CN112220969B - Photocuring 3D printing preparation method of degradable meniscus support - Google Patents

Abstract

The invention relates to a photocuring 3D printing preparation method of a degradable meniscus scaffold, belonging to the preparation of biomedical tissue engineering scaffold materials. The photosensitive resin for printing the meniscus support comprises the following raw material components in parts by weight: 10-80 parts of urethane acrylate, 20-80 parts of acrylate monomer, 0.5-3.0 parts of free radical photoinitiator and 0.001-0.5 part of defoaming agent. Adding photosensitive resin into a resin groove of a 3D printer, printing and lifting a meniscus support blank according to set printing parameter equipment, and carrying out ethanol cleaning, ultraviolet curing and oven curing to obtain a meniscus support sample. The meniscus scaffold provided by the invention has good biocompatibility and mechanical properties similar to those of natural menisci, and the photocuring printing has the characteristics of high precision and high speed and can accurately control the three-dimensional structure and the porous structure of the scaffold.

Description

Photocuring 3D printing preparation method of degradable meniscus support

Technical Field

The invention belongs to the technical field of tissue engineering, particularly relates to preparation of a biomedical tissue engineering scaffold material, and particularly relates to a photocuring 3D printing preparation method of a degradable tissue engineering meniscus scaffold.

Background

Menisci, a white cartilage tissue interposed between articular surface cartilages, have a smooth surface that significantly reduces friction between the articular surfaces during movement. In recent years, with the prevalence of physical exercise and the aging population, the incidence of meniscal damage has increased year by year, and more patients are afflicted with this disease. However, since the meniscus site lacks blood supply, innervation, and lymphatic return, the ability to self-repair after injury is poor; if the traditional Chinese medicine is not treated in time, osteoarthritis can develop, ordinary people seriously affect daily life, and professional athletes break careers.

Tissue engineering meniscal techniques, i.e., the construction of a substitute that can repair damaged menisci, can achieve good therapeutic results. Three basic elements of tissue engineering are scaffold material, seed cells and growth factors. Despite the great advances made in this technology in recent years, there are still many issues to be solved, such as the choice of stent material and the construction of the structure. The scaffold material provides a good environment for adhesion, proliferation and differentiation of seed cells, and effectively guides the growth of the new tissue to a preset shape. The stent material must have: (1) good biocompatibility; (2) the porous structure and the reasonable three-dimensional structure are favorable for the adhesion, proliferation and differentiation of cells, and realize the free diffusion of nutrient substances and the free growth of the cells; (3) good mechanical properties, the mechanical properties of the scaffold should be similar to those of natural menisci, the compressive modulus should be in the range of 75-150 kPa, and the tensile modulus should be in the range of 75-150 MPa [ Biomaterials, 126, 18-30, 2017 ].

The stent material can be classified into a substitution type and a degradation type. Wherein the substitute meniscus graft is mainly made of polyvinyl alcohol hydrogel, polycarbonate polyurethane and other materials with good biocompatibility, in vivo stability and excellent biomechanical property. The degradable meniscus scaffold can be divided into natural material and synthetic material meniscus scaffolds according to different material sources. Natural hydrogel polymer Materials such as collagen, silk fibroin, chitosan, alginate, gelatin, hyaluronic acid and the like are applied to the study of meniscus scaffolds (Advanced Materials, 29, 2017) due to good biocompatibility]. Collagen meniscus implant (trade name CMI) developed by Ivy Sports Medicine, Germany^®) Can effectively improve the clinical symptoms of patients and the clinical scores of knee joints. But the mechanical property of the material can not maintain the normal function of the knee joint and the degradation is faster, so the material is only suitable for the case that the meniscus is locally damaged [ the journal of biomedical engineering, 35, 488-one 492, 2018 ]]。

