CN101185774A - Preparation of medical bioavailability bracket material and uses thereof - Google Patents
Preparation of medical bioavailability bracket material and uses thereof Download PDFInfo
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- CN101185774A CN101185774A CNA2007101656319A CN200710165631A CN101185774A CN 101185774 A CN101185774 A CN 101185774A CN A2007101656319 A CNA2007101656319 A CN A2007101656319A CN 200710165631 A CN200710165631 A CN 200710165631A CN 101185774 A CN101185774 A CN 101185774A
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Abstract
The invention relates to a preparation and using method of medical biocompatible stent material. The medical biocompatible stent material is prepared by applying the small intestine submucosal and the tendon tissue of people or animals as the raw materials. The invention has good biocompatibility, appropriate porosity and mechanical strength and stable spatial structure, and the invention contains various growth factors to induce the tissue for regeneration and reconstruction, thereby improving the success rate of the repair of the various tissue defects and the reconstruction of tissue structures, therefore, the invention is an ideal tissue engineering material. The medical biocompatible stent material which is provided by the invention is applicable to the preparation of various tissue engineering equipments and the application in the repair of various tissue defects and injuries, including skin, blood vessels, nerves, tendons, bones, cartilages, abdominal walls, urinary tracts, oral mucosa and so on, and the invention can be used in the treatment of trauma, abdominal external hernia, anal fistula, rectovaginal fistula, rectum anterior protrusion, bone and cartilage defects, vascular injuries, nerve injuries, tendon injuries, oral mucosal injuries, relaxed pelvic floor, urinary tract injuries and other diseases.
Description
Technical field
The present invention relates to a kind of preparation and using method thereof of medical bioavailability bracket material.This medical bioavailability bracket material is that application human or animal's tela submucosa intestini tenuis and tendon tissue is raw material, prepare submucous layer of small intestine substrate and collagen respectively, then the acetic acid solution of submucous layer of small intestine substrate, the acetic acid solution and the chondroitin sulfate solution mixing system of collagen are formed fully.This material has excellent biological compatibility, more suitable porosity and mechanical strength, more stable space structure, contain various somatomedin, can promote cell proliferation, adhesion, induced tissue is regenerated and is reinvented, and has significantly improved the success rate of repairing various tissue defects, reinventing organizational structure, is even more ideal tissue engineering material.Medical bioavailability bracket material provided by the present invention is fit to be applied to prepare various organizational project equipment, be applied to repair various tissue defects and damage, comprise skin, blood vessel, nerve, tendon, bone, cartilage, stomach wall, urinary tract, oral mucosa etc., diseases such as treatment wound, abdominal external hernia, anal fistula, recto-vaginal fistula, encysted rectum, bone and cartilage defect, blood vessel injury, nerve injury, tendon injury, oral mucosa lesion, relaxed pelvic floor, urinary tract damage.
Background technology
Bioavailability bracket material is the emphasis of Tissue Engineering Study, can be used to repair the damaged and defective of tissues such as skin, stomach wall, urethra, bladder, bone, blood vessel, nerve, tendon, mucosa, has broad clinical application prospect.
Ideal bioavailability bracket material should have following characteristic simultaneously: 1. the favorable tissue compatibility; 2. degradability; 3. the former effect of nonreactive; 4. Shi Yi porosity; 5. good mechanical intensity; 6. keep cellular morphology and the phenotype of growth on it, promote the adhesion and the reproduction of cell, induced tissue regeneration.
The maximum bioavailability bracket material of research is a pig intestinal mucosa lower floor substrate at present.Pig intestinal mucosa lower floor substrate is a kind of natural heterogenous cell epimatrix, shows better biocompatibility, antimicrobial acivity and tissue regeneration ability in the organizational project structure zooscopy of skin, bladder mucosa, bone, vascular patch, nerve, tendon etc.But it is used major part and also is in animal experiment stage, and successful clinical practice report is less.
