (3), the thrombin concentration is 5mg/mL, and the other component contents and preparation steps are the same as example 1.

FIG. 16 is a scanning electron microscope image of the fibers of the double-sided scaffold provided in example 13, as shown in FIG. 16, the concentration of thrombin also affects the degree of cross-linking, and the decrease in the solubility of thrombin results in a decrease in the degree of cross-linking of the fiber network and an increase in the pore size.

Comparative example 1

This comparative example provides a double-sided scaffold, which is different from example 1 only in that, in step (1), chitosan was not added, the collagen content was increased to 6 wt%, and the contents of other components and the preparation steps were the same as example 1.

As a result: the stability of the whole fiber is lowered, and the swelling phenomenon occurs in water.

Comparative example 2

This comparative example provides a double-sided scaffold, which is different from example 1 only in that, in step (1), collagen was not added, the chitosan content was increased to 6 wt%, and the contents of other components and the preparation steps were the same as example 1.

As a result: the cell adhesion properties of the whole fiber are reduced.

Comparative example 3

This comparative example provides a double-sided stent, which is different from example 1 only in that 0.50 wt% of polyethylene oxide is replaced with 0.50 wt% of polylactic acid, and the contents of other components and the preparation procedure are the same as example 1.

As a result: the degradation products of polylactic acid in the later period can cause local pH drop, and the adverse effect is generated on the cell regeneration of tissue engineering.

Test example 2

Characterization of cells on scaffold Material

The present test example provides a tissue regeneration assisting material, and a method for preparing the tissue regeneration assisting material includes the steps of:

(1) stem cell extraction and culture: soaking a third molar tooth in 75% ethanol, taking out pulp tissue, and cutting pulp tissue to 1cm³Adding 3mg/mL collagenase type I and 4mg/mL dispase in a volume 5 times the volume of the small pieces, incubating at 37 ℃ for 2-4 hours, filtering with a 70 μm cell sieve to collect a single cell solution, and completely culturing with a-MEMCulturing in culture medium.

(2) Wetting the double-sided scaffold with PBS in an environment at 37 ℃;

(3) inoculating human dental pulp mesenchymal stem cells to one side of the chitosan collagen composite fiber layer of the double-sided bracket, and culturing at 37 ℃ for 2-3 days to obtain the tissue regeneration auxiliary material;

fig. 17 to 18 are electron micrographs of the morphology of cells on the functional layer and the dense layer of the double-sided scaffold provided in example 1, respectively. FIGS. 19-22 are fluorescence images of the morphology of cells on the functional, dense layer of the double-sided scaffolds provided in example 1. As shown in FIGS. 19-22, the cytoskeletal fluorochrome is TRITC Phalloidin (rhodamine-labeled Phalloidin), the cells and the dye are DAPI (i.e., 4', 6-diamidino-2-phenylindole, i.e., 4', 6-diamidino-2-phenylindole), and the excitation wavelengths are F-actin 561nm and 405nm, respectively. As shown in fig. 17-22, the cells have better cell morphology in the functional layer and the dense layer, and both have good biocompatibility.

Test example 3

Physical characterization of scaffold materials

The double-sided scaffolds prepared in the examples were physically characterized by the following specific test methods:

(1) fiber diameter: using SEM (Hitachi, model: SU8220) to collect pictures of different positions of the stent material, and counting the diameters of 100 fibers in a measuring mode;

(2) infrared: fourier transform infrared spectrometer (manufacturer: Perkin Elmer Instrument Co., Ltd., model: S-One) performs Fourier transform infrared spectrum detection on the material by using a 200i spotlight, and the detection range is 4000-600 cm^-1；

(3) DSC: mechanical refrigeration differential scanning calorimeter (manufacturer: Perkin Elmer apparatus, Inc., instrument model: Diamond DSC), with detection temperature range of 20-200 deg.C and heating rate of 10 deg.C/min;

in fig. 23 and 24, the fiber diameter distributions prepared by PEO in different proportions are gradually increased in fiber diameter with increasing PEO content, but the uniformity of the fibers is gradually decreased. The infrared results of fig. 25 show that there is a shift in characteristic peaks due to hydrogen bonding interaction between collagen and chitosan. Among them, the DSC of fig. 26 indicates that, consistent with the infrared results, there is a shift in characteristic peak due to hydrogen bond interaction between collagen and chitosan.

Test example 4

Activity assay

(1) The cell proliferation test is carried out on the double-sided scaffold prepared in example 1, and the specific test method comprises the following steps: dropwise adding the cell suspension into a double-sided bracket material, adding a complete culture medium, and detecting the DNA content at different time points by using a Quant-IT Pico Green ds DNA Regent kit; the time points are 3 days, 5 days, 7 days, 14 days and 21 days respectively;

in fig. 27, the cell proliferation curves of cells on 2D and scaffold materials are shown, and it can be seen that the three-dimensional scaffold material promotes cell proliferation and has good biocompatibility.

