WO2013042869A1

WO2013042869A1 - Medical dressing using silkworm gland-derived hydrolysate and method of manufacturing the same

Info

Publication number: WO2013042869A1
Application number: PCT/KR2012/005914
Authority: WO
Inventors: Heui Sam Lee; Hae Yong Kweon; Kang Sun Ryu; Pil Don Kang; Kwang Young Park; Mi Ja Kim; Young Soo Choi; Ho Jin Kim; Jung Wook Hwang
Original assignee: Republic Of Korea (Management : Rural Development Administration); College Of Medicine Pochon Cha University Industry-Academic Cooperation Foundation
Priority date: 2011-09-20
Filing date: 2012-07-25
Publication date: 2013-03-28

Abstract

This invention relates to a medical dressing using a silkworm gland-derived hydrolysate having superior cell proliferation effects thus exhibiting superior wound treatment effects and moisturizing power, and to a method of manufacturing the same, including 1) dissolving a silkworm gland-derived hydrolysate thus preparing a hydrolysate solution, 2) adding a crosslinking agent to the hydrolysate solution and reacting the hydrolysate solution with stirring at 15 ~ 30℃ for 0.5 ~ 2 hr, and 3) performing rapid lyophilization.

Description

MEDICAL DRESSING USING SILKWORM GLAND-DERIVED HYDROLYSATE AND METHOD OF MANUFACTURING THE SAME

The present invention relates to a medical dressing using a silkworm gland-derived hydrolysate and a method of manufacturing the same, and more particularly, to a medical dressing using a silkworm gland-derived hydrolysate which is superior in terms of treating wounds, and to a method of manufacturing the same.

The skin is responsible for performing an immune function and constitutes the greatest surface area of the human body. The skin also plays a role in protecting the human body from a variety of harmful conditions, including external microorganisms or UV light, chemicals, etc., and in suppressing the evaporation of moisture from the human body, thereby preventing dehydration and adjusting the body temperature. Furthermore, the skin has to be resistant to physical stimuli and to have elasticity and should sufficiently perform its functions over the life span of the individual.

When the skin is damaged due to burns or a variety of external wounds, it loses its protective actions, undesirably causing dysfunctions, and also many side effects and bacterial infections are caused by the loss of moisture, etc., making it difficult to treat the affected portions or generating additional side effects such as secondary dysfunction or damage, which have a negative influence on prolonging the life span.

Thus, to rapidly treat the wounds and minimize a variety of secondary side effects, treating wounds using an appropriate dressing is essential.

Currently, dressings commercially available for self-cure are provided in a fixed form by applying an adhesive onto nonwoven fabric or a synthetic resin film and adhering a liquid-absorbing pad at a position corresponding to the wounded portion, and are thus inexpensive and simple to use. However, a non-waterproof dry dressing does not make a wet condition adapted for treating a wound, and furthermore absorbs an exudate necessary for treating a wound. Also, a waterproof film dressing is problematic because it does not properly control an exudate.

Even for the waterproof type, a conventional dressing is designed so that an exudate is moved perpendicular to the wound portion and is thus continuously absorbed over a range that exhibits an ability to absorb liquid, making it impossible to appropriately control storage of the exudate.

In order to satisfy the opposite conditions of a wet condition on the wound portion and a dry condition around the wound portion, there are known many dressings having controlled moisture permeability, including a dressing using a material having high moisture permeability such as a porous material or the like as a coating, a dressing having a hydrophilic material or the like to increase moisture permeability of an adhesive layer applied on a coating, a dressing having an adhesive partially applied, etc. However, these dressings increase moisture permeability at both the wound portion and the peripheral portion thereof, or only at the wound portion, and research into maintaining the dry condition around the wound portion while maintaining the wet condition on the wound portion has not yet been reported.

Prior techniques related thereto include Korean Unexamined Patent Application Publication No. 10-2009-0034173 (published date: April 07, 2009, entitled Silk Dressing) and Japanese Unexamined Patent Application Publication No. 2002-128691 (published date: May 09, 2002, entitled Sericin-containing Material, Method of Preparing the same and Use thereof).

