CN115025286B - Biomimetic mineralized 3D printing PLA bracket and manufacturing method thereof - Google Patents
Biomimetic mineralized 3D printing PLA bracket and manufacturing method thereof Download PDFInfo
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Abstract
The invention discloses a method for manufacturing a biomimetic mineralized 3D printing PLA bracket, which comprises the following steps: and (3) printing to obtain a PLA bracket with a porous structure, performing plasma modification on the PLA bracket, then sterilizing, then placing the PLA bracket into an I-type collagen solution for incubation, cleaning and sterilizing the PLA bracket after the completion of the plasma modification, placing the PLA bracket into simulated body fluid for incubation, and cleaning and sterilizing the PLA bracket after the completion of the plasma modification. The invention also discloses a PLA bracket prepared by the preparation method. The purpose of the method is to provide a biomimetic mineralized 3D printing PLA bracket and a manufacturing method thereof, which are modified based on a plasma technology, so that not only can the surface roughness of PLA material be increased, but also the cell adhesion proliferation is promoted, and the protein adhesion is promoted through the introduction of active groups, so that the biological recognition site is increased, and the bone ingrowth is promoted.
Description
Technical Field
The invention relates to the field of bone repair materials, in particular to a 3D printing PLA bracket and a manufacturing method thereof.
Background
Bone defects are a common clinical problem, and with the continuous development of modern society, the bone defects caused by wounds, infections, tumors, severe osteoporosis fracture and the like are increasingly generated. It is counted that up to 100 tens of thousands of patients are treated for bone grafting per year due to trauma in the united states. Bone defects caused by congenital diseases, traffic injuries, sports injuries and the like in China are up to 350 ten thousand per year. But in the current clinical treatment, the failure rate of the bone repair and reconstruction operation is up to 25 percent, and the occurrence rate of the operation complications is up to 30 to 60 percent. Thus, repair and reconstruction of bone defects is a great challenge to orthopedics today.
The current treatment strategy of the bone defect not only needs to fill and repair the defective bone, but also ensures complete healing of the defective part and avoids bone nonunion, thereby maximally recovering the limb functions of the patient. Current clinical treatments mainly include autologous bone grafting, allogeneic/xenogeneic bone grafting, angiotheca grafting, masquelet technology and Ilizarov technology. The above treatments have advantages, but also have certain disadvantages, so that it is difficult to satisfy the clinical treatment needs despite the abundance of the current treatments.
The application of the bone tissue engineering material provides a new method for repairing bone defects. Bone tissue engineering is widely used for repairing bone defects, and is widely known to be composed of three key elements, namely a scaffold material, a signal molecule and seed cells. The ideal bone tissue engineering material not only has proper degradation rate, good biocompatibility and bioactivity, but also has certain mechanical property to provide defect structural support, and finally meets the requirements of simple preparation and convenient operation. Even though the current manufacturing process and composite forms are continuously advanced, the scaffold, factors and cells are continuously integrated and compounded, and an ideal repairing material is still difficult to obtain.
Along with the rapid development of computer-aided technology, tissue engineering materials and 3D printing technology, the tissue engineering materials based on 3D printing are widely applied to various fields of biomedicine, and the advantages of rapid molding, individual customization, accurate control and the like of the 3D printing technology are mainly attributed to the fact that the 3D printing technology. Aiming at the bone defect repair and reconstruction problem, the 3D printing of PLA material has gained a great deal of attention. However, PLA materials are difficult to generate a good interface reaction due to their hydrophobicity and lack of biological recognition sites, resulting in poor osteogenic properties, and thus it is difficult to meet the requirements of bone tissue engineering materials for a single application.
Disclosure of Invention
The invention aims to solve the technical problem of providing a biomimetic mineralized 3D printing PLA bracket and a manufacturing method thereof, which are modified based on a plasma technology, so that not only can the surface roughness of PLA material be increased to promote cell adhesion proliferation, but also protein adhesion is promoted by introducing active groups, thereby increasing biological recognition sites and promoting bone ingrowth.
