Synthetic method of bionic gradient mineralized collagen-polycaprolactone composite material

Abstract

The invention relates to the technical field of biomedical material synthesis, in particular to a synthetic method of a bionic gradient mineralized collagen-polycaprolactone composite material, which comprises the following steps: a) activating the surface of PCL (polycaprolactone) through argon plasma to generate carboxyl groups with the density of more than or equal to 5 * 10 / cm; b) carrying out amination modification on nano-hydroxyapatite (nHA) by adopting APTES, wherein the surface potential of the modified nHA is greater than or equal to + 15 mV; c) covalently grafting aminated nHA to the surface of PCL through an amido bond under the conditions that the molar ratio of EDC / NHS is 1: 2 and the pH is 5.5 + / -0.2, and the grafting density is greater than or equal to 3 * 10 particles / mu m; the method has the beneficial effects that the gradient mineralization structure is subjected to bionic optimization: the concentration and the flow velocity (the inner / outer layer flow velocity ratio is 1: 3) of mineralization liquid are accurately controlled through a microfluidic gradient generator, linear gradual change (the gradient slope is 1.5 + / -0.3 wt% / 100 [mu] m, R is equal to 0.97) of the mineralization degree from 20-30 wt% of the inner layer to 50-60 wt% of the outer layer is realized, the mechanical gradient characteristics of the natural bone are simulated, the compression modulus is increased from 0.8 GPa of the inner layer to 3.2 GPa of the outer layer in a gradient manner, and the mechanical property of the natural bone is improved. The mechanical matching property of the material and the host bone is obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of oral tooth regeneration material synthesis, in particular to a method for synthesizing a biomimetic gradient mineralized collagen-polycaprolactone composite material. Background Art

[0002] Existing tooth tissue regeneration materials have the following limitations:

[0003] Uncontrollable degradation rate: The degradation rate of traditional polycaprolactone (PCL) materials is too slow (degradation rate <30% in 12 weeks), which cannot match the dentin regeneration cycle; homogeneous copolymer design makes it difficult to balance degradation rate and mechanical strength; gradient structure mismatch: materials with a single mineralization degree cannot simulate the gradient characteristics of the natural dentin-pulp interface, resulting in a lack of coordination between mechanical support and degradation rate, affecting the staged regeneration of tissues; insufficient bioactivity: lack of a growth factor controlled release system, low cell adhesion rate (<60%), and easy to cause bacterial infection, limiting clinical translation.

[0004] Therefore, we proposed a synthesis method of biomimetic gradient mineralized collagen-polycaprolactone composite materials to solve the above problems. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention discloses a method for synthesizing a biomimetic gradient mineralized collagen-polycaprolactone composite material, the technical solution adopted is comprising the following steps:

[0006] a) Activate the PCL surface by argon plasma to generate carboxyl groups with a density of ≥5×10¹ 5 / cm²;

[0007] b) APTES was used to modify nanohydroxyapatite (nHA) by amino modification, and the surface potential of the modified nHA was ≥ +15 mV;

[0008] c) Aminated nHA was covalently grafted onto the PCL surface via amide bonds at an EDC / NHS molar ratio of 1:2 and pH 5.5 ± 0.2, with a grafting density of ≥ 3 × 10 4 Particles / μm²;

[0009] d) Using a microfluidic gradient generator, sequentially inject an inner layer mineralization solution (parameters: Ca²⁺5mM + PO4³⁻3mM + collagen 1mg / mL, pH 7.0) and an outer layer mineralization solution (parameters: Ca²⁺15mM + PO4³⁻9mM + collagen 0.5mg / mL, pH 7.8), controlling the mineralization degree to linearly change from 20-30wt% in the inner layer to 50-60wt% in the outer layer, with a gradient slope of 2.5wt% / 100μm;

[0010] e) During the mineralization process, the pH and collagen concentration are regulated so that hydroxyapatite preferentially grows along the axial direction of the collagen fibers, with a (002) crystal plane orientation of ≥85%.

[0011] As a preferred technical solution of the present invention, the argon plasma treatment parameters in step a) are power 50 W and time 5 min, and the contact angle of the PCL surface after treatment is ≤35°.

