CN113559324B

CN113559324B - Application of nano compound liquid in chondrocyte self-repair and regenerative medicine aesthetics

Info

Publication number: CN113559324B
Application number: CN202110775346.9A
Authority: CN
Inventors: 杜剑波
Original assignee: Shanghai Dinglingling Information Technology Co ltd
Current assignee: Shanghai Dinglingling Information Technology Co ltd
Priority date: 2021-07-08
Filing date: 2021-07-08
Publication date: 2022-06-24
Anticipated expiration: 2041-07-08
Also published as: CN113559324A

Abstract

The invention provides a nano compound liquid for cartilage repair, which comprises PBS buffer solution, hyaluronic acid and a nano microsphere particle compound, wherein the nano microsphere particle compound comprises microsphere particles, a filling agent and bone morphogenetic protein analogues. The invention further discloses a preparation method of the nano compound liquid for cartilage repair. The invention utilizes the bone morphogenetic protein analogue active peptide to prepare the nano microsphere particle compound with the slow release function and the corresponding nano compound liquid, the induction of cartilage cells is realized by utilizing the prepared compound liquid, and the compound liquid can be widely applied to the damage of joints and cartilage and the cosmetic repair by activating a human body self-healing system based on a liquid layering hydrodynamic soft stripping (water stripping) technology, has extremely high popularization value, and can be widely applied to the autologous repair of cartilage cells and the aesthetic of regenerative medicine.

Description

Application of nano compound liquid in chondrocyte self-repair and regenerative medicine aesthetics

Technical Field

The invention relates to the field of biomedical engineering, in particular to a nano compound liquid for cartilage injury regeneration and application of the nano compound liquid in chondrocyte self-repair and regenerative medicine aesthetics.

Background

In recent years, articular cartilage injuries caused by trauma, inflammation, degeneration and the like are quite common in clinic, and are accompanied by obvious symptoms such as joint pain, joint deformity, even joint dysfunction and the like, so that the health condition and the life quality of the people are seriously affected.

Since articular cartilage is not innervated by blood vessels, lymphatic vessels, and nerves, it is difficult to restore its original structure and state once abrasion or trauma has occurred. Articular cartilage contains only a single cell type, chondrocytes. The growth of chondrocytes itself is restricted, and thus, articular cartilage is difficult to repair itself after it is damaged. The avascular microenvironment of cartilage results in restricted nutrient exchange or cartilage precursor cell circulation in the articular cartilage, impeding the healing process in the damaged area. Although surgical treatments such as stem cell stimulation, allograft and autograft may be helpful in some respects to restore cartilage function, these conventional treatments have limitations including cartilage degradation, poor integration with host tissues, secondary surgery, etc.

In addition, cartilage defects and damage can cause post-traumatic Osteoarthritis (OA), increasing the difficulty of healing cartilage damage. Therefore, repair of articular cartilage damage has been one of the major clinical problems. The cartilage repairing treatment method includes microfracture technology, tissue engineering technology, cartilage and periosteum transplantation technology and gene therapy technology. The microfracture technology is characterized in that bone marrow mesenchymal stem cells, growth factors and the like released by punching holes in a cartilage defect area are utilized to form a microscopic blood clot, the microscopic blood clot is differentiated into fibrocartilage tissues, and traditional treatment methods for promoting cartilage regeneration, such as microfracture, mosaic plasty, autologous chondrocyte transplantation, allogeneic bone/cartilage transplantation and the like, can relieve pain and improve joint functions, however, the generated new cartilage usually forms type I collagen, lacks the characteristics of hyaline cartilage of type II collagen, and has poor long-term clinical effect. The artificial joint replacement has the problems of prosthesis abrasion, corrosion, looseness and the like, and is limited in applicable population. Therefore, the existing repair methods aiming at the articular cartilage damage only temporarily relieve symptoms, but not cure the articular cartilage damage, and functional cartilage is not regenerated. In addition, osteochondral transplantation and autologous chondrocyte transplantation are also known. Osteochondral transplantation is a method in which cartilage conforming to the size of an injured site is removed from a secondary site in a knee joint and then implanted into a cartilage defect. Although surgery is successful and eliminates the risk of immune reactions, the cartilage in the secondary site is weaker. Autologous chondrocyte transplantation (ACI) is a method in which normal living cartilage tissue is removed from a secondary site in the knee joint, chondrocytes are cultured in a laboratory, proliferated to a sufficient amount, and implanted into a cartilage defect site, and the new state of chondrocytes is similar to that of natural articular cartilage. If the joint pathology is severe, the clinician may perform a rescue type of surgery, such as an osteotomy or total joint replacement.