(2) The cell morphology of the double-sided scaffold prepared in example 1 after 28 days of osteogenic differentiation was tested, and the specific test method was: adding an AM/PI dye into a culture medium of the differentiated stem cell patch, and observing by using a laser confocal microscope (manufacturer: Perkin Elmer instrument, model: UltraVIEW VoX); fixing the differentiated stem cell double-sided scaffold material with 2.5% glutaraldehyde for 2 hours, dehydrating with alcohol gradient, and then obtaining an SEM sample which can be observed by means of freeze drying or carbon dioxide drying;

28A-28D are SEM and fluorescence images of cells on the material after osteogenic differentiation for 28 days, the morphology of the cells on the scaffold underwent osteogenic differentiation, and the scaffold material also had a clear network structure. The results of AM and PI show that the osteoblastic differentiated cells have better activity.

(3) The activity of phosphatase in the osteogenic differentiation process on the double-sided scaffold prepared in example 1 was tested by the following specific test method: samples at different time points were subjected to three times of freezing and thawing to lyse cells, centrifuged at 1000rpm to take the supernatant, and the supernatant was examined for ALP activity (Solebao, BC2145) using an enzyme-labeling apparatus (manufacturer: American MD, model: SpectraMax M5);

fig. 29 is a graph showing the activity of alkaline phosphatase in osteogenic differentiation media for stem cells cultured on two-dimensional planar and double-sided scaffolds, indicating that the activity of alkaline phosphatase is higher during osteogenic differentiation on scaffolds than in two-dimensional planar osteogenic differentiated cells.

(4) The double-sided scaffold prepared in example 1 was subjected to cytotoxicity test by the following specific test method: the double-sided scaffold material is soaked in a serum-free culture medium, the culture medium of 7 days, 14 days, 21 days and 28 days is respectively taken to prepare a complete culture medium, gingival epithelial cells and dental pulp mesenchymal stem cells are cultured, the cytotoxicity detection is carried out by using CCK-8, and the fluorescence of the supernatant is detected by using an enzyme labeling instrument (manufacturer: American MD company, model: SpectraMax M5).

In fig. 30A and 30B, cytotoxicity tests were performed, the material immersion liquid for 4 weeks did not affect dental pulp mesenchymal stem cells and gingival epithelial cells, and the cell activity was not significantly different from that of the control group.

(5) The specific test method for detecting the osteogenesis and odontogenesis indexes of the double-sided scaffold prepared in the example 1 comprises the following steps: transplanting the stem cell double-sided scaffold material to the subcutaneous part of a nude mouse, embedding wax blocks at the transplanted part after three months, and staining tissue sections;

wherein, FIG. 31 is a diagram of HE and immunohistochemistry of ex-situ mineralization of stem cell double-sided scaffold material under the skin of nude mice. As shown in FIG. 31, it is demonstrated that ectopic mineralization can be successfully performed under the skin of mice, and the detection of osteogenesis and odontogenesis indexes is positive. However, the process relies on the addition of exogenous stem cells, and the immunohistochemical results of the scaffold material without the addition of exogenous stem cells showed negative, but angiogenesis was observed inside the local scaffold, indicating that the scaffold has good biocompatibility. The results of immunohistochemistry of osteogenesis and odontoblasts of the scaffold added with exogenous cells are positive, which indicates that the scaffold material loaded with stem cells has a certain application prospect for periodontal repair. FIG. 32 is a graph showing HE staining of heart, liver, spleen, lung and kidney after subcutaneous implantation in nude mice. As shown in fig. 32, no abnormality was found, indicating that the stem cell double-sided patch provided by the present invention has better biological safety.

Test example 5

Double-sided bracket material for repairing periodontal tissue defect

Cell extraction and culture: soaking and washing teeth with PBS buffer solution containing double antibody, taking out dental pulp tissue, and cutting into 1mm pieces³The tissue mass was enzymatically digested with collagenase type I and dispase, and then filtered through a cell sieve to obtain a single cell suspension. Culturing cells by using alpha-MEM containing 10% FBS, changing the liquid after 5 days for the first time, changing the liquid every three days, and freezing and storing the cells for later use after the cells are fused to 70% -80%.

Dental pulp mesenchymal stem cells were seeded on one side of the double-sided scaffold material provided in example 1 after sterilization, and cultured in a medium for 2 to 3 days. The cleaned root surface is tightly attached through the flap-turning operation, and the periodontal defect is treated.

Wherein, fig. 33 is a drawing of periodontal CT and HE slices after rat periodontal defect repair with stem cell double-sided scaffold material, which illustrates the repair effect of rat periodontal defect, and the repair effect of the tooth-derived stem cell-loaded patch is better than that of the non-cell-loaded patch material than that of the untreated patch material. The stem cells and the scaffold material play a certain role in promoting tissue repair at the defect part. FIG. 34 is a CT view of periodontal tissues after repair of periodontal defects of a mini pig with a stem cell double-sided scaffold material, showing the repair effect of periodontal defects of a mini pig, which is consistent with the repair effect of periodontal defects of a rat.

The applicant states that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A dual-sided stand, comprising: chitosan collagen composite fiber layer and hydroxyapatite modified chitosan collagen composite fiber layer.