[0012] As a preferred technical solution of the present invention, in step d), the channel width of the microfluidic gradient generator is 100 μm, and the flow rate ratio of the inner layer and the outer layer mineralization liquid is 1:3.

[0013] As a preferred technical solution of the present invention, the mineralization temperature of the inner layer is 37° C., and the mineralization time is 24 hours; the mineralization temperature of the outer layer is 42° C., and the mineralization time is 72 hours.

[0014] As a preferred technical solution of the present invention, the bionic gradient mineralized composite material has a tensile strength of ≥55 MPa, an interface bonding force of ≥40 nN, and a compression modulus that increases gradually from 0.8 GPa in the inner layer to 3.2 GPa in the outer layer.

[0015] As a preferred technical solution of the present invention, in step d), the pH of the inner layer mineralization liquid is 7.0±0.2, the pH of the outer layer mineralization liquid is 7.8±0.2, and the slope of the mineralization gradient is 1.5±0.3wt% / 100μm.

[0016] As a preferred technical solution of the present invention, the swelling rate of the bionic gradient mineralized composite material is 52±2%, and the 7-day osteoblast proliferation rate is ≥240%.

[0017] Beneficial effects of the present invention:

[0018] 1. Precise degradation control: Through molecular weight design (Mn=10-20kDa) and the introduction of PEG / PLA / PGA copolymers, a 12-week degradation rate of 60-80% is achieved while maintaining a tensile strength of ≥40MPa, resolving the contradiction of traditional materials: "fast degradation leads to weak mechanical properties, while strong mechanical properties lead to slow degradation";

[0019] 2. Bionic gradient mineralization: The surface layer (60wt% HA) provides initial mechanical support (tensile strength ≥55MPa), the inner layer (20wt% HA) rapidly degrades (70% in 12 weeks) to release pores, and the gradient of the middle layer adapts to the dentin regeneration process. Microfluidic parameter optimization (channel 100μm, flow rate ratio 1:5) enables the mineralization gradient slope to reach 2.5wt% / 100μm, which is close to the natural dentin structure and promotes directional infiltration of dental pulp tissue. DETAILED DESCRIPTION

[0020] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0021] The present invention discloses a method for synthesizing a biomimetic gradient mineralized collagen-polycaprolactone composite material, and the technical solution adopted is as follows:

[0022] 1) Covalent coupling process of nHA and PCL

[0023] a) PCL surface activation

[0024] PCL film was treated with argon plasma (50W, 5min) and PCL with molecular weight (Mn=10-20kDa) was used to accelerate the degradation rate (target: 60-80% degradation rate in 12 weeks). Carboxyl groups (density ≥5×10¹) were introduced on its surface. 5 / cm²), the activation effect was verified by contact angle measurement (108°→32°) and XPS analysis (oxygen content 18%→42%), and PCL-PEG-PCL triblock copolymers were synthesized. The hydrophilicity of PEG was used to enhance the degradation rate while maintaining a tensile strength ≥40 MPa. PCL-PLA / PGA copolymers were introduced to accelerate degradation through ester bond hydrolysis.

[0025] b) Amino modification of nHA

[0026] Nanohydroxyapatite (nHA) with an aspect ratio of 20:1 and a diameter of 50 nm was selected and reacted in a 2% v / v 3-aminopropyltriethoxysilane (APTES) ethanol solution for 2 h. The successful amination was confirmed by Zeta potential analysis (surface potential changed from -25 mV to +18 mV).

[0027] c) Covalent grafting

[0028] Aminated nHA was covalently grafted onto the PCL surface via amide bonds at a molar ratio of 1:2 (EDC (50 mM) / NHS (100 mM)) and pH 5.5 ± 0.2. The grafting yield was 31.5 wt% as confirmed by thermogravimetric analysis (TGA), and the grafting density was ≥3 × 10 4 Particles / μm².

[0029] 2) Gradient mineralization construction

[0030] a) Inner low mineralization zone (20wt%HA)

[0031] Mineralization solution: 5mM Ca²⁺ + 3mM PO₄³⁻ + 1mg / mL collagen (pH 7.0), mineralized at 37°C for 24 hours. Under these conditions, collagen fibers are moderately mineralized, retaining their porous structure (swelling ratio 65±3%).

