CN110051884A - A kind of bio-sponge and preparation method based on Acelluar small intestinal submucosa - Google Patents
A kind of bio-sponge and preparation method based on Acelluar small intestinal submucosa Download PDFInfo
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Abstract
The invention discloses a kind of bio-sponge and preparation method based on Acelluar small intestinal submucosa, including prepare de- cell intestinal mucosa lower layer, pepsin enzymatic hydrolysis, cleaning, freeze-drying, crush and cross-linking step.The present invention is by disclosing a kind of bio-sponge and preparation method based on Acelluar small intestinal submucosa, and using intestinal mucosa lower layer as source, after de- cell processing, immune rejection is minimum.Crosslinking Treatment is carried out using nontoxic crosslinking agent simultaneously, ensure that the biological safety of the product of acquisition.The product is used not only for wound repair and excellent scaffold materials of tissue-engineered skin, and not only source is abundant; securely and reliably, cheap and easy to get, and its ingredient the Nomenclature Composition and Structure of Complexes and the mankind are seemingly; ethnics Problem is not present simultaneously, is convenient for scale, industrialization preparation.
Description
Technical field
The present invention relates to technical field of biological materials, more particularly to a kind of biology based on Acelluar small intestinal submucosa
Sponge and preparation method.
Background technique
Burn, wound, diabetes, chronic ulcer etc. often result in the defect of skin.Intractable, chronic trauma and tissue defect
The change of caused pain, physiological function missing, appearance drastically influences the freedom of action and quality of life of patient, and further
Lead to psychology and social concern.Dermatoplasty is being faced as tissue defect and the most effective treatment means of Skin pigment abnormalities
It is but limited by source in bed and influences treatment process.Organization engineering skin is then most potential treatment method.
Current scaffold materials of tissue-engineered skin mainly has chemical synthesis class material such as: polylactic acid (PLA) gathers left lactic acid
(PLLA) etc., although material properties is stable, structure is uniform, satisfactory mechanical property, biocompatibility is poor, and self and of the same race
Heteroplastic transplantation is often become inevitable choice by limitation, animal derived xenogenic biological materials such as source, ethics and infection diseases.
Animal derived xenogenic biological materials source is abundant, is a kind of good regenerating tissues stock support, and not different securely and reliably
The ethnics Problem of body biomaterial implantation is convenient for scale, industrialization preparation.
After natural material is sloughed cell, it is set to have good mechanical property, good through certain crosslinking agent processing
Good three-dimensional structure, preferable histocompatbility and lower immunogenicity, is the common method of tissue tissue engineering research.But
These methods have certain problems at present: current crosslinking agent is mostly the reagents such as glutaraldehyde, EDC/NHS, these reagent sheets
Body also can cause environmental pollution, and subsequent processing increases cost.Most importantly the residual of reagent will cause serious cell toxicant
Property, greatly influence clinical use, and the product obtained by chemical crosslinking, mostly without containing the life conducive to tissue cell growth
The nutriments such as the long factor cannot function as a kind of ideal biogenic material.
Therefore, inventing a kind of and dermis of skin has approximate immanent structure, and abundant containing a large amount of collagenous fibres
Growth factor has good biocompatibility, while biological safety is good and is conducive to the biology sea of the growth of sertoli cell
The problem of silk floss is those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides one kind to be based on Acelluar small intestinal submucosa (small intestinal
Submucosa, SIS) bio-sponge and preparation method, using intestinal mucosa lower layer as source, after de- cell processing,
Immune rejection is minimum.Crosslinking Treatment is carried out using nontoxic crosslinking agent simultaneously, ensure that the biology of the product of acquisition
Safety.The product is used not only for wound repair and excellent scaffold materials of tissue-engineered skin, and not only source is filled
Point, it is securely and reliably, cheap and easy to get, and its ingredient the Nomenclature Composition and Structure of Complexes and the mankind are seemingly, while ethnics Problem is not present, convenient for into
Professional etiquette modelling, industrialization preparation.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of bio-sponge based on Acelluar small intestinal submucosa, bio-sponge are changed using SIS as raw material by being crosslinked
Property method and utilize fa-PEG (Four-arm polyethylene glycol with succinimydil
Glutarateterminated branches) it is crosslinked, it has prepared with three-D space structure, rich in growth factor
Novel Scaffold Materials for Tissue Engingeering.