Studies show that, though pig intestinal mucosa lower floor substrate has the favorable tissue compatibility, in the external adhesion and the reproduction that can promote cell, keep cellular morphology and phenotype, but it lacks good porosity and uniform hole, and can not keep certain structure and mechanical strength in the degradation in vivo process, limit tool clinical practice and curative effect.Therefore, how to improve the key issue that the hole of submucous layer of small intestine substrate and biomechanical strength are its clinical practices of decision.
The present invention has improved the processing technique of submucous layer of small intestine substrate innovatively, improved the dissolution rate of submucous layer of small intestine substrate, by collagen solution, chondroitin sulfate cellulose solution are mixed with the small intestinal submucosa matrix solution, prepared a kind of new medical bioavailability bracket material; This medical bioavailability bracket material has excellent biological compatibility, more suitable porosity and mechanical strength, more stable space structure, contain various somatomedin, can promote cell proliferation, adhesion, induced tissue is regenerated and is reinvented, and has significantly improved the success rate of repairing various tissue defects, reinventing organizational structure, is even more ideal tissue engineering material.
Medical bioavailability bracket material provided by the present invention can be applied to prepare various organizational project equipment, be widely used in repairing various tissue defects and damage, comprise skin, blood vessel, nerve, tendon, bone, cartilage, stomach wall, urinary tract, oral mucosa etc., thus various diseases such as treatment wound, abdominal external hernia, anal fistula, recto-vaginal fistula, encysted rectum, bone and cartilage defect, blood vessel injury, nerve injury, tendon injury, oral mucosa lesion, relaxed pelvic floor, urinary tract damage.
Summary of the invention
The invention provides a kind of preparation and application of medical bioavailability bracket material, the preparation method of described medical bioavailability bracket material may further comprise the steps:
A. prepare submucous layer of small intestine substrate step, described step is the submucous layer of small intestine tissue of separation of human or animal, soaks 16h in the solution that contains ethylenediaminetetraacetic acid (100mmol/L) and sodium hydroxide (10mmol/L) (pH value is 11～12); Rinse well with deionized water then, place the solution (pH value is 0～1) of hydrochloric (1mmol/L) and sodium chloride (1mmol/L) to soak 6～8h; With in sodium chloride (1mmol/L) phosphate buffer (PBS), soaking 16h behind the deionized water rinsing; Then with in PBS solution (pH value is 7～7.4), soaking 2h behind the deionized water rinsing; Reuse deionized water rinsing 2h; Each step of said process all at room temperature carries out, and the ratio of material and liquor capacity all remained on 1: 100.
B. lyophilization, pulverising step, described step are to use vacuum freeze mixed solution is carried out lyophilization, use pulverizing homogenate equipment then and carry out pulverization process, and described step can repeat 2 ~ 3 times;
C. dissolving step, described step are to use dilute acid soln submucous layer of small intestine stromatolysis after handling is pulverized in lyophilization,
D. prepare collagen stroma solution step, described step is the tendon of people or animal or cartilage, skin histology to be shredded the back immerse 0.05mol/L acetic acid, puts in 4 ℃ of refrigerators, shakes, and behind the 48h, moves in the sterilization centrifuge tube; The centrifugal 30min of 4000r/min, draw supernatant, the ratio that adds 5g NaCl in the 100mL supernatant adds the NaCl crystal, centrifugal, abandoning supernatant stays the collagen stroma precipitation, use vacuum freeze liquid and carry out being dissolved in the 0.05mol/L acetic acid behind the lyophilization 48h, make collagen stroma solution;
E. blend step, described step is mixed in proportion submucous layer of small intestine matrix solution and collagen stroma solution,
F. add chondroitin sulfate C solution step, described step is that the chondroitin sulfate C drips of solution is added in the above-mentioned mixed solution, and constantly stirs, and the whole mass concentration that makes chondroitin sulfate C is 10 ~ 80g/L;
G. forming step, described step is that mixed solution is poured in ready-formed glass or the stainless steel mould, rolls flatly, the refrigerator pre-freeze of putting into-80 ℃ is more than 24 hours.
