CN102018991A - Nano silver-porcine acellular dermal matrix (PADM) biological dressing and preparation method thereof - Google Patents
Nano silver-porcine acellular dermal matrix (PADM) biological dressing and preparation method thereof Download PDFInfo
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- CN102018991A CN102018991A CN2010105699796A CN201010569979A CN102018991A CN 102018991 A CN102018991 A CN 102018991A CN 2010105699796 A CN2010105699796 A CN 2010105699796A CN 201010569979 A CN201010569979 A CN 201010569979A CN 102018991 A CN102018991 A CN 102018991A
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Abstract
The invention discloses a nano silver-porcine acellular dermal matrix (PADM) biological dressing and a preparation method thereof. The biological dressing comprises PADM and nano-silver particles assembled on the PADM, wherein, the content of the nano-silver particles is 435.66-447.66 mu g/g, and the particle size of the nano-silver particles is 55-75nm. The preparation method of the biological dressing comprises the following steps: (1) reducing silver nitrate with trisodium citrate to prepare nano silver solution; (2) carrying out gradient dehydration with alcohol and ultrasonic treatment on the PADM; and (3) placing the treated PADM obtained from the step (2) in the nano silver solution for self-assembly, thus finally obtaining the nano silver-PADM biological dressing. The biological dressing obtained in the invention has the advantages of strong antibacterial property, high biological safety and good tissue compatibility; and simultaneously, the process is simple, and the use is convenient.
Description
Technical field
The invention belongs to medical instruments field, be specifically related to a kind of nanometer silver-pig acellular dermal matrix biological dressing and preparation method thereof.
Background technology
Skin is the barrier of keeping homeostasis and stoping the microorganism invasion.Rebuilding or recovering skin barrier is the final goal of burn treating, and before reaching this target, the wound-surface cover of a function admirable can temporarily play the effect of skin barrier function, and an environment that helps wound healing is provided.A kind of ideal wound-surface cover should have: 1. good adhesion, permeability, can control water evaporates again, and have the water evaporates rate of similar normal skin; 2. reduce nutrient substance and lose, stop bacterial invasion and restricting bacterial on wound surface, to be grown surely through wound surface; 3. ease the pain; 4. good compliance has better elastic and pliability, can be suitable for human body different anatomic position with bodily form flap coverage; 5. effectively prevent from only to use the cicatrization and the wound surface that after body thin skin sheet is transplanted, cause to shrink; 6. the new life of cell guiding impels from the body fibroblast and grows in the xenogenesis dermis scaffold; Promote wound healing, to reach flat appearance, the good purpose of function; It is convenient to store, use; 7. to organizing nonirritant, avirulence, no antigen, non-carcinogenesis; 8. can tolerate high pressure or other sterilization method commonly used and handle, can be in room temperature long preservation, and can mass production.
Pig acellular dermal matrix (PADM) has been removed and can have been brought out the cell component of immunological rejection in recent years, and is widely used in wound repair.Pigskin structure and application on human skin structural similarity, the skin anatomical physiology characteristic is also basic identical, and the source is wide, cost is low, traditional biological dressing such as heterogenous skin, xenogenesis Corii Sus domestica play a good role for the treatment of II degree burn wound, but also existence is frequent as wound surface easy infection, dressing change frequency, has the shortcomings such as danger of cross infection again.And various synthetic dressings itself do not have germ-resistant ability.This makes its extensive use clinically be subjected to certain limitation.
The research (CN 1775300A) of pair nano silver bionic dressing is also arranged, but it is comparatively complicated to the processing as the chitosan film of substrate, it is also comparatively difficult to obtain uniform film body in processing procedure, simultaneously it is a membrane structure, is unfavorable for that fibroblast and vascular endothelial cell are according to due histology's mode skin corium of growing into.
Summary of the invention
The objective of the invention is susceptible defective when solving the pig acellular dermal matrix as biological dressing.
In order to achieve the above object, the invention provides a kind of nanometer silver-pig acellular dermal matrix biological dressing, comprise pig acellular dermal matrix and the nano silver particles that is assembled on the pig acellular dermal matrix, wherein the content of nano silver particles is 435.66-447.66 μ g/g, and the size of nano silver particles is 55-75nm.
