CN103893816B - Method for preparing composite amniotic extracellular matrix dressing of bacterial cellulose containing plant ingredients - Google Patents
Method for preparing composite amniotic extracellular matrix dressing of bacterial cellulose containing plant ingredients Download PDFInfo
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- CN103893816B CN103893816B CN201410061448.4A CN201410061448A CN103893816B CN 103893816 B CN103893816 B CN 103893816B CN 201410061448 A CN201410061448 A CN 201410061448A CN 103893816 B CN103893816 B CN 103893816B
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Abstract
The invention discloses a method for preparing composite amniotic extracellular matrix dressing of bacterial cellulose containing plant ingredients, and relates to a technology for preparing a medical material. The method comprises the following steps: selecting a bacterial strain capable of secreting bacterial cellulose to activate and prepare seed mash; then mixing the seed mash of which the bacterial strain concentration is 30-50wt% with culture medium containing natural rosemary freeze-dried powder and then carrying out static culture in a culture container to obtain a bacterial cellulose membrane containing natural plant ingredients; taking amniotic extracellular matrix to soak into the culture medium; paving on the surface of the bacterial cellulose membrane after completely wetting; continuing to carry out static culture for 1-6 hours; carrying out purification and sterilization to obtain the composite amniotic extracellular matrix dressing of the bacterial cellulose containing the natural plant ingredients. The method is simple and feasible in preparation process, low in cost, and wide in material source, and the prepared dressing has the properties, such as sterilization, moisturizing, absorbing wound exudates, accelerating wound healing and the like, and is applicable to various chronic skin wounds.
Description
Technical field
The present invention relates to the preparing technical field of medical material, refer to a kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition especially.
Background technology
Amnion is from cytotrophoblast derivation, is the internal layer of two-layer fetal membrane.Normal amnion is thin and bright, and not containing structures such as blood vessel, nerve and lymphatic vessels, thickness is 20 ~ 500 μm.Amnion has 5 Rotating fields and can be divided into: epithelial lining, basement membrane layer, tight zone, fibroblast layer, spongy layer.Remove in the characteristics of amniotic extracellular matrix material after cell containing Multiple components such as collagen, glycoprotein, protein-polysaccharide, integrin and laminated bodies, express the associated protein of multiple somatomedin and mRNA thereof, abundant nutritive ingredient can be provided for the propagation of cell, differentiation, be conducive to the growth and breeding of cell; Basement membrane layer is thicker, is conducive to attaching and the migration of epidermic cell, promotes epithelization, prevents its apoptosis; The collegen filament of tight zone and spongy layer form loose 3 D stereo configuration; Histocompatibility is good, does not express human leucocyte antigen, without induction rejection; Easily obtain, and plasticity-is good, is easy to processing treatment.Large quantity research confirms, amnion has good effect as biological dressing for skin injury such as treatment large-area burns, scald etc.But because amnion is thin and soft, insulation poor water retention property, the surface of a wound ooze out and be mostly that Yi Rong loses, biomechanical property is poor, and operation acquires a certain degree of difficulty, and makes it be restricted in biological dressing application simultaneously.
Bacteria cellulose is a kind of natural biopolymer, has hyperfine reticulated structure, is combined into the thick fibrous bundle of 40 ~ 60 nanometers by the fento of diameter 3 ~ 4 nanometer, and is intertwined to form flourishing hyperfine network structure.And its chemical purity is very high, due to physical strength, good fluid absorbent and performance of keeping humidity high when bacteria cellulose has good biocompatibility, a hygrometric state, make it in medical biotechnology Materials, show great advantage, can be used as desirable degradable solid support material and be applied to the numerous areas such as wound dressings, drug carrier material.The simple bacteria cellulose of external employing has been reported as moist dressing, and industrialization is used for clinical.