H. lyophilization step, described step are to use vacuum freeze mixed solution is carried out lyophilization,
I. cross-linking step, described step are to use the composite material of methods such as cross-linking agent, ultraviolet radiation after to lyophilization to carry out crosslinking Treatment,
J. the step of washing and eliminating residual cross-linking agent and regulate pH value, described step are to use the material of neutral buffered liquid after to crosslinking Treatment to wash, and use Freamine hatch to pH value be 7.35 ~ 7.55,
K. introduce the step of antibiotic coating,
L. sterilization steps.
Described animal can be pig, cattle, sheep, rabbit, Mus, preferably pig and cattle, and being more preferably the described ratio of pig is 1: 1 ~ 1: 5.
Described crosslinking Treatment is to use ultraviolet radiation to handle.
Described crosslinking Treatment is to be immersed in 0.005% glutaraldehyde solution, puts 4 ℃ of refrigerator 24h hours.
Described cross-linking agent comprises that various that the present technique field is used always can make the collagen fiber in the collagen tissue more stable; can make tropocollagen molecule that the interior intramolecular crosslinking of indivedual helical regions takes place; between contiguous different molecular crosslinked or procollagen and other intermolecular cross-linking material; it can be the glutaraldehyde of any concentration; formaldehyde; carbodiimides; 1; hexamethylene-diisocyanate (HDI); genipin; 1; 4-two (3; the 4-hydroxy benzenes)-2; 3 dimethylbutanes (NDGA); epoxide; the dicarboxylic acid compound that contains disulphide functional group; acyl azide and diphenylphosphine hydrochlorate (DP-PA) and glyoxalic acid etc., preferably 0.005% glutaraldehyde.
Described neutral buffered liquid can be phosphate buffer, borate buffer solution, TRIS buffer (Tris buffer).Proof buffer used in the invention process is a sodium phosphate buffer.
Described buffered with amino acid liquid can be glycine-HCl buffer, glycine-NaOH buffer, glycine-Tris buffer, histidine buffering liquid, Aminoacetamide (glycine amide) buffer, glycylglycine (glycylglycine) buffer.Its effect is to adjust pH value to 7.35 ~ 7.55 of taking off the tissue after cell is handled, and it selects to decide according to the pH value that takes off the tissue after cell is handled.Used proof acidic amino acid buffer is glycine-HCl buffer in the invention process.
Described antibiotic coating preparation method is the mode by spraying, soaking or instil, antibiotic homogenizing aqueous solution is incorporated in the tissue after the molding, through evaporation and/or after using desiccant and removing water basically, add at least a amphiphatic molecule solution soluble in water by same method again, obtain antibiotic coating through evaporation and/or after using the desiccant drying again.
Described antibiotic can be an aminoglycoside antibiotics, tetracycline antibiotics, lincosamides, quinolone antibiotic, the nitro glyoxaline antibiotic, the sulfonamides antibiotic, methylfuran class antibiotic, specifically the mixture of itrofurans antibiotic or above-mentioned antibiotic combination in any can be gentamycin, metronidazole, sisomicin, netilmicin, streptomycin, tobramycin, spectinomycin, vancomycin, ciprofloxacin, Moxifloxacin, clindomycin, lincomycin, tetracycline, shuttle chlortetracycline, oxytetracycline and the mixture of enumerating tetracycline or above-mentioned antibiotic combination in any.Proof antibiotic used in the invention process is the compositions of sulmycin and metronidazole.
Described amphiphatic molecule can be alkyl sulfate, alkylsulfonate, alkyl aryl sulfate, di alkylaryl sulfate, alkylaryl sulfonates, di alkylaryl sulfonate, cycloalkyl sulfate, cycloalkyl sulfonate, alkyl-cycloalkyl sulfate.Proof amphiphatic molecule used in the invention process is a sodium lauryl sulphate.