The manufacturing method of the biomimetic mineralized 3D printing PLA bracket comprises the following steps:
a PLA scaffold with a porous structure was obtained by 3D printing,
performing plasma modification on the PLA bracket obtained by 3D printing, then sterilizing the PLA bracket subjected to plasma modification,
placing the sterilized PLA scaffold into a type I collagen solution for first incubation, taking the PLA scaffold out of the type I collagen solution after the first incubation is completed, performing first cleaning and first sterilization,
and (3) placing the PLA bracket subjected to the first sterilization into the simulated body fluid for the second incubation, taking the PLA bracket out of the simulated body fluid after the second incubation is completed, and performing the second cleaning and the second sterilization.
The preparation method of the invention comprises the following specific steps of plasma modification of the PLA bracket obtained by 3D printing:
placing the PLA bracket obtained by 3D printing in a vacuum cavity of a plasma cleaning machine, vacuumizing the vacuum cavity to below 10Pa, then filling air into the vacuum cavity to adjust the pressure to 240Pa, setting the frequency of the plasma cleaning machine to be 13.56MHz after the pressure in the vacuum cavity is stable, setting the discharge power to be 20W and the treatment time to be 30 minutes, and then starting the plasma cleaning machine, wherein the plasma cleaning machine generates glow discharge plasma to treat the PLA bracket obtained by 3D printing.
The preparation method comprises the following specific steps of: the plasma modified PLA scaffold was sterilized by cobalt 60 for 60 minutes.
The preparation method comprises the specific steps of putting the sterilized PLA scaffold into a type I collagen solution for first incubation: the sterilized PLA scaffold was immersed in a type I collagen solution having a concentration of 2 mg/mL for 24 hours, and the temperature of the type I collagen solution was set to 4 ℃.
The manufacturing method of the invention comprises the following specific steps: and (3) rinsing the PLA bracket for three times by distilled water, wherein the specific steps of the first sterilization are as follows: the PLA stent was uv sterilized for 60 minutes.
The preparation method of the invention comprises the specific steps of placing the PLA scaffold after the first sterilization into simulated body fluid for the second incubation:
firstly, preparing simulated body fluid, putting 800mL of distilled water into a beaker, heating to 36 ℃ under magnetic stirring, and then sequentially adding 7.996g of NaCl reagent and 0.350g of NaHCO 3 Reagent, 0.224g KCl reagent, 0.228g K 2 HPO 4 ·3H 2 O reagent, 0.305g MgCl 2 ·6H 2 O reagent, 0.278g CaCl 2 Reagent and 0.071g of Na 2 SO 4 Adding reagents, adding next reagent after each reagent is completely dissolved, adding tris buffer solution to adjust pH value to 7.40 after all reagents are completely dissolved, adjusting temperature to 36.5 ℃, fixing volume to 1000mL to obtain simulated body fluid,
the PLA scaffold after the first sterilization was then placed into the resulting simulated body fluid for 24 hours of infiltration.
The manufacturing method of the invention comprises the following specific steps of: and (3) rinsing the PLA bracket for three times by distilled water, wherein the specific steps of the second sterilization are as follows: the PLA stent was uv sterilized for 60 minutes.
The biomimetic mineralized 3D printing PLA bracket is prepared according to the preparation method.
The bionic mineralized 3D printing PLA bracket is cylindrical, a first through hole, a second through hole and a third through hole are formed in the PLA bracket, the first through hole is arranged along the axial direction of the PLA bracket, the second through hole and the third through hole are both perpendicular to the axial direction of the PLA bracket, the second through hole is perpendicular to the third through hole, the first through hole, the second through hole and the third through hole are all arranged in a plurality, the plurality of first through holes are arranged in an array, the plurality of second through holes are also arranged in an array, and the plurality of third through holes are also arranged in an array.
The biomimetic mineralized 3D printing PLA bracket provided by the invention has the advantages that the first through hole, the second through hole and the third through hole are square holes, the side length of each square hole is 350 mu m, and the interval between every two adjacent square holes is 350 mu m.