The slow release of the medicine is to combine the micromolecule medicine with the macromolecule carrier by a physical or chemical method, and the micromolecule medicine is continuously released in proper concentration in vivo through control modes such as diffusion, permeation and the like, which is beneficial to improving the bioavailability of the medicine, reducing toxic and side effects, relieving the pain of patients after taking the medicine for many times, and further improving the clinical medication level. At present, the main developed drug sustained-release polymer carrier materials mainly include natural polymer materials, synthetic polymer materials and inorganic materials.

At present, chitosan is often used as a carrier of polypeptide, protein and antitumor drugs, drug release is regulated through self degradation, the drugs can be released at a relatively stable rate within several weeks or months, effective blood concentration is maintained, administration times of the half-life period of the drugs are reduced, and the chitosan is suitable for the drugs with short half-life period and long-term use.

With the development of tissue engineering technology, the damage repair of articular cartilage enters a new height, and the technology mainly comprises three elements of seed cells, a bracket material and cell growth factors, wherein the bracket material is used for simulating extracellular matrix, providing a proper microenvironment for the growth, proliferation and differentiation of cells at the defect part, and promoting the repair and regeneration of the articular cartilage by utilizing a natural biological process of interaction between the cells and biomolecules. Therefore, the success of tissue engineering depends to a large extent on the extent to which the scaffold mimics the extracellular matrix (ECM) of the corresponding tissue in structure and composition.

Injectable hydrogels have received a great deal of attention as candidate materials for cartilage repair. The aim is to directly inject hydrogel precursor liquid loaded with cells or growth factors into the defect part of the articular cartilage for in-situ filling, and then rapidly polymerize the hydrogel precursor liquid in a short time to form solid gel. The advantage of this strategy is that irregular shaped defects can be easily filled by minimally invasive surgery, not only can fusion well with the defect site, but also cells and bioactive factors can be uniformly incorporated in the hydrogel, and the injectable nano-compound solution of nanoparticles is used for cartilage repair as schematically shown in fig. 1.

In order to further improve the mechanical strength and stability of injectable hydrogels, some researchers have added fillers such as microsphere particles (e.g., gelatin, sodium alginate, chitosan microspheres), nanoparticles (e.g., hydroxyapatite, metal particles, clay), and nanofibers (e.g., albumin, electrospun PCL, silica nanofibers [45]) as injectable hydrogel materials for articular cartilage repair. The composite hydrogel has the excellent characteristics of pore communication, high compression modulus, good formability, reasonable degradability and the like, and is a matrix suitable for cartilage tissue engineering.

Autologous cartilage transplantation has advantages and disadvantages, has no immunological rejection, has disadvantages of low differentiation and low biological activity, and can be used for treating cartilage injury of small and medium size, and complication such as infection and postoperative pain due to invasive operation of cartilage cells. The source of allogeneic periosteum transplantation is sufficient, but immunological rejection reaction is easy to occur. Both approaches have limited clinical use. The application of gene therapy technology in cartilage injury still stays at the theoretical level at present, and the extraction, transfection and clinical mature application of genes are still explored.

Advanced skin ecology awakens the field of cell regeneration medicine and aesthetics of 'water stripping', which is the top technology from Europe and America, and professional anti-aging and wrinkle-removing technology activates a self-healing system of a human body to enable the operated part to 'grow reversely' within 28 days. The number of cells regenerated in this period is greater than the number of cells lost, and the subcutaneous reticular fibers are corrected and reconstructed. The "stripping" is to separate the distance between cells by means of the water expansion force, and after entering into the subcutaneous part, the water will avoid the capillary and nerve net, so as to complete the cell stripping without forming wound surface.

Bone Morphogenetic Proteins (BMPs) were first discovered and named by Urist. BMPs are a group of highly conserved functional proteins with similar structures, belonging to the transforming growth factor-P (TGF-P) superfamily. Recent studies have found that BMPs have various biological functions. The most interesting function of their developers is their ability to induce bone and cartilage formation in vivo.