According to embodiment 1, the invention provides and use the specific embodiments that pig small intestine tissue and tendon tissue or dermal tissue, cartilaginous tissue prepare medical bioavailability bracket material.
According to embodiment 2, the invention provides the specific embodiments that provides to choose small intestine and tendon tissue or dermal tissue, cartilaginous tissue to prepare medical bioavailability bracket material.
According to embodiment 3, the invention provides to provide and use the specific embodiments that calf intestinal tissue and tendon tissue or dermal tissue, cartilaginous tissue prepare medical bioavailability bracket material.
According to embodiment 4, the invention provides the biocompatibility feature of described medical bioavailability bracket material, i.e. avirulence, nonirritant, non-immunogenicity, no hemolytic, biocompatibility is good.
According to embodiment 5, the invention provides the porosity and the mechanical characteristics of described medical bioavailability bracket material, promptly porosity is (90 ± 5.8) %, and yield stress is (10.31 ± 0.65) MPA, and strain is 57.2 1 ± 7.55.
According to embodiment 6, the invention provides the various growth factor contents of described medical bioavailability bracket material, be that basic fibroblast growth factor content is about (101.8 ± 2.163) ng/L, VEGF content is about (89.8 ± 2.053) ng/L, platelet derived growth factor content is about (45.8 ± 4.834) ng/L, and β transforming growth factor content is about (35.8 ± 3.065) ng/L.
According to embodiment 7, embodiment 8, embodiment 9, embodiment 10, embodiment 11, embodiment 12, embodiment 13, embodiment 14, embodiment 15, embodiment 16, embodiment 17, embodiment 18 and embodiment 19, the inventor also provides the application in the preparation tissue engineering material.
Described tissue engineering material is meant the function that is used to repair, safeguard, promote behind various tissues of human body or the organ injury and the biological substitution thing of form.
According to embodiment 7, the inventor provides described medical bioavailability bracket material to be used for the treatment of the organization engineering skin of skin injury, burn, wound and the application in the wound dressing in preparation.
According to embodiment 8, the inventor provides described medical bioavailability bracket material to be used for the treatment of application in the embedded material of abdominal external hernia and other abdominal-wall defect diseases in preparation, and the specific embodiments of described medical bioavailability bracket material treatment abdominal external hernia also is provided.
Described abdominal external hernia is meant that abdominal viscera passes through the disease that stomach wall is weak or the formation enclosed mass is given prominence to body surface by damaged place, specifically, is meant the abdominal-wall defect disease that indirect inguinal hernia, direct inguinal hernia, femoral hernia, incisional hernia and any reason cause.
According to embodiment 9, the inventor provides described medical bioavailability bracket material to be used for the treatment of application in the embedded material of anal fistula in preparation, and the specific embodiments of described medical bioavailability bracket material treatment anal fistula also is provided.
Described anal fistula is meant a kind of chronic infection pipeline that anal skin and rectum/anal canal communicate, and specifically, is meant the simple property of low level anal fistula, low level complexity anal fistula, high-order simple property anal fistula, high-order complexity anal fistula.
According to embodiment 10, the inventor provides described medical bioavailability bracket material to be used for the treatment of application in the embedded material of recto-vaginal fistula in preparation, and the specific embodiments of described medical bioavailability bracket material treatment recto-vaginal fistula also is provided.
Described recto-vaginal fistula is meant the pathologic passage between rectum and the vagina.
According to embodiment 11, the inventor provides described medical bioavailability bracket material to be used for the treatment of application in the embedded material of encysted rectum in preparation, and the specific embodiments of described medical bioavailability bracket material treatment encysted rectum also is provided.
Described encysted rectum is also referred to as the preceding bulging of rectum, is also referred to as the rectum front wall hernia, is also referred to as the posterior vaginal wall hernia, is meant because of the weak caused disease of rectum lower end antetheca, recto-vaginal septum and posterior vaginal wall.
According to embodiment 12, the inventor provides described medical bioavailability bracket material to be used for the treatment of application in the embedded material of hemorrhoid in preparation, and the specific embodiments of described medical bioavailability bracket material treatment hemorrhoid also is provided.