The preparation of a meniscus scaffold made of a synthetic material with excellent mechanical properties is a hot spot of current research. Polyurethane, polycaprolactone, polylactic acid and the like are widely applied to the research of the meniscus scaffold due to good biocompatibility, excellent mechanical properties and easy processability. A polycaprolactone/polyurethane meniscus scaffold manufactured by Orteq, UK has been commercialized (under the trade name Atifit)^®） [Biomaterials, 35, 3527-3540, 2014]. The polycaprolactone moiety can be completely degraded by ester hydrolysis within five years, and the urethane moiety can be slowly phagocytosed by macrophages or gradually incorporated into the surrounding tissues [ Knee Surgery, 26, 2227-]. Song et al prepared hydroxyapatite hybrid polyurethane type scaffolds with bioactive surfaces, adjusted their mechanical properties by controlling their porosity and used as meniscal grafts [ Macromolecular Materials and Engineering, 304, 2019]. However, none of the above materials allow for precise control of scaffold pore structure and customization of a meniscal scaffold of a specific three-dimensional shape to a patient, and the recent advent of 3D printing technology has made possible the fabrication of artificial meniscal materials and precise control of microstructure.

Currently, a meniscus scaffold manufactured by 3D printing is mainly manufactured by using natural materials or synthetic materials such as polycaprolactone, polylactic acid and the like and through Fused Deposition Modeling (FDM) (chinese patents CN110354309A, CN105013011A, CN110478527A and CN 211271414U). The mechanical strength of the meniscus is different from that of a natural meniscus, the printing precision is not high, and the precise regulation and control of the meniscus structure cannot be realized.

Disclosure of Invention

The invention aims to provide a photocuring 3D printing preparation method of a degradable tissue engineering meniscus scaffold, and provides a new technological method for tissue engineering meniscus research. The tissue engineering meniscus prepared by the method has good mechanical property, biocompatibility and biodegradability, the mechanical property of the tissue engineering meniscus is similar to that of a natural meniscus, the printing precision is high, and the meniscus scaffold with an individualized specific three-dimensional structure and a porous structure can be customized.

The invention is realized by the following technical scheme:

a photocuring 3D printing preparation method of a degradable meniscus scaffold comprises the following steps:

s1, synthesizing urethane acrylate with a specific structural formula I;

s2, preparing photosensitive resin according to the formula proportion by utilizing the polyurethane acrylate prepared in the step S1, wherein the photosensitive resin comprises the following raw material components in parts by weight:

10-80 parts of urethane acrylate;

20-80 parts of an acrylate monomer;

0.5-3.0 parts of free radical photoinitiator;

0.001-0.5 part of defoaming agent;

s3, adding the photosensitive resin prepared in the step S2 into a resin tank of 3D printing equipment, setting printing process parameters, and printing to obtain a meniscus support blank;

s4, removing the support of the meniscus support blank, and then putting the meniscus support blank into ethanol for ultrasonic cleaning for 10 min;

s5, placing the meniscus support blank cleaned in the step S4 into a purple box, and curing for 30min by using 280 and 350nm ultraviolet light;

and S6, placing the meniscus support blank subjected to ultraviolet curing into an oven at 60-100 ℃ and curing for 0.5-3 h to finally obtain a meniscus support sample.

Preferably, the urethane acrylate having the specific structure I in step S1 has the structural formula:

wherein R1 is one of isophorone, hexamethylene, dicyclohexyl methyl; r2 is one or more of ethyl, propyl and n-butyl; r3 is one of hydrogen or methyl; m and n are both natural numbers of 10-100.

Preferably, the preparation method of the polyurethane acrylate comprises the following steps:

1) mixing the compound II, the compound III and the compound IV, adding a catalyst, and carrying out polycondensation reaction for 2-14 h at the temperature of 20-100 ℃ to obtain the isocyanate group-terminated polyurethane resin, wherein the structural formula of the compound II is shown in the specification,

the structural formula of the compound III is shown as,

the structural formula of the compound IV is shown as,

；

2) reacting the isocyanate group-terminated polyurethane resin obtained in the step 1) with hydroxyethyl methacrylate or hydroxyethyl acrylate at 50-100 ℃ for 2-8 h, and adding hydroquinone serving as a polymerization inhibitor during the reaction to obtain methacrylate or acrylate-terminated polyurethane acrylate.

Preferably, the catalyst is a tertiary amine or organometallic catalyst.