BMP-2 is one of the most widely studied BMPs with the highest osteogenesis inducing activity. At present, a chemical synthesis method is used in China to synthesize a novel short peptide P17-BMP-2 containing 17 amino acids. The short peptide overcomes the defects of rhBMP-2 prepared by gene engineering technology at home and abroad. It is similar to BMP2 in bone induction activity, and has simple preparation process. Solves the problems that the rhBMP-2 can not be produced in a large scale and the safety is difficult. The P17-BMP-2 only contains 17 amino acids, can effectively overcome antigenicity brought by macromolecules and other biological side effects, and has an amino acid sequence of IVAPPGYHAFYCHGECP. The short peptide has simple structure, more stable biological property and better activity. Moreover, modification of the peptide chain is very convenient. It can be said that the nucleotide sequence inherits the activity of BMP-2, overcoming its disadvantages.

The guanidyl contained in the arginine-rich cell-penetrating peptide can be combined with phosphate and sulfate which are negatively charged on the surface of a cell membrane, so that the arginine-rich cell-penetrating peptide can efficiently penetrate through the cell membrane. Meanwhile, the cell-penetrating peptide has low cytotoxicity, so that the cell-penetrating peptide has application in the cell-penetrating field (Suncheng and the like, research progress of the cell-penetrating peptide. the poly-arginine (rR)3R2 hybridized by non-natural D-type arginine (R) and natural L-type arginine (R) can efficiently enter cells with low toxicity, and simultaneously has certain enzymolysis resistance and serum degradation resistance, so that the poly-arginine is concerned.

Bone defects caused by trauma, tumors, infection and the like are increasing year by year worldwide, and have greater and greater influence on the health of people. In our country, there are approximately 400 million patients that need to be treated because of bone defects each year. In addition to bone grafts required for disease-induced bone defects, bone grafts are often required for plastic shaping in cosmetic and plastic surgery. Bone graft materials have become the largest graft in demand next to blood transfusion, and have tended to increase year by year.

Disclosure of Invention

Aiming at the problems of the prior art that the cartilage repair transplantation material is in short supply and the effect is not ideal enough, the invention provides the biological combination preparation for cartilage repair, the biological combination preparation can be suitable for cartilage repair by the tissue engineering technology, the repair effect on cartilage injury is good, and the treatment is time-saving and quick.

In order to solve the technical problems, the invention adopts the following technical scheme:

the sequence of the known novel short peptide P17-BMP-2 is optimized by modification so as to increase the penetrability of the peptide.

Preferably, amino acid modifications are introduced into the sequence of P17-BMP-2 that are known to aid in membrane penetration.

Preferably, the polypeptide is modified with an amino acid having a membrane-penetrating property such as arginine, leucine or tryptophan.

Preferably, the sequence of the modified P17-BMP-2 short peptide molecule is XXIVXPPXYHAFYCHGECP, wherein X can be one selected from arginine, leucine and tryptophan.

Preferably, the length of the modified P17-BMP-2 short peptide molecule is 19 amino acids, and the preferred sequence can be RRIVLPPWYHAFYCHGECP.

The invention discloses a nano compound liquid for cartilage repair, which comprises the following components in part by weight: PBS buffer solution, hyaluronic acid and a nano microsphere particle compound, wherein the nano microsphere particle comprises microsphere particles, a filling agent and bone morphogenetic protein analogues.

Preferably, the microsphere particles are selected from chitosan microspheres, and the filling agent is selected from bovine serum albumin.

Preferably, the bone morphogenetic protein analog is prepared by artificial synthesis, and has the sequence of RRIVLPPWYHAFYCHGECP.

The invention discloses a cosmetic product which comprises the compound liquid.

The invention discloses a preparation method of a nano compound liquid, which comprises the following steps:

(1) preparing microsphere particles: weighing chitosan, dissolving in 2% v/v acetic acid, dripping into n-octanol, stirring at 1500rpm for 30 min; centrifuging, and discarding the upper aqueous phase; adding isopropanol, centrifuging and discarding the supernatant; adding double distilled water, centrifuging, discarding supernatant, sterilizing with ethylene oxide, and storing for use;

(2) dissolving BSA in hydrochloric acid aqueous solution, filtering for sterilization, and adding bone morphogenetic protein analogues to obtain bone morphogenetic protein analogue preserving fluid;

(3) weighing the microsphere particles obtained in the step (1), adding the preservation solution prepared in the step (2), placing in a shaking table, and fully and uniformly oscillating;

(4) centrifuging, washing by double distilled water, and centrifuging to obtain the nano microsphere particle compound.