Described hemorrhoid be the pathological change that props up of supporting structure, vascular plexus and the arteriovenous anastomosis of anus pad (anal canal blood vessel pad) and (or) unusual displacement, can cause hemorrhage, a series of clinical symptoms such as deviate from.Specifically, be meant internal hemorrhoid, external hemorrhoid, mixed hemorrhoid.
According to embodiment 13, the inventor provides described medical bioavailability bracket material to repair the application of the damaged embedded material of vascular tissue in preparation.
According to embodiment 14, the inventor provides described medical bioavailability bracket material to repair the application of the damaged embedded material of nervous tissue in preparation.
According to embodiment 15, the inventor provides described medical bioavailability bracket material to repair the application of the damaged embedded material of tendon tissue in preparation.
According to embodiment 16, the inventor provides described medical bioavailability bracket material in the application for preparing the damaged embedded material of reparation bone and cartilaginous tissue.
According to embodiment 17, the inventor provides described medical bioavailability bracket material to be used for the application of the embedded material that oral mucosas tissue repairs in preparation, and the specific embodiments of described medical bioavailability bracket material treatment reconstruction of oral defect disease also is provided.
According to embodiment 18, the inventor provides described medical bioavailability bracket material to be used for repairing the application of the embedded material of urinary tract damage in preparation.
According to embodiment 19, the inventor provides the application of the embedded material that described medical bioavailability bracket material rebuilds at the bottom of preparation is used for basin, the specific embodiments of relaxed pelvic floor diseases such as described medical bioavailability bracket material treatment female incontinence and uterine prolapse also is provided.
Rebuild at the bottom of the described basin and be meant that Reconstruction is exactly by modus operandi too lax tissue to be suspended in midair, repairs at the bottom of the basin, reconstruction pelvic floor tissue framework.
Description of drawings
Fig. 1 is the scanning electron microscope picture of material provided by the present invention and the submucous layer of small intestine contrast for preparing with conventional method;
Fig. 2 is the stereoscan photograph of the tissue engineered human dermis of material preparation provided by the present invention;
Fig. 3 is that material provided by the present invention is repaired the damaged histology pictures of vascular tissue and the color Doppler inspection reaches the submucous layer of small intestine contrast for preparing with conventional method;
Fig. 4 is that material provided by the present invention is repaired the damaged histology pictures of tendon tissue;
Fig. 5 is that material provided by the present invention is repaired the damaged stereoscan photograph of osseous tissue;
Fig. 6 is that material provided by the present invention is repaired the damaged histology pictures of urinary tract.
The specific embodiment
Describe the present invention in detail below in conjunction with drawings and Examples, described embodiment is used to describe the present invention, rather than restriction the present invention.
Embodiment
Embodiment 1: application pig small intestine tissue and tendon tissue or dermal tissue, cartilaginous tissue prepare medical bioavailability bracket material
Fresh small intestinal is taken from the pig of enclosed grazing, finishes cleaning course in 4h.Remove mucous layer, placenta percreta and the flesh layer of small intestinal, continue in this process with 40 ℃ of water flushings.
16h is soaked in isolated pig intestinal mucosa lower floor in the solution that contains ethylenediaminetetraacetic acid (100mmol/L) and sodium hydroxide (10mmol/L) (pH value is 11～12); Rinse well with deionized water then, place the solution (pH value is 0～1) of hydrochloric (1mmol/L) and sodium chloride (1mmol/L) to soak 6～8h; With in sodium chloride (1mmol/L) phosphate buffer (PBS), soaking 16h behind the deionized water rinsing; Then with in PBS solution (pH value is 7～7.4), soaking 2h behind the deionized water rinsing; Reuse deionized water rinsing 2h; Each step of said process all at room temperature carries out, and the ratio of material and liquor capacity all remained on 1: 100.After material-80 ℃ pre-freeze processing, to use vacuum freeze liquid and carry out lyophilization, sublimation drying is 48h; Use pulverizing homogenate equipment then and carry out pulverization process, use the submucous layer of small intestine stromatolysis after 6% acetic acid solution is handled the lyophilization pulverizing.Lyophilization pulverizing and dissolving step repeat 2 times.