Rosmarinus officinalis (Rosmarinus officinatis L.) has another name called You Ancao, and another name tansy is Labiatae Rosmarinus plant, perennial evergreen undershrub.Most important in rosemary extract is rosemary antioxidant and rosemary ethereal oil.Rosemary natural anti-oxidant Stability Analysis of Structures, be not easy to decompose, resistance to 190 DEG C of high temperature, its antioxidant effect is good, and does not have toxicity.Containing the multiple compounds such as phenol, acid, ketone, terpene in rosemary plant, at food, medicine, to be industrially widely used.Rosemary extract has good antimicrobial property, all has good inhibition to streptococcus aureus, intestinal bacteria, Bacillus subtilus, Hansenula anomala bacterium, mould, aspergillus niger, flavus and Penicillium citrinum; Rosemary extract is used for the treatment of the reconstruction that chronic wounds can reduce inflammation, strengthen contraction of wounds, contributes to epithelium and granulation tissue, and can improve the formation of wound blood vessels and collagen protein.In sum, bacteria cellulose containing natural rosemary extract composition is combined with characteristics of amniotic extracellular matrix material the matrix material obtaining having three-dimensional net structure, the moist dressing having histocompatibility, antibacterial, moisturizing, absorbing wound exudate, accelerating wound healing and satisfactory mechanical property concurrently can be prepared.
This patent adopts fermentable cultural method In-situ reaction characteristics of amniotic extracellular matrix material.Bacteria cellulose material containing natural rosemary extract composition and characteristics of amniotic extracellular matrix material, to be interacted by the collegen filament of bacteria cellulose nanofiber and characteristics of amniotic extracellular matrix material tight zone and spongy layer and nanofiber runs through mutually, formed and link matrix material closely.Preparation is simple in the present invention, cost is low, material source is extensive, and the dressing of preparation has the performances such as sterilization, moisturizing, absorbing wound exudate and accelerating wound healing, is applicable to all kinds of chronic cutaneous wound.
Summary of the invention
The object of this invention is to provide a kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.Relate to a kind of technology of preparing of medical material.Preparation is simple in the present invention, cost is low, material source is extensive, the dressing of preparation has the performances such as sterilization, moisturizing, absorbing wound exudate and accelerating wound healing, is applicable to all kinds of chronic cutaneous wound such as diabetic foot ulcer, venous leg ulcers, pressure sore, skin donor site wound.
The invention discloses a kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition, comprising: choose and the activation of secreting bacteria cellulosic bacterial strain can be prepared into seed mash, is then that the seed mash of 30 ~ 50wt% mixes the bacteria cellulose film being placed on quiescent culture in culture vessel and obtaining containing natural plant composition with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration; Get characteristics of amniotic extracellular matrix film to soak in the medium, after its complete wetting, be laid in bacteria cellulose film upper surface; Continue quiescent culture 1 ~ 6h, purified process, after sterilizing, obtain a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.
As preferred technical scheme:
Wherein, a preparation method for bacteria cellulose compound characteristics of amniotic extracellular matrix dressing as above containing natural plant composition, the described cellulosic bacterial strain of energy secreting bacteria refers to one or more in acetobacter xylinum, rhizobium, Sarcina, Rhodopseudomonas, achromobacter, Alcaligenes, aerobacter or Azotobacter.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, the described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 50 ~ 70wt% containing natural Rosmarinus officinalis lyophilized powder 10 ~ 50g, Sodium phosphate dibasic 4 ~ 16g, potassium primary phosphate 4 ~ 16g, yeast extract paste 4 ~ 20g, peptone 4 ~ 20g in the aqueous ethanolic solution of every 1L.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, described quiescent culture refers to that seed mash mixes with the fermention medium containing natural Rosmarinus officinalis lyophilized powder and is placed on quiescent culture 5 ~ 7d in culture vessel, wherein seed mash mix with the fermention medium containing collagen solution after liquid level in culture vessel be 0.5 ~ 3cm.Under normal circumstances, bacteria cellulose film floats on the top of nutrient solution, and bacteria cellulose can consume a part of substratum in fermentation culture process, and a part of substratum is lost by evaporation simultaneously.Method described in this patent is by controlling nutrient solution consumption at zone of reasonableness, and substratum consumption crosses the biosynthesizing that major general affects bacteria cellulose, and affecting characteristics of amniotic extracellular matrix material at most is effectively laid in bacteria cellulose film upper surface excessively.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, the described bacteria cellulose film containing natural plant composition refers to the hydrogel bacteria cellulose film that quiescent culture obtains, thickness is 1 ~ 3cm, the three-dimensional network nanofiber of wherein bacteria cellulose is evenly attached with the chemical substance containing natural rosemary extract composition.Bacterium a large amount of amplification also biosynthesizing Mierocrystalline cellulose fento first in the medium in fermenting process, chemical substance containing natural rosemary extract composition is attached on Mierocrystalline cellulose fento, along with longer fermentation times is formed cellulose nano-fibrous, and evenly adhere to rosemary extract composition on nanofiber; Final formation contains the gluey bacteria cellulose film of water-setting of natural rosemary extract composition.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, described characteristics of amniotic extracellular matrix material refers to safe source, treated removal cell, fat and soluble antigen and the de-cell biological amnion of dry sterilization.Fresh amnion takes from the placenta of healthy Cesarean esction puerpera, and hepatitis B, the third liver, syphilis and adaptive immune deficit syndrome are got rid of in antenatal Serological testing, and process of drawing materials all completes under aseptic technique; Treated removal cell, fat, soluble antigen, general adopt freeze-drying to carry out drying and through irradiation sterilization.
