JPH0759840A - Wound healing material - Google Patents

Abstract

PURPOSE:To coat a lesion, to guide a blood capillary, etc., in an early period and to prevent bacterial infection by depositing the material which are extracted from the serum, plasma or platelet and bind to heparin to a base material contg. collagen or the collagen and the heparin. CONSTITUTION:This wound healing material is formed of the base material contg. the fraction eluted by a 0.5MNaCl soln. and/or 1.0MNaCl soln. by bonding the liquid extracted from the serum, plasma or platelet and the collagen and heparin thereto. The wound healing material strongly supports cell proliferation and has high bioaffinity when the wound heeling material is applied in the vital body and, therefore, the material contributes to the formation of the granulation layer and skin formation and accelerates heeling when the material is applied as, for example, the artificial skin. Further, the wound healing material is lined by a backing layer having air permeability, by which the generation of the leakage of the body fluid, bacterial infection, etc., is minimized when the material is applied as the artificial skin.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a wound healing material used for tissue repair and wound healing.

[0002]

2. Description of the Related Art A wound coating temporarily applied to an affected area for the purpose of protecting the affected area due to a disease or a wound such as a burn, a skin-wound and a skin-exfoliated wound, and a traumatic skin defect wound and promoting healing. Conventionally, gauze, absorbent cotton, etc. have been used as materials, but these have low bacterial infection prevention, and when the wound surface is dry to absorb exudate quickly, it may cause pain, bleeding, etc. when removed. Accompanied. Although an ointment or the like is also used in combination, in this case, on the contrary, absorption of exudate is insufficient and the wound surface becomes excessively wet.

As an alternative to these, particularly when applied to a wide range of wound surfaces, artificial synthetic resin sheets such as silicone gauze, silicone rubber and nylon and teflon having a velor-like surface structure are used. Coating films of materials and freeze-dried pig skins, fibrin films, non-woven fabrics of chitin, collagen films, polyamino acid sponges, mucopolysaccharide complex collagen films, and other bio-derived materials are also known. However, among these, the coating film of the artificial material leaves various problems in terms of adhesion to the affected area, water vapor permeability, cracking, etc., while the coating film of the biological material has good biocompatibility. However, many of them have antigenicity, and also have drawbacks such as bacterial infection and deterioration due to exudate, and there is a problem that the material is difficult to obtain.

More recently, a composite membrane consisting of a nylon mesh treated with collagen and a silicone membrane has been developed and put into practical use. Although it has good adhesion and has a proper water permeability, it adheres to the wound surface and the granulation tissue is covered. There was a drawback that it penetrated into the covering film. On the other hand, drugs for the purpose of wound healing have been developed, such as basic fibroblast growth factor (basic FG).
F [basic fibroblast growth factor]), platelet-derived growth factor (PDGF)
t]), CHL-Cu complex (Glycyl-L-Histidyl-L-Lysin
e-Cu Complex) and heparin-copper complex. All of these are known as angiogenic factors. However, when applied to the wound surface, these angiogenic factors flow out from the wound surface and are halved. For this reason, it is necessary to increase the frequency of application, or it is contained in another matrix (for example, synthetic resin) for use. Also,
Since these angiogenic factors are very expensive, a platelet-derived cell growth factor containing a plurality of β-TGF and PDGF as in JP-A-63-79588 is used.

Although some effects have been obtained without using these angiogenic factors, it cannot be said that sufficient effects have been obtained. For example, it is described in JP-A-63-68174 that the collagen-heparin complex has excellent biocompatibility and proliferation of endothelial cells when used as a biocompatible material for artificial blood vessels and artificial organs. ing. JP-A-60-222425 describes a wound healing agent comprising a suspension of collagen and chemotactic inducible glucosaminoglycan. In addition, JP-A-6
3-54328 has a wound dressing consisting of a mixture of fibrillar collagen and heparin or heparin-like glycosamine glycans and platelet-derived growth factor or fibroblast growth factor.

[0006]

Since the conventional wound healing agents or artificial skin have their respective problems as described above, as a treatment for an affected area when skin tissue is lost due to a burn or the like, autologous transplantation is used. (Autograft) is currently considered the best method. However, it is very difficult when the skin defect portion covers a wide area, and even if it is applicable, it needs to be repeated many times over a long period of time.

[0007] In addition, allograft is applied as a method of temporarily covering the affected area instead of autografting. Allogeneic transplants have the advantage that they are less susceptible to infection because blood vessels are secured, but the epidermis part eventually falls off. Therefore, it is desired to develop a wound healing agent or artificial skin that can prevent bacterial infection by covering the affected area at an early stage to cover the affected area instead of autologous transplantation or allograft transplantation.

