Mineralizable guided bone regeneration membrane based on 3D printing as well as preparation method and application of mineralizable guided bone regeneration membrane

Abstract

The invention provides a mineralizable guided bone regeneration membrane based on 3D printing as well as a preparation method and application thereof, and relates to the technical field of bone tissue regeneration. The preparation method of the mineralizable guided bone regeneration membrane comprises the steps of precursor solution preparation, DLP 3D printing, surface mineralization and the like. The structural adaptability of the membrane is guaranteed through the digital light processing 3D printing technology, the bioactivity of osteoblasts is activated through the ZIF-8 mineralization layer, dynamic balance of mechanical support and bone regeneration of the membrane is maintained through an intelligent degradation system, finally, three-in-one bone repair regulation of structure-function-metabolism is achieved, and the membrane has excellent clinical transformation potential.

Description

Technical Field

[0001] The present invention relates to the technical field of bone tissue regeneration, and particularly to a mineralizable guided bone regeneration membrane based on 3D printing, a preparation method thereof, and an application thereof. Background Art

[0002] In the field of clinical medicine, how to effectively repair bone tissue defects caused by trauma, inflammation, congenital malformations, or tumor resection has always been a major challenging topic and research focus. As an important solution for bone defect repair, the guided bone regeneration technology creates a physical barrier interface at the bone defect site through the precise application of a guided bone regeneration membrane (GBR membrane). This bioengineering strategy can selectively inhibit the invasion and interference of surrounding soft tissues (including fibroblasts and epithelial cells), thereby creating a favorable microenvironment for bone tissue regeneration and ensuring the smooth progress of the bone repair process dominated by osteoblasts.

[0003] Traditional GBR membranes are generally made of collagen or polytetrafluoroethylene and obtained after being formed into a flat sheet structure through a molding or electrospinning process. These traditional GBR membranes generally have problems such as poor plasticity and low interface fitting degree. During the operation, they need to be repeatedly hand-cut and adjusted to fit the shape of the bone defect, which easily leads to poor sealing between the barrier membrane and the edge of the bone defect (fitting gap > 1 mm), resulting in delayed bone healing or even regeneration failure. After the operation, due to the existence of the gap, the soft tissue infiltration rate is high (30%). In addition, the mechanical properties of traditional collagen membranes are weak, and the tensile strength is only about 5 MPa, which cannot resist the operation stress during the operation, is prone to tearing or collapse, and is difficult to maintain the three-dimensional spatial stability of large bone defects, especially prone to collapse and deformation in the load-bearing area.

[0004] For the repair of complex bone defects, autologous bone transplantation is the "gold standard" for clinical repair of long bone defects, but its donor source is limited (such as insufficient bone harvesting from the ilium and fibula), and bone tissue needs to be obtained through a second operation, which not only increases the trauma and pain of the patient, but also the incidence of donor site complications (such as hematoma, infection, chronic pain) exceeds 20%. Allogeneic bone transplantation faces the core problem of immune rejection. Even after decellularization or lyophilization treatment, residual antigens may still induce an inflammatory response, resulting in graft absorption or fibrous encapsulation. At the same time, it also has problems such as biosafety risks (such as virus transmission) and high postoperative infection rates. The above defects together make it difficult for existing repair schemes to meet the clinical needs of personalized and functional bone regeneration.

[0005] Therefore, it is necessary to develop a GBR membrane with precise adaptability, controllable degradation, biosafety, and good osteogenic efficacy and mechanical properties to overcome the problems existing in the prior art. Summary of the Invention

[0006] In view of this, the present invention provides a mineralizable guided bone regeneration membrane based on 3D printing, its preparation method and application. This method ensures structural adaptability through digital light processing (DLP) 3D printing technology, activates the biological activity of osteoblasts with a ZIF-8 mineralized layer, and maintains the dynamic balance between mechanical support and bone regeneration with an intelligent degradation system, ultimately achieving a trinity of "structure-function-metabolism" bone repair regulation and having excellent clinical transformation potential.

[0007] The first aspect of the present invention is to provide a preparation method of a mineralizable guided bone regeneration membrane based on 3D printing, including the following steps:

[0008] Prepare a precursor solution, then design an STL model based on CT scan data, obtain a printed membrane by DLP 3D printing using the precursor solution as the raw material, and immerse the printed membrane in an SBF solution for surface mineralization to obtain a mineralizable guided bone regeneration membrane based on 3D printing.

[0009] Furthermore, the precursor solution includes the following raw materials:

[0010] 8 wt.% - 12 wt.% of tetra-armed polyethylene glycol modified with o-nitrobenzyl alcohol (PEGNB), 1 wt.% - 3 wt.% of methacrylated hyaluronic acid (HAMA), 0.5 wt.% - 1 wt.% of a photoinitiator, the content of ZIF-8 powder is 2 wt.% - 10 wt.%, and the balance is deionized water.

