WO2001032850A1

WO2001032850A1 - Surface treatment with polypeptides to improve fibroblast adhesion to hyaluronan

Info

Publication number: WO2001032850A1
Application number: PCT/CN1999/000174
Authority: WO
Inventors: Min Hu; Shaowei Li
Original assignee: Min Hu; Shaowei Li
Priority date: 1999-10-29
Filing date: 1999-10-29
Publication date: 2001-05-10
Also published as: AU6459299A

Abstract

The present invention relates to hyaluronan (HyA), which are extracellar media, they have special chemical proptery to confer the cell adhesion and can promote cell growth. The limit a lot the use in the artificial biomaterial tissue engineering as they are soluble material in water. The present invention provides a new method to achieve bifunctions, one of which is to decrease HyA solubility by polypeptides, and the other is to promote cell adhesion through the effection of the cell surface adhesion molecular receptor, using a new method to treat surfaces using polypeptides to improve cell adhesion to HyA, HyA being first crosslinked by glutaraldehyde to strand forms, then being treated on the surfaces by polylysine, glycine or glutamate respectively. The modified HyA are incubated together with fibroblast in vitro for utility experiments. The cell adhesion and proliferation are assayed by histology and immunohistochemistry test. It is showed: (1) polylysine can remarkably improve fibroblast adhesion to HyA; (2) HyA can be crosslinked by glutaraldehyde so as to decrease biodegradation; (3) it is verified both in vivo and in vitro that the modified HyA have high biological MHC, they are new complex biomaterials which have potential use in industrial scale.

Description

Polypeptidized surface treatment to improve the adhesion of cellulose to hyaluronic acid Field of the invention

The present invention relates to a chemical process for surface treatment of a secondary polymer. The present invention particularly relates to the use of a peptide polymer as a surface treatment to improve the adhesion of hyaluronic acid (Hyaluronan: HyA) and fibroblasts to enhance the compatibility between HyA and the cell body. Background of the invention

Hyaluronic acid (HyA) as an external medium (Matfix Component) can promote the growth and adhesion of biomass, but because of its water-soluble properties, it has difficulties and limitations in its application. Hyaluronic acid is a high molecular weight polysaccharide, a polysaccharide chain consisting of disaccharide units consisting of L-glucosaldehyde and N-ethylene-L-glucosamine repeatedly. As a medium, it exists in all biological connective tissues. In addition to its known morphological properties to support and maintain tissues or organs, hyaluronic acid (HyA) has also recently been found to promote the directional movement and proliferation of cells. These characteristics make HyA play an important role in the widespread use of biochemical morphology engineering. It has been found that HyA exerts these effects through three cell surface adhesion molecule receptors called CD44, RHAMM, and B ICAM-1. In addition to cell surface receptors, HyA also regulates cellular behavior through intracellular HyA binding proteins. Due to the characteristics of purified HyA, extensive research is currently underway to better utilize HyA's potential multiple functions. In addition, purified HyA is water-soluble at room temperature. How to make HyA insoluble in water and reduce its rapid hydrolysis is a need for surgery, pharmaceuticals, and many industrial products, which requires chemical cross-linking of HyA. Although many attempts have been made on the cross-linking method of HyA, it is still in the exploratory stage. Therefore, it is still necessary for HyA to further improve its functions in terms of chemical properties in order to further enhance its biochemical compatibility, so that it can produce higher efficiency in industrial and biochemical products. Summary of invention The present invention is characterized by using a surface treatment method to improve the hyaluronic acid HyA's bio-compatibility and adhesion, so as to increase and strengthen its colonization in industry, biochemical products and biochemical tissues. efficacy. Detailed description of the invention

The present invention strengthens the cell adhesion of hyaluronic acid (HyA) by surface treatment to improve its biochemical properties and expand its range of use and functions. In the process of surface treatment, the present invention cross-links HyA cord with glutaraldehyde, and then treats it by the following method. Glutaraldehyde (Glut) is a traditional cross-linking agent widely used in electron microscopy, protein chemistry, and immunohistochemistry. As the most widely used cross-linking agent for cross-linking biological materials, glutaraldehyde is also used to stabilize allogeneic tissue grafts and strengthen tissue repairs, such as heart valve, blood vessel, and ligament transplants. Although there are concerns about cytotoxicity in the cross-linking of biomaterials using glutaraldehyde, short-term glutaraldehyde-treated autologous organs were observed when glutaraldehyde-crosslinked pericardium did not show calcification and contracture after transplantation. (Tissue) The graft has once again become an important object of biochemical research. Recent research has discovered that the cell surface adhesion molecule receptors are related to certain peptide growth factors and cytokines. Positively-charged coating materials such as polylysine are usually coated on the surface of plastic and glassware to improve cell adhesion for in vitro research. The present invention utilizes the properties of polylysine to treat the direct coating of the graft. The invention first cross-links HyA Strands with glutaraldehyde, and then performs surface coating treatment with four different amino acids and peptides. After the fibroblasts were planted on the coating surface, the histological and immunohistochemical staining methods were used to detect and evaluate the adhesion and growth of the chemically modified HyA cord to the fibroblasts. It also explores certain biological characteristics. The present invention is illustrated in more detail by the following examples:

Example 1: Preparation of HyA

(1) Ion exchange of sodium-hyaluronic acid

Inject 0.15 g of sodium hyaluronate into a high-pressure steam-sterilized dialysis tube, dialyze the cation exchange resin, and rinse the sample with sterile water. The liquefied HyA is then transferred to DMSO. After stirring for another two days, HyA was drawn into a 10 ml syringe. (2) Preparation and cross-linking of HyA cord

A 25gaugt (0.5mm) injection needle was connected to a 10ml syringe with deionized HyA, and then HyA was pushed into a beaker containing 100% alcohol to form a bar, and stored at 4 ° C overnight. Place HyA cord on 5mm filter paper and air dry. The samples in the experimental group were immersed in biological-grade glutaraldehyde solutions at concentrations of 0.5%, 5%, 25%, and 50%, and stored at 4 ° C for 1, 2, 4, 8, 12, 24 , 48, 72 hours. After leaching with distilled water, the HyA cord was dialyzed against PBS for 48 hours to reduce residual glutaraldehyde, and then stored in PBS overnight. Example 2: Surface treatment with amino acids and peptides

Poly-L-lysine solution of freshly prepared 10% dextrose glutamate, 1% glycine 5mg / lml concentrations of poly-L-lysine, and / ml concentration 10m _g. HyA strips were immersed in one of four solutions for 1 hour. After surface treatment, rinse with distilled water for later use. Example 3: Seeding fibroblasts on the surface of HyA cord

Take the growth phase fibroblasts (cells that divide for about 3 days) and treat with protease for 15 minutes to form a suspension. After 10 minutes at 2000 rpm, the supernatant was removed, DMEM was added, and the cells were suspended. At this time there are about 5 cells 4-6X10 per cubic centimeter. Aspirate and push the cell culture solution 3-5 times with a syringe with a sterile flat-tip needle to suspend the cells evenly. Gently inject fibroblasts into a Petri dish containing HyA cords. Then inject 1 ml of culture solution. The cells inoculated with HyA were incubated at 37 ° C in an incubator, and observed with an inverted microscope every 24 hours for 7 consecutive days, and then divided into two experimental groups. The group is ready to be implanted into living tissue. Example 4: HyA cord transplantation with fibroblasts

Experimental organisms were anesthetized with 4% chloroform. Observation was performed using NIH laboratory animal use and care standards. Take 4 segments (2 cm in length) of HyA cords inoculated with fibroblasts, implant them into the subcutaneous chest of the right side of the experimental organism, and incision with 4 nylon suture. Example 5: Collection and preparation of samples

After 2-4 weeks, remove the biological sample and fix it in formaldehyde buffered fixative for 2 hours. Routine processing samples: Paraffin embedded, made into 10mm sections. The sections are then dewaxed and dehydrated for later use. The samples in the culture vessel were immersed in PBS buffer for 30 minutes, and fixed in 10% neutral formaldehyde solution for 1 hour. Then immerse in PBS for 30 minutes. Example 6: Immunohistochemical staining