The present invention also provides the preparation method of a kind of nanometer silver-pig acellular dermal matrix biological dressing, and this method may further comprise the steps:
(1) preparation of nano silver particles: configuration concentration is 0.5 * 10
-3-1.5 * 10
-3Mol/LAgNO
3Solution stirs and heating, after the boiling, and Dropwise 5-15mg/mL Na
3C
6H
5O
72H
2O solution reacted after 20-40 minute, and natural cooling filters, and obtains nano-class silver colloidal solution; Described AgNO
3Solution and Na
3C
6H
5O
72H
2The volume ratio of O solution is 48-52: 1;
(2) processing of pig acellular dermal matrix: with the pig acellular dermal with the alcohol-pickled dehydration of 75-95% 6-18 hour, carry out ultrasonic 2-6 minute after, from soak, take out under the sterile working, dry under the ultra violet lamp;
(3) preparation of nanometer silver-pig acellular dermal matrix biological dressing: the pig acellular dermal matrix after handling in the step (2) is soaked in the nano-class silver colloidal solution that obtains in the step (1) fully, ultrasonic 2-4 minute, and at 2-6 ℃ of following cryopreservation 0.5-4 days, wherein storage temperature was preferred 4 ℃; Take out the pig acellular dermal matrix after soaking, promptly obtain aseptic nanometer silver-pig acellular dermal matrix with normal saline flushing.
Wherein, the AgNO of configuration in the step (1)
3The solution preferred concentration is 1 * 10
-3Mol/L, Na
3C
6H
5O
72H
2O solution preferred concentration is 10mg/mL, AgNO
3Solution and Na
3C
6H
5O
72H
2The preferred volume ratio of O solution is 50: 1.
The processing procedure of the described pig acellular dermal matrix of step (2) is preferably: the pig acellular dermal matrix was placed 75% ethanol 6 hours, placed 85% ethanol after the taking-up again 6 hours, taking out places 95% ethanol to carry out processed in 6 hours at last, ultrasonic 2 minutes of 95% ethanol that will have the pig acellular dermal matrix then carried out ultrasonic 2 minutes after two minutes at interval again; Perhaps the pig acellular dermal matrix is placed 75% ethanol after 6 hours, ultrasonic 2 minutes, take out, again the pig acellular dermal matrix is placed 85% ethanol after 6 hours, ultrasonic 2 minutes, take out, at last the pig acellular dermal matrix is placed 95% ethanol after 6 hours, ultrasonic 2 minutes.After taking out the pig acellular dermal matrix, the ultra violet lamp time is preferably 3-5 hour.
Nanometer silver-pig acellular dermal matrix biological dressing with obtaining in the step (3) is immersed in and carries out ultrasonic 2-4 minute in the normal saline, washes with normal saline after ultrasonic the finishing; After the flushing, be immersed in once more in the nanometer silver solution that obtains in the step (1) and after ultrasonic 2-4 minute, at 2-6 ℃ of following cryopreservation 0.5-4 days, wherein storage temperature was preferred 4 ℃; Soak the back and take out, promptly obtain nanometer silver-pig acellular dermal matrix biological dressing with normal saline flushing.
The nanometer silver that finally obtains-pig acellular dermal matrix biological dressing places physiological saline solution to carry out low temperature (2-6 ℃) and preserves, 4 ℃ of preferred storage temperatures.
Nanometer silver of the present invention-pig acellular dermal matrix biological dressing has following advantage:
1, the skin texture of pig is similar to people's skin, has therefore intactly kept the morphosis and the constituent of cell as the PADM of substrate, has the low characteristics of antigenicity; Can induce simultaneously fibroblast with regeneration capacity and vascular endothelial cell according to due histology's mode skin corium of growing into;
2, the nano silver particles that is assembled on the PADM can constantly act on regenerated bacteria, has lasting, the characteristics that sterilize efficiently, can reduce the dressing change frequency of this dressing, has reduced the danger of cross infection, has reduced the misery when changing dressings simultaneously;
3, nano-silver biological dressing can not produce drug resistance, does not have irritated phenomenon and takes place, and has lasting anti-infection ability and excellent biological compatibility and stability; And nano silver particles is little as the nano-scale particle volume, has great contact area, can control effectively under the prerequisite of wound surface antibacterial, has greatly reduced the consumption of noble metal silver;
4, the pig acellular dermal matrix is dewatered and supersound process simultaneously, can remove compositions such as original cell effectively, thereby the reduction rejection, and can make and disperse self assembly on the pig acellular dermal matrix nano silver particles by pig acellular dermal matrix short texture more equably; Adopt the ethanol gradient to soak, can make that dehydration is more abundant; Repeated ultrasonic is handled, and makes the short texture of pig acellular dermal matrix to be more prone to the combining nano silver particles;
5, in the process of nano silver particles self assembly on the pig acellular dermal matrix, adopt physical absorption and the common combination of chemical bond dual mode, make nano silver particles more evenly be assembled on the pig acellular dermal matrix securely: the pig acellular dermal matrix directly is immersed in the nanometer silver solution, carries out physical absorption; After the physical absorption, carry out supersound process, make nano silver particles can pass through the interaction of silver-nitrogen key, be assembled in more securely on the pig acellular dermal matrix by bonding action; Adopt and repeatedly soak and supersound process, make that the absorption of nano silver particles is tightr.
