Preparation method of oral cavity repairing filmTechnical Field
The invention belongs to the technical field of biomedical material preparation; in particular to a preparation method of an oral cavity repairing film.
Background
Tissue Engineering (Tissue Engineering) is a new subject which is emerging in recent years, and has a good development prospect and a wide application market. Acellular Dermal Matrix (ADM) is an excellent biomedical material and is widely used because of its good biocompatibility. At present, acellular dermal matrixes are mainly derived from terrestrial animals, and due to the limitations of spread of diseases such as foot-and-mouth disease, mad cow disease and the like and religious belief, the search for medical collagen from aquatic organisms as a substitute source becomes more and more important.
At present, most oral cavity repairing materials adopt a method of acellular matrix, the product specification and model are single, the plasticity is poor, or land-source collagen is adopted, the source is limited, the oral cavity repairing materials can only be extracted from specific animals or parts, the risk of potential disease pathogenicity exists, and the cost is relatively expensive.
The main component of the acellular dermal matrix is collagen, and the research proves that the aquatic collagen can be utilized and has the potential of preparing biological materials. However, the aquatic collagen has the disadvantages of poor mechanical properties, poor thermal stability, poor degradation resistance and the like, which greatly limits the further development of the aquatic collagen on medical materials. At present, acellular dermal matrices crosslinked with Glutaraldehyde (GA) have been studied, which have excellent mechanical properties, thermal stability and degradation resistance, but are limited in their use for human skin repair due to the toxicity of Glutaraldehyde (GA).
The fish skin collagen I has rich collagen content, simple extraction and low immunogenicity, but has fast degradation rate in human body and poorer spinnability and mechanical property, and in order to solve the problem, the high molecular material PCL and the fish collagen are blended, PEO is added to assist in spinning to improve the porosity of the membrane, and the degradable oral cavity repairing membrane material is prepared.
Disclosure of Invention
The invention aims to provide a preparation method of an oral cavity repairing film.
The invention is realized by the following technical scheme:
the invention relates to a preparation method of an oral repair film, which comprises the following steps:
step 1, preparing an electrospinning solution:
preparing 10% wt solution by using 40% acetic acid as a solvent and fish collagen and PCL as solutes, adding PEO (polyethylene oxide) with the mass of 8% of the solutes as a spinning aid, and performing ultrasonic treatment; after the high molecular substances are completely dissolved, adding phycocyanin, stirring at low speed, and carrying out electrospinning to obtain an electrospinning solution;
step 2, preparing a membrane: the conductivity and viscosity of the electrospinning solution are used as indexes to ensure the consistency among production batches. The experimental result shows that the fiber diameter is increased along with the increase of the viscosity, and the adhesion phenomenon occurs; the phenomenon of beading and the like occurs due to the uneven sprayed fibers caused by the excessively low conductivity. When the viscosity of the electrospinning solution is 108-125mPa & S and the conductivity is 28us/cm-44ms/cm, uniform and smooth fibers are obtained; the fiber diameter is between 100 and 300 nm; electrospinning parameters: the voltage is 18kV, the solution flow rate is 0.6mL/h, the spinning chamber humidity is 40-50%, and the spinning chamber temperature is 20-30 ℃. Because the difference of collagen extraction processes exists in batches, and factors such as stirring time, temperature and the like in the preparation process of the electrospinning solution also influence the solution state, a method for judging the spinnability of the solution through conductivity and viscosity is provided, so that the consistency of products in different batches is ensured.
Step 3, washing the prepared fiber with 0.1 mol/L PBS and 0.01mol/L PBS in sequence, and removing acetic acid residue and PEO to obtain a fiber membrane;
and 4, freezing the treated fibrous membrane in vacuum, drying, and sterilizing by using ethylene oxide to obtain the collagen/PCL-phycocyanin composite membrane.
And 5, carrying out vacuum freeze drying on the treated membrane, and sterilizing by using ethylene oxide.