A preparation method for bacteria cellulose compound characteristics of amniotic extracellular matrix dressing as above containing natural plant composition, the described bacteria cellulose film upper surface be laid on containing natural plant composition refers to that the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.Characteristics of amniotic extracellular matrix material has 5 Rotating fields and can be divided into: epithelial lining, basement membrane layer, tight zone, fibroblast layer, spongy layer.Wherein the collegen filament of tight zone and spongy layer form loose 3 D stereo configuration, are conducive to the cellulose nano-fibrous network structure mutually run through with its formation in bacteria cellulose film.Basement membrane layer contains IV type and V Collagen Type VI, ln, the various chelating albumen of fibronectin bletilla, can promote the differentiation of seed cell, propagation and migration.Therefore, when preparing dressing, the spongy layer of characteristics of amniotic extracellular matrix material is contacted with bacteria cellulose film upper surface, be conducive to the cellulose nano-fibrous loose collegen filament inside entering spongy layer of bacteria cellulose film, maintain the biological activity of basement membrane layer simultaneously.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, described purification process refers to that matrix material soaks 12 ~ 24h at the aqueous sodium hydroxide solution 30 ~ 60 DEG C of 1 ~ 3wt%, and by washed with de-ionized water to neutral.The sodium hydroxide of low concentration is selected to process matrix material at low temperatures in this patent, sodium hydroxide solution soaks the residual media thoroughly can removed tropina and stick on bacteria cellulose film, guarantee that the intracellular toxin of matrix material meets the requirement of medical implanting material, simultaneously low-concentration sodium hydroxide can prevent from affecting in treating processes all polyproteins and somatomedin that contain in characteristics of amniotic extracellular matrix material.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, the described bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition refers to the matrix material of bacteria cellulose film containing natural plant composition and characteristics of amniotic extracellular matrix, wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formation links matrix material closely.
A kind of preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as above, in use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in described a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.
Another object of the present invention is to provide a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition, it is characterized in that: comprise bacteria cellulose, Rosmarinus officinalis and characteristics of amniotic extracellular matrix.
Compared with prior art, the invention has the beneficial effects as follows:
This patent adopts fermentable cultural method In-situ reaction characteristics of amniotic extracellular matrix material.Bacteria cellulose material containing natural rosemary extract composition and characteristics of amniotic extracellular matrix material, to be interacted by the collegen filament of bacteria cellulose nanofiber and characteristics of amniotic extracellular matrix material tight zone and spongy layer and nanofiber runs through mutually, formed and link matrix material closely.Ensureing, under the histocompatibility of matrix material, bioactive prerequisite, effectively to enhance the mechanical property of material, anti-microbial property; The reconstruction that contained natural rosemary extract composition can reduce wound inflammation, strengthen contraction of wounds, contributes to epithelium and granulation tissue, and the formation of wound blood vessels and collagen protein can be improved.Preparation is simple in the present invention, cost is low, material source is extensive, the dressing of preparation has the performances such as sterilization, moisturizing, absorbing wound exudate and accelerating wound healing, is applicable to all kinds of chronic cutaneous wound such as diabetic foot ulcer, venous leg ulcers, pressure sore, skin donor site wound.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1:
Choosing and the cellulosic acetobacter xylinum activation of secreting bacteria can be prepared into seed mash, is then that the seed mash of 50wt% mixes with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration.The described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 70wt% containing natural Rosmarinus officinalis lyophilized powder 30g, Sodium phosphate dibasic 16g, potassium primary phosphate 16g, yeast extract paste 20g, peptone 20g in the aqueous ethanolic solution of every 1L.Mixed solution is placed in culture vessel, and the liquid level of mixed solution in culture vessel is 3cm, and quiescent culture 5d obtains the bacteria cellulose film of water-setting glue containing natural rosemary extract composition that film thickness is 3cm.