[0008]

The above object can be achieved by the wound healing material of the present invention having the following constitution. (1) Serum, plasma or platelet extract is bound to a heparin column under physiological conditions using a heparin column to give a 0.5M NaCl solution and / or 1.0M Na.
A wound healing material comprising a fraction eluted with a Cl solution and a base material containing collagen and heparin. (2) The wound healing material according to (1) above, wherein the eluted fraction is eluted under the condition of a 1.0 M NaCl solution, and the 0.5 M NaCl solution is excluded. (3) The wound healing material according to the above (1) or (2), wherein the collagen is atelocollagen obtained by using bovine dermis-derived collagen as a raw material and removing the telopeptide of the antigenic group at the molecular end with proctase or pepsin.

(4) The collagen is fibrotic atelocollagen obtained by reconstituting a collagen derived from bovine dermis as a raw material and adding a neutral buffer solution to the collagen.
(3) The wound healing material described. (5) The wound healing material according to the above (1) to (4), wherein heparin is contained in the collagen-containing substrate, and the fibrotic atelocollagen and heparin are bonded by reversible cross-linking. Further, the present invention is achieved by the following configurations. (6) An artificial skin comprising a water vapor transmission control layer and a wound healing layer, wherein the wound healing layer binds serum, plasma or platelet extract to heparin under physiological conditions using a heparin column, An artificial skin comprising a substrate containing fibrotic atelocollagen and denatured atelocollagen, containing a fraction eluted with a 1.0 M NaCl solution.

(7) An artificial skin comprising a water vapor permeation control layer and a wound healing layer, which is bound to heparin under physiological conditions in a serum, plasma or platelet extract using a heparin column. Let's go further, 1.0M
The artificial according to (6) above, which comprises a fibrotic atelocollagen and / or denatured atelocollagen and heparin and / or heparin-like glycosaminoglycan base material containing a fraction obtained by elution under conditions of NaCl solution. Skin. (8) The above-mentioned collagen is atelocollagen obtained by using collagen derived from bovine dermis as a raw material, removing an antigenic group, and thermally denaturing it in a temperature range of 40 to 90 ° C.
The artificial skin according to (7). (9) The artificial skin according to the above (6) to (8), wherein the water vapor transmission control layer is a silicone elastomer.

The wound healing material of the present invention induces serum, plasma or platelets, particularly platelet aggregation, and the extract containing various cell growth factors released at this time is passed through a heparin column under physiological conditions to heparin. Combined with the column,
It consists of a collagen substrate containing fractions eluted under conditions of 0.5 M NaCl solution and 1.0 M NaCl solution.
Serum, plasma or platelet extract in the present invention is bound to heparin and heparin through a heparin column under physiological conditions to obtain 0.5M NaCl solution and 1.0M NaC.
The fraction eluted under the condition of the 1-solution will be specifically described. In the present invention, specifically, the physiological condition is p
It means using physiological saline in the range of H6 to 8 or physiological saline and a pH buffer solution.

[0012] Human or rat serum or platelet extract was added to a heparin column, washed thoroughly with phosphate buffer-physiological saline (PBS solution), and washed with 0.5 M NaC.
Fraction eluted with the 1 solution
The fraction eluted with 1.0 M NaCl solution was designated as Fr.
Action II. Further, the fraction eluted with the 1.6 M NaCl solution is referred to as Fraction III.

When these fractions are analyzed by electrophoresis, they are mainly PDGF as a component of Fraction I.
(MW 30,000), prothrombi
n, MW 72,000), fibronectin (fibronect
in, MW 250,000), vitronectin (vitronect
in, MW 75,000) and the like.

The component of Fraction II is β-thromboglobulin (β-TG, MW 8,90).
0), low-affinity platelet factor 4: LA-PF4, M
W 9,200), platelet basic protein (PBP, MW 10,000)
0), hepatocyte growth factor (hepatocyte)
growth factor: HGF, MW 85,000) and the like.

The component of Fraction III is platelet factor 4: PF4, MW 1
2,000), anti thrombin III (anti thrombi
n III: AT III, MW 65,000).