Description of drawings
Fig. 1 is nano silver particles transmission electron microscope figure of the present invention;
Fig. 2 is the cross section HE colored graph of nanometer silver of the present invention-pig acellular dermal matrix biological dressing;
Fig. 3 is nanometer silver-pig acellular dermal matrix biological dressing and pig acellular dermal matrix to the bacteriostatic tests of escherichia coli (A) comparison diagram as a result;
Fig. 4 is nanometer silver-pig acellular dermal matrix biological dressing and the pig acellular dermal matrix biocidal property comparison of test results figure to staphylococcus aureus (B);
Fig. 5 is nanometer silver-pig acellular dermal matrix biological dressing and pig acellular dermal matrix to the stability tests of escherichia coli (a) comparison diagram as a result;
Fig. 6 is nanometer silver-pig acellular dermal matrix biological dressing and pig acellular dermal matrix to the stability test of staphylococcus aureus (b) comparison diagram as a result;
The growth conditions figure of Fig. 7 for adopting variable concentrations nanometer silver-pig acellular dermal matrix biological dressing lixiviating solution l cell (L-929 cell) to be cultivated 2 days;
The growth conditions figure of Fig. 8 for adopting variable concentrations nanometer silver-pig acellular dermal matrix biological dressing lixiviating solution l cell (L-929 cell) to be cultivated 4 days;
The growth conditions figure of Fig. 9 for adopting variable concentrations nanometer silver-pig acellular dermal matrix biological dressing lixiviating solution l cell (L-929 cell) to be cultivated 7 days;
Among Fig. 3 to Fig. 6,1,2,3 is the antibacterial ring of nanometer silver-pig acellular dermal matrix biological dressing; 4 is the antibacterial ring of pig acellular dermal matrix;
Among Fig. 7, a1 is that 100% lixiviating solution was cultivated 2 days, and a2 is that 50% lixiviating solution was cultivated 2 days, and the negative matched group of a3 was cultivated 2 days;
Among Fig. 8, b1 is that 100% lixiviating solution was cultivated 4 days, and b2 is that 50% lixiviating solution was cultivated 4 days, and the negative matched group of b3 was cultivated 4 days;
Among Fig. 9, c1 is that 100% lixiviating solution was cultivated 7 days, and c2 is that 50% lixiviating solution was cultivated 7 days, and the negative matched group of c3 was cultivated 7 days.
The specific embodiment
1, the preparation of nano silver particles:
(1) 1 * 10
-3Mol/LAgNO
3Solution: accurately take by weighing 0.085gAgNO
3Place beaker,, be transferred in the 500mL volumetric flask standardize solution with inferior boiling water dissolving.Remove by filter in the container that is added to behind the impurity with inferior boiling water rinse;
(2) 10mg/mLNa
3C
6H
5O
72H
2O solution: accurately take by weighing 0.1gNa
3C
6H
5O
72H
2O places beaker and with inferior boiling water dissolving, is transferred in the 10mL volumetric flask standardize solution.Remove by filter impurity;
(3) heating and stirring fill AgNO
3The container of solution after the boiling, drips Na
3C
6H
5O
72H
2O solution reacted about 30 minutes, broke away from thermal source, and natural cooling promptly obtains mean diameter and be the nano silver particles solution about 70nm.