Accordingly, the present invention has been made keeping in mind the above problems occurring in the related art, and an object of the present invention is to provide a medical dressing which exhibits superior wound treatment effects while maintaining a wet condition on the wound portion, and a method of manufacturing the same. In addition, a medical dressing having an absorption rate adapted for a variety of wound states and conditions and a method of manufacturing the same are provided.

The technical problem according to the present invention is not limited to the above object, and the other objects will be obviously understood by those having ordinary skill in the art from the following description.

In order to accomplish the above object, the present invention provides a medical dressing, comprising a silkworm gland-derived hydrolysate.

The hydrolysate may be extracted from a supernatant obtained by centrifuging a solution of glands obtained from a lyophilized silkworm.

The medical dressing may further comprise collagen. As such, the collagen may be derived from a mammal or human umbilical cord. Particularly useful is type I collagen.

The amount of the collagen may be one to three times the weight of the hydrolysate.

In addition, the present invention provides a method of manufacturing a medical dressing, comprising 1) dissolving a silkworm gland-derived hydrolysate thus preparing a hydrolysate solution, 2) adding a crosslinking agent to the hydrolysate solution, and reacting the hydrolysate solution with stirring at 15 ~ 30℃ for 0.5 ~ 2 hr, and 3) performing rapid lyophilization.

In 2), collagen may be further added to the hydrolysate solution. As such, the amount of the collagen may be one to three times the weight of the hydrolysate solution.

The silkworm gland-derived hydrolysate in 1) may be obtained by lyophilizing a silkworm to separate glands, dissolving the separated glands to prepare a gland solution, centrifuging the gland solution to obtain a supernatant, adding an alkali to the supernatant, hydrolyzing the supernatant, and performing neutralization and desalting.

As such, hydrolyzing may be performed at 15 ~ 30℃.

The crosslinking agent used in 2) may be glutaraldehyde, which is preferably used in an amount of 0.1 ~ 0.5 vol% based on the volume of the hydrolysate solution.

According to the present invention, a medical dressing includes a silkworm gland-derived hydrolysate usable as a material having superior properties such as skin coating effects, moisturization, antioxidative activity, skin affinity, etc., so that a wet condition is maintained, and cells can freely move between pores thereof, thus enabling treatment of small and large wounds.

Also, the silkworm gland-derived hydrolysate having superior cell proliferation effects is used, thus imparting wound treatment effects and providing wet conditions adapted for treating the wound, thereby facilitating the treatment of the wound.

FIGS. 1A to 1J illustrate scanning electron microscope (SEM) images of medical dressings according to the present invention;

FIG. 2 illustrates the biologically active concentration (IC₅₀: the half maximal inhibitory concentration) determined after measuring the antioxidative activity of a water-soluble hydrolysate solution derived from ultrafine powder of silkworm glands compared to vitamin C;

FIG. 3 illustrates the antioxidative activity of the mixture solution of collagen and water-soluble hydrolysate derived from ultrafine powder of silkworm glands;

FIG. 4 illustrates the ability of the medical dressing including collagen and water-soluble hydrolysate derived from ultrafine powder of silkworm glands to maintain the outer shape thereof in a solvent;

FIG. 5A illustrates the swelling ratio of the medical dressing of the invention containing 0.1 wt% of glutaraldehyde;

FIG. 5B illustrates the swelling ratio of the medical dressing of the invention containing 0.5 wt% of glutaraldehyde;

FIG. 6A illustrates the stability of the medical dressing of the invention containing 0.1 wt% of glutaraldehyde;

FIG. 6B illustrates the stability of the medical dressing of the invention containing 0.5 wt% of glutaraldehyde;

FIG. 7A illustrates the degradation of the medical dressing of the invention containing 0.1 wt% of glutaraldehyde;

FIG. 7B illustrates the degradation of the medical dressing of the invention containing 0.5 wt% of glutaraldehyde;

FIGS. 8 and 9 illustrate the regeneration of skin tissue on the wound portion of the back of a mouse over time;

FIG. 10 illustrates the regeneration of skin tissue on the wound portion of the back of a mouse over time, as compared to using H&E dyeing; and

FIG. 11 illustrates the collagen synthesis and regeneration of skin tissue on the wound portion of the back of a mouse over time, as compared to using trichrome dyeing.