[0032] b) Outer layer high mineralization area (30-50wt%)

[0033] Mineralization solution formula: 15mM Ca²⁺ + 9mM PO₄³⁻ + 0.5mg / mL collagen (pH 7.8), mineralization at 42°C for 72 hours. Increasing temperature and ion concentration promotes rapid deposition of HA, forming a dense mineralized layer.

[0034] c) Surface layer (60wt% HA): degradation rate of 30% in 12 weeks, providing initial mechanical support

[0035] d) Gradient transition control

[0036] A microfluidic gradient generator (channel width 100 μm, flow rate ratio 1:3) was used to achieve a linear gradient of mineralization solution concentration. The mineralization gradient slope was 2.5 wt% / 100 μm, and Micro-CT three-dimensional reconstruction showed a linearity R²=0.97.

[0037] 3) HA crystal orientation regulation

[0038] By regulating the pH of the mineralization solution (7.0→7.8) and the collagen concentration (1→0.5 mg / mL), HA was induced to grow along the axial direction of the collagen fibers. XRD analysis showed that the (002) crystal plane orientation was ≥85%, which is better than traditional random mineralized materials (orientation <50%).

[0039] As a preferred technical solution of the present invention, when the EDC concentration is increased to 50 mM, the nHA grafting amount increases from 15.2 wt% to 31.5 wt% (TGA data), and the tensile strength increases from 22±2 MPa to 58±3 MPa.

[0040] As a preferred technical solution of the present invention, under the condition of EDC concentration of 50mM, the grafting density reached 3.2×10 4 particles / μm², interface bonding force 45±3nN.

[0041] As a preferred technical solution of the present invention, when the pH of the outer mineralization liquid is increased from 7.4 to 7.8, the mineralization degree increases from 45wt% to 58wt%, and the HA orientation degree increases from 72% to 85% (XRD analysis).

[0042] As a preferred technical solution of the present invention, when the flow rate ratio is 1:3, the mineralization gradient slope is 1.5wt% / 100μm (R²=0.97), which is better than 1:1 (R²=0.82) or 1:5 (discontinuous gradient).

[0043] Animal experiment verification

[0044] The material of the present invention was implanted into the tibial defect of beagle dogs (8 mm in diameter). After 6 weeks, Micro-CT showed that the new bone volume accounted for 62%, and tissue sections showed that the mineralized layer was seamlessly connected to the host bone (HE staining);

[0045] Mechanical testing showed that the interface bonding strength was 4.3±0.5MPa, close to the natural bone-bone interface (4.8±0.3MPa).

[0046] PCL surface activation and nHA grafting process optimization

[0047] Table 1: Effects of different plasma treatment parameters on PCL surface properties

[0048] Processing conditions (power / time) Contact angle (°) <![CDATA[Surface carboxyl density (×10¹ 5 / cm²)]]> XPS oxygen content (%) Unprocessed 108±2 0 18±1 30W / 5min 65±3 3.2±0.3 32±2 50W / 5min 32±1 5.1±0.4 42±1 70W / 5min 28±2 5.3±0.5 43±2 (but the material is coked)

[0049] Table 2: Effect of EDC concentration on nHA grafting amount

[0050] EDC concentration (mM) NHS concentration (mM) Grafting amount (wt%, TGA) Tensile strength (MPa) Interface bonding strength (nN) 20 40 15.2±1.3 38±2 22±3 50 100 31.5±2.1 58±3 45±3 80 160 33.8±2.5 60±4 (but the swelling rate drops to 50%) 46±4

[0051] Gradient mineralization parameter optimization

[0052] Table 3: Effect of pH of mineralization solution on HA crystal orientation

[0053] Mineralization solution pH (002) crystal plane orientation (%) Mineralization (wt%) Swelling rate (%) 7.0 72±2 25±1 65±3 7.4 80±3 45±2 58±2 7.8 85±1 58±3 52±2 8.2 86±2 (but collagen fibers are broken) 60±4 40±3

[0054] Table 4: Effect of microfluidic flow rate ratio on mineralization gradient linearity

[0055] Flow rate ratio (inner layer: outer layer) Gradient slope (wt% / 100μm) Linearity (R²) Mineralized layer bonding strength (MPa) 1:1 0.8±0.2 0.82±0.05 3.1±0.3 1:3 1.5±0.3 0.97±0.02 4.3±0.5 1:5 2.2±0.4 0.75±0.06 3.8±0.4 (discontinuous gradient)

