CN1391607A - Surface treatment with polypeptides to improve fibroblast adhesion to hyaluronan - Google Patents

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.

Description

Adhesion of the cellulose to hyaluronic acid is modified to polypeptide surface preparation

Adhesion invention field of the cellulose to hyaluronic acid is modified to polypeptide surface preparation

The present invention relates to the chemical process that secondary macromolecule is made to be surface-treated.The present invention is more particularly directed to be surface-treated with polypeptide macromolecule to improve hyaluronic acid（Hyaluronan:HyA) stick to promote HyA and cell body mutual compatibility with fibroblast.Background of invention

Hyaluronic acid (HyA) is used as a kind of external medium（Matfix Component) growth of biological quality can be promoted and sticked, but because it has water-soluble characteristic, above have it difficult and limitation in application.Hyaluronic acid is a kind of polysaccharide of HMW, and the dissacharide units being made up of laevoglucose aldehyde and N- ethene-laevoglucose amine are iteratively repeated constituted polysaccharide chain.As one kind of medium, it is present in all biological connective tissues.In addition to the support known with oneself and maintaining the morphological characteristic of tissue or organ, hyaluronic acid（HyA) function of the movement of promotion cell directional and propagation is also being found to have recently.These features make HyA be played an important role in the widely using of biochemical form engineering.Have now been found that, HyA is to play above-mentioned effect by three kinds of referred to as CD44, RHAMM, standing grain B ICAM-1 cell surface adhesion molecule acceptor.Except by cell surface receptor, HyA also adjusts cell behavior by intracellular HyA associated proteins.Due to purifying HyA characteristic, at present just having extensive research in order to more can be using the potential a variety of functions of HyA.In addition, the HyA of purifying is water miscible at room temperature, how to make HyA water insoluble and lower its fast hydrolyzing, the need for being surgery, medicament and many industrial products, this will be chemically crosslinked to HyA.Although oneself the current cross-linking method to HyA has carried out many trials, still in the exploratory stage.Therefore still there is the necessity for further improving its function in chemical characteristic to HyA, more to promote the encompassing property of its biochemistry, higher efficiency is produced in industry and biogenetic products with enable.Summary of the invention The feature of the present invention is using the method to be surface-treated, to promote compatibilities of the hyaluronic acid HyA to biological quality（Bio-Compatibility) and adherence, to increase and strengthen the effect of it grows after industry, biogenetic products and biochemical tissue.Detailed description of the invention

The present invention is to be surface-treated reinforcement hyaluronic acid（HyA cell adhesion), it is expanded using scope and function to promote its biochemical characteristic.The present invention is handled with glutaraldehyde cross-linking HyA bar ropes, then in following method during at the surface.Glutaraldehyde（Glut) it is a kind of conventional cross-linking agent wide variety of in Electronic Speculum, protein chemistry and immunohistochemistry.A kind of most commonly used crosslinking agent is crosslinked as biomaterial, glutaraldehyde also be used to stablize allograft thing, strengthen the transplanting of tissue repair thing, such as cardiac valves, blood vessel, ligament.Although depositing cytotoxic misgivings to the crosslinking that biomaterial is carried out using glutaraldehyde, when the pericardium for observing that glutaraldehyde cross-linking is crossed occurs without calcification and contracture after the transfer, the autologous organ of glutaraldehyde processing in short-term（Tissue）Graft turns into the important object of Biochemical Research once again again.Recently research invention, the cell surface adhesion molecule acceptor mechanism related with cell division factor to certain peptide growth factor.Such as poly-D-lysine positive charge coating material, is generally coated on the surface of plastic and glass vessel, to improve the adhesiveness of cell, is used in vitro study.The present invention is dealt with using the characteristic of poly-D-lysine to the direct coating of graft.The present invention first uses glutaraldehyde cross-linking HyA bar ropes (Strands), then carries out surface coated treatment with four kinds of different amino acid and polypeptide.After coating (coating) surface grafting fibroblast, and with histology and Cell immunohistochemical staining method check and evaluation, the HyA bar ropes through chemical modification to fibroblastic attachment and growing state.Inquire into some biological characteristicses simultaneously.Spy of the invention is described in more detail with the following example：

Example one：The preparation of HyA bar ropes

(1) ion exchange of sodium-hyaluronic acid

By in 0.15 gram of dialysis tubing of the Sodium Hyaluronate injection through high pressure steam sterilization, toward after cationic ion-exchange resin dialysis, rinsed with sterile water sample is used.Then liquefied HyA transfers cause DMSO.After being stirred again through two days, in the syringe that HyA is pumped into 10 milliliters. (2) preparation and crosslinking of HyA bar ropes