Unless otherwise stated, the meanings of the terms, descriptions, etc., disclosed in the present specification may be those that are typically used in the art to which the present invention belongs.

Hereinafter, a method of manufacturing a medical dressing using a silkworm gland-derived hydrolysate is described in detail.

1. Preparation of hydrolysate solution

A hydrolysate solution is prepared by dissolving silkworm glands. As such, because the glands dissolve well in water, water is preferably used to dissolve the glands.

Any material may be included in the hydrolysate so long as it is separated from the silkworm glands, and preferably those separated from ultrafine powder of silkworm glands are used.

The hydrolysate is obtained by hydrolyzing silk proteins, which dissolve at room temperature, present in the glands including fibroin and sericin mixed together, and thus is in a state of a mixture of both fibroin and sericin. In particular, the hydrolysate is water-soluble so that it is easily dissolved in water.

The preparation of such a hydrolysate is specified below.

First, silkworms are lyophilized to separate glands therefrom.

As such, Bombix mori may be used as the silkworms, but the present invention is not limited thereto. All species of silkworm able to obtain glands may be used, and in particular, matured silkworms are preferably used in terms of superior cell proliferation effects, short breeding time and low personnel expenses, in order to acquire a hydrolysate.

Second, the separated glands are processed into ultrafine powder, which is then dissolved in water, followed by performing centrifugation, thus recovering a supernatant.

The case where the glands are in ultrafine powder form enables easy dissolution at room temperature (15 ~ 30℃) so as to minimize changes in components thereof.

Also the reason why the glands in ultrafine powder form are dissolved in water is that the area of contact with water molecules is maximized upon dissolution, thus facilitating the dissolution, and also that only the gland proteins dissolved at room temperature may be separated. More preferably, the particle size of the ultrafine powder of the glands may be 600 ~ 800 mesh.

Third, an alkali is added to the supernatant, and then the supernatant is hydrolyzed.

An example of the alkali is sodium hydroxide (NaOH). This is because the addition of NaOH to the supernatant enables the decomposition into peptides having small molecular weight.

Preferably the final concentration of NaOH is 0.2 ~ 1 M, and more preferably 1 M, after which heating is performed at room temperature (15 ~ 30℃) for about 0.5 ~ 1 hr, preferably 30 min, so that hydrolysis takes place. Concretely, because all of the silkworm glands may decompose at room temperature, conventional problems of properties of a hydrolysate resulting from high-temperature treatment partially deteriorating may be solved, and it renders it possible to separate the hydrolysate even though a harmful solvent such as LiBr is not used.

Fourth, neutralization and desalting are carried out after hydrolysis, thus obtaining a silkworm gland-derived hydrolysate.

The neutralization is conducted by adding an acid, preferably phosphoric acid until the pH is about 7.0 ~ 7.5.

The desalting may be conducted using any typical desalting process. In the case of a polymer having a molecular weight in the thousands, a variety of methods that make use of the difference in molecular size between the polymer and the salt, for example, gel filtration, ultrafiltration, dialysis, etc., may be utilized.

The hydrolysate obtained as above may be easily added to a serum-free medium and may exhibit superior cell proliferation effects and thus may be used as a serum replacement, and may also be utilized as a material having such superior properties as skin coating effects, moisturization, antioxidative activity, skin affinity and so on.

2. Manufacturing of the medical dressing of the invention

A crosslinking agent is added to the above hydrolysate solution, after which the reaction is ran with stirring at 15 ~ 30℃ for 0.5 ~ 2 hr, followed by performing rapid lyophilization, thus manufacturing a medical dressing.