Preferably, the acrylate monomer in step S2 is one or a combination of hydroxyethyl acrylate, isobornyl acrylate, diethylene glycol diacrylate, dipropylene glycol diacrylate, (ethoxylated) trimethylolpropane triacrylate and pentaerythritol tetraacrylate.

Preferably, the radical photoinitiator in step S2 is one or more of benzil derivatives, α -hydroxyalkylbenzones, and acylphosphorus oxide cleavage type photoinitiators.

Preferably, the defoaming agent is BYK-055 defoaming agent.

Preferably, the method for preparing the photosensitive resin in step S2 includes the steps of:

1) weighing the following raw materials according to the formula proportion: urethane acrylate, an acrylate monomer, a free radical photoinitiator and a defoaming agent;

2) pouring urethane acrylate, an acrylate monomer, a free radical photoinitiator and a defoaming agent into a stirrer in sequence, and stirring at a low speed of 400 r/min for 2-4 h to obtain transparent photosensitive resin.

The invention has the beneficial effects that:

(1) the meniscus scaffold provided by the invention is degradable, good in biocompatibility, matched with biomechanical characteristics and free of cytotoxicity.

(2) The meniscus support is prepared by adopting a photocuring 3D printing mode, the printing precision is high, and the maximization bionics of the meniscus shape and the accurate control of the internal three-dimensional structure and the pore structure can be realized.

(3) The meniscus scaffold printed by the invention is similar to the natural meniscus, and is an ideal material for meniscus transplantation and repair.

Drawings

FIG. 1 shows a graph of the mechanical tensile modulus of a degradable polyurethane meniscal scaffold of the present application;

FIG. 2 shows a histogram of the mechanical tensile modulus of natural menisci versus degradable menisci;

FIG. 3A is a photograph showing the staining of cell nuclei and skeletal structure on the surface of a material observed by a confocal laser microscope;

FIG. 3B is a photograph showing the experiment of cell death and viability on the surface of the material observed by confocal laser microscopy;

FIGS. 4A and 4B show photographs of 3 month post-operative NMR T2 images of degradable polyurethane menisci of the present application implanted in the knee joint of a New Zealand white rabbit;

FIGS. 5A to 5D show photographs of the dissections at 3 months after the in vivo implantation of New Zealand white rabbits.

Detailed Description

The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

Example 1

In a 250 mL round-bottomed flask equipped with mechanical stirring, a nitrogen inlet tube, a thermometer and a dropping funnel, 16.8 g of Hexamethylene Diisocyanate (HDI) was added, and then a mixture of 20.0 g of polyethylene glycol diol (molecular weight 1000) and 90.0 g of polycaprolactone diol (molecular weight 3000) was dropwise added to the three-necked flask while maintaining the temperature of the reaction system in the flask at 50 ℃. After the addition was complete, the mixture was allowed to react for 2 h. Adding catalyst stannous octoate (600 ppm of the mass of the reactant) into the system to promote the reaction to fully occur. The extent of reaction was monitored by Fourier infrared and when the characteristic infrared absorption peak of the isocyanate groups did not decrease any more, a mixture of 0.1 g of hydroquinone and 11.6 g of hydroxyethyl acrylate was added dropwise to the system while maintaining the temperature of the system at 80 ℃. After the dropwise addition is finished, the reaction is continued until the characteristic absorption peak of the isocyanate group in the infrared spectrogram completely disappears, and the polyurethane acrylate is obtained, wherein the designed number average molecular weight is 2768 and is named as PUA-1.