[0011] Even further, the precursor solution includes the following raw materials:

[0012] 10 wt.% of PEGNB modified with o-nitrobenzyl alcohol, 2 wt.% of HAMA, 0.6 wt.% of a photoinitiator, 10 wt.% of ZIF-8 powder, and the balance is deionized water.

[0013] Furthermore, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0014] Furthermore, the accuracy of the 3D scanner is ±0.05 mm, the ultraviolet light intensity is 14 - 15 mW / cm 3 , the exposure time is 55 - 65 s / layer, and the layer thickness is 50 μm;

[0015] Furthermore, the preparation method of the SBF solution is as follows:

[0016] Dissolve 7.996 g of NaCl, 0.350 g of NaHCO3, 0.224 g of KCl, 0.231 g of K2HPO4·3H2O, 0.305 g of MgCl2·6H2O, 0.278 g of CaCl2, 0.071 g of Na2SO4, and 6.057 g of tris(hydroxymethyl)aminomethane (Tris-base) in 900 mL of deionized water. Adjust the pH to 7.4 with 1 M HCl or NaOH and then make up the volume to 1 L with deionized water to obtain the SBF solution.

[0017] Furthermore, the surface mineralization temperature is 36.5°C - 37.5°C, preferably 37°C, and the surface mineralization time is 8 - 10 days, preferably 10 days. During this period, the SBF solution is replaced every 3 days.

[0018] The second aspect of the present invention is to provide a 3D printing-based mineralizable guided bone regeneration membrane prepared according to the above method.

[0019] The third aspect of the present invention is to provide an application of the 3D printing-based mineralizable guided bone regeneration membrane in bone tissue regeneration, and the 3D printing-based mineralizable guided bone regeneration membrane is the one described in the above solution.

[0020] The present invention has achieved a breakthrough upgrade of bone defect repair materials through multi-dimensional technological innovation, and its technical advantages are concentrated in the following aspects:

[0021] (1) Precise adaptation: The present invention constructs a personalized three-dimensional model based on the patient's CT image data and uses DLP 3D printing technology to achieve a manufacturing accuracy of the micron level (error < 0.1 mm), completely solving the interface mismatch problem caused by the "one-size-fits-all" of traditional membrane materials;

[0022] (2) Osteogenic efficacy: The present invention constructs a hydroxyapatite active layer with a thickness of 50 - 100 μm (uniformity error < 5%) through ZIF-8-induced surface biomimetic mineralization, which can increase the osteoblast differentiation efficiency by 40% and enhance the alkaline phosphatase (ALP) activity by 2 times;

[0023] (3) Degradation controllability: The PEGNB / HAMA hydrogel system of the present invention regulates the degradation period (6 - 12 months) through the crosslinking density, realizes the dynamic matching of the material degradation rate and the bone regeneration process, and avoids the risk of secondary surgery;

[0024] (4) Mechanical properties: The tensile strength of the ZIF-8 reinforced composite membrane of the present invention breaks through 10 MPa, which is 200% higher than that of the traditional collagen membrane. It can stably maintain the structural integrity of large bone defects, and the clinical repair period is shortened to 70% of the traditional method;

[0025] (5) Biosafety: The proliferation activity of bone marrow mesenchymal stem cells (BMSCs) on the surface of the material of the present invention is increased by 50%. After in vivo implantation, there is no abnormal expression of inflammatory factors or toxic reactions, showing excellent potential for clinical translation. Brief Description of the Drawings

[0026] The present invention will be further described below in conjunction with the drawings.

[0027] Figure 1 It is a physical diagram of the precursor solutions for Examples 1-3 and Comparative Example 1;

[0028] Figure 2 It is an infrared spectrum (FTIR) diagram of the products for Examples 1-3 and Comparative Example 1;

[0029] Figure 3 It is an X-ray diffraction (XRD) diagram of the products for Examples 1-3 and Comparative Example 1;

[0030] Figure 4 It is a rheological analysis diagram of the products for Examples 1-3 and Comparative Example 1;

[0031] Figure 5 It is a CAD model (STL format) of the product;

[0032] Figure 6 It is a 3D printed PEGNB / HAMA and ZIF-8 / P / H GBR membrane;

[0033] Figure 7 It is a Calcein-AM staining result diagram;

[0034] Figure 8 It is an ALP and ARS staining result diagram;

[0035] Figure 9 It is a Micro-CT diagram;

[0036] Figure 10 It is a HE and Masson staining result diagram. Detailed Embodiments

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0038] The first aspect of the present invention is to provide a preparation method of a mineralizable guided bone regeneration membrane based on 3D printing, specifically including the following steps:

[0039] S1. Preparation of precursor solution

[0040] Dissolve PEGNB, HAMA, and photoinitiator in deionized water, homogenize, and then add ZIF-8 powder and disperse evenly to obtain a precursor solution, where the content of PEGNB is 8 wt.% - 12 wt.%, the content of HAMA is 1 wt.% - 3 wt.%, the content of photoinitiator is 0.5 wt.% - 1 wt.%, and the content of ZIF-8 powder is 2 wt.% - 10 wt.%; the photoinitiator is LAP; the precursor solution is stored at 4°C;

[0041] In some preferred embodiments of the present invention, the precursor solution comprises the following raw materials:

[0042] PEGNB modified with o-nitrobenzyl alcohol 10 wt.%, HAMA 2 wt.%, photoinitiator 0.6 wt.%, and the content of ZIF-8 powder is 10 wt.%, and the balance is deionized water.

[0043] S2, DLP 3D printing

[0044] Design an STL model based on CT scan data, and obtain a printed film by DLP 3D printing using the precursor solution as the raw material; the accuracy of the 3D scanner is ±0.05 mm, the ultraviolet light intensity is 14 - 15 mW / cm 3 , preferably 15 mW / cm 3 , the exposure time is 55 - 65 s / layer, preferably 65 s / layer, and the layer thickness is 50 μm;

[0045] S3, surface mineralization

[0046] Immerse the printed film in SBF solution for surface mineralization to obtain a 3D printing-based mineralizable guided bone regeneration membrane; the preparation method of the SBF solution is as follows: Dissolve 7.996 g of NaCl, 0.350 g of NaHCO3, 0.224 g of KCl, 0.231 g of K2HPO4·3H2O, 0.305 g of MgCl2·6H2O, 0.278 g of CaCl2, 0.071 g of Na2SO4, and 6.057 g of tris(hydroxymethyl)aminomethane (Tris-base) in 900 mL of deionized water, adjust the pH to 7.4 with 1M HCl or NaOH and then make up the volume to 1 L with deionized water to obtain the SBF solution; the SBF solution is stored in the dark at 4°C and the validity period is 7 days; the surface mineralization temperature is 36.5°C - 37.5°C, preferably 37°C, the surface mineralization time is 8 - 10 days, preferably 10 days, and the SBF solution is replaced every 3 days during this period.

[0047] After step S3 of the present invention, there is also a sterilization and packaging process, specifically: irradiating and sterilizing the 3D printing-based mineralizable guided bone regeneration membrane, and then performing aseptic packaging to obtain the finished product; the dose of γ-ray irradiation sterilization is 25 kGy.

[0048] The second aspect of the present invention is to provide a 3D printing-based mineralizable guided bone regeneration membrane prepared according to the above method.

[0049] The third aspect of the present invention is to provide an application of a 3D printing-based mineralizable guided bone regeneration membrane in bone tissue regeneration.

[0050] To further illustrate the present invention, the following examples are used for detailed description. The raw materials used in the following examples of the present invention are all commercially available.

[0051] Unless otherwise specified, all tests are repeated 3 times, and the results are expressed as averages.

[0052] Example 1 A method for preparing a 3D printing-based mineralizable guided bone regeneration membrane, the steps are as follows:

[0053] S1. Dissolve PEGNB, HAMA, and LAP in deionized water, homogenize, and then add ZIF-8 powder and disperse evenly to obtain a precursor solution, which is stored at 4°C. The content of PEGNB is 10 wt.%, the content of HAMA is 2 wt.%, the content of LAP is 0.6 wt.%, and the content of ZIF-8 powder is 10 wt.%.

[0054] S2. Design an STL model based on the bone defect CT scan data (3D scanning accuracy ±0.05 mm), and use the precursor solution as the raw material to obtain a printed membrane with a length of 2.5 cm by DLP 3D printing. The DLP 3D printing layer thickness is set to 50 μm, and the ultraviolet light intensity is 15 mW / cm 3 , the exposure time for each layer is 65 s, and the total printing time is 1.5 h;

[0055] S3. Immerse the printed membrane in SBF solution at 37°C for 10 days, and replace the SBF solution every 3 days. A uniform hydroxyapatite layer is formed on the membrane surface to obtain a 5% ZIF-8 / PEGNB / HAMA 3D printing-based mineralizable guided bone regeneration membrane, denoted as 10% ZIF-8 / P / H.

[0056] After testing, the tensile strength of the 10% ZIF-8 / P / H membrane is 12.5 MPa, which is significantly higher than 4.8 MPa of the traditional collagen membrane.

[0057] Example 2

[0058] Same as Example 1, with the difference that the content of ZIF-8 powder in the precursor solution is 2 wt.%, denoted as 2% ZIF-8 / P / H.