2, the processing of pig acellular dermal matrix (PADM):
(1) PADM that buys was placed 75% ethanol 6 hours;
(2) take out, PADM was placed 85% ethanol 6 hours again;
(3) take out, PADM was placed 95% ethanol 6 hours again;
(4) will have ultrasonic 2 minutes of the 95% ethanol liquid of PADM, 2 minutes at interval, ultrasonic again 2 minutes;
(5) in superclean bench PADM is taken out, place aseptic container, ultra violet lamp promptly obtained aseptic Corii Sus domestica substrate in 3 hours.
3, the preparation of nanometer silver-pig acellular dermal matrix biological dressing:
(1) in superclean bench, filters nano silver particles solution;
(2) in superclean bench pretreated PADM is fully immersed in the filtering nano silver particles solution, after ultrasonic 2 minutes, 4 ℃ of refrigerators were preserved 0.5 day;
(3) the cell Corii Sus domestica substrate of taking off that will handle well in superclean bench was taken out, with normal saline flushing and immersion, ultrasonic 2 minutes;
(4) PADM that step (3) is obtained is fully immersed in the nanometer silver solution, ultrasonic 2 minutes, preserves 0.5 day for 4 ℃;
(5) PADM is taken out, with normal saline flushing for several times, promptly obtain aseptic nanometer silver-pig acellular dermal matrix biological dressing;
(6) nanometer silver-pig acellular dermal matrix biological dressing that obtains is soaked in the physiological saline solution 4 ℃ of cryopreservation.
1, the preparation of nano silver particles:
(1) 0.5 * 10
-3Mol/LAgNO
3Solution: accurately take by weighing 0.042gAgNO
3Place beaker,, be transferred in the 500mL volumetric flask standardize solution with inferior boiling water dissolving.Remove by filter in the container that is added to behind the impurity with inferior boiling water rinse;
(2) 5mg/mLNa
3C
6H
5O
72H
2O solution: accurately take by weighing 0.05gNa
3C
6H
5O
72H
2O places beaker and with inferior boiling water dissolving, is transferred in the 10mL volumetric flask standardize solution.Remove by filter impurity;
(3) heating and stirring fill AgNO
3The container of solution after the boiling, drips Na
3C
6H
5O
72H
2O solution reacts after about 20 minutes, breaks away from thermal source, natural cooling, and promptly obtaining mean diameter is the nano silver particles solution of 55-75nm.
2, the pretreatment of pig acellular dermal matrix:
(1) place 75% ethanol after 6 hours the PADM that buys, ultrasonic 2 minutes
(2) take out, again PADM is placed 85% ethanol after 6 hours, ultrasonic 2 minutes;
(3) take out, again PADM is placed 95% ethanol after 6 hours, ultrasonic 2 minutes;
(4) in superclean bench PADM is taken out, place aseptic container, ultra violet lamp promptly obtained aseptic PADM in 4 hours.
3, the preparation of nanometer silver-pig acellular dermal matrix biological dressing:
(1) in superclean bench, filters nano silver particles solution;
(2) in superclean bench pretreated PADM is fully immersed in the filtering nano silver particles solution, after ultrasonic 4 minutes, 4 ℃ of refrigerators were preserved 1 day;
(3) in superclean bench, will take off cell Corii Sus domestica substrate and take out,, promptly obtain aseptic nanometer silver-pig acellular dermal matrix biological dressing with normal saline flushing and immersion.
1, the preparation of nano silver particles:
(1) 1.5 * 10
-3Mol/LAgNO
3Solution: accurately take by weighing 0.085gAgNO
3Place beaker,, be transferred in the 500mL volumetric flask standardize solution with inferior boiling water dissolving.Remove by filter in the container that is added to behind the impurity with inferior boiling water rinse;
(2) 15mg/mL Na
3C
6H
5O
72H
2O solution: accurately take by weighing 0.15gNa
3C
6H
5O
72H
2O places beaker and with inferior boiling water dissolving, is transferred in the 10mL volumetric flask standardize solution.Remove by filter impurity;
(3) heating and stirring fill AgNO
3The container of solution after the boiling, drips Na
3C
6H
5O
72H
2O solution reacted about 40 minutes, broke away from thermal source, natural cooling, and promptly obtaining mean diameter is the nano silver particles solution of 55-75nm.
2, the pretreatment of pig acellular dermal matrix:
(1) place 75% ethanol after 6 hours the PADM that buys, ultrasonic 2 minutes;
(2) take out, again PADM is placed 85% ethanol after 6 hours, ultrasonic 2 minutes;
(3) take out, PADM was placed 95% ethanol 6 hours again;
(4) will have ultrasonic 2 minutes of the 95% ethanol liquid of PADM, 2 minutes at interval, ultrasonic again 2 minutes;
(5) in superclean bench PADM is taken out, place aseptic container, ultra violet lamp promptly obtained aseptic PADM in 5 hours.