The invention mixes phycocyanin, PCL and fishskin collagen, breaks through the blending technology, improves the porosity of the material, effectively combines the excellent mechanical property of the high molecular material, the excellent repairability of the fishcollagen and the antibacterial and anti-inflammatory functions of the phycocyanin, and fully exposes the hydrophilic sites, thereby facilitating the creeping growth of the implanted cells.
Preferably, in the step 1, the solute is a mixture of PCL and fishskin collagen in a mass ratio of 2: 8.
Preferably, in step 1, the spinning aid is 8% by mass of polyethylene oxide.
Preferably, in step 1, the temperature of the ultrasonic treatment is 37 ℃ and the time is 30 min.
Preferably, in the step 1, the addition amount of the phycocyanin is 5 mg/mL; the time of the low-speed stirring is 12 h.
Preferably, in step 2, the fiber diameter is 100-300 nm.
Preferably, in step 2, the electrospinning parameters: the voltage is 18kV, the solution flow rate is 0.6mL/h, the spinning chamber humidity is 40-50%, and the spinning chamber temperature is 20-30 ℃.
Preferably, in step 3, the swelling ratio of the maintenance membrane is 786.53%, the hydrolytic stability (37 ℃ 5d) is 85% (no hydrolytic stability/0 hydrolytic stability of pure collagen membrane), the tensile strength is 15.5MPa (1.7 of pure collagen membrane), the elongation is 37% (6.3 of pure collagen membrane), the elastic modulus is 184MPa (15.2 of pure collagen membrane), and the cytotoxicity is less than 1 grade.
Preferably, the drying temperature is 37 ℃ and the drying time is 72 h.
The invention has the following advantages:
(1) one of the raw materials adopted by the preparation method of the invention is the fish skin collagen with wide source and high purity, which is mostly leftovers in the fishery development process, the cost is low, the extraction process is simple, the production cost is greatly reduced, and the treatment problem of partial byproducts is solved.
(2) The method judges the spinnability of the solution according to the indexes of the conductivity and the viscosity of the electrospinning solution, formulates a standardized preparation and inspection process, ensures the uniformity of the electrospinning membrane prepared in each batch, saves time compared with a detection finished product, is convenient to operate, and can be monitored at any time.
(3) The invention completes the blending of collagen/PCL/phycocyanin for the first time; the oral cavity repairing film prepared by combining the antibacterial and anti-inflammatory activities of the phycocyanin and the repairing function of the collagen is beneficial to repairing the wound surface, has certain antibacterial and anti-inflammatory effects and reduces the infection of the wound.
(4) The method of the invention designs a gradient cleaning process, effectively removes the chemical reagent residue in the process of preparing the membrane material by electrostatic spinning, and ensures the biological safety of the membrane material.
Detailed Description
The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.
Examples
The embodiment relates to a preparation method of an oral cavity repairing film, which comprises the following steps:
step 1, preparing an electrospinning solution: preparing 10 wt% solution with 40% acetic acid as solvent and solute, adding PEO 8% of the solute by mass as spinning aid, and performing ultrasonic treatment; after the high molecular substances are completely dissolved, adding phycocyanin, stirring at low speed, and carrying out electrospinning to obtain an electrospinning solution;
step 2, preparing a membrane: the conductivity and viscosity of the electrospinning solution are used as indexes to ensure the consistency among production batches. The experimental result shows that the fiber diameter is increased along with the increase of the viscosity, and the adhesion phenomenon occurs; the phenomenon of beading and the like occurs due to the uneven sprayed fibers caused by the excessively low conductivity. When the viscosity of the electrospinning solution is 108-125mPa & S and the conductivity is 28us/cm-44ms/cm, uniform and smooth fibers are obtained; the fiber diameter is between 100 and 300 nm; electrospinning parameters: the voltage is 18kV, the solution flow rate is 0.6mL/h, the spinning chamber humidity is 40-50%, and the spinning chamber temperature is 20-30 ℃. Because the difference of collagen extraction processes exists in batches, and factors such as stirring time, temperature and the like in the preparation process of the electrospinning solution also influence the solution state, a method for judging the spinnability of the solution through conductivity and viscosity is provided, so that the consistency of products in different batches is ensured.