[0059] After testing, the tensile strength of the membrane is 8.5 MPa.

[0060] Example 3

[0061] Same as Example 1, with the difference that the content of ZIF-8 powder in the precursor solution is 5 wt.%, denoted as 5% ZIF-8 / P / H.

[0062] After testing, the tensile strength of the membrane is 10.5 MPa.

[0063] Comparative Example 1

[0064] Same as Example 1, with the difference that the precursor solution does not contain ZIF-8 powder, denoted as PEGNB / HAMA.

[0065] After testing, the tensile strength of the membrane is 7.8 MPa.

[0066] Test Example 1 Compatibility test

[0067] The compatibility of the materials obtained in Examples 1-3 and Comparative Example 1 was tested. The experimental method is as follows:

[0068] BMSCs culture:

[0069] Rat BMSCs (passage 3) were seeded on the surface of the mineralized membrane at a density of 4×10 4 / cm 2 and cultured for 7 days. Calcein-AM staining was used to detect cell viability. The blank control group was denoted as Blank. The results are as Figure 7 shown.

[0070] Osteogenic differentiation assessment:

[0071] ALP staining (day 7) and ARS staining (day 14) were performed. The blank control group was denoted as Blank. The results are as Figure 8 shown.

[0072] Test Example 2 Animal experiment

[0073] Rabbit radius defect model:

[0074] Eighteen New Zealand rabbits were randomly divided into 3 groups, denoted as the blank group, the PEGNB / HAMA group, and the 5% ZIF-8 / P / H group.

[0075] A 1.4 cm radius segment was surgically removed and the corresponding GBR membrane was implanted. The rabbits were sacrificed 12 weeks after surgery.

[0076] Detection indexes:

[0077] Micro-CT was used to analyze bone volume (BV) and bone mineral density (BMD). The blank control group was denoted as Blank, and the results were as Figure 9 shown.

[0078] HE and Masson staining were used to evaluate soft tissue infiltration and new bone formation. The blank control group was denoted as Blank, and the results were as Figure 10 shown.

[0079] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A preparation method of a mineralizable guided bone regeneration membrane based on 3D printing, characterized in that, It includes the following steps: Prepare a precursor solution, then design an STL model based on CT scan data, use the precursor solution as raw material to obtain a printed film by DLP 3D printing, immerse the printed film in an SBF solution for surface mineralization, and obtain a 3D printing-based mineralizable guided bone regeneration membrane.

2. The preparation method according to claim 1, wherein, The precursor solution includes the following raw materials: 8 wt.% - 12 wt.% of tetra-arm polyethylene glycol modified with o-nitrobenzyl alcohol, 1 wt.% - 3 wt.% of methacrylated hyaluronic acid, 0.5 wt.% - 1 wt.% of photoinitiator, 2 wt.% - 10 wt.% of ZIF-8 powder, and the balance is deionized water.

3. The preparation method according to claim 2, wherein The precursor solution includes the following raw materials: 10 wt.% of tetra-arm polyethylene glycol modified with o-nitrobenzyl alcohol, 2 wt.% of methacrylated hyaluronic acid, 0.6 wt.% of photoinitiator, 10 wt.% of ZIF-8 powder, and the balance is deionized water.

4. The preparation method according to claim 1, wherein, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

5. The preparation method according to claim 1, characterized in that, The accuracy of the 3D scanner is ±0.05 mm.

6. The preparation method according to claim 1, wherein During the post-curing process of the DLP 3D printing, the intensity of the ultraviolet light used is 14 - 15 mW / cm 3 , the exposure time is 55 - 65 s / layer, and the layer thickness is 50 μm.

7. The preparation method according to claim 1, characterized in that The preparation method of the SBF solution is as follows: Dissolve 7.996 g of NaCl, 0.350 g of NaHCO3, 0.224 g of KCl, 0.231 g of K2HPO4·3H2O, 0.305 g of MgCl2·6H2O, 0.278 g of CaCl2, 0.071 g of Na2SO4, and 6.057 g of tris(hydroxymethyl)aminomethane in 900 mL of deionized water, adjust the pH to 7.4 with 1M HCl or NaOH, and then make up the volume to 1 L with deionized water to obtain the SBF solution.

8. The preparation method according to claim 1, wherein The surface mineralization temperature is 37 °C and the surface mineralization time is 10 days.

9. A mineralizable guided bone regeneration membrane based on 3D printing, characterized in that, The 3D printing-based mineralizable guided bone regeneration membrane is prepared by the method according to any one of claims 1-8.

10. Application of a mineralizable guided bone regeneration membrane based on 3D printing in bone tissue regeneration, characterized in that, The 3D printing-based mineralizable guided bone regeneration membrane is a 3D printing-based mineralizable guided bone regeneration membrane prepared by the method according to any one of claims 1-8.

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