The crosslinking agent is used to chemically bind linear polymer compound molecules to each other to form a three-dimensional reticulated polymer compound, and functions to impart mechanical strength, such as hardness or elasticity, and chemical stability to resin.

Hence, any crosslinking agent known in the art may be used, and preferably glutaraldehyde (GA) is used. Particularly in order to obtain an absorption rate adapted for a variety of states and conditions of wounds, GA is used in an amount of 0.1 ~ 1.0 vol%, preferably 0.1 ~ 0.5 vol% based on the volume of the hydrolysate solution.

The medical dressing of the invention manufactured as above, including the silkworm gland-derived hydrolysate, may further contain collagen in order to maintain the outer shape thereof and attain higher moisturizing power.

As such, any collagen may be used so long as it may maintain the outer shape of the dressing, and preferably useful is collagen derived from mammals or human umbilical cords. Particularly useful is type I collagen having the greatest amount of proline among 19 kinds of collagen.

If the collagen is mixed in an amount less than one times the weight of the hydrolysate solution, the ability of the dressing to maintain its outer shape may be slightly decreased due to the water. In contrast, the case where the collagen is mixed in an amount greater than three times the weight of the hydrolysate solution is very similar to when collagen is added in an amount of three times the weight of the hydrolysate solution, and thus the monetary benefit is negatively impacted. Hence, the collagen is preferably mixed in an amount one to three times the weight of the hydrolysate solution.

The medical dressing of the invention has overcome the drawbacks of conventional nonwoven fabric or gauze dressings, including dry conditions, low absorption performance, etc., and enables the cells to move between pores thereof and thus may be widely applied to not only minor wounds but also large wounds. Moreover, this dressing may efficiently supply nutrients necessary for growth and proliferation of cells and provides wet conditions appropriate to wounds, thus facilitating the treatment of wounds, resulting in excellent wound treatment effects.

Below, the present invention will be more fully described via the following examples and test examples but such examples do not limit the scope of the invention.

<Example 1> Preparation of Water-soluble Hydrolysate derived from Ultrafine Powder of Silkworm Glands

Matured silkworms were prepared as a silk material and then lyophilized.

Glands were separated from the lyophilized material and then processed into ultrafine powder using a cryogenic mill.

The ultrafine powder of silkworm glands processed as above was dissolved while being stirred at room temperature (15 ~ 30℃) for 30 min, and then centrifuged thus recovering only a supernatant, after which NaOH was added in a final concentration of 1 M to the supernatant, and the supernatant was then stirred at 24℃ for 30 min.

Subsequently, the stirred solution was neutralized to pH 7 using phosphoric acid, and then desalted using ultrafiltration.

Subsequently, lyophilization was conducted, thus obtaining a water-soluble hydrolysate derived from the ultrafine powder of silkworm glands used in the present invention.

<Examples 2 to 8> Manufacturing of Medical Dressing of the Invention

The water-soluble hydrolysate powder derived from the ultrafine powder of silkworm glands obtained in Example 1 was prepared.

The water-soluble hydrolysate powder was dissolved in 4% (w/v) distilled water based on the weight of the water-soluble hydrolysate powder, thus preparing a water-soluble hydrolysate solution. Separately, 1 wt% of a collagen solution was prepared as in Example 9 as will be described later.

The water-soluble hydrolysate solution and the collagen solution were mixed as shown in Table 1 below.