Fresh tendon is taken from the pig heel string of enclosed grazing, shreds the back and immerses 0.05mol/L acetic acid, puts in 4 ℃ of refrigerators, shakes, and behind the 48h, moves in the sterilization centrifuge tube; The centrifugal 30min of 4000r/min draws supernatant, and the ratio that adds 5g NaCl in the 100mL supernatant adds the NaCl crystal, and visible volume precipitation is separated out, the centrifugal 10min of 1000r/min once more, and abandoning supernatant, the gained precipitation is collagen stroma; Use vacuum freeze liquid and carry out being dissolved in the 0.05mol/L acetic acid behind the lyophilization 48h, make collagen stroma solution.The used raw material of this preparation collagen stroma solution step can also be pig dermis tissue, cartilaginous tissue, and processing method is organized identical (omiting) herein with heel string.
Submucous layer of small intestine matrix solution and collagen stroma solution are pressed 1: 1 mixed; The chondroitin sulfate C drips of solution is added in this mixed solution, stirring and evenly mixing, the whole mass concentration that makes chondroitin sulfate C is 80g/L.Then mixed solution is poured in ready-formed glass or the stainless steel mould, rolled flatly, the refrigerator pre-freeze of putting into-80 ℃ is more than 24 hours.Use vacuum freeze lyophilization 12 ~ 24 hours, use ultraviolet radiation 24 ~ 48 hours then the material after the lyophilization is carried out crosslinking Treatment, if it is not crosslinked thorough, again material is put into 0.005% glutaraldehyde solution, put 4 ℃ of refrigerator 24h hours, with filtering tap water rinsing 2 hours, change distilled water then into and put 4 ℃ of refrigerators 24 hours, remove not in conjunction with remaining glutaraldehyde.The washing of application sodium phosphate buffer; Material after will washing is then hatched in glycine-HCl buffer to eliminate remaining fixative and to reduce pH value, is 7.35 ~ 7.55 until pH value.
Tissue after the above-mentioned processing is soaked in the metronidazole homogenizing aqueous solution of the gentamycin sulfate that mixes 50mg/ml and 50mg/ml, takes out after 10 minutes, drying is soaked in tissue in the sodium dodecyl sulfate solution of 50mg/ml lyophilization 24 hours then.
Material after the above-mentioned processing is soaked 8h in containing 20% alcoholic solution of 0.1% peracetic acid, cleans 2h, procedurally then cool to-80 ℃ with the PBS solution that contains 0.05% Hydrazoic acid,sodium salt, after the lyophilization with radiation gamma (25～35kGy) sterilizations.
Embodiment 2: should choose small intestine and tendon tissue or dermal tissue, cartilaginous tissue prepare medical bioavailability bracket material
Present embodiment is raw materials used to be fresh human corpse's small intestine and tendon tissue or dermal tissue and cartilaginous tissue, and all the other preparation methoies are identical with embodiment 1.
Embodiment 3: application calf intestinal tissue and tendon tissue or dermal tissue, cartilaginous tissue prepare medical bioavailability bracket material
Present embodiment is raw materials used to be small intestine and tendon tissue or dermal tissue and the cartilaginous tissue of the cattle of enclosed grazing, and all the other preparation methoies are identical with embodiment 1.
Embodiment 4: medical bioavailability bracket material provided by the present invention is carried out evaluation of its biocompatibility
Ratio in film surface area and lixiviate medium volume is 3ml/cm
2Embodiment 1, embodiment 2, material that embodiment 3 provided are joined in the respective media (a kind ofly be normal saline, another is the DMEM complete culture solution), in 37 ℃ of calorstats, leave standstill and be prepared into lixiviating solution in 24 hours, in 24 hours, carry out following biological experiment.