By safe source, treated removal cell, fat and soluble antigen and the characteristics of amniotic extracellular matrix material of dry sterilization soak in the medium, are laid in bacteria cellulose film upper surface after its complete wetting.Wherein, the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.Continue quiescent culture 1h, the aqueous sodium hydroxide solution 60 DEG C that the matrix material obtained is placed in 1wt% soaks 12h, and by washed with de-ionized water to neutral, obtains a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.Wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formed and link matrix material closely.In use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in this dressing.
Embodiment 2:
Choose can the cellulosic rhizobium of secreting bacteria and Rhodopseudomonas activation be prepared into seed mash, be then that the seed mash of 40wt% mixes with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration.The described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 70wt% containing natural Rosmarinus officinalis lyophilized powder 20g, Sodium phosphate dibasic 14g, potassium primary phosphate 14g, yeast extract paste 18g, peptone 18g in the aqueous ethanolic solution of every 1L.Mixed solution is placed in culture vessel, and the liquid level of mixed solution in culture vessel is 2cm, and quiescent culture 5d obtains the bacteria cellulose film of water-setting glue containing natural rosemary extract composition that film thickness is 2.5cm.
By safe source, treated removal cell, fat and soluble antigen and the characteristics of amniotic extracellular matrix material of dry sterilization soak in the medium, are laid in bacteria cellulose film upper surface after its complete wetting.Wherein, the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.Continue quiescent culture 2h, the aqueous sodium hydroxide solution 50 DEG C that the matrix material obtained is placed in 2wt% soaks 12h, and by washed with de-ionized water to neutral, obtains a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.Wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formed and link matrix material closely.In use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in this dressing.
Embodiment 3:
Choose can the cellulosic Sarcina of secreting bacteria and Alcaligenes activation be prepared into seed mash, be then that the seed mash of 30wt% mixes with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration.The described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 60wt% containing natural Rosmarinus officinalis lyophilized powder 10g, Sodium phosphate dibasic 12g, potassium primary phosphate 12g, yeast extract paste 16g, peptone 16g in the aqueous ethanolic solution of every 1L.Mixed solution is placed in culture vessel, and the liquid level of mixed solution in culture vessel is 1cm, and quiescent culture 6d obtains the bacteria cellulose film of water-setting glue containing natural rosemary extract composition that film thickness is 1cm.
By safe source, treated removal cell, fat and soluble antigen and the characteristics of amniotic extracellular matrix material of dry sterilization soak in the medium, are laid in bacteria cellulose film upper surface after its complete wetting.Wherein, the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.Continue quiescent culture 3h, the aqueous sodium hydroxide solution 30 DEG C that the matrix material obtained is placed in 3wt% soaks 16h, and by washed with de-ionized water to neutral, obtains a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.Wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formed and link matrix material closely.In use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in this dressing.
Embodiment 4:
Choosing and the activation of the cellulosic acetobacter xylinum of secreting bacteria, aerobacter and Azotobacter can be prepared into seed mash, is then that the seed mash of 50wt% mixes with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration.The described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 60wt% containing natural Rosmarinus officinalis lyophilized powder 50g, Sodium phosphate dibasic 10g, potassium primary phosphate 10g, yeast extract paste 10g, peptone 10g in the aqueous ethanolic solution of every 1L.Mixed solution is placed in culture vessel, and the liquid level of mixed solution in culture vessel is 0.5cm, and quiescent culture 6d obtains the bacteria cellulose film of water-setting glue containing natural rosemary extract composition that film thickness is 2cm.
By safe source, treated removal cell, fat and soluble antigen and the characteristics of amniotic extracellular matrix material of dry sterilization soak in the medium, are laid in bacteria cellulose film upper surface after its complete wetting.Wherein, the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.Continue quiescent culture 3h, the aqueous sodium hydroxide solution 30 DEG C that the matrix material obtained is placed in 1wt% soaks 24h, and by washed with de-ionized water to neutral, obtains a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.Wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formed and link matrix material closely.In use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in this dressing.