Example 2

In a 250 mL round-bottomed flask equipped with mechanical stirring, a nitrogen inlet tube, a thermometer and a dropping funnel, 16.8 g of Hexamethylene Diisocyanate (HDI) was added, and then a mixture of 40.0 g of polyethylene glycol diol (molecular weight 1000) and 90.0 g of polycaprolactone diol (molecular weight 3000) was dropwise added to the three-necked flask while maintaining the temperature of the reaction system in the flask at 80 ℃. After the addition was complete, the mixture was allowed to react for a further 14 h. Adding catalyst stannous octoate (600 ppm of the mass of the reactant) into the system to promote the reaction to fully occur. The extent of reaction was monitored by Fourier infrared and when the characteristic infrared absorption peak of the isocyanate groups did not decrease any more, a mixture of 0.1 g of hydroquinone and 7.0 g of hydroxyethyl acrylate was added dropwise to the system while maintaining the temperature of the system at 80 ℃. After the dropwise addition is finished, the reaction is continued until the characteristic absorption peak of the isocyanate group in the infrared spectrogram completely disappears, and the polyurethane acrylate is obtained, wherein the designed number average molecular weight is 5125 and is named as PUA-2.

Example 3

In a 250 mL round-bottom flask equipped with mechanical stirring, nitrogen inlet, thermometer and dropping funnel, 22.2 g of isophorone diisocyanate (IPDI) was added, and then a mixture of 20.0 g of polyethylene glycol diol (molecular weight 1000) and 80.0 g of polycaprolactone diol (molecular weight 4000) was added dropwise into the three-necked flask, while maintaining the temperature of the reaction system in the flask at 100 ℃. After the addition was complete, the mixture was allowed to react for a further 4 h. Adding catalyst stannous octoate (600 ppm of the mass of the reactant) into the system to promote the reaction to fully occur. The extent of reaction was monitored by Fourier infrared and when the characteristic infrared absorption peak of the isocyanate groups did not decrease any more, a mixture of 0.1 g of hydroquinone and 13.9 g of hydroxyethyl acrylate was added dropwise to the system, during which the temperature of the system was maintained at 50 ℃. And after the dropwise addition is finished, continuing the reaction until the characteristic absorption peak of the isocyanate group in the infrared spectrogram completely disappears, thus obtaining the polyurethane acrylate, wherein the designed number average molecular weight is 2268 and is named as PUA-3.

Example 4

In a 250 mL round-bottomed flask equipped with mechanical stirring, a nitrogen inlet, a thermometer and a dropping funnel, 22.2 g of isophorone diisocyanate (IPDI) was added, and then a mixture of 30.0 g of polyethylene glycol diol (molecular weight 1000) and 100.0 g of polycaprolactone diol (molecular weight 2000) was added dropwise into the three-necked flask while maintaining the temperature of the reaction system in the flask at 80 ℃. After the addition was complete, the mixture was allowed to react for a further 4 h. Adding catalyst stannous octoate (600 ppm of the mass of the reactant) into the system to promote the reaction to fully occur. The extent of reaction was monitored by Fourier infrared and when the characteristic infrared absorption peak of the isocyanate groups did not decrease any more, a mixture of 0.1 g of hydroquinone and 4.6 g of hydroxyethyl acrylate was added dropwise to the system, during which the temperature of the system was maintained at 60 ℃. After the dropwise addition is finished, the reaction is continued until the characteristic absorption peak of the isocyanate group in the infrared spectrogram completely disappears, and the polyurethane acrylate is obtained, wherein the designed number average molecular weight is 7842 and is named as PUA-4.

Preparation of photosensitive resin

Example 5

Firstly, weighing the following raw materials according to the formula proportion: the photosensitive resin comprises the following raw material components in parts by weight:

polyurethane acrylate PUA-170 parts

29 parts of hydroxyethyl acrylate

1 part of 2-hydroxy-2-methyl-1-phenyl acetone

BYK-0550.01 parts

Then, the components are poured into a stirrer in sequence, and are stirred uniformly at a low speed to obtain transparent photosensitive resin, wherein the stirring speed is 400 r/min, and the stirring time is 2-4 h.

Example 6

Firstly, weighing the following raw materials according to the formula proportion: the photosensitive resin comprises the following raw material components in parts by weight:

polyurethane acrylate PUA-250 parts

44 parts of hydroxyethyl acrylate

Trimethylolpropane triacrylate 5 parts

1 part of 2-hydroxy-2-methyl-1-phenyl acetone

BYK-0550.01 parts

Then, the components are poured into a stirrer in sequence, and are stirred uniformly at a low speed to obtain transparent photosensitive resin, wherein the stirring speed is 400 r/min, and the stirring time is 2-4 h.