The bionic mineralized 3D printing PLA bracket and the manufacturing method are different from the prior art in that the PLA bracket is obtained through 3D printing, has a porous structure, structurally simulates the spatial configuration of a bionic cancellous bone, combines the PLA bracket with an I-type collagen solution and simulated body fluid in sequence after being modified by a plasma technology, prepares a bionic micro-surface structure by means of fixing the I-type collagen and a hydroxyapatite-like coating, increases cell recognition sites, and realizes the constitutive bionic natural bone of the bracket. Therefore, the invention is modified based on a plasma technology, not only can increase the surface roughness of PLA material so as to promote cell adhesion and proliferation, but also can promote protein adhesion through the introduction of active groups so as to increase biological recognition sites and promote bone ingrowth.
The invention is further described below with reference to the accompanying drawings.
Drawings
FIG. 1 is a front view (and also a rear view) of a biomimetic mineralized 3D printed PLA scaffold embodiment I of the present invention;
FIG. 2 is a left side view (and also a right side view) of a biomimetic mineralized 3D printed PLA scaffold embodiment I of the invention;
FIG. 3 is a top view (and bottom view) of a biomimetic mineralized 3D printed PLA scaffold embodiment I of the invention;
FIG. 4 is a perspective view of a biomimetic mineralized 3D printed PLA scaffold embodiment I of the present invention;
FIG. 5 is a front view (and also a rear view) of a biomimetic mineralized 3D printed PLA scaffold embodiment II in the present invention;
FIG. 6 is a left side view (and also a right side view) of a biomimetic mineralized 3D printed PLA scaffold embodiment II in the present invention;
FIG. 7 is a top view (and bottom view) of a biomimetic mineralized 3D printed PLA scaffold embodiment II in accordance with the present invention;
FIG. 8 is a perspective view of a biomimetic mineralized 3D printed PLA scaffold embodiment II in the present invention;
fig. 9 is a flowchart of a method for manufacturing a biomimetic mineralized 3D printed PLA scaffold in the present invention.
Detailed Description
As shown in fig. 9, the method for manufacturing the biomimetic mineralized 3D printing PLA scaffold in the invention comprises the following steps:
a PLA scaffold with a porous structure was obtained by 3D printing,
performing plasma modification on the PLA bracket obtained by 3D printing, then sterilizing the PLA bracket subjected to plasma modification,
placing the sterilized PLA scaffold into a type I collagen solution for first incubation, taking the PLA scaffold out of the type I collagen solution after the first incubation is completed, performing first cleaning and first sterilization,
and (3) placing the PLA bracket subjected to the first sterilization into the simulated body fluid for the second incubation, taking the PLA bracket out of the simulated body fluid after the second incubation is completed, and performing the second cleaning and the second sterilization.
The preparation method of the invention comprises the following specific steps of plasma modification of the PLA bracket obtained by 3D printing:
placing the PLA bracket obtained by 3D printing in a vacuum cavity of a plasma cleaning machine, vacuumizing the vacuum cavity to below 10Pa, then filling air into the vacuum cavity to adjust the pressure to 240Pa, setting the frequency of the plasma cleaning machine to be 13.56MHz after the pressure in the vacuum cavity is stable, setting the discharge power to be 20W and the treatment time to be 30 minutes, and then starting the plasma cleaning machine, wherein the plasma cleaning machine generates glow discharge plasma to treat the PLA bracket obtained by 3D printing.
The preparation method comprises the following specific steps of: the plasma modified PLA scaffold was sterilized by cobalt 60 for 60 minutes.
The preparation method comprises the specific steps of putting the sterilized PLA scaffold into a type I collagen solution for first incubation: the sterilized PLA scaffold was immersed in a type I collagen solution having a concentration of 2 mg/mL for 24 hours, and the temperature of the type I collagen solution was set to 4 ℃.
The manufacturing method of the invention comprises the following specific steps: and (3) rinsing the PLA bracket for three times by distilled water, wherein the specific steps of the first sterilization are as follows: the PLA stent was uv sterilized for 60 minutes.