3, the preparation of nanometer silver-pig acellular dermal matrix biological dressing:
(1) in superclean bench, filters nano silver particles solution;
(2) pretreated PADM is fully immersed in the nano silver particles solution in superclean bench, and ultrasonic 4 minutes, 4 ℃ of refrigerators were preserved 2 days;
(3) in superclean bench, will take off cell Corii Sus domestica substrate and take out,, promptly obtain aseptic nanometer silver-pig acellular dermal matrix biological dressing with normal saline flushing and immersion.
Effect embodiment
Nanometer silver-pig acellular dermal matrix the biological dressing that obtains among the embodiment 1 is carried out following effect detection:
1, electron microscopic structure
Figure 1 shows that the projection electron microscope figure of the nanometer silver that embodiment 1 obtains, from figure, observe the nano silver particles diameter and be about 55-75nm; The cross section HE colored graph of the nanometer silver that Fig. 2 obtains for embodiment 1-pig acellular dermal matrix biological dressing.
2, silver content
By the flame atomic absorption spectrometry silver content: with the accurate solution of inferior boiling water stepwise dilution silver label is the accurate solution of silver label of 0,0.2,0.4,0.6,0.8,1.0,1.2,1.5 μ g/mL, under selected condition, measure, and drawing curve, the regression equation that obtains is A=0.0555C+0.0007, R2=0.9997 obtains by regression equation that silver content is 441.66 ± 6.00 μ g/g in the nanometer silver-pig acellular dermal matrix biological dressing of embodiment 1 preparation.
3, fungistatic effect
Adopt staphylococcus aureus (ATCC6538) and escherichia coli (8099) biological dressing to be carried out the mensuration of bacteriostatic test, weigh the fungistatic effect of nanometer silver-pig acellular dermal matrix biological dressing by the antibacterial ring of measuring bacteriostatic test, in conjunction with Fig. 3 and Fig. 4, obtain the inhibition zone diameter of nanometer silver-pig acellular dermal matrix biological dressing bacteriostatic test greater than 12mm by vernier caliper measurement, as shown in table 1, illustrate that this biological dressing has fungistatic effect, and the pig acellular dermal matrix there is not fungistatic effect.
Table 1 bacteriostatic test result (mm, x ± s, n=4)
4, stability
Adopt staphylococcus aureus (ATCC6538) and escherichia coli (8099) biological dressing to be carried out the mensuration of stability test, place 4 ℃ of refrigerators and aseptic condition to store down a part of nanometer silver-pig acellular dermal matrix medical dressing and pig acellular dermal matrix medical dressing, do bacteriostatic test after 45 days again, repeat 4 times, observe its fungistatic effect.Weigh the stability of nanometer silver-pig acellular dermal matrix biological dressing by measuring antibacterial ring, in conjunction with Fig. 5 and Fig. 6, obtain the inhibition zone diameter of nanometer silver-pig acellular dermal matrix biological dressing bacteriostatic test greater than 12mm by vernier caliper measurement, as shown in table 2, illustrate that the bacteriostasis property of this biological dressing is stable.
Table 2 stability test result (mm, x ± s, n=4)
5, hydrophilic
By the hydrophilic of water absorption rate and the dressing of water retention mensuration, as shown in table 3, the experimental group that this experiment is done and the water absorption rate of matched group and water retention indifference illustrate that nanometer silver-pig acellular dermal matrix biological dressing also has good hydrophilicity.
Table 3 water absorption rate and water retention (%, x ± s, n=6)
6, cytotoxicity
The preparation of 100% nanometer silver-pig acellular dermal matrix biological dressing lixiviating solution: nanometer silver-pig acellular dermal matrix biological dressing is cut into the long-pending skin graft of certain surface, puts into conical flask, adding by surface area and volume ratio is 3cm
2: the DMEM culture fluid of 1mL, place 37 ℃ of electro-heating standing-temperature cultivator lixiviates 24 hours, promptly obtain 100% lixiviating solution;
The preparation of 50% nanometer silver-pig acellular dermal matrix biological dressing lixiviating solution: nanometer silver-pig acellular dermal matrix biological dressing is cut into the long-pending skin graft of certain surface, puts into conical flask, adding by surface area and volume ratio is 3cm
2: the DMEM culture fluid of 2mL, place 37 ℃ of electro-heating standing-temperature cultivator lixiviates 24 hours, promptly obtain 50% lixiviating solution;
Negative control group: DMEM culture fluid.