Soak in 0.3% hydrogen peroxide for 30 minutes and rinse with PBS. The samples were then blocked with horse serum for 30 minutes. Then incubate with 1: 200 diluted PCNA antibody overnight. Samples were processed with a biotin complex kit, developed with DAB, and mounted on slides. Other samples were stained with bromophenol blue or HE. Use a Baxter cytometer to count the number of cells on a HyA cord within 1 cm. Growing cells were marked by PCVA positive staining. Use "O" to "++++", (0 = negative result, + = 1-25%; ++ = 26-50%; +++ = 51 -75%; ++++ = 76- 100%) classification method to evaluate the shade ratio of shade in the sun. Results of the example The 50% glutaraldehyde 72-hour crosslinked HyA cord is better than the other low concentration short-time incubation groups. Stable and insoluble in water. They can maintain their shape even after soaking in PBS solution or distilled water for 2 days, even more than 3 months. Crosslink the group with glutaraldehyde at a low concentration (such as 25%), and its cords are in PBS Dissolve in 5 minutes to 12 hours successively. And all the crosslinked cords with concentration lower than 25% are immediately dissolved in PBS solution or distilled water. Although 50% glutaraldehyde crosslinked HyA cords are quite stable, they are planted. Fibroblasts cannot be attached to the surface of cords. After surface treatment with polylysine, the ability of HyA cords to adhere to cells was significantly enhanced, especially with L-polylysine and d-glutamine Acid and glycine coatings do not promote cell attachment. Poly-L-lysine promotes cell attachment most effectively, with HyA strips per cm 50-100 cells are attached to the surface; the next is poly-d-lysine, which has 40-80 cells per centimeter. At the same time, both types of poly-lysine can allow cells to grow on the surface of HyA cords for at least one month . Positive results of treatment of fibroblasts on cords with PCNA staining [(Table 1) Adhesion and growth on four different amino groups Comparison of cells treated with HyA strands, one of the acids or peptides], has a slightly lower staining intensity than cells grown at the bottom of the culture dish. The growth of cells adhered to the surface of HyA strands of polylysine-coated houses was significantly better than that of other groups. PCNA is mainly stained in the nucleoli, while other stains are stained in the cytoplasm. The histocompatibility of HyA cords cross-linked and planted with glutaraldehyde has been confirmed by in vitro and in vivo experiments. In vivo, no obvious inflammation and necrosis were seen after 4 weeks of implantation in living tissues of rats. Observed under a light microscope, the HyA cord remained the shape at the time of implantation, and the cells grew well along the cord. PCNA staining showed that cells proliferated actively on the surface of HyA cords treated with poly-L-lysine and poly-L-lysine, whose grade was "++". There was connective tissue infiltration around the graft, but no inflammatory cells, macrophages, or lymphocytes were localized. As an extracellular medium, in addition to its viscosity and pressure resistance and the role of a buffer chamber, HyA has recently discovered its properties with tissues. Most natural and chemically synthesized HyA are only used in liquid or paste formulations or mixed with cell culture fluids for research and observation in vitro or injection into the body. Because HyA is water-soluble at room temperature, its application is greatly limited, and it is usually only used for clinical treatment of filling cavity of liquid contents, such as a vitreous substitute, or a medium cavity such as a synovial cavity. It is not easy to use as a substitute for soft tissue. In the invention, a solid and semi-solid HyA is formed, which is insoluble in water after cross-linking, and can be used more conveniently. Glutaraldehyde is a widely used reagent for cross-linking biochemical materials. The invention increases collagen HgA insolubility by increasing the concentration of glutaraldehyde. After 72 hours of immersion in 50% glutaraldehyde solution, the morphological properties of HyA cords were quite stable. The low concentration or short time incubation is very easy to dissolve in PBS solution or water. In order to avoid the toxic effect of glutaraldehyde on the cells, the glutaraldehyde cross-linked HyA strips need to be dialyzed and rinsed in PBS solution. Fibroblasts were observed to grow well along HyA cords cross-linked with 50% glutaraldehyde, and HyA cords were planted in vivo without inflammation and necrosis. This shows that all or most The residual glutaraldehyde has been removed by dialysis and rinsing, and any residual aldehyde groups will cause damage to living cells and tissues. In the present invention, HyA strips are first cross-linked with glutaraldehyde at a high concentration, and then surface-treated with polylysine to achieve the effect of strengthening the cross-linking effect. Therefore, surface treatment with polylysine can not only enhance the histocompatibility of the material and promote cell adhesion to the material, but also strengthen the cross-linking by further cross-linking the material and slow down the material during transplantation. Degradation in tissue. The present invention successfully uses the surface treatment technology to improve the function and characteristics of HyA, and also enhances the coordination and transduction process of protein kinases based on polylysine, and utilizes the advantages of polylysine coating characteristics to enhance HyA. Adhesion. Polylysine can stimulate the activity of protein kinase CK2. Therefore, polylysine itself or / and HyA regulate activation through intracellular signal transduction, and it is even possible to regulate cell activity, including cell adhesion and proliferation, by dividing gene protein-activated kinases. Polylysine also has a cross-linking function. Unlike some types of relatively weak hydroxy and hydroxy bond cross-linking agents, L-taurine or L-methyl lysine ester cross-links HyA to form Very strong amino bond. This bond is more resistant to hydrolysis than Shiff-base bonds. Therefore, the HyA cord treated with the polylysine surface of the present invention greatly enhances the adhesion of cells to the HyA surface and promotes cell proliferation. L-polylysine is more effective than d-lysine. The present invention also uses the mechanism of action between HyA material and polylysine. The modified HyA strand can be used in self-representation or allogeneic transplantation, and as a valuable biological material carrying drugs. For different cell lines, HyA and collagen media can achieve the excellent properties of adhesion and anti-solubility.

Claims

Claim

1. A hyaluronic acid characterized by:

Contains glutaraldehyde and cross-linked into a strand;

Containing polylysine; and

Contains fibroblasts and has surface layer adhesion.

2. The hyaluronic acid according to claim 1, further comprising a glutamic acid surface treatment agent.

3. A hyaluronic acid, characterized in that it contains a solubilizer.

4. The hyaluronic acid according to claim 3, further comprising a surface treating agent.

5. The hyaluronic acid according to claim 4, wherein the surface treatment agent is a polypeptide surface treatment agent.

6. The hyaluronic acid according to claim 4, wherein said polypeptide surface treating agent is polylysine.

7. The hyaluronic acid according to claim 3, wherein said hyaluronic acid is in a crosslinked state.

8. The hyaluronic acid according to claim 3, wherein the reducing solvent is glutaraldehyde.

The hyaluronic acid according to claim 5, wherein the polypeptide surface treating agent is polylysine.

10. The hyaluronic acid according to claim 5, wherein said polypeptide surface treating agent is glycine.