The preparation method of the invention comprises the specific steps of placing the PLA scaffold after the first sterilization into simulated body fluid for the second incubation:
firstly, preparing simulated body fluid, putting 800mL of distilled water into a beaker, heating to 36 ℃ under magnetic stirring, and then sequentially adding 7.996g of NaCl reagent and 0.350g of NaHCO 3 Reagent, 0.224g KCl reagent, 0.228g K 2 HPO 4 ·3H 2 O reagent, 0.305g MgCl 2 ·6H 2 O reagent, 0.278g CaCl 2 Reagent and 0.071g of Na 2 SO 4 Adding reagents, adding next reagent after each reagent is completely dissolved, adding tris buffer solution to adjust pH value to 7.40 after all reagents are completely dissolved, adjusting temperature to 36.5 ℃, fixing volume to 1000mL to obtain simulated body fluid,
and then placing the PLA scaffold subjected to the first sterilization into the prepared simulated body fluid for infiltration for 24 hours, so that a bone-like apatite coating structure is constructed on the surface of the PLA scaffold.
The manufacturing method of the invention comprises the following specific steps of: and (3) rinsing the PLA bracket for three times by distilled water, wherein the specific steps of the second sterilization are as follows: the PLA stent was uv sterilized for 60 minutes.
In the preparation of the simulated body fluid according to the invention, tris buffer is added to adjust the pH, i.e.50 mmol/L (CH 2 OH)3CNH 2 And HCl buffer at a concentration of 0.1mol/L to adjust the pH to 7.40. When the simulated body fluid was fixed to 1000mL, the concentration of the simulated body fluid was 1.0mol/L. The ion concentration of each component in the simulated body fluid approximates the ion concentration of human plasma, see table 1 below.
TABLE 1 comparative Table simulating the ion concentration of each component in body fluids and human plasma
| Na + | K + | Ca + | Mg + | Cl - | HCO 3 - | HPO 4 2- | HSO 4 2- | |
| Human blood plasma | 142.0 | 5.0 | 2.5 | 1.5 | 103.0 | 27.0 | 1.0 | 0.5 |
| Simulated body fluid | 142.0 | 5.0 | 2.5 | 1.5 | 148.0 | 4.2 | 1.0 | 0.5 |
The biomimetic mineralized 3D printing PLA bracket is prepared according to the preparation method. The specific structure of the biomimetic mineralized 3D printed PLA scaffold in the invention is described in detail below.
As shown in fig. 1 and in combination with fig. 2-8, the biomimetic mineralized 3D printing PLA stent in the present invention, wherein the PLA stent is cylindrical, and a first through hole 1, a second through hole 2 and a third through hole 3 are provided on the PLA stent. The first through holes 1 are arranged along the axial direction of the PLA stent, that is, the first through holes 1 penetrate through the upper and lower end surfaces of the PLA stent. The second through hole 2 and the third through hole 3 are both arranged perpendicular to the axial direction of the PLA bracket, and the second through hole 2 is perpendicular to the third through hole 3, that is, the second through hole 2 and the third through hole 3 are both outer circumferential surfaces penetrating through two opposite sides of the PLA bracket. It can be seen that the first through hole 1 penetrates through the upper and lower end surfaces of the PLA holder in the Z direction, the second through hole 2 penetrates through the outer circumferential surfaces of the opposite sides of the PLA holder in the X direction, and the third through hole 3 penetrates through the outer circumferential surfaces of the opposite sides of the PLA holder in the Y direction. The first through holes 1, the second through holes 2 and the third through holes 3 are all arranged in a plurality, the first through holes 1 are arranged in an array, the second through holes 2 are also arranged in an array, and the third through holes 3 are also arranged in an array.
The biomimetic mineralized 3D printing PLA bracket provided by the invention has the advantages that the first through hole 1, the second through hole 2 and the third through hole 3 are square holes, the side length of each square hole is 350 mu m, and the interval between every two adjacent square holes is 350 mu m.
1-4 show an embodiment of a biomimetic mineralized 3D printed PLA scaffold, the length of the PLA scaffold in the embodiment is 3mm, and the diameter is 12 mm. Fig. 5-8 show a second embodiment of a biomimetic mineralized 3D printed PLA scaffold, where the PLA scaffold in this embodiment has a length of 12 mm and a diameter of 5 mm. The first embodiment of the biomimetic mineralized 3D printed PLA scaffold was prepared for in vitro experiments, while the second embodiment was applied to animal experiments, i.e. prepared for animal experiments. When the invention is applied to a human body, the PLA bracket matched with the human body can be prepared according to the shape of the specific bone defect part of the human body.