Collagen which can be used in the present invention includes:
Although not particularly limited, type I collagen obtained by treating bovine dermis with protease in an acidic solution is preferable. This collagen is generally a transparent and uniform solution in an acidic solution with a concentration of 0.3% or less under low ionic strength, but under neutral conditions, in the living body by appropriately selecting ionic strength, temperature, etc. It is possible to form fibers similar to the state of. Type I collagen is a rod-shaped molecule with a length of 3000Å (Å = 1 × 10 ^-8 m) and a diameter of 15Å, which has a three-stranded helix structure with a molecular weight of 300,000 in a non-crosslinked single molecule state, and has telopeptides at both ends It has a non-helical structure consisting of a few 10 amino acid residues. This telopeptide site occupies most of the antigenicity of native collagen and can be easily removed by hydrolysis with a protease such as pepsin.

In the present invention, the base material containing collagen may contain heparin or the like. The present invention is further achieved by artificial skin in which the denatured collagen helix content of the artificial skin is 0 to 80%. The present invention is further achieved by artificial skin in which the content ratio of denatured collagen in the artificial skin is 5 to 30% by weight based on the whole. The content ratio of pepsin in the artificial skin of the present invention is preferably 0.1 to 2.0% by weight, more preferably 0.5 to 1.0% by weight. Further, the denatured collagen used in the present invention has a triplex helix content molecularly peculiar to collagen of 0 to 80.
%, Preferably 0-50%, more preferably 30-50
%, For denaturation of collagen, for example, heat treatment,
It can be performed by chemical treatment or physical treatment, and most preferably, the collagen solution is heat treated.

When the present invention is used as a wound healing material, the wound healing material contains the above-mentioned atelocollagen in a pH 3 solution (hydrochloric acid, etc.).
To 0.3 (w / v)%, and then add phosphate buffer-physiological saline (PBS solution) to this solution and treat it in a 37 ° C thermostat for 4 hours to convert atelocollagen into fibrils. It becomes cloudy. This suspension was centrifuged, concentrated to a predetermined concentration, and freeze-dried. This substrate is vacuumed at 110 ° C.
The heat treatment was performed for 2 to 8 hours. On the other hand, Fraction I or Fraction II separated by the above-mentioned method is impregnated. Fraction II is more preferable. The method for producing the wound healing material of the present invention is not limited at all, and serum, plasma or platelet extract is bound to heparin under physiological conditions using a heparin column to give 0.5.
The fraction eluted with the M NaCl solution and / or the 1.0 M NaCl solution may be mixed with the substrate containing collagen.

Hereinafter, the present invention will be described in more detail with reference to examples.

【Example】

Example 1 Rat Serum Preparation 25 ml of rat blood was collected in Autosep (manufactured by Terumo Corp.), left at room temperature for 2 hours, and then left at 4 ° C. overnight.
This solution was centrifuged at 3000 rpm for 15 minutes,
Filtration through a 0.45 μm filter gave 10 ml of serum.

(Example 2) Preparation of rat platelet extract 75 ml of rat blood was added to 0.1 volume of 0.15M NaCl-7.
Collect with 7mM EDTA (pH 7.4) solution, 1000r
The platelet-rich plasma was collected by centrifugation at pm for 15 minutes. After further centrifugation at 3000 rpm for 15 minutes, the precipitate (platelet) was added with 0.01M PB (pH 7.0) -0.15M N.
The aCl solution was washed twice. This solution is 1 mM P
MSF (phenylmethyl sulfonyl fluoride) -7.7 mM EDTA-0.01M PB (pH 7.
The suspension was suspended in 1 ml of 0) -0.15M NaCl solution, sonicated for 15 minutes, and centrifuged at 3000 rpm for 15 minutes. The resulting solution was filtered through a 0.45 μm pore size filter to obtain 0.8 × 10 ¹⁰ platelet extract.

(Example 3) Separation of heparin-binding substance The serum and platelet components separated in Examples 1 and 2 were treated with bovine collagen-sepharose 4B and bovine denatured collagen.
Pour onto Sepharose 4B. Heparin-
Run on a Sepharose CL-6B column. 0.15M N
After thoroughly washing with aCl-0.01M PB (pH 7.0) solution, the fractions eluted with 0.5M NaCl solution were fractionated.
ction I. Fraction II was then eluted with a 1.0 M NaCl solution. Further, the fraction eluted with a 1.6 M NaCl solution was designated as Fraction III.

Example 4 Impregnation with a heparin-binding substance
Preparation of artificial skin Atelocollagen powder (manufactured by Koken Co., Ltd.) was dissolved in dilute hydrochloric acid having a pH of 3.0 at a temperature of 4 ° C. to prepare 0.3 to 0.4 (w / v)%. A heparin sodium solution (derived from porcine small intestinal mucosa) was added to this solution, and a phosphate buffer of pH 7.4 was added while maintaining it at 4 ° C. to give a final concentration of 0.2 (w / v)% atelocollagen (30 mM Na ₂ HPO). ₄ , 100 mM NaC
l) a collagen-heparin solution (0.5 mg heparin / 100 mg collagen).