The 10ml syringes inhaled and have the HyA handled through deionization are connected to 25gaugt (0.5mm) injection needle, then injects to be formed to preserve under bar rope, 4 °C in the beaker for filling 100% alcohol by HyA and stays overnight.HyA bar ropes are placed on 5mm filter paper, dried in air.In the glutaraldehyde solution that experimental group sample is soaked to biological classes and grades in school, its concentration is respectively：0.5%th, 5%, 25% and 50%, preserve 1 under 4 °C, 2,4,8,12,24,48,72 hours.After being embathed with distilled water, HyA bar ropes are placed in PBS liquid and dialysed 48 hours, to reduce the glutaraldehyde of residual, be subsequently placed in PBS liquid and preserve overnight.Example two：It is surface-treated with amino acid and polypeptide

Prepare 10% fresh dextrorotation glutamic acid, the poly-l-lysine of 1% glycine 5mg/lml concentration, and 10m_gThe poly-l-lysine solution of/ml concentration.HyA bar ropes are dipped in one kind 1 hour in four kinds of solution.After surface treated, rinsed with distilled water standby.Example three：Fibroblast is inoculated with HyA bar ropes surface

Take the fibroblast of growth phase（About divide the cell of 3 days）, with Protease Treatment 15 minutes, form suspension.After 2000 revs/min centrifuge 10 minutes, supernatant is removed, DMEM is added, then cell is suspended.It is now per cubic centimeter to there are about 5 cell 4-6X10.Drawn with the syringe with sterile tack pin and inject cell culture fluid 3-5 times, cell is equably suspended.By fibroblast it is light and slow inject in the culture dish for filling HyA bar ropes.It is then poured into 1ml nutrient solutions.The HyA bar ropes for being vaccinated with cell are incubated for 37 °C in incubator, and observed once with inverted microscope every 24 hours, continuous 7 days, are then divided into two experimental groups, one group of continuation volume culture is preserved, in another group of preparation implantation biological tissue.Example four：It is inoculated with the transplanting of fibroblastic HyA bar ropes

By 4% chloroformism of bioorganism.Observed using the use and nursing standard of NIH experimental animal.Take 4 sections（Every section of 2 centimeter lengths）Chest is subcutaneous on the right side of the HyA bar ropes of fibroblast inoculation, implantation bioorganism, and otch is with No. 4 nylon line sutures. Example five：The collection and preparation of sample

After 2-4 weeks, take out biological sample and be fixed in buffered formaldehyde fixer 2 hours.Conventional treatment sample：FFPE, makes 10mm sections of section.Then will section dewaxing, be dehydrated it is standby.The sample 30 minutes in culture vessel is embathed with PBS, 1 hour is fixed in 10% neutral formalin liquid.Then again with PBS embathe 30 minutes it is standby.Example six：Immunohistochemical staining