The bionic mineralized 3D printing PLA bracket and the manufacturing method are different from the prior art in that the PLA bracket is obtained through 3D printing, has a porous structure, structurally simulates the spatial configuration of a bionic cancellous bone, combines the PLA bracket with an I-type collagen solution and simulated body fluid in sequence after being modified by a plasma technology, prepares a bionic micro-surface structure by means of fixing the I-type collagen and a hydroxyapatite-like coating, increases cell recognition sites, and realizes the constitutive bionic natural bone of the bracket. Therefore, the invention is modified based on a plasma technology, not only can increase the surface roughness of PLA material so as to promote cell adhesion and proliferation, but also can promote protein adhesion through the introduction of active groups so as to increase biological recognition sites and promote bone ingrowth.
When the PLA scaffold is 3D printed, setting basic printing parameters, namely: the underfill had a thickness of 1mm, a packing density of 20%, a printing speed of 30mm/s, a nozzle temperature of 210 ℃, a hot bed temperature of 50 ℃, a printing material extrusion amount of 100%, and a nozzle aperture of 0.3mm.
The invention takes the bionic idea as an entry point, and designs and prepares the bionic bone material based on the natural structure and composition of the bone so as to construct the cellular tissue microenvironment. Bone structure is known to be mainly compact bone and cancellous bone, cancellous bone is a three-dimensional porous loose structure, and porosity is more than 70%. The bone mainly comprises 35% of organic components and 65% of inorganic components, nearly 90% of the organic matrix consists of I-type collagen, and more than 60% of the inorganic matters are hydroxyapatite. Therefore, polylactic acid (PLA) approved by the national food and drug administration (CFDA) and the American Food and Drug Administration (FDA) is used as a raw material, and a 3D printing technology is adopted to build the structure of the three-dimensional porous bracket bionic natural bone; the surface property and the topology morphology of the scaffold are improved by adopting plasma modification, an I-type collagen and hydroxyapatite-like bionic composition structure is constructed, and the bionic composite bone repair scaffold which is formed by combining the bone structure bionics and the bone composition bionics is established, so that the bionic composite bone repair scaffold is characterized by combining personalized structural design, stable and accurate configuration and microenvironment component regulation.
In summary, the biomimetic mineralized 3D printing PLA scaffold is manufactured through 3D printing, the porosity of the scaffold is 84.1% (higher than 80%) and the pore diameter is 350 mu m, the spatial configuration of the biomimetic cancellous bone is simulated structurally, the biomimetic mineralized PLA scaffold is combined with the type I collagen and simulated body fluid sequentially after being modified by a plasma technology, a biomimetic micro-surface structure is manufactured through the fixation of the type I collagen and the means of the hydroxyapatite-like coating, the cell recognition site is increased, and the biomimetic natural bone on the composition of the scaffold is realized. In vitro experiments prove that the biomimetic mineralized 3D printing PLA scaffold does not change the macroscopic structure and mechanical property of the scaffold, and the modified scaffold has good hydrophilicity and certain mechanical property, can effectively promote cell adhesion, proliferation and differentiation, promotes the expression of osteogenic genes ALP, OPN, col-1, OCN and RunX2, and has the performance of repairing rabbit radius critical bone defects.
The beneficial effects of the invention are as follows:
(1) The three-dimensional porous structure scaffold is designed and prepared through a 3D printing technology, the porosity of the scaffold is higher than 80%, the pore diameter is 350 mu m, and the spatial configuration of the bionic cancellous bone is simulated structurally.
(2) The bone repair material with the component bionic is prepared by a simple and easy modification method on the premise of not influencing the inherent characteristics of the material and introducing toxic substances through plasma modification, type I collagen loading and simulated body fluid mineralization.
(3) The bionic composite bone repair stent which is combined with personalized structural design, stable and accurate configuration and micro-environment component regulation is realized.