Preferably, the method further comprises the following steps: (5) dissolving a hyaluronic acid stock solution in a PBS buffer solution;

(6) and (4) dissolving the nano microsphere particles prepared in the step (4) in the buffer solution prepared in the step (5).

The invention discloses a nano compound liquid for cartilage repair, which is prepared by adopting the method.

The invention discloses application of the nano compound liquid in preparing a beauty product.

Preferably, the cosmetic product is a cartilage damage cosmetic repair product.

Advantageous effects

The invention discloses a bone morphogenetic protein analogue, wherein the analogue polypeptide can induce the formation of osteocytes, the invention further utilizes the active peptide to prepare nano microsphere particles with a slow release function and corresponding nano compound liquid, the prepared compound liquid is utilized to realize the repair and regeneration of chondrocytes, and the analogue can be widely applied to the damage of joints and cartilages and the cosmetic repair, and has extremely high popularization value.

Drawings

Fig. 1 is a schematic view of an injectable nano-compound solution with nanoparticles added for cartilage repair;

FIG. 2 is the result of electron microscope examination of the prepared microspheroidal particles;

FIG. 3 is an immunohistochemical assay of type II collagen differentiation of BMSC cells into chondrocytes;

FIG. 4 is a result of observation of cultured MSC cells by an optical microscope, wherein A is an observation result after 1 day of culture and B is a result after 7 days of culture;

FIG. 5 is the result of AKP detection in MSC cells, wherein the abscissa is the number of days of cell culture and the ordinate is the AKP content in μ g/mL;

fig. 6 is the result of alizarin red staining about 2 weeks after MSC cell induction.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1 preparation of microspheroidal particles and identification of microsphere morphology

1) Weighing 200mg of chitosan, and dissolving in 8ml of 2% v/v acetic acid solution;

2) gradually dripping the solution into 150ml of n-octanol, and stirring for 30 min;

3) adding 10ml of 8% w/vTPP solution, stirring for 30min at 1200 rpm;

4) centrifuging at 4000rpm, and discarding the upper aqueous phase;

5) adding 8ml of isopropanol, centrifuging at 2500 rpm, and removing supernatant;

6) adding 8ml of double distilled water, centrifuging at 2500 rpm, and removing supernatant;

7) repeating the steps 5 and 6 for 2-3 times;

8) sterilizing with ethylene oxide to obtain chitosan microsphere particles, and storing;

9) and scanning and observing the distribution and the form of the prepared microspheres by adopting an SEM electron microscope.

The electron microscope observation result shows that the prepared microspheres have uniform particle size, similar form and uniform dispersion, are good slow-control carriers, and the electron microscope inspection result is shown in figure 2.

Example 2 preparation of Nanoparticulate particle composite

(1) The synthetic bone morphogenetic protein analog was dissolved in PBS and had the sequence RRIVLPPWYHAFYCHGECP.

(3) weighing the microsphere particles prepared in the example 1, adding the preservation solution prepared in the step (2), placing in a shaking table, and fully and uniformly oscillating;

Example 3 Induction of chondrocyte differentiation in vitro by Nanoparticulate composites

Taking BMSC cells and mixing with 6X10³The density of each hole is inoculated on a 16-hole cell culture plate, and when the cells are fused to about 70%, the nano microsphere particle compound prepared in the example 2 is added into the culture medium, and the concentration is about 0.5-2 mg/L. Culturing is continued for 15-20 days, and after 15 days of culturing, a part of the cells are taken out every day to perform a detection test of type II collagen immunohistochemistry, so as to identify the condition of BMSC cell differentiation into chondrocytes. Wherein, after about 18 days of culture, positive cells can be identified by an immunohistochemical test, which shows that some BMSC cells have differentiated into chondrocytes after being induced by the nanoparticle complex in vitro, wherein the detection result of type II collagen immunohistochemistry is shown in figure 3.

EXAMPLE 4 preparation of a Nanocomposition solution

(1) Dissolving sodium hyaluronate in water at room temperature to form a transparent solution with the mass fraction of 0.1%, adding cation exchange resin, stirring overnight, and filtering to obtain a hyaluronic acid stock solution;

(2) dissolving hyaluronic acid stock solution in PBS solution, and respectively storing according to the volume ratio of 2: 1;

(3) dissolving the nano microsphere particles prepared in the example 2 in the solution, and uniformly mixing according to the mass-volume ratio of 1:5 (g/mL);

(4) storing in two syringes connected in parallel, and aseptic packaging.