Table 1

		Glutaraldehyde	Water-soluble Hydrolysate Powder of Ex. 1 : Collagen
Ex. 1	FIG.1A	0.1%	1 : 0
C.Ex.1	FIG.1B	0.1%	0 : 1
Ex. 2	FIG.1C	0.1%	1 : 1
Ex. 3	FIG.1D	0.1%	3 : 1
Ex. 4	FIG.1E	0.1%	1 : 3
Ex. 5	FIG.1F	0.5%	1 : 0
C.Ex.2	FIG.1G	0.5%	0 : 1
Ex. 6	FIG.1H	0.5%	1 : 1
Ex. 7	FIG.1I	0.5%	3 : 1
Ex. 8	FIG.1J	0.5%	1 : 3

Subsequently, to respective solutions prepared as above was added 0.5% (w/v) or 0.1% (w/v) glutaraldehyde as a crosslinking agent, and the resulting solutions were reacted with uniform stirring at 20℃ for 1 hr, frozen at -80℃, and then dried, thus manufacturing medical dressings.

The medical dressings were observed using SEM. The results are illustrated in FIGS. 1A to 1J. Specifically, as illustrated in FIG. 1J, the medical dressing of the invention is provided in the form of a three-dimensional reticulated structure by chemically binding the water-soluble hydrolysate derived from ultrafine powder of silkworm glands of Example 1 with the collagen using the crosslinking agent. The case where 0.5% GA was added exhibited the greatest pores (gaps between the particles) when observed with SEM.

<Example 9> Preparation of Collagen derived from Human Umbilical Cord

The frozen umbilical cord was thawed at room temperature. The umbilical cord was cut to a length of 1 ~ 2 ㎝, and then washed with purified water.

Treatment with 70% ethanol solution was performed, followed by carrying out the reaction at 4℃ for 24 hr.

Subsequently, the reaction product was washed with purified water, treated with 3% H₂O₂ solution, and then stirred using a magnetic bar at 4℃ for 12 ~ 24 hr.

The resulting solution was washed at least two times with purified water, added with 0.5 M acetic acid solution, and then treated using a blender and a homogenizer so that tissue was milled.

The resulting solution was treated with pepsin, reacted at 4℃ for 24 hr, and then centrifuged at 10,000 rpm and 4℃ for 30 min.

After centrifugation, the pH of the supernatant was adjusted to 7 using NaOH, thus deactivating the pepsin enzyme.

The solution having the adjusted pH was treated with sodium chloride (NaCl), stirred until sodium chloride was thoroughly dissolved, and then allowed to stand at 4℃ for 12 ~ 24 hr so that the collagen was precipitated via salting out.

Subsequently, centrifugation was conducted at 10,000 rpm and 4℃ for 30 min, after which the slated-out collagen pellets were separated, desalted using a ultrafiltration system, and then concentrated. Finally, sterile filtration and lyophilization were performed, and the product was then stored.

<Comparative Example 1> Manufacturing of Medical Dressing 1 composed exclusively of Collagen Solution

The collagen of Example 9 was prepared in the form of a 1% (w/v) solution, mixed with 0.1% (w/v) glutaraldehyde, reacted with uniform stirring at 20℃ for 1 hr, frozen at -80℃, and then dried, thus manufacturing a medical dressing 1 composed exclusively of a collagen solution (Table 1).

<Comparative Example 2> Manufacturing of Medical Dressing 2 composed exclusively of Collagen Solution

A medical dressing 2 composed exclusively of a collagen solution was manufactured in the same manner as in Comparative Example 1, with the exception that 0.5% (w/v) GA was used (Table 1).

<Test Example 1> Measurement of Antioxidative activity

1% (10 mg/ml) water-soluble hydrolysate solution of Example 1 and 1% (10 mg/ml) collagen solution were prepared to analyze antioxidative activity.

As such, antioxidative activity was evaluated using DPPH assay.

Concretely, 40 ㎕ of each of the water-soluble hydrolysate solution and the collagen solution was prepared, and 160 ㎕ of 0.2 mM DPPH dissolved in methanol was added thereto, after which the reaction was carried out at 30℃ for 30 min under dark conditions.

After completion of the reaction, the absorbance of the sample was measured at 517 nm using a microplate reader.

A total of three measurements were performed, and the biologically active concentration (IC₅₀: the half maximal inhibitory concentration) was determined using the values of a blank and a control.