Then, leave it in a constant temperature bath at 37 ° C. for 4 hours,
A fibrotic atelocollagen-heparin (FC-Hp) solution was prepared. The FC.Hp solution was concentrated by centrifugation under sterile conditions to adjust the concentration to 2 (w / v)%.
On the other hand, sterilized atelocollagen powder was redissolved in sterile distilled water to 6.6 (w / v)% and left in a constant temperature bath at 60 ° C for 30 minutes to cause heat denaturation and denatured atelocollagen solution. And This solution was poured into a stainless steel pad, rapidly cooled at −30 ° C. or lower to be sufficiently frozen, and then freeze-dried under a vacuum of −40 ° C./less than 0.1 Torr. Fractio on the matrix of this collagen sponge
It was impregnated with n I (10 ml of 84.3 μg / 0.15 M NaCl solution).

(Example 5) Impregnation with a heparin-binding substance
Preparation of artificial skin A collagen sponge was prepared in the same manner as in Example 4. Fraction II (2.4 μg / 0.1
It was impregnated with 10 ml of 5M NaCl).

Comparative Example Impregnated with a heparin-binding substance
Preparation of artificial skin A collagen sponge was prepared in the same manner as in Example 4. Fraction III (1.9 μm / 0.
Impregnated with 15 M NaCl 10 ml).

(Test Example 1) Rat Subcutaneous Implantation Test The matrices prepared in Examples 4 to 6 are subcutaneously implanted into rats and pathologically examined for histology. Subcutaneous implantation (implantation)
For use, about 200 g of SD rat female rat is used. Before embedding, after anesthetizing with 5-fold diluted Nembutal, the rat's back surface was wetted with isodine solution for surgery (manufactured by Meiji Seika Co., Ltd.), and the back surface was treated with isodine so as not to leave uncut hair with a razor for shaving. Disinfect with ethanol. From each cut,
The incision is widened to create a void within the loose connective tissue beneath the rat cutaneous muscle. Insert the sample into this void so that the entire sample lies flat. Sew the cut end with a nylon thread with square needles. Sew three stitches at the cut end. The same specimen is similarly implanted in another rat. On day 1, 3, 7-14 after implantation, animals are killed with ether or 2-fold diluted Nembutal. Make sure that the implant remains in the tissue,
Cut the skin tissue on the back muscle of the rat to a size of 8 cm × 12 cm or larger. This tissue is placed in a 10% neutral buffered formalin solution, left to stand overnight for fixation, and then subjected to pathological tissue search.

The pathological tissue search starts with cutting out a specimen from the tissue. Tissue should be 0.5c to ensure that the sample is included.
Cut out into strips measuring m x 2.5 cm. This is clarified with ethanol, then xylene, and finally replaced with paraffin. After the replacement, the tissue containing the sample is placed in a heated solution of solid paraffin and rapidly cooled to complete the paraffin embedding. The embedded tissue is sliced with a rotary microtome manufactured by Yamato Co., Ltd. to obtain a paraffin section having a thickness of 4 μm.
After deparaffinizing this, pathological tissue staining is performed by an arbitrary staining method to complete the preparation. Hematoxylin-eosin (HE) staining was adopted as the pathological tissue staining. From the findings of pathological tissues, Fraction I and Fraction
When artificial skin impregnated with II was used, the invasion of fibroblasts and blood vessels was good. However, in the artificial skin impregnated with Fraction III, the invasion of fibroblasts and the like was poor, and it was rather inhibited.

[0028]

As described above, according to the present invention, collagen or a substrate containing collagen and heparin is loaded with a substance extracted from serum, plasma or platelets that binds to heparin. Since it is a characteristic wound healing material, it strongly supports cell growth when applied in vivo, and has high biocompatibility, so that when it is applied as artificial skin, for example, early granulation A layer can be formed or an epidermis can be formed to promote healing. Furthermore, the wound healing material of the present invention can be lined with a breathable support layer, for example, a support layer made of silicone, whereby when applied as, for example, artificial skin, leakage of body fluid, occurrence of bacterial infection, etc. occur. It can be kept to a minimum.

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Claims (1)

[Claims] 1. A serum, plasma, or platelet extract is bound to a heparin column under physiological conditions using a heparin column to give a 0.5 M NaCl solution and / or 1.0.
A wound healing material comprising a fraction eluted with a M NaCl solution and a base material containing collagen and heparin.