Example 5 Induction of differentiation of in vitro osteocytes by Nanocompositions

(1) Taking out the culture plate for culturing the mouse MSC cells from the incubator, discarding the culture solution, washing with PBS for 3 times, adding 1ml of trypsin digestion solution finished product into each hole, and digesting the cells;

(2) observing by an optical microscope, and immediately and completely stopping digestion when the cell gap is increased and the cells become round;

(3) transferring the cells into the centrifuge tube, adding the culture medium again, beating by blowing, repeating for multiple times until the cells float under the lens, and transferring the washing liquid into the centrifuge tube;

(4) centrifuging, removing supernatant, adding culture solution, blowing, mixing, and culturing in a 37 deg.C carbon dioxide incubator;

(5) the nano-composite solution prepared in example 4 was added to a concentration of about 1-2.5 mg/L. Continuing culturing, performing osteogenesis induction when cell fusion reaches more than 75%, and performing induction liquid change every three days;

(6) measuring alkaline phosphatase (according to the operation instruction of the alkaline phosphatase measuring kit) on the induced cells, wherein 1mL of cultured cells at different induction culture time points are taken, and are subjected to microscopic dilution to the same cell concentration, and then are measured by using the kit;

(7) cells approximately 2 weeks after osteogenic induction were stained with alizarin red and observed under an optical microscope.

The results of the observation of the cultured MSC cells by the optical microscope are shown in fig. 4, in which a is the observation after 1 day of culture in which the cells are already spindle-shaped, and B is the observation after 7 days of culture in which the growth of the cells is accelerated, fusion occurs, and most of the cells are spindle-shaped.

The results of the AKP detection in the cells are shown in FIG. 5, and the AKP content in the induced cells gradually increases with time.

Alizarin red staining results for cells at about 2 weeks after induction are shown in fig. 6, which indicates that calcium nodules have formed in the induced MSC cells.

The induced differentiation of osteocytes can be successfully realized by adopting the nano compound liquid and the nano microsphere particle compound provided by the application. Therefore, the method can be widely applied to joint and cartilage damage and cosmetic repair, and has high popularization and application values.

The present invention is illustrated by the above examples, but the present invention is not limited to the above process steps, which means that the present invention must not be dependent on the above process steps. It will be apparent to those skilled in the art that any modifications to the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific forms, etc., are within the scope and disclosure of the present invention.

Claims

1. A nano-composite fluid for cartilage repair, the composite fluid comprising: the bone morphogenetic protein comprises PBS buffer solution, hyaluronic acid and a nano microsphere particle compound, wherein the nano microsphere particle compound comprises microsphere particles, a filling agent and a bone morphogenetic protein analogue, the bone morphogenetic protein analogue is prepared in an artificial synthesis mode, and the sequence of the bone morphogenetic protein analogue is RRIVLPPWYHAFYCHGECP.

2. The compound fluid of claim 1, wherein the microsphere particles are selected from chitosan microspheres and the bulking agent is selected from bovine serum albumin.

3. A cosmetic preparation comprising the complex liquid according to any one of claims 1-2.

4. The method for preparing the nano-compound liquid as claimed in claim 1, which is characterized by comprising the following steps:

(1) preparing microsphere particles: weighing chitosan, dissolving in 2% v/v acetic acid, dripping into n-octanol, stirring at 1500rpm for 30 min; centrifuging, and discarding the upper aqueous phase; adding isopropanol, centrifuging and discarding the supernatant; adding double distilled water for centrifugation, abandoning the supernatant, and storing for later use, wherein the epoxy brick is manufactured by a sintered method and is sterilized;

(4) centrifuging, washing by double distilled water, and centrifuging to obtain a nano microsphere particle compound; the bone morphogenetic protein analogue is prepared by adopting an artificial synthesis mode, and the sequence of the bone morphogenetic protein analogue is RRIVLPPWYHAFYCHGECP.

5. The method of claim 4, further comprising: (5) dissolving a hyaluronic acid stock solution in a PBS buffer solution;

(6) and (4) dissolving the nano microsphere particle compound prepared in the step (4) in the buffer solution prepared in the step (5).

6. A nano-compound liquid for cartilage repair, which is prepared by the method of claims 4-5.

7. Use of the nano-composite liquid according to claim 6 for preparing a cosmetic product.

8. Use according to claim 7, characterized in that the cosmetic product is a cartilage damage cosmetic repair product.