A kind of bio-sponge based on Acelluar small intestinal submucosa disclosed by the invention, the sea with loose 3D structure
Silk floss inside has hole, is interconnected, grows into convenient for cell, exchange nutriment conducive between cell, between cell and tissue.Together
When bio-sponge better mechanical property of the invention, be conducive to clinical manipulation；Furthermore compared with other artificial synthesized products, this hair
Bright disclosed bio-sponge growth factor rich in, has good biocompatibility, is more advantageous to the life of sertoli cell
It is long.
It is a kind of that tissue reconstruction, wound repair and injection are used in preparation based on the bio-sponge of Acelluar small intestinal submucosa
Application in the material of filling.
A kind of preparation method of the bio-sponge based on Acelluar small intestinal submucosa, comprising the following steps:
(1) de- cell intestinal mucosa lower layer is prepared
Fresh pig small intestine contents are squeezed out, tunica muscularis intestini tenuis, mucous layer and subsidiary fat constituent are removed；And it is anti-with tap water
After multiple flushing, distilled water rinsing, the rear Iodophor cleaning and sterilizing with 2% and the de- iodine of the alcohol using 75% are recycled；
Further utilize hypotonic solution soaking and washing；Hypertonic salt solution is recycled to handle 12-24h；Discard hypertonic salt solution
Afterwards, the detergent oscillation treatment 30min-2h for being 1-5% with concentration by intestinal mucosa lower layer；Finally cleaned with physiological saline,
SIS can be obtained；
(2) pepsin digests
The pepsin solution of 1%-5% is added into SIS, 4 DEG C of stirrings digest 12-15h；
(3) it cleans
By the SIS after step (2) enzymatic hydrolysis, precipitating is filtered to take, wash with distilled water by SIS precipitating benefit；
(4) it is lyophilized
The SIS that step (3) obtains is deposited in freeze dryer, 12-15h is lyophilized；
(5) it crushes
SIS after freeze-drying is deposited in micronizer and crushes 3-5min, de- cell SIS powder can be obtained；
(6) it is crosslinked
Distilled water is added in the de- cell SIS powder that step (5) obtains, forming concentration is 0.01g/ml-0.5g/ml's
Solution is then added the fa-PEG powder with quality such as de- cell SIS powder, is sufficiently mixed；And solution is placed in -20 DEG C of refrigerators
Middle freezing 6h；
(7) it is lyophilized
12-15h is lyophilized in SIS after step (6) is crosslinked in freeze dryer.
The invention discloses a kind of preparation methods of bio-sponge based on Acelluar small intestinal submucosa, wherein going to eliminate
Amount, which removes, removes tunica muscularis intestini tenuis, mucous layer and subsidiary fat constituent, is only left small intestine matrix, to offer convenience in next step；
The remaining influence of mucous membrane tissue, adipose tissue is avoided to take off cellular processes simultaneously.In addition, SIS is by occurring free radical with fa-PEG
The polymerization reaction of initiation obtains the crosslinking sponge based on SIS and fa-PEG.
Further, in step (1), hypotonic solution is sterile distilled water.
Further, in step (1), hypertonic salt solution is 0.5-3mol/L sodium chloride solution.
Hypertonic salt solution disclosed by the invention is sodium chloride, and wherein sodium chloride is cheap and easy to get, and nontoxic, effect is reliable.
Further, in step (1), detergent is SDS solution.
It can be seen via above technical scheme that compared with prior art, the present invention provides one kind based on de- cell small intestine
The bio-sponge and preparation method of submucosa have following technological merit:
(1) bio-sponge disclosed by the invention based on Acelluar small intestinal submucosa has the sponge of loose 3D structure,
Inside there is hole, is interconnected, is grown into convenient for cell, exchange nutriment conducive between cell, between cell and tissue.Simultaneously originally
The bio-sponge better mechanical property of invention is conducive to clinical manipulation；
(2) bio-sponge disclosed by the invention have with the approximate immanent structure of dermis of skin, contain a large amount of collagen fibre
Dimension and growth factor abundant have good biocompatibility, are more advantageous to the growth of sertoli cell；SIS-PEG biology
The raw material SIS main component of sponge includes I type, type III, VI collagen type and elastin laminin, glycosaminoglycan, albumen
The ingredient of the important role in wound healing such as glycan, growth factor.These ingredients are promoting reepithelialization and increasing
It grows, plays a significant role in terms of improving blood vessel microenvironment.Therefore compared with simple filler such as hyaluronic acid, SIS-PEG
Bio-sponge is better able to the reconstruction for promoting to organize after wound repair and plombage；
(3) this is nontoxic, nonirritant using fa-PEG for bio-sponge disclosed by the invention, has good water solubility,
And having the crosslinking agent of good intermiscibility with many organic matter components to make de- cell SIS crosslinking, obtained bio-sponge has
Three-D space structure, and ensure that the biological safety of bio-sponge, which is used not only for wound repair, and
Excellent scaffold materials of tissue-engineered skin；
(4) bio-sponge disclosed by the invention is source using intestinal mucosa lower layer, and not only source is abundant, safely may be used
Lean on, it is cheap and easy to get, and its ingredient the Nomenclature Composition and Structure of Complexes and the mankind are seemingly, while ethnics Problem is not present, be convenient for scale,
Industrialization preparation.