As illustrated in FIG. 2, IC₅₀ of vitamin C was 0.1±0.1 ㎍/㎖, IC₅₀ of the silkworm gland-derived hydrolysate was 73.8±7.4 ㎍/㎖, and IC₅₀ of cocoon-derived sericin (available from S) was 202.9±34.1 ㎍/㎖, from which the antioxidative activity of the silkworm gland-derived hydrolysate according to the present invention was evaluated to be higher.

As illustrated in FIG. 3, compared to vitamin C used as a positive control, the antioxidative activity of the mixture including 2% (20 mg/ml) water-soluble hydrolysate solution and 1% (10 mg/ml) collagen solution mixed at 1:1 was about 75%, which is evaluated to be the highest among the mixtures of hydrolysate and collagen.

Such a high antioxidative activity shows the usability of the dressing of the invention as a biocompatible medical dressing, and the water-soluble hydrolysate or the mixture of water-soluble hydrolysate and collagen can be utilized in a medical dressing.

<Test Example 2> Ability of the Medical Dressing of the Invention to Maintain Outer Shape Thereof

A dressing manufactured for medical use has to maintain its morphology and shape in a variety of solvents. Hence, a solvent was added to the manufactured dressing to observe the outer shape and properties of the dressing.

The medical dressings of Examples 1 to 8 were prepared, and comparative dressings of Comparative Examples 1 and 2 were also prepared. 1 ㎖ of a solvent (distilled water or 40% ethanol) was added thereto, and changes in morphology and properties were observed.

As illustrated in FIG. 4, the dressing including the water-soluble hydrolysate and collagen at a 1:3 ratio was the greatest in terms of maintaining the outer shape thereof in the above solvent. Accordingly, the mixture of water-soluble hydrolysate and collagen at a 1:3 ratio can be seen to be the best as the dressing of the invention.

<Test Example 3> Porosity of the Medical Dressing of the Invention

In order to evaluate the porosity of the medical dressings of Examples 1 to 8, the weight (W) of each support was first determined, and the support was then placed in a tube containing a predetermined amount of water (V₁), after which the amount (V₂) of water increased by the support was recorded.

Next, the support was removed, and the remaining amount (V₃) of water was recorded, and the porosity (e) was determined using the following equation.

e (%) = [(V₁-V₃)/(V₂-V₃)] ×100

The results are given in Table 2.

Table 2

Porosity (Cross-linker)	Scaffold composition(wt% ratio)Water-soluble Hydrolysate:Collagen	Average Porosity(%)
0.1% Glutaraldehyde	1:0	78.6 ± 10.1
	0:1	65.5 ± 1.7
	1:1	79.5 ± 2.5
	3:1	78.1 ± 2.7
	1:3	61.2 ± 3.4
0.5% Glutaraldehyde	1:0	81.2 ± 1.7
	0:1	64.6 ± 2.9
	1:1	79.5 ± 0.7
	3:1	79.9 ± 4.9
	1:3	64.1 ± 3.6

As is apparent from Table 2, the porosity of the water-soluble hydrolysate was 78.6 ± 10.1%, which was higher than when only the collagen was contained. As the proportion of the water-soluble hydrolysate increased, there were no changes in porosity, but the porosity was gradually decreased in proportion to the increase in the proportion of collagen. Also, the concentration of the crosslinking agent did not have a great influence on the porosity.

Changes in porosity depending on the proportion of collagen are considered to aid the movement of cells between pores and the attachment of the cells to a wide area.

<Test Example 4> Swelling Ratio of the Medical Dressing of the Invention

The swelling ratio of the medical dressings of Examples 1 to 8 was measured.

As a control, the swelling ratio of the medical dressings of Comparative Examples 1 and 2 was measured.

As illustrated in FIGS. 5A and 5B, the swelling ratios were not greatly different depending on the concentration of the crosslinking agent. However, the total swelling ratio was slightly lower at 0.5% GA than at 0.1% GA.