Embodiment 5:
Choose can the cellulosic acetobacter xylinum of secreting bacteria and achromobacter activation be prepared into seed mash, be then that the seed mash of 40wt% mixes with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration.The described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 50wt% containing natural Rosmarinus officinalis lyophilized powder 40g, Sodium phosphate dibasic 4g, potassium primary phosphate 4g, yeast extract paste 4g, peptone 4g in the aqueous ethanolic solution of every 1L.Mixed solution is placed in culture vessel, and the liquid level of mixed solution in culture vessel is 2cm, and quiescent culture 7d obtains the bacteria cellulose film of water-setting glue containing natural rosemary extract composition that film thickness is 2cm.
By safe source, treated removal cell, fat and soluble antigen and the characteristics of amniotic extracellular matrix material of dry sterilization soak in the medium, are laid in bacteria cellulose film upper surface after its complete wetting.Wherein, the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.Continue quiescent culture 6h, the aqueous sodium hydroxide solution 40 DEG C that the matrix material obtained is placed in 3wt% soaks 18h, and by washed with de-ionized water to neutral, obtains a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.Wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formed and link matrix material closely.In use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in this dressing.
Embodiment 6:
The subcutaneous implantation experiment of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition
Experimental technique: get healthy SD rat 30, male or female, be divided into experimental group and control group, sterilization, adopts ether inhalation anesthesia, and normal sterile operates, and cut skin of back, 51 × 1cm are implanted at experimental group back
2dressing (the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition respectively prepared by Example 1-5), namely control group is sewed up after cutting.Observe the postoperative generalized case of animal and wound healing situation.Within postoperative 1,2,4 week, put to death each treated animal, take out implantation dressing glutaraldehyde and fix, conventional embedded section, HE dyes, om observation implant peripheral lymphocyte, macrophages infiltration situation.
Experiment shows: can normally take food after operation animal on the same day is clear-headed, wound is without Inflammatory responses such as redness, sepages, and all surface of a wound all can well heal, and operative incision infects without 1 example, animal is without the toxic reaction such as death, convulsions, apoplexy, implant surrounding soft tissue has no the performances such as blackout, necrosis, suppuration, hydrops, and postoperative 1 week of histological observation, has large amount lymphocyte around implant, when 2 weeks, only a small amount of lymphocyte, when 4 weeks, exists without lymphocyte substantially.Confirm dressing abiology toxicity of the present invention, have good histocompatibility.
Embodiment 7
Bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition is to the Experiment on therapy of diabetic foot ulcer rat
Use high lipid food to coordinate the rats with type 2 diabetes of streptozotocin induction, use scald apparatus to make dark II degree of scald induced skin ulcer and make diabetic foot ulcer rat model.Rat is SD male rat, SPF level, body weight (180 ± 10) g, totally 30, often organize 5, totally 6 groups, wherein one group is contrast, another 5 groups of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition used embodiment 1-5 respectively and prepare.The preparation method of high lipid food is: first, by barley 15%, and wheat 31%, corn 15%, wheat bran 15%, after fish meal 6% is uniformly mixed in proportion, after adding dregs of beans, oil, salt mixing, basal feed is made in extrusion forming, then, by basal feed 73%, lard 20%, milk powder 2%, cholesterol 1%, sucrose 4% is uniformly mixed rear extrusion forming in proportion and makes high lipid food, and finished product is that cylinder is block.After high lipid food feeds 8 weeks, the citric acid-sodium citrate damping fluid that 1.5% streptozotocin is dissolved in pH value 4.5 is injected by the dosage left lower quadrant of 35mg/kg.Use SQL-5Q scald apparatus, temperature 90 DEG C, duration of contact 10S, pressure 9.8N, area 4cm
2, scald in back part skin after anesthesia.After modeling success, the surface of a wound is cleaned with Iodophor, with the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing external application surface of a wound containing natural plant composition of the present invention, then wrap up with sterile gauze, within 1 day, change dressings 1 time, add up to indicator-specific statistics the average healing with ulcer area and blood leukocytes, the results are shown in Table 1.