[0056] Example 3: Biological performance verification

[0057] Table 5: Proliferation and differentiation of MC3T3 cells on different mineralized materials

[0058] Material Type 7-day proliferation rate (%) ALP activity (U / mg) OCN gene expression (relative fold) Unmineralized PCL 100±5 12±2 1.0±0.1 Homogeneous mineralized material (30wt%) 180±8 35±3 2.1±0.2 Gradient material of the present invention 245±10 98±5 3.5±0.3

[0059] Table 6: Comparison of bone defect repair effects in beagle dogs (6 weeks)

[0060] Group New bone volume percentage (%) Interface bonding strength (MPa) Bone density (g / cm³) Blank control group 15±3 - 0.25±0.02 Traditional homogeneous mineralized materials 38±4 2.8±0.4 0.52±0.03 Gradient material of the present invention 62±5 4.3±0.5 0.89±0.04 natural bone - 4.8±0.3 1.12±0.05

[0061] Components not described in detail herein are prior art.

[0062] Although the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by ordinary technicians in this field, various changes can be made without departing from the purpose of the present invention. Modifications or deformations that do not involve creative work are still within the scope of protection of the present invention.

Claims

1. A method for synthesizing a biomimetic gradient mineralized collagen-polycaprolactone composite material, characterized in that: The following steps are involved: a) Activate the PCL surface by argon plasma to generate carboxyl groups with a density of ≥5×10¹ 5 / cm²; b) APTES was used to modify nanohydroxyapatite (nHA) by amino modification, and the surface potential of the modified nHA was ≥ +15 mV; c) Aminated nHA was covalently grafted onto the PCL surface via amide bonds at an EDC / NHS molar ratio of 1:2 and pH 5.5 ± 0.2, with a grafting density of ≥ 3 × 10 4 Particles / μm²; d) Using a microfluidic gradient generator, sequentially inject an inner layer mineralization solution (parameters: Ca²⁺5mM + PO4³⁻3mM + collagen 1mg / mL, pH 7.0) and an outer layer mineralization solution (parameters: Ca²⁺15mM + PO4³⁻9mM + collagen 0.5mg / mL, pH 7.8), controlling the mineralization degree to linearly change from 20-30wt% in the inner layer to 50-60wt% in the outer layer, with a gradient slope of 2.5wt% / 100μm; e) During the mineralization process, the pH and collagen concentration are regulated so that hydroxyapatite preferentially grows along the axial direction of the collagen fibers, with a (002) crystal plane orientation of ≥85%.

2. The method for synthesizing the biomimetic gradient mineralized collagen-polycaprolactone composite material according to claim 1, wherein: The argon plasma treatment parameters in step a) are power 50 W and time 5 min, and the contact angle of the PCL surface after treatment is ≤35°.

3. The method for synthesizing the biomimetic gradient mineralized collagen-polycaprolactone composite material according to claim 1, characterized in that: In the step d), the channel width of the microfluidic gradient generator is 100 μm, and the flow rate ratio of the inner layer and the outer layer mineralization liquid is 1:

3.

4. The method for synthesizing the biomimetic gradient mineralized collagen-polycaprolactone composite material according to claim 1, characterized in that: The mineralization temperature of the inner layer is 37° C., and the mineralization time is 24 hours; the mineralization temperature of the outer layer is 42° C., and the mineralization time is 72 hours.

5. A biomimetic gradient mineralized composite material prepared according to any one of claims 1 to 4, characterized in that: The bionic gradient mineralized composite material has a tensile strength of ≥55 MPa, an interface bonding force of ≥40 nN, and a compression modulus that increases gradually from 0.8 GPa in the inner layer to 3.2 GPa in the outer layer.

6. The method according to claim 1, wherein: In the step d), the pH of the inner layer mineralized liquid is 7.0±0.2, the pH of the outer layer mineralized liquid is 7.8±0.2, and the slope of the mineralization gradient is 1.5±0.3 wt% / 100 μm.

7. A biomimetic gradient mineralized composite material prepared according to any one of claims 1 to 6, characterized in that: The swelling rate was 52±2%, and the osteoblast proliferation rate was ≥240% after 7 days.