The dressing including water-soluble hydrolysate and collagen at a 1:3 ratio exhibited the lowest swelling ratio. The swelling ratio was lower in proportion to the increase in the amount of collagen.

This means that the dressing is formed very densely and absorption of the solution into the dressing is very slow. On the other hand, a dressing with high swelling ratio is thought to absorb the solution well and efficiently supply nutrients necessary for growth and proliferation of cells into the dressing.

Accordingly, the selection of the dressing on a swelling ratio basis is considered to vary depending on the end uses in terms of tissue engineering.

<Test Example 5> Stability of the Medical Dressing of the Invention

In order to measure the extent of protein release of the water-soluble hydrolysate and collagen, the stability of the dressings of Examples 1 to 8 depending on the use of the crosslinking agent was evaluated.

As a control, the stability of the medical dressings of Comparative Examples 1 and 2 was evaluated.

Specifically, 10 ㎖ of water was added to each support, and the amount of protein contained in water was measured over time.

The resulting measurement sample was uniformly mixed with a Bradford reagent and then reacted at room temperature for 10 min.

After completion of the reaction, the absorbance of the sample was measured at 595 nm, and the amount of protein released from the support was measured over time.

As illustrated in FIGS. 6A and 6B, as the amount of water-soluble hydrolysate was larger, the protein release increased over time.

This is considered to be because non-crosslinked water-soluble hydrolysate was released in the water-soluble hydrolysate than in collagen. The dressing including the water-soluble hydrolysate and collagen at a 1:3 ratio was more stable compared to the other dressings having different mixing ratios.

<Test Example 6> Degradation of the Medical Dressing of the Invention

The degradation of the dressing including the mixture of water-soluble hydrolysate and collagen using collagenase was evaluated. Respective dressings were pretreated for one day with PBS (pH 7.4), treated with collagenase 10U^-1 to measure the degree of degradation, and then observed at 37℃ over time.

After the lapse of a predetermined period of time, the support was dried and weighed. The degradation of the support was calculated using the following equation:

% weight loss = 100 × (W₀ - W_t)/W₀

wherein W₀ is the weight of the lyophilized support and W_t is the weight of the dried support after a predetermined period of time.

As illustrated in FIGS. 7A and 7B, the degradation was different depending on the amount of collagen contained in the dressing.

The case where the water-soluble hydrolysate and the collagen were contained at a 3:1 ratio exhibited the highest degradation over time.

<Test Example 7> Non-inferiority of the Medical Dressing of the Invention (Mouse Model)

A wound model was formed on the back portion of a five-week-old female mouse (BALB/c-nu Slc) using a biopsy punch having a diameter of 8 mm, and to prevent self-cure a chimney having a diameter of 11 mm was inserted around the wound as shown in FIG. 8. Then, the dressing of the invention was applied thereon, and whether the skin tissue of the wound portion regenerated over time was evaluated.

As a negative control, no treatment was performed, and as a positive control, commercially available Mediform was used.

As test groups, the dressings of the invention including only the water-soluble hydrolysate, only the collagen, and the mixture of water-soluble hydrolysate and collagen at a 1:3 ratio were used.

Respective dressings were applied on the wound model, after which, in order to prevent the introduction of bacteria or dust in the air into the peripheral region of the wound tissue, the wound was fixed with Tegaderm, and whether the skin tissue was regenerated over time was evaluated.

As illustrated in FIG. 8, the regeneration of skin tissue after ten days was observed to more efficiently occur in the test groups than in the negative control.

<Test Example 8> Histological Analysis of Non-inferiority of Medical Dressing of the Invention (Mouse Model)

15 days after the dressing was applied, Tegaderm and the chimney were cautiously removed and the mouse was fixed in 4% neutral buffered formalin with shaking for 24 hr. Thereafter, the regeneration of the skin was observed in a state of the blood being removed. As illustrated in FIG. 9, the dressing including the water-soluble hydrolysate and collagen at a 1:3 ratio regenerated the skin to an extent very similar to the positive control.