It should be noted that, the positional or positional relationship indicated by the terms such as "center", "upper", "lower", "front", "rear", "left", "right", "middle", etc. are based on the positional or positional relationship shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the apparatus or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "connected," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (7)
1. The manufacturing method of the biomimetic mineralized 3D printing PLA bracket is characterized by comprising the following steps:
a PLA scaffold with a porous structure was obtained by 3D printing,
performing plasma modification on the PLA bracket obtained by 3D printing, then sterilizing the PLA bracket subjected to plasma modification,
placing the sterilized PLA scaffold into a type I collagen solution for first incubation, taking the PLA scaffold out of the type I collagen solution after the first incubation is completed, performing first cleaning and first sterilization,
placing the PLA scaffold after the first sterilization into the simulated body fluid for the second incubation, taking the PLA scaffold out of the simulated body fluid after the second incubation is completed, performing the second cleaning and the second sterilization,
the specific steps of plasma modification of the PLA bracket obtained by 3D printing are as follows:
placing the PLA bracket obtained by 3D printing in a vacuum cavity of a plasma cleaning machine, vacuumizing the vacuum cavity to below 10Pa, then filling air into the vacuum cavity to adjust the pressure to 240Pa, setting the frequency of the plasma cleaning machine to be 13.56MHz after the pressure in the vacuum cavity is stable, setting the discharge power to be 20W and the treatment time to be 30 minutes, then starting the plasma cleaning machine, generating glow discharge plasma by the plasma cleaning machine to treat the PLA bracket obtained by 3D printing,
the method comprises the specific steps of placing the sterilized PLA scaffold into a type I collagen solution for first incubation: placing the sterilized PLA scaffold into 2 mg/mL type I collagen solution for infiltration for 24 hours, setting the temperature of the type I collagen solution to be 4 ℃,
the specific steps of putting the PLA scaffold after the first sterilization into simulated body fluid for the second incubation are as follows:
firstly, preparing simulated body fluid, putting 800mL of distilled water into a beaker, heating to 36 ℃ under magnetic stirring, and then sequentially adding 7.996g of NaCl reagent and 0.350g of NaHCO 3 Reagent, 0.224g KCl reagentK of 0.228g 2 HPO 4 ·3H 2 O reagent, 0.305g MgCl 2 ·6H 2 O reagent, 0.278g CaCl 2 Reagent and 0.071g of Na 2 SO 4 Adding reagents, adding next reagent after each reagent is completely dissolved, adding tris buffer solution to adjust pH value to 7.40 after all reagents are completely dissolved, adjusting temperature to 36.5 ℃, fixing volume to 1000mL to obtain simulated body fluid,
the PLA scaffold after the first sterilization was then placed into the resulting simulated body fluid for 24 hours of infiltration.
2. The method according to claim 1, wherein the specific steps of sterilizing the plasma modified PLA stent are: the plasma modified PLA scaffold was sterilized by cobalt 60 for 60 minutes.
3. The method of claim 2, wherein,
the specific steps of the first cleaning are as follows: the PLA stent was rinsed three times with distilled water,
the specific steps of the first sterilization are as follows: the PLA stent was uv sterilized for 60 minutes.
4. The method of claim 3, wherein,
the specific steps of the second cleaning are as follows: the PLA stent was rinsed three times with distilled water,
the specific steps of the second sterilization are as follows: the PLA stent was uv sterilized for 60 minutes.
5. A biomimetic mineralized 3D printed PLA scaffold, made according to the manufacturing method of any one of claims 1-4.
6. The biomimetic mineralized 3D printed PLA scaffold as in claim 5, wherein: the PLA support is cylindricly, be equipped with first through-hole, second through-hole and third through-hole on the PLA support, the axial that the PLA support was followed to first through-hole is arranged, the axial that second through-hole and third through-hole all perpendicular to PLA support is arranged, second through-hole perpendicular to third through-hole, first through-hole, second through-hole and third through-hole all are established to a plurality of, and a plurality of first through-holes are array arrangement, and a plurality of second through-holes are array arrangement too, and a plurality of third through-holes are array arrangement too.
7. The biomimetic mineralized 3D printed PLA scaffold as in claim 6, wherein: the first through holes, the second through holes and the third through holes are square holes, the side length of each square hole is 350 mu m, and the interval between every two adjacent square holes is 350 mu m.
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