2. The double-sided stent according to claim 1, wherein the chitosan collagen composite fiber layer is prepared by electrostatic spinning of chitosan collagen spinning solution containing RGD;

preferably, the chitosan collagen spinning solution containing RGD comprises, by mass: 1-3% of chitosan, 1-15% of collagen, 0.25-0.75% of polyoxyethylene and the balance of solvent;

preferably, the concentration of RGD in the chitosan collagen spinning solution containing RGD is 0.02-1 mg/mL;

preferably, the chitosan has a molecular weight of 10⁴-10⁵Da；

Preferably, the collagen is type i collagen, preferably type i collagen of bovine tendon;

preferably, the polyethylene oxide has a molecular weight of 10⁴-10⁵Da；

Preferably, the solvent is an aqueous acetic acid solution, preferably an 85-95 wt% aqueous acetic acid solution;

preferably, the chitosan collagen composite fiber layer modified by hydroxyapatite comprises the chitosan collagen composite fiber layer and hydroxyapatite deposited between the chitosan collagen composite fiber layers;

preferably, the mass of the hydroxyapatite accounts for 5-20% of the total mass of the chitosan collagen composite fiber layer modified by the hydroxyapatite.

3. The double-sided stent of claim 1 or 2, wherein the fiber diameter of the double-sided stent is 0.5-1.5 μ ι η;

preferably, the pore diameter of the chitosan collagen composite fiber layer is less than 10 μm;

preferably, the pore diameter of the chitosan collagen composite fiber layer modified by the hydroxyapatite is 20-30 μm;

preferably, the double-sided scaffold further comprises fibrinogen and thrombin;

preferably, the content of fibrinogen in the double-sided scaffold is 1-20 wt%;

preferably, the content of thrombin in the double-sided scaffold is 10-30U/cm²。

4. The method for preparing a double-sided stent according to any one of claims 1 to 3, comprising the steps of:

5. The preparation method of the double-sided stent according to claim 4, wherein in the step (1), the specific preparation method of the chitosan collagen composite fiber layer is as follows: mixing chitosan, collagen, polyoxyethylene, RGD and a solvent to obtain a spinning solution, preparing chitosan collagen composite fiber by an electrostatic spinning technology, washing and drying to obtain a chitosan collagen composite fiber layer;

preferably, the parameters of the electrospinning technique are: using a 20-23-gauge needle, extruding at a rate of 0.1-1cm/min, under-5-0 kV, and under-15-25 kV;

preferably, after the soaking, washing is needed, wherein the washing is performed for at least 3 times by using ethanol, and then the washing is performed for at least 3 times by using water;

preferably, the drying is freeze drying, the drying temperature is-90 to-70 ℃, and the drying time is 12 to 24 hours.

6. The method for preparing a double-sided stent according to claim 4 or 5, wherein in the step (2), the concentration of the hydroxyapatite deposition solution is 0.1-10 mg/mL;

preferably, in the step (2), the soaking needs to be performed by shaking on a shaking table, the shaking frequency is 70-90rpm, and the shaking time is 20-60 min;

preferably, in the step (2), after the soaking, washing is further performed, wherein ethanol with a volume concentration of 40-60% is used for washing, and the washing frequency is at least 3 times.

7. The method for preparing a double-sided bracket according to any one of claims 4-6, characterized in that in the step (3), the specific steps of adhesion are as follows: after the chitosan collagen composite fiber layer and the hydroxyapatite-modified chitosan collagen composite fiber layer are bonded, adding a fibrinogen solution and thrombin from one side of the chitosan collagen composite fiber layer for reaction to obtain the double-sided bracket;

preferably, the concentration of the fibrinogen solution is 1-9mg/mL, and the solvent is normal saline;

preferably, the concentration of thrombin is 5-50U/mL;

preferably, the reaction temperature is 30-40 ℃, and the reaction time is 20-30 h;

preferably, the reaction is oscillated in a constant temperature shaker at a frequency of 70-90 rpm.

8. Use of a double-sided scaffold according to any one of claims 1-3 for the preparation of a tissue regeneration assisting material;

preferably, the tissue is periodontal tissue.

9. A tissue regeneration assisting material, comprising stem cells and the double-sided scaffold according to any one of claims 1 to 3;

preferably, the stem cells are dental stem cells;

preferably, the number of passage of the stem cells is less than 5;

preferably, the load of the stem cells is 10⁷-10⁹Individual cell/cm²。

10. The method for preparing a tissue regeneration assisting material according to claim 9, wherein the method for preparing a tissue regeneration assisting material comprises:

(a) wetting the double-sided stent of any one of claims 1-3;

(b) inoculating the stem cells to one side of the chitosan collagen composite fiber layer of the double-sided bracket, and culturing to obtain the tissue regeneration auxiliary material;

preferably, in step (a), the wetting is with PBS buffer;

preferably, in the step (a), the temperature of the wetting is 35-40 ℃, and the time of the wetting is 2-4 h;

preferably, in step (b), the stem cells are inoculated in an amount of 10⁷-10⁹Individual cell/cm²；

Preferably, in step (b), the temperature of the culture is 35-40 ℃ and the culture time is 2-3 days.