L cell (L-929 cell) was cultivated after 24 hours, discarded original fluid, added 100% respectively, 50% nanometer silver-pig acellular dermal matrix biological dressing lixiviating solution, every hole 100 μ L, every group of kind 8 holes continue to cultivate, and the L-929 cellular morphology is observed under the inverted microscope.
Cultivated 2 days: as shown in Figure 6,100% lixiviating solution group cell is less relatively, 50% and negative control group between difference less.Clear fusiformis or polygon or the rhombus of being of the cell outline of adherent growth on the integral body, the rounded or irregular shape of the cell of suspension.
Cultivated 4 days: as shown in Figure 7,100% lixiviating solution group, 50% lixiviating solution group and negative control group are compared, and the cell fusion degree is less, is 70-80% probably, and the cell fusion degree is bigger on the integral body, and it is normal that each organizes adherent cellular morphology.
Cultivated 7 days: as shown in Figure 8, cell all merges, and distinguish and diminishes between each group, and visible cell comes off, and cell rounding, diminishes, and kytoplasm, karyon are concentrated.
After 2,4,7 days, respectively take out a culture plate, carry out vitro cytotoxicity test (seeing Table 4), it is the 3-(4 of 5g/L that every hole adds 20 μ L concentration, 5-dimethyl-2-thiazole)-2,5-diphenyl bromination tetrazolium salts solution (MTT) was hatched 4 hours for 37 ℃, suction is abandoned and is added 150 μ L dimethyl sulfoxide (DMSO) behind the culture fluid and placed under the room temperature 15～20 minutes, shook 10 minutes, and adopted microplate reader to measure each hole absorbance value (OD value), and calculate the average of each group at the 490nm wavelength.From table as can be known, the absorbance there was no significant difference (P＞0.05) between the 2nd day, the 4th day, the 7th day 100%, 50% lixiviating solution group and the negative control group.
Table 4 vitro cytotoxicity measurement result
Table 5 is relative appreciation rate and the evaluation of poison level, evaluate the cytotoxicity of each material according to " the biomaterial for medical purpose biological property testing standard of non-direct contact blood " 6 grades of toxicity grading methods, the poison level of two groups of variable concentrations lixiviating solution is 1 grade, meets the cytotoxicity requirement of implant into body biomaterial.
Table 5 is appreciation rate (RGR) and the evaluation of poison level relatively
7, primary cutaneous irritant test:
Tried 6 of Cavia porcelluss, tested preceding 24 hours, the unhairing district of 1 3cm * 3cm area is respectively selected in the spinal column both sides, carries out the left and right sides unhairing with the device of shedding, and can do the experiment of skin thorn after observing after one day the skin zero damage in the district that finds to shed.Be soaked in 2.5cm * 2.5cm filter paper piece in the lixiviating solution of nanometer silver-pig acellular dermal matrix biological dressing to saturated, stick in the test position.Lixiviate medium normal saline is as negative control.Material sticks behind skin, uses 3cm * 3cm gauze piece to cover the outermost layer immobilization with adhesive tape immediately.Stick fix 24 hours after, remove and stick thing, stick the district and blot with warm water cleaning, observe erythema and the edema situation of removing 4,24,48 and 72 hours skin behind the patch thing, calculate PiI (PII).Edema and erythema do not appear in ginseng test group guinea pig skin, and the skin nonirritant (see Table 6) of this dressing to Cavia porcellus is described.
Table 6 skin primary stimulus result of the test, PiI (PII) and scoring situation (n=6)
8, thermal source experiment:
The normal saline lixiviating solution of preparation nanometer silver-pig acellular dermal matrix biological dressing: nanometer silver-pig acellular dermal matrix biological dressing is cut into the long-pending skin graft of certain surface, puts into conical flask, adding by surface area and normal saline volume ratio is 3cm
2: a certain amount of normal saline culture fluid of 1mL places 37 ℃ of electro-heating standing-temperature cultivator lixiviates 24 hours, preparation normal saline lixiviating solution.