Detailed description of the invention
Fig. 1 show de- cell SIS powder；
Fig. 2 show the SIS-PEG bio-sponge of different size；Wherein, A, B are the SIS-PEG of 24 orifice plates production
Bio-sponge；C, D is the SIS-PEG bio-sponge of six orifice plates production；
Fig. 3 is SIS-PEG bio-sponge scanning electron microscope (SEM) photograph；
Fig. 4 is that SIS-PEG bio-sponge is used for wound healing check experiment result；
Fig. 5 is that SIS-PEG transplants the immunofluorescence picture for promoting vascular proliferation situation after skin wound；
Fig. 6 is that SIS-PEG transplants the promotion vascular proliferation situation statistics after skin wound.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
The embodiment of the invention discloses a kind of bio-sponges based on Acelluar small intestinal submucosa, and bio-sponge is with SIS
It for raw material, is crosslinked, has been prepared with three-D space structure, rich in life by cross-linked modification method and using fa-PEG
The long factor and the Novel Scaffold Materials for Tissue Engingeering that ethnics Problem is not present.
The preparation method of bio-sponge, comprising the following steps:
(1) de- cell intestinal mucosa lower layer (SIS) is prepared
Fresh pig small intestine contents are squeezed out, the mesenterium for being attached to surface is removed.Using scissors, scalpel etc. is removed small
Myenteron, mucous layer and subsidiary fat constituent, as far as possible removal are clean, avoid remaining, after tap water repeated flushing, reuse distillation
Water rinses 3 times, each 15min.Using 2% Iodophor cleaning and sterilizing, taking off iodine using alcohol, after impregnated with sterile distilled water it is clear
It washes；1.8mol/L sodium chloride solution is recycled to handle 18h；It discards after sodium chloride solution and is with concentration by intestinal mucosa lower layer
2.5% SDS solution concussion processing processing 1h；It is finally cleaned with physiological saline, SIS can be obtained；
(2) pepsin digests
2.5% pepsin solution is added into SIS, at room temperature stirring enzymatic hydrolysis 13.5h；
(3) it cleans
By the SIS after step (2) enzymatic hydrolysis, precipitating is filtered to take, wash with distilled water by SIS precipitating benefit；
(4) it is lyophilized
The SIS that step (3) obtains is deposited in freeze dryer (Wuxi moral composes DP-D504) and freezes 12h；
(5) it crushes
SIS after freeze-drying is subjected to crushing 4min using IKA vertical reciprocating type pulverizer, de- cell SIS powder can be obtained
End, as shown in Figure 1；
(6) it is crosslinked
Distilled water is added in the de- cell SIS powder that step (5) obtains, forming concentration is 0.01g/ml-0.5g/ml's
Solution is then added the fa-PEG powder with quality such as de- cell SIS powder, is sufficiently mixed；And solution is placed in -20 DEG C of refrigerators
Middle freezing 6h；
(7) it is lyophilized
12-15h is lyophilized in SIS after step (6) is crosslinked in freeze dryer, as shown in Figure 2.