1. cell toxicity test (mtt assay)
Former goat bone marrow stroma stem cell of being commissioned to train foster grows to the logarithmic growth after date, with 4 * 10
3The cell density of/ml is inoculated in 96 orifice plates, every hole 100 μ l, cultivate and carry out lixiviating solution displacement (medium is DMEM complete culture solution person) after 24 hours, establish lixiviate stock solution group, 1/2 liquid group, 1/5 liquid group, negative control group (DMEM complete culture solution) and blank group (the DMEM complete culture solution that does not add cell) respectively, every group 10 hole.Continue to cultivate after 72 hours, add 0.5%MTT20 μ l, hatch and end after 4 hours to cultivate, abandon supernatant, add DMSO150 μ l/ hole, shook immediately 5 minutes, measure every hole OD value on the enzyme-linked immunosorbent assay instrument.
2. pyrogen testing
It is (male to get 3 of the new zealand white rabbits of raising under the qualified the same terms of body temperature screening, healthy adult, body weight 1.5kg～2.5kg), under the tranquility, respectively surveyed body temperature in preceding 75 minutes, 45 minutes, 15 minutes respectively at injection and get average 3 times, the meansigma methods of twice body temperature in back is a normal body temperature.Then from the lixiviating solution (2ml/kg) of auricular vein injection prepared fresh, to inject back 1 hour, 2 hours, 3 hours and respectively survey body temperature next day 1 time, elevated temperature subtracts normal body temperature for the high order in the body temperature of injection back.
3. hemolytic test
Each 3 pipe of three kinds of material groups (normal saline soaks body fluid), negative control group (normal saline) and positive controls (distilled water), every pipe adds respective liquid 10ml, put in 37 ℃ of water baths preheating after 30 minutes, every pipe adds dilution anticoagulant Sanguis Leporis seu oryctolagi 0.2ml (heart extracting blood, the potassium oxalate anticoagulant, the normal saline dilution), the rearmounted 37 ℃ of water bath relayings continuation of insurance of mixing temperature is 1 hour gently, centrifugal absorption supernatant, mensuration is respectively managed absorbance under the ultraviolet spectrophotometer 540nm wavelength, normal saline and distilled water that the same terms is handled down are blank, calculate hemolysis rate.
4. hypersensitive test
Adopt the healthy albefaction Cavia porcellus that just grows up of same outbreeding strain to test, divide experimental group, negative control group and positive controls, each 3 animal is respectively at every the plucked scapula inside line of animal intradermal injection SIS lixiviating solution, normal saline and each 0.1ml of 5% formalin.The local speckle that pastes in intradermal injection one week back was induced in 48 hours, and two all local excitation are 24 hours afterwards, removed to observe every animal skin of abdomen respectively at 24 hours, 48 hours, 72 hours three time points behind the thing of applying ointment or plaster and excite position erythema and edema response situation.
The gained data are carried out statistical analysis with the SAS6.12 statistical software.Measurement data represents that with arithmetical average ± standard deviation (x ± s), group difference adopts paired data t check, is that difference has significance with p＜0.05.
5. result
5.1 cell toxicity test
Inverted phase contrast microscope is observed down, three kinds of material lixiviating solution groups and the growth of negative control group cell attachment, form is good, prolongs visible cell quantity in time and increases gradually, and forming a plurality of clones, three material groups are compared with negative control group and are not seen notable difference (p＞0.05).
5.2 pyrogen testing
In 3 experimental rabbits, fervescence all is lower than 0.6 ℃, and fervescence adds up to 0.90 ℃ (＜1.4 ℃), meets the standard-required of biomaterial pyrogen testing, and the material that embodiment 1, embodiment 2 and embodiment 3 are provided does not all have the pyrogenicity reaction.
5.3 hemolytic test hemolytic test
The hemolysis rate that is calculated the material that gained embodiment 1, embodiment 2 and embodiment 3 provided by formula is respectively