The average healing of table 1 pair diabetic foot ulcer rat compares (n=5)
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | |
| The average healing/sky | 18.5±1.4 | 17.8±0.8 | 18.4±1.2 | 19.2±1.0 | 17.0±1.2 |
Embodiment 8:
The hospital clinical trial of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition
Bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition prepared by the embodiment of the present invention 1 is through hospital clinical trial treatment totally 102 examples.Wherein relate to diabetic foot ulcer 37 example, venous leg ulcers 21 example, sore or carbuncle furuncle 44 example, its result for the treatment of refers to table 2.
In table 2, the curative effect determinate standard of various diabetic foot ulcer, venous leg ulcers, sore or carbuncle furuncle is:
1. fully recover: constitutional symptom disappears, local swelling is dissipated, or fester absorbs dissipation, or sore face healing after bursting, and blood leukocytes sum normally.
2. effective: constitutional symptom disappears, and swelling, abscess or the open sore part reduce more than 70%, blood leukocytes sum is normal.
3. effective: constitutional symptom alleviates, swelling, abscess or the open sore part reduce more than 30%, and less than 70%, blood leukocytes sum is close to normal.
4. invalid: not reach effective standard.
The clinical experiment of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing of table 2 containing natural plant composition
| Case type | Case load | Recovery from illness | Effective | Effectively | Invalid | Curative ratio |
| Diabetic foot ulcer | 37 | 36 | 1 | 0 | 0 | 97.3% |
| Venous leg ulcers | 21 | 19 | 1 | 1 | 0 | 90.5% |
| Sore or carbuncle furuncle | 44 | 42 | 1 | 1 | 0 | 95.5% |
Wherein concrete case has:
Week certain, man, 68 years old, diabetic history 20 years, fasting plasma glucose 14 mnol/l, early 2 hours blood glucose 17.9 mmol/L, glycolated hemoglobin 11.2 mg/dl after the meal, diabetic foot ulcer three grades, red swelling of the skin, fester, deep ulcer, forms ulcer chamber, ulcer area 145.5 mm
2, blood leukocytes sum 1.8 × 10
9/ l, neutrophil leucocyte number 77%.The surface of a wound is cleaned with Iodophor, remove necrotic tissue and purulent secretion, clog in chamber and the external application surface of a wound with the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition of the present invention, then wrap up with sterile gauze, lifting affected limb, does not make foot pressurized, within 1 day, changes dressings 1 time, 12 days courses for the treatment of of continuous treatment one, ulcer area reduces to 64.5 mm
2, blood leukocytes sum is down to 0.7 × 10
9/ l, neutrophil leucocyte number 55%.Continued treatment to 24 day, muscle groups tissue layer, lipid layer, skin corium, epidermal area recover original weave construction, cure completely.
Wang, female, 47 years old, fasting plasma glucose 8.1 mmol/L, early 2 hours blood glucose 10.4 mmol/L, glycolated hemoglobin 8.7 mg/dl after the meal, left sufficient diabetic foot ulcer one-level, ulcer area 123.3 mm
2, wound festers, blood leukocytes sum 1.6 × 10
9/ l, neutrophil leucocyte number 70%.The surface of a wound is cleaned with Iodophor, remove necrotic tissue and purulent secretion, with the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing external application surface of a wound containing natural plant composition of the present invention, then wrap up with sterile gauze, lifting affected limb, does not make foot pressurized, within 1 day, changes dressings 1 time, continuous treatment 7 days local symptoms disappear, and blood leukocytes sum is down to 0.5 × 10
9/ l, neutrophil leucocyte number 48%, ulcer surface heals completely.
Jiao, female, 67 years old, sore, intractable ulcer patient, left lower extremity ulcer 5 wheat harvesting period, local presents swollen, hard tubercle, and risen root bundle, sore look red, there is purulent core on sore top, and pain is obvious, and blood leukocytes sum and neutrophil leucocyte number increase, blood leukocytes sum 1.4 × 10
9/ l, neutrophil leucocyte number 73%.The surface of a wound is cleaned with Iodophor, remove necrotic tissue and purulent secretion, adopt of the present invention containing on the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing external application ulcer surface of natural plant composition, then wrap up with sterile gauze, within 1 day, change dressings 1 time, medication is after 7 days, and redness obviously alleviates, ulcer surface reduces, and blood leukocytes sum is down to 0.7 × 10
9/ l, neutrophil leucocyte number 60%.Continue medication to the 14th day, left lower extremity is without red, swollen, and without pain, ulcer wound surface heals, and blood leukocytes sum is down to 0.6 × 10
9/ l, neutrophil leucocyte number 53%, does not stay obvious scar, treatment recovery from illness.