Step 3, washing the prepared fiber with 0.1 mol/L PBS and 0.01mol/L PBS in sequence, and removing acetic acid residue and PEO to obtain a fiber membrane;
and 4, freezing the treated fibrous membrane in vacuum, drying, and sterilizing by using ethylene oxide to obtain the collagen/PCL-phycocyanin composite membrane.
And 5, carrying out vacuum freeze drying on the treated membrane, and sterilizing by using ethylene oxide.
The invention mixes phycocyanin, PCL and fishskin collagen, breaks through the blending technology, improves the porosity of the material, effectively combines the excellent mechanical property of the high molecular material, the excellent repairability of the fishcollagen and the antibacterial and anti-inflammatory functions of the phycocyanin, and fully exposes the hydrophilic sites, thereby facilitating the creeping growth of the implanted cells.
Further, in the step 1, the solute is formed by mixing PCL and fish skin collagen according to the mass ratio of 2: 8.
Further, in step 1, the spinning aid is% polyethylene oxide.
Further, in the step 1, the temperature of the ultrasonic treatment is 37 ℃ and the time is 30 min.
Further, in the step 1, the addition amount of the phycocyanin is 5 mg/mL; the time of the low-speed stirring is 12 h.
Further, in step 2, the fiber diameter is 100-300 nm.
Further, in step 2, the electrospinning parameters: the voltage is 18kV, the solution flow rate is 0.6mL/h, the spinning chamber humidity is 40-50%, and the spinning chamber temperature is 20-30 ℃.
Further, in step 3, the swelling ratio of the maintenance film is 786.53%, the hydrolytic stability (5 d at 37 ℃) is 85% (no hydrolytic stability/0 hydrolytic stability of the pure collagen film), the tensile strength is 15.5MPa (1.7% of the pure collagen film), the elongation is 37% (6.3% of the pure collagen film), the elastic modulus is 184MPa (15.2% of the pure collagen film), and the cytotoxicity is less than 1 grade.
Further, the drying temperature is 37 ℃, and the drying time is 72 h.
The collagen/PCL-phycocyanin composite membrane prepared by the embodiment has an obvious inhibition effect on proliferation of mouse macrophage RAW264.7, and the cell growth inhibition rate of RAW264.7 is 105.1% by taking lipopolysaccharide as a control; the material is cut into a film sample with the diameter of 0.6cm, the film sample is pasted at the center of a flat plate, the film sample is placed in an incubator upside down, the diameter of an inhibition zone for escherichia coli is measured to be 17.3mm after the film sample is cultured for 24 hours at 37 ℃, and the material is proved to have good anti-inflammatory and antibacterial effects.
In summary, the following steps: one of the raw materials adopted by the preparation method of the invention is the fish skin collagen with wide source and high purity, which is mostly leftovers in the fishery development process, the cost is low, the extraction process is simple, the production cost is greatly reduced, and the treatment problem of partial byproducts is solved. The method judges the spinnability of the solution according to the indexes of the conductivity and the viscosity of the electrospinning solution, formulates a standardized preparation and inspection process, ensures the uniformity of the electrospinning membrane prepared in each batch, saves time compared with a detection finished product, is convenient to operate, and can be monitored at any time. The invention completes the blending of collagen/PCL/phycocyanin for the first time; the oral cavity repairing film prepared by combining the antibacterial and anti-inflammatory activities of the phycocyanin and the repairing function of the collagen is beneficial to repairing the wound surface, has certain antibacterial and anti-inflammatory effects and reduces the infection of the wound. The method of the invention designs a gradient cleaning process, effectively removes the chemical reagent residue in the process of preparing the membrane material by electrostatic spinning, and ensures the biological safety of the membrane material.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.