According to the GB/T16175-1996 pyrogen testing, selected 3 rabbit in preceding 7 days in test, male and female are not limit, and doe does not have pregnant, and preceding 7 days planted agents of thermometric raise with same feedstuff in same environmental condition, and the rabbit body weight does not have and alleviates during this period, and spirit, appetite, drainage etc. are no abnormal.After 3 days rabbit is fixed in the holder.Beginning is measured for the 1st time after 30 hours, surveys 1 time every 30 hours later on, surveys altogether 2 times.The difference of body temperature is no more than 0.2 ℃, is the normal body temperature of this rabbit with the meansigma methods of these 2 body temperature.And the body temperature that used rabbit the same day is in 38.0-39.6 ℃ scope, and regular using warming therapy difference is no more than 1 ℃ between each rabbit.Met the requirements back 15 minutes in the rabbit normal body temperature, slowly inject the normal saline lixiviating solution of 38 ℃ of nanometer silvers of preheating-pig acellular dermal matrix biological dressing from ear vein, dosage is 10mL/kg.Injection back is every 1 hour take temperature 1 time, and the highest in 6 body temperature to deduct normal body temperature 1 time be experimental rabbits fervescence value.3 rabbit body temperatures raise and all are lower than 0.6 ℃, and 3 rabbit body temperature rising summations are lower than 1.4 ℃, and the security performance better (table 7) of dressing be described.
Table 7 thermal source experimental result
9, Intradermal irritant test:
Nanometer silver-pig acellular dermal matrix biological dressing vegetable oil lixiviating solution preparation: nanometer silver-pig acellular dermal matrix biological dressing is cut into the long-pending skin graft of certain surface, puts into conical flask, adding by surface area and volume ratio is 3cm
2: the vegetable oil culture fluid of 1mL places 37 ℃ of electro-heating standing-temperature cultivator lixiviate 24h, preparation vegetable oil lixiviating solution.
24h before the experiment is tried rabbit spinal column both sides and respectively cuts and shave the 5cm * 25cm zone rabbit hair, should avoid injured skin.Skin with the exposure of 75% (V/V) ethanol disinfection.Respectively select 10 points in rabbit spinal column both sides, every some interval 2cm, every some intradermal injection dosage 0.2mL.One side preceding 5 is selected the normal saline lixiviating solution of injection nanometer silver-pig acellular dermal matrix biological dressing, and back 5 injections are with batch negative control normal saline; 5 select vegetable oil lixiviating solution and the negative control vegetable oil of injecting nanometer silver-pig acellular dermal matrix biological dressing respectively before and after the opposite side.Injection site and surrounding skin tissue reaction were observed in 24,48,72 hours in the injection back, and the list of references standard is kept the score and estimated.The result tests not show speckle of animal skin, and no edema forms, and illustrates that this dressing does not have the Intradermal zest to rabbit skin, experiment effect more satisfactory (table 8).
Table 8 Intradermal irritant test result, PiI (PII) and scoring situation (every group of 30 injection points)
10, acute general toxicity test:
Healthy mice is divided into experimental group at random and contrasts two groups, 5 every group.The experimental group animal is by the normal saline lixiviating solution of tail vein injection nanometer silver-pig acellular dermal matrix biological dressing, and dosage is 50mL/kg.Control animals is by the normal saline of tail vein injection with lot number, and dosage is 50mL/kg.Injection back is in general state, toxicity performance and the dead animal number of 24,48,72 hours observed and recorded experimental grouies and control animals.Observation index list of references standard.Lixiviating solution injection back 24,48,72 hours, the mice body weight has no significant change, and does not also have death and toxic reaction, and behavior of animal and physiological situation are all normal, and the security performance better (table 9) of dressing is described.
The acute general toxicity result of the test of table 9
Discussion about nanometer silver antimicrobial mechanism: the nanometer materials specific surface area is big, and be inversely proportional to size, because reducing of particle diameter, the atomic number that is in surface layer increases rapidly, cause former coordination deficiency, unsaturated bond increases, and the surface energy of atom increases, and the chemism of nano level ultramicron is strengthened.After nanometer silver-pig acellular dermal matrix bionic dressing covers wound surface, nano silver particles with contain-enzyme of SH combines, make the enzyme loss of activity and cause pathogenic bacteria death, thereby reach germ-resistant purpose.