Claims (9)
1. the preparation method of the bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition, it is characterized in that: choose and the activation of secreting bacteria cellulosic bacterial strain can be prepared into seed mash, is then that the seed mash of 30 ~ 50wt% mixes the bacteria cellulose film being placed on quiescent culture in culture vessel and obtaining containing natural plant composition with the fermention medium containing natural Rosmarinus officinalis lyophilized powder by bacterial strain concentration; Get characteristics of amniotic extracellular matrix film to soak in the medium, after its complete wetting, be laid in bacteria cellulose film upper surface; Continue quiescent culture 1 ~ 6h, purified process, after sterilizing, obtain a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.
2. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, is characterized in that: the described cellulosic bacterial strain of energy secreting bacteria refers to one or more in acetobacter xylinum, rhizobium, Sarcina, Rhodopseudomonas, achromobacter, Alcaligenes, aerobacter or Azotobacter.
3. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, it is characterized in that: the described fermention medium containing natural Rosmarinus officinalis lyophilized powder refers to that wherein aqueous ethanolic solution concentration is 50 ~ 70wt% containing natural Rosmarinus officinalis lyophilized powder 10 ~ 50g, Sodium phosphate dibasic 4 ~ 16g, potassium primary phosphate 4 ~ 16g, yeast extract paste 4 ~ 20g, peptone 4 ~ 20g in the aqueous ethanolic solution of every 1L.
4. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, it is characterized in that: the bacteria cellulose film containing natural plant composition refers to the hydrogel bacteria cellulose film that quiescent culture obtains, thickness is 1 ~ 3cm, the three-dimensional network nanofiber of wherein bacteria cellulose is evenly attached with the chemical substance containing natural rosemary extract composition.
5. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, it is characterized in that: described characteristics of amniotic extracellular matrix material refers to safe source, treated removal cell, fat and soluble antigen and the de-cell biological amnion of dry sterilization.
6. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, is characterized in that: the described bacteria cellulose film upper surface be laid in containing natural plant composition refers to that the spongy layer of characteristics of amniotic extracellular matrix material contacts with the bacteria cellulose film upper surface containing natural plant composition.
7. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, it is characterized in that: described purification process refers to that matrix material soaks 12 ~ 24h at the aqueous sodium hydroxide solution 30 ~ 60 DEG C of 1 ~ 3wt%, and by washed with de-ionized water to neutral.
8. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, it is characterized in that: the described bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition refers to the matrix material of bacteria cellulose film containing natural plant composition and characteristics of amniotic extracellular matrix, wherein characteristics of amniotic extracellular matrix is positioned at the upper strata of dressing, bacteria cellulose film containing natural plant composition is positioned at the lower floor of dressing, and the collegen filament in the tight zone of characteristics of amniotic extracellular matrix material and spongy layer run through mutually with containing the nanofiber in the bacteria cellulose film of natural plant composition, formation links matrix material closely.
9. the preparation method of a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition as claimed in claim 1, it is characterized in that: in use, the characteristics of amniotic extracellular matrix being positioned at dressing upper strata contacts with skin wound in described a kind of bacteria cellulose compound characteristics of amniotic extracellular matrix dressing containing natural plant composition.
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| CN111481735A (en) * | 2019-01-25 | 2020-08-04 | 华中科技大学同济医学院附属协和医院 | Medical antibacterial wound-protecting hydrogel dressing and preparation method thereof |
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| CN1618954A (en) * | 2003-05-01 | 2005-05-25 | 四川大学华西医院 | Bioderived amnion, composite bioderived amnion and its preparation method |
| CN102250378A (en) * | 2011-03-30 | 2011-11-23 | 东华大学 | Bacterial cellulose/polymer composite film and preparation method thereof |
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| CN1618954A (en) * | 2003-05-01 | 2005-05-25 | 四川大学华西医院 | Bioderived amnion, composite bioderived amnion and its preparation method |
| CN102250378A (en) * | 2011-03-30 | 2011-11-23 | 东华大学 | Bacterial cellulose/polymer composite film and preparation method thereof |
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