Embodiment 2
The preparation method of bio-sponge, comprising the following steps:
(1) de- cell intestinal mucosa lower layer (SIS) is prepared
Fresh pig small intestine contents are squeezed out, the mesenterium for being attached to surface is removed.Using scissors, scalpel etc. is removed small
Myenteron, mucous layer and subsidiary fat constituent, as far as possible removal are clean, avoid remaining, after tap water repeated flushing, reuse distillation
Water rinses 3 times, each 15min.Using 2% Iodophor cleaning and sterilizing, taking off iodine using alcohol, after impregnated with sterile distilled water it is clear
It washes；Recycle the processing of 0.5mol/L sodium chloride solution for 24 hours；It discards after sodium chloride solution and is with concentration by intestinal mucosa lower layer
5% SDS solution concussion processing processing 30min；It is finally cleaned with physiological saline, SIS can be obtained；
(2) pepsin digests
1% pepsin solution is added into SIS, at room temperature stirring enzymatic hydrolysis 15h；
(3) it cleans
By the SIS after step (2) enzymatic hydrolysis, precipitating is filtered to take, wash with distilled water by SIS precipitating benefit；
(4) it is lyophilized
The SIS that step (3) obtains is deposited in freeze dryer (Wuxi moral composes DP-D504) and freezes 15h；
(5) it crushes
SIS after freeze-drying is subjected to crushing 3min using IKA vertical reciprocating type pulverizer, de- cell SIS powder can be obtained
End；
(6) it is crosslinked
Distilled water is added in the de- cell SIS powder that step (5) obtains, forming concentration is 0.01g/ml-0.5g/ml's
Solution is then added the fa-PEG powder with quality such as de- cell SIS powder, is sufficiently mixed；And solution is placed in -20 DEG C of refrigerators
Middle freezing 6h；
(7) it is lyophilized
12-15h is lyophilized in SIS after step (6) is crosslinked in freeze dryer.
Embodiment 3
The preparation method of bio-sponge, comprising the following steps:
(1) de- cell intestinal mucosa lower layer (SIS) is prepared
Fresh pig small intestine contents are squeezed out, the mesenterium for being attached to surface is removed.Using scissors, scalpel etc. is removed small
Myenteron, mucous layer and subsidiary fat constituent, as far as possible removal are clean, avoid remaining, after tap water repeated flushing, reuse distillation
Water rinses 3 times, each 15min.Using 2% Iodophor cleaning and sterilizing, taking off iodine using alcohol, after impregnated with sterile distilled water it is clear
It washes；3mol/L sodium chloride solution is recycled to handle 12h；Discarding intestinal mucosa lower layer concentration after sodium chloride solution is 1%
SDS solution concussion processing processing 2h；It is finally cleaned with physiological saline, SIS can be obtained；
(2) pepsin digests
5% pepsin solution is added into SIS, at room temperature stirring enzymatic hydrolysis 12h；
(3) it cleans
By the SIS after step (2) enzymatic hydrolysis, precipitating is filtered to take, wash with distilled water by SIS precipitating benefit；
(4) it is lyophilized
The SIS that step (3) obtains is deposited in freeze dryer (Wuxi moral composes DP-D504) and freezes 14h；
(5) it crushes
SIS after freeze-drying is subjected to crushing 5min using IKA vertical reciprocating type pulverizer, de- cell SIS powder can be obtained
End；
(6) it is crosslinked
Distilled water is added in the de- cell SIS powder that step (5) obtains, forming concentration is 0.01g/ml-0.5g/ml's
Solution is then added the fa-PEG powder with quality such as de- cell SIS powder, is sufficiently mixed；And solution is placed in -20 DEG C of refrigerators
Middle freezing 6h；
(7) it is lyophilized
12-15h is lyophilized in SIS after step (6) is crosslinked in freeze dryer.
Embodiment 4
Taking diameter is that 1cm is placed in Electronic Speculum fixer with a thickness of 1 bio-sponge of 1mm embodiment and fixes 2h.
By 30%, 50%, 70%, 80%, 90% ethanol concentration gradient be dehydrated 1 time, 5min/ times, 100% ethanol dehydration
2 times, 5min/ times.
Tert-butyl alcohol warm water water-bath to ice crystal dissolves: preparing 50%, 70%, 90%, 95% concentration gradient with dehydrated alcohol
The tert-butyl alcohol is replaced ethyl alcohol each 1 time, 100% tert-butyl alcohol 3 times, 10min/ times from low to high by concentration gradient.
Sample is laid in culture dish, is put on copper mesh after natural drying, metal spraying is fixed on sample carrier, scanning electricity
Sem observation is simultaneously taken pictures.
The scanning electron microscope of SIS-PEG bio-sponge is as shown in Figure 3.
Embodiment 5
The full thickness dermal Nude mice model that diameter is 1cm, simulated skin defect are manufactured with punch.
Control group (Control group) is set after manufacturing full thickness dermal, this group of surface of a wound covers hyaluronic acid dressing, laggard
Row wrapping.Be arranged experimental group (SIS-PEG bio-sponge group), this group of surface of a wound covers SIS-PEG bio-sponge, after wrapped up.
Test result is as Figure 4-Figure 6.