For histological analysis, the fixed skin tissue was dehydrated for 1 hr each while increasing the concentration in the range from 50% alcohol up to 100% alcohol, and then substituted for 1 hr using a xylene solution.

The substituted tissue was placed in an oven at 60℃ for 24 hr so that a paraffin solution penetrated therein, and the tissue was placed in a paraffin block frame and then embedded with novel paraffin.

The embedded tissue was cut to a thickness of 5 ~ 6 mm using a tissue cutter and then placed on a slide glass, and to prevent the tissue from being removed from the slide glass, the tissue was dried at 37℃ for 24 hr and then dyed (ematoxyline-Eosin).

As illustrated in FIG. 10, in the case of the dressing including the water-soluble hydrolysate and collagen at a 1:3 ratio, novel tissue regenerated on subcutaneous tissue starting on the third day after wound treatment. On 15^th day, during the regeneration procedure for treatment, the formation of deformed portions of the tissue regeneration had remarkably increased instead of creating normal tissue, compared to the negative control.

As a result of dyeing with trichrome, as illustrated in FIG. 11, in the case of the dressing including the water-soluble hydrolysate and collagen at a 1:3 ratio, keratinization of skin was decreased over time, compared to the negative control, and the collagen fibers under the cuticle were more dense, which means that the structure and elasticity of the skin were maintained and regenerated into the original state of the skin.

The medical dressing of the invention includes the silkworm gland-derived hydrolysate usable as a material having superior properties such as skin coating effects, moisturization, antioxidative activity, skin affinity, etc., thus maintaining wet conditions and enabling the movement of cells between pores. Hence, this dressing can be applied to small and larger wounds.

The use of the silkworm gland-derived hydrolysate having superior cell proliferation effects enables the wound to be treated and imparts appropriate wet conditions to the wound, thus facilitating the treatment of wounds.

Although the preferred examples and test examples of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

A medical dressing, comprising a silkworm gland-derived hydrolysate.
The medical dressing of claim 1, wherein the silkworm gland is in ultrafine powder form having a size of 600 ~ 800 mesh.
The medical dressing of claim 1, wherein the hydrolysate is water-soluble.
The medical dressing of claim 1, wherein the hydrolysate is extracted from a supernatant obtained by centrifuging a solution of glands obtained from a lyophilized silkworm.
The medical dressing of any one of claims 1 to 4, further comprising collagen.
The medical dressing of claim 5, wherein the collagen is used in an amount one to three times a weight of the water-soluble hydrolysate.
The medical dressing of claim 5, wherein the collagen is derived from a mammal.
The medical dressing of claim 5, wherein the collagen is derived from a human umbilical cord.
The medical dressing of claim 5, wherein the collagen is type 1 collagen.
A method of manufacturing a medical dressing, comprising:

1) dissolving a silkworm gland-derived hydrolysate, thus preparing a hydrolysate solution;

2) adding a crosslinking agent to the hydrolysate solution, and reacting the hydrolysate solution with stirring at 15 ~ 30℃ for 0.5 ~ 2 hr; and

3) performing rapid lyophilization.
The method of claim 10, wherein the silkworm gland-derived hydrolysate in 1) is obtained by lyophilizing a silkworm to separate glands, dissolving the separated glands to prepare a gland solution, centrifuging the gland solution to obtain a supernatant, adding an alkali to the supernatant, hydrolyzing the supernatant, and performing neutralization and desalting.
The method of claim 11, wherein the hydrolyzing is performed at 15 ~ 30℃.
The method of claim 10, wherein the crosslinking agent used in 2) is glutaraldehyde.
The method of claim 10, wherein the crosslinking agent used in 2) is 0.1 ~ 0.5 vol% of glutaraldehyde based on a volume of the hydrolysate solution.
The method of any one of claims 10 to 14, wherein in 2) collagen is further added to the hydrolysate solution.
The method of claim 15, wherein an amount of the collagen is one to three times a weight of the hydrolysate solution.