Example 7

Firstly, weighing the following raw materials according to the formula proportion: the photosensitive resin comprises the following raw material components in parts by weight:

polyurethane acrylate PUA-370 parts

Isobornyl acrylate 15 parts

14 parts of hydroxyethyl acrylate

1 part of 2-hydroxy-2-methyl-1-phenyl acetone

BYK-0550.01 parts

Then, the components are poured into a stirrer in sequence, and are stirred uniformly at a low speed to obtain transparent photosensitive resin, wherein the stirring speed is 400 r/min, and the stirring time is 2-4 h.

Example 8

Firstly, weighing the following raw materials according to the formula proportion: the photosensitive resin comprises the following raw material components in parts by weight:

polyurethane acrylate PUA-450 parts

20 parts of isobornyl acrylate

19 parts of hydroxyethyl acrylate

Pentaerythritol tetraacrylate 10 parts

1 part of 2-hydroxy-2-methyl-1-phenyl acetone

BYK-0550.01 parts

Then, the components are poured into a stirrer in sequence, and are stirred uniformly at a low speed to obtain transparent photosensitive resin, wherein the stirring speed is 400 r/min, and the stirring time is 2-4 h.

Examples 9-12 printing of meniscal scaffolds

Step 1, model printing is performed on the photosensitive resin prepared in the above embodiments 5 to 8 by using DLP 3D printing equipment, and the model has a smooth surface and high fineness; the printing parameters of the 3D printer are set as needed, and in embodiments 9 to 12 of the present application, the printing parameters of the 3D printer are the same.

And 2, removing the support of the sample blank, putting the sample blank into ethanol for ultrasonic treatment for 10min, then putting the sample blank into an ultraviolet box, curing the sample blank for 15 min by adopting ultraviolet light of 300nm, finally putting the sample blank into an oven at 80 ℃, and curing the sample blank for 1 h to finally obtain the meniscus support.

Mechanical property evaluation of the meniscal scaffold.

The mechanical properties of the printed meniscal scaffolds were evaluated according to the GB/T2567-2008 test standard including tensile strength and tensile modulus.

	Example 9	Example 10	Example 11	Example 12
					Tensile Strength (MPa)	22.5	25.6	19.5	22.3
Elongation at break	45.2%	78.7%	56.7%	89.2%
					Tensile modulus (MPa)	123	80	108	78

As shown in fig. 1, showing a graph of the mechanical tensile modulus of the degradable polyurethane meniscus scaffold of the present application, fig. 2 is a bar graph comparing the mechanical tensile modulus of a natural meniscus to that of a degradable meniscus, showing that the mechanical tensile modulus of a degradable meniscus is very close to that of a natural meniscus.

Fig. 3A and fig. 3B are photographs respectively showing that rabbit adipose-derived mesenchymal stem cells are planted on the degradable polyurethane meniscus scaffold in vitro, and cell nuclei, skeleton structure staining detection and cell death and survival experiments on the surface of the scaffold are observed through a laser confocal microscope, which shows that the cells are in good survival state on the surface of the material, the attachment is good, and the cell skeleton is fully extended on the surface of the material.

Fig. 4A and 4B show photographs of 3-month post-operative nmr T2 images of a degradable polyurethane meniscus implanted in a new zealand white rabbit knee, showing that the degradable meniscus scaffold is structurally intact in the joint space, well positioned, with no apparent destruction of articular cartilage, and with ingrowth of autologous tissue around the scaffold.

Fig. 5A to 5D show anatomical photographs of the implanted new zealand white rabbit after 3 months of operation, showing that the medial femoral condyle cartilage and the medial tibial plateau cartilage have no obvious osteoarthritis, the articular cartilage is in a good state, the structure of the meniscus scaffold is complete, autologous tissues grow into the pores of the scaffold, and degradation traces are visible at the edge.