3d after material transplanting
Control group: surface of a wound exposure, exudation are obvious；
SIS-PEG bio-sponge group: surface of a wound area reduces, no exudation；
7d after material transplanting
Control group: the surface of a wound still has exposure, a little to ooze out, and surface of a wound area is reduced；
SIS-PEG bio-sponge group: surface of a wound incrustation, no exudation, surface of a wound area are obvious compared with initial reduction；
14d after material transplanting
Control group: surface of a wound area reduces, but healing completely not yet；
SIS-PEG bio-sponge group: the surface of a wound heals substantially.
There are the growth factors such as VEGF and bFGF in SIS-PEG, angiogenesis can be promoted, SIS-PEG can promote the surface of a wound
Skin heart is newborn, and the growth factors such as this and the VEGF and bFGF that wherein contain are closely related.
The present invention provides a kind of bio-sponge and preparation method based on Acelluar small intestinal submucosa, it is glutinous with chitterlings
Film lower layer is source, and after de- cell processing, immune rejection is minimum.It is carried out simultaneously using nontoxic crosslinking agent
Crosslinking Treatment, obtained bio-sponge have three-D space structure, and ensure that the biological safety of the product of acquisition.The product
It is used not only for wound repair and excellent scaffold materials of tissue-engineered skin, not only source is abundant, securely and reliably, valence
It is honest and clean to be easy to get, and its ingredient the Nomenclature Composition and Structure of Complexes and the mankind are seemingly, while ethnics Problem is not present, and are convenient for scale, industry
Change preparation.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (6)
1. a kind of bio-sponge based on Acelluar small intestinal submucosa, which is characterized in that the bio-sponge is with small intestinal mucosa
Lower layer SIS is raw material, is crosslinked by cross-linked modification method and using fa-PEG, and having prepared has three-dimensional space knot
Structure, the Novel Scaffold Materials for Tissue Engingeering rich in growth factor.
2. it is according to claim 1 it is a kind of based on the bio-sponge of Acelluar small intestinal submucosa preparation be used for tissue weight
It builds, the application in the material of wound repair and injection fillers.
3. a kind of preparation method of bio-sponge based on Acelluar small intestinal submucosa according to claim 1, special
Sign is, comprising the following steps:
(1) de- cell intestinal mucosa lower layer is prepared
Fresh pig small intestine contents are squeezed out, tunica muscularis intestini tenuis, mucous layer and subsidiary fat constituent are removed；And it is rushed repeatedly with tap water
After washing, distilled water rinsing is recycled, the rear Iodophor cleaning and sterilizing with 2% and the alcohol using 75% take off iodine；
Further utilize hypotonic solution soaking and washing；Hypertonic salt solution is recycled to handle 12-24h；It, will after discarding hypertonic salt solution
The detergent oscillation treatment 30min-2h that intestinal mucosa lower layer is 1-5% with concentration；It is finally cleaned, can be obtained with physiological saline
To SIS；
(2) pepsin digests
The pepsin solution of 1%-5% is added into the SIS, 4 DEG C of stirrings digest 12-15h；
(3) it cleans
By the SIS after step (2) enzymatic hydrolysis, precipitating is filtered to take, wash with distilled water by SIS precipitating benefit；
(4) it is lyophilized
The SIS that step (3) obtains is deposited in freeze dryer, 12-15h is lyophilized；
(5) it crushes
The SIS after freeze-drying is deposited in micronizer and crushes 3-5min, de- cell SIS powder can be obtained；
(6) it is crosslinked
Distilled water is added in the de- cell SIS powder that step (5) obtains, forming concentration is 0.01g/ml-0.5g/ml's
Solution is then added the fa-PEG powder with quality such as the de- cell SIS powder, is sufficiently mixed；And solution is placed in -20 DEG C
6h is freezed in refrigerator；
(7) it is lyophilized
12-15h is lyophilized in SIS after step (6) is crosslinked in freeze dryer.
4. a kind of preparation method of bio-sponge based on Acelluar small intestinal submucosa according to claim 3, special
Sign is, in step (1), the hypotonic solution is sterile distilled water.
5. a kind of preparation method of bio-sponge based on Acelluar small intestinal submucosa according to claim 3, special
Sign is, in step (1), the hypertonic salt solution is 0.5-3mol/L sodium chloride solution.
6. a kind of preparation method of bio-sponge based on Acelluar small intestinal submucosa according to claim 3, special
Sign is, in step (1), the detergent is SDS solution.
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