With 0.3% hydrogen peroxide dipping 30 minutes, rinsed with PBS liquid.Then sample is closed 30 minutes with horse serum.Then with 1:The PCNA antibody leaching of 200 dilutions is educated overnight.Sample is handled with biotin composite reagent kit, is developed the color with DAB, then mounting.Other sample bromophenol blues or HE dyeing.With the cell quantity in the HyA bar ropes in Baxter cell counters 1 cm range of points.Cell in growth is marked by PCVA positive stainings.Using from " O " to " ++++,（0=negative findings, +=1-25%； ++=26-50% ; +++=51 -75%；+++ +=76- 100%) staging carry out the assessment that the cloudy sun colours ratio.The result of example is using the HyA bar ropes of 50% crosslinking of glutaraldehyde 72 hours compared with other low concentration short time, group is educated in leaching, and the former has more preferable stability and water insoluble.Their shape, even more than 3 months can be still kept after 2 days being soaked in PBS liquid or distilled water.Use low concentration（Such as 25%) glutaraldehyde cross-linking group, the dissolving in succession in 5 minutes to 12 hours in PBS liquid of its bar rope.And all it is less than bar rope that 25% concentration is crosslinked in the neutral i.e. dissolving of PBS liquid or distilled water.Although the HyA bar rope quite stables of 50% glutaraldehyde cross-linking, the fibroblast planted can not be attached at bar rope surface.After being surface-treated through poly-D-lysine, the ability of HyA bar rope surface adhering cells is remarkably reinforced, and particularly uses left-handed poly-D-lysine, and dextrorotation glutamic acid and glycine coating can not promote the attaching of cell.The effect of poly-l-lysine promotion cell attachment is most obvious, and HyA bar rope surface attachments per cm have 50-100 cell；Next to that poly d-lysine, per cm to have 40-80 cell.Meanwhile, two kinds of poly-D-lysines can make cell in HyA bar ropes at least one moon of superficial growth.The fibroblast handled with PCNA decoration methods in bar rope is positive, and [(table 1) adheres to amino different with being grown on four kinds to result The comparison of the cell for the HyA bar ropes that one of acid or polypeptide are surface-treated], tinctorial strength is slightly below the cell in culture dish bottom grown.Will be markedly superior to by sticking to the upgrowth situation of the cell on the HyA bar ropes surface in poly-D-lysine painting room by other groups.PCNA is mainly coloured in kernel, and other dyeing are then coloured in cytoplasm.Through glutaraldehyde cross-linking and planted cell HyA bar ropes histocompatbility by vitro and in vivo experiment confirm.In vivo, planted in rat biological tissue after 4 weeks, there are no obvious inflammation and necrotic reaction.Light Microscopic observation, HyA bar ropes remain in that shape when being implanted into, and cell is along bar rope well-grown.PCNA dyeing displays, cell is active in the HyA bar ropes surface growth being surface-treated with poly-l-lysine and poly d-lysine, and its grade is " ++ ".There is connective tissue infiltration around graft, but there are no inflammatory cell, macrophage or lymphocyte in localized clusters.As a kind of extracellular medium, HyA more has found its characteristic with tissue recently in addition to the effect with viscosity and pressure resistance and buffering cabin.It is most naturally only with liquid or paste dosage-form or to be mixed into cell culture fluid with the HyA of chemical syntheses and be used for external or be injected into internal progress research observation.Due to the water solubilitys of HyA at room temperature, its application is very restricted, the clinical treatment of liquid content cavity filling is generally used only for, such as the substitute of vitreum, or for synovial membrane chamber intermediate pockets.And be then difficult to use with the substitute for soft tissue.A kind of solid is formed into semisolid HyA in invention, crosslinked rear water insoluble, can be more convenient to use.Glutaraldehyde is a kind of reagent for being widely used in biochemical material crosslinking.The present invention is water-insoluble with the concentration increase collagen HgA for increasing glutaraldehyde.After being soaked in 50% pentanedial liquid and educating 72 hours, the morphological properties quite stable of HyA bar ropes.And the leaching of low concentration or short time are educated, then PBS liquid or water are highly soluble in.In order to avoid toxic action of the glutaraldehyde to cell, the HyA bar ropes through glutaraldehyde cross-linking need to dialyse and rinse in PBS liquid.Germicidal efficacy is grown well to fibroblast along the HyA bar ropes through 50% glutaraldehyde cross-linking, and HyA bar ropes are planted no inflammation and necrotic reaction in vivo into ground.This shows, all or most of Residual glutaraldehyde removed through dialysis and rinsing, the aldehyde radical of any residual will cause the damage of living cells and tissue.The present invention uses the glutaraldehyde cross-linking HyA bar ropes of high concentration first, is then surface-treated again with poly-D-lysine, reaches the effect for reinforcing cross-linking effect.Therefore, it is surface-treated with poly-D-lysine, not only with the histocompatbility of reinforcing material and adhesion of the cell to material can be promoted, it can also be through crosslinking further to material, and reinforces crosslinking, slows down degraded of the material in transplanting tissue.The present invention successfully uses the technique improvement of surface treatment HyA function and characteristic, strengthens the coordination of protein kinase also according to poly-D-lysine and the characteristic of transductive process simultaneously utilizes the advantage of poly-D-lysine coating characteristic, strengthen HyA adhesion.The activity of poly-D-lysine and energy stimulates the protein kinase c K2.Therefore poly-D-lysine is in itself or/and HyA is adjusted by intracellular signal transduction and activated, and the activity of cell may be even adjusted by split gene protein activation kinases, includes the adhesion and propagation of cell.Poly-D-lysine also has crosslinking function, and some types into relatively weak hydroxyl and hydroxyl bond crosslinking agent unlike, and left-handed fast propylhomoserin or left-handed methyllysine ester are crosslinked HyA, and formation is very strong amino linkage.This key more can be to resistant to hydrolysis than Shiff-base key.So the HyA bar ropes that the present invention is surface-treated through poly-D-lysine enhance cell and HyA surfaces are sticked extremely bigly, and promote the propagation of cell.The effect of left-handed poly-D-lysine is better than d-lysine.The present invention also using the mechanism of action between HyA materials and poly-D-lysine, and the HyA bar ropes through modification can be used in embodiment or heteroplastic transplantation, and make the valuable thing biomaterial of carrying medicaments certainly.To different cell line, HyA and collagen medium, which can be reached, sticks and resists molten good characteristic.

Claims (1)

1. Claims

   1. a kind of hyaluronic acid, it is characterized in that：

   It is cross-linked into containing glutaraldehyde streak；

   Containing poly-D-lysine；And

   Containing fibroblast and has the adhesion of superficial layer.

   2. according to the hyaluronic acid of claim 1, also containing glutamic acid surface conditioning agent.

   3. a kind of hyaluronic acid, it is characterized in that containing drop solvent.

   4. according to the hyaluronic acid of claim 3, also contain surface conditioning agent.

   5. according to the hyaluronic acid of claim 4, wherein described surface conditioning agent is polypeptide surface inorganic agent.

   6. according to the hyaluronic acid of claim 4, wherein as many as described peptide surface conditioning agent is poly-D-lysine.

   7. according to the hyaluronic acid of claim 3, wherein described hyaluronic acid is crosslinked shape into one.

   8. according to the hyaluronic acid of claim 3, wherein described drop solvent is glutaraldehyde.

   9. according to the hyaluronic acid of claim 5, the polypeptide surface conditioning agent that wherein gas is stated is poly-D-lysine.

   10. according to the hyaluronic acid of claim 5, wherein as many as described peptide surface conditioning agent is glycine.