The tissue engineering meniscus prepared by the method has good mechanical property, biocompatibility and biodegradability, the mechanical property of the tissue engineering meniscus is similar to that of a natural meniscus, the printing precision is high, and the meniscus scaffold with an individualized specific three-dimensional structure and a porous structure can be customized.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (7)

1. A photocuring 3D printing preparation method of a degradable meniscus scaffold is characterized by comprising the following steps:

s1, synthesizing urethane acrylate with a specific structural formula I;

s2, preparing photosensitive resin according to the formula proportion by utilizing the polyurethane acrylate prepared in the step S1, wherein the photosensitive resin comprises the following raw material components in parts by weight:

10-80 parts of urethane acrylate;

20-80 parts of an acrylate monomer;

0.5-3.0 parts of free radical photoinitiator;

0.001-0.5 part of defoaming agent;

s3, adding the photosensitive resin prepared in the step S2 into a resin tank of 3D printing equipment, setting printing process parameters, and printing to obtain a meniscus support blank;

s4, removing the support of the meniscus support blank, and then putting the meniscus support blank into ethanol for ultrasonic cleaning for 10 min;

s5, placing the meniscus support blank cleaned in the step S4 into a UV box for curing for 30 min;

s6, placing the meniscus support blank subjected to ultraviolet curing into a 60-100 ℃ oven, and curing for 0.5-3 h to finally obtain a meniscus support sample;

the structural formula of the urethane acrylate having the specific structure I in step S1 is:

wherein R1 is one of isophorone base, hexamethylene and dicyclohexyl methyl; r2 is one or more of ethylene, propylene and n-butylene; r3 is one of hydrogen or methyl; m andn is a natural number of 10 to 100.

2. The photo-curing 3D printing preparation method of the degradable meniscus scaffold according to claim 1, wherein the preparation method of the urethane acrylate comprises the following steps:

1) mixing the compound II, the compound III and the compound IV, adding a catalyst, and carrying out polycondensation reaction for 2-14 h at the temperature of 20-100 ℃ to obtain the isocyanate group-terminated polyurethane resin, wherein the structural formula of the compound II is shown in the specification,

the structural formula of the compound III is shown as,

the structural formula of the compound IV is shown as,

wherein, R1 is one of isophorone base, hexamethylene and dicyclohexyl methyl; r2 is one or more of ethylene, propylene and n-butylene; m and n are both natural numbers of 10-100;

2) reacting the isocyanate group-terminated polyurethane resin obtained in the step 1) with hydroxyethyl methacrylate or hydroxyethyl acrylate at 50-100 ℃ for 2-8 h, and adding hydroquinone serving as a polymerization inhibitor during the reaction to obtain methacrylate or acrylate-terminated polyurethane acrylate.

3. The photo-cured 3D printing preparation method of the degradable meniscus scaffold according to claim 2, wherein the catalyst is a tertiary amine or organometallic catalyst.

4. The method for preparing a degradable meniscus scaffold by photocuring 3D printing according to claim 1, wherein the acrylate monomer in step S2 is one or more of hydroxyethyl acrylate, isobornyl acrylate, diethylene glycol diacrylate, dipropylene glycol diacrylate, (ethoxylated) trimethylolpropane triacrylate and pentaerythritol tetraacrylate.

5. The photo-curing 3D printing preparation method of the degradable meniscus scaffold according to claim 1, wherein the radical type photo-initiator in step S2 is one or more of benzil derivatives, alpha-hydroxyalkylbenzones or acyl phosphorus oxide cleavage type photo-initiators.

6. The photo-curing 3D printing preparation method of the degradable meniscus scaffold, according to claim 1, wherein the antifoaming agent is BYK-055 antifoaming agent.

7. The photo-curing 3D printing preparation method of the degradable meniscus scaffold according to claim 1, wherein the preparation method of the photosensitive resin in the step S2 comprises the following steps:

1) weighing the following raw materials according to the formula proportion: urethane acrylate, an acrylate monomer, a free radical photoinitiator and a defoaming agent;

2) pouring urethane acrylate, an acrylate monomer, a free radical photoinitiator and a defoaming agent into a stirrer in sequence, and stirring at a low speed of 400 r/min for 2-4 h to obtain transparent photosensitive resin.

Images