To sum up, nanometer silver-pig acellular dermal matrix biological dressing combines the advantage of nanometer silver and pig acellular dermal matrix, not only has the strong antibiotic performance, do not develop immunity to drugs, and biological safety height, histocompatibility is good, has played very big effect aspect the painful and infection of patient alleviating.Biological dressing technology of the present invention is simple, easy to use simultaneously, can have goodish potential applicability in clinical practice in clinical application.
Claims (8)
1. nanometer silver-pig acellular dermal matrix biological dressing, it is characterized in that: comprise pig acellular dermal matrix and assembling nano silver particles thereon, the content of described nano silver particles is 435.66-447.66 μ g/g, and the size of described nano silver particles is 55-75nm.
2. the preparation method of nanometer silver-pig acellular dermal matrix biological dressing, it is characterized in that: this preparation method may further comprise the steps:
(1) preparation of nano silver particles: configuration concentration is 0.5 * 10
-3-1.5 * 10
-3Mol/LAgNO
3Solution stirs and heating, after the boiling, and Dropwise 5-15mg/mLNa
3C
6H
5O
72H
2O solution reacted after 20-40 minute, and natural cooling filters, and obtains nano-class silver colloidal solution; Described AgNO
3Solution and Na
3C
6H
5O
72H
2The volume ratio of O solution is 48-52: 1;
(2) processing of pig acellular dermal matrix: with the pig acellular dermal with the alcohol-pickled dehydration of 75-95% 6-18 hour, carry out ultrasonic 2-6 minute after, from soak, take out under the sterile working, dry under the ultra violet lamp;
(3) preparation of nanometer silver-pig acellular dermal matrix biological dressing: the pig acellular dermal matrix after handling in the step (2) is soaked in the nano-class silver colloidal solution that obtains in the step (1) fully, ultrasonic 2-4 minute, and at 2-6 ℃ of following cryopreservation 0.5-4 days; Take out the pig acellular dermal matrix after soaking, promptly obtain nanometer silver-pig acellular dermal matrix biological dressing with normal saline flushing.
3. according to the preparation method of the described biological dressing of claim 2, it is characterized in that: the processing procedure of the described pig acellular dermal matrix of step (2) is: the pig acellular dermal matrix was placed 75% ethanol 6 hours; Placed 85% ethanol after the taking-up again 6 hours; Take out and to place 95% ethanol to handle in 6 hours at last, then immersion is had ultrasonic 2 minutes of 95% ethanol of pig acellular dermal matrix, carried out again ultrasonic 2 minutes after two minutes at interval.
4. according to the preparation method of the described biological dressing of claim 2, it is characterized in that: the processing procedure of the described pig acellular dermal matrix of step (2) is: place 75% ethanol after 6 hours the pig acellular dermal matrix, and ultrasonic 2 minutes; Take out, again the pig acellular dermal matrix is placed 85% ethanol after 6 hours, ultrasonic 2 minutes; Take out, at last the pig acellular dermal matrix is placed 95% ethanol after 6 hours, ultrasonic 2 minutes.
5. according to the preparation method of the arbitrary described biological dressing of claim 2 to 4, it is characterized in that: the AgNO of configuration in the step (1)
3Solution concentration is 1 * 10
-3Mol/L, Na
3C
6H
5O
72H
2The O solution concentration is 10mg/mL, AgNO
3Solution and Na
3C
6H
5O
72H
2The volume ratio of O solution is 50: 1.
6. according to the preparation method of the arbitrary described biological dressing of claim 2 to 4, it is characterized in that: the ultra violet lamp time described in the step (2) is 3-5 hour.
7. according to the preparation method of the arbitrary described biological dressing of claim 2 to 4, it is characterized in that: with the nanometer silver-pig acellular dermal matrix biological dressing that obtains in the step (3), be immersed in and carry out ultrasonic 2-4 minute in the normal saline, wash with normal saline after ultrasonic the finishing; After the flushing, be immersed in once more in the nanometer silver solution that obtains in the step (1) and after ultrasonic 2-4 minute at 2-6 ℃ of following cryopreservation 0.5-4 days; Take out, promptly obtain nanometer silver-pig acellular dermal matrix biological dressing with normal saline flushing.
8. according to the preparation method of the arbitrary described biological dressing of claim 2 to 4, it is characterized in that: the storage temperature of pig acellular dermal matrix in nanometer silver solution described in the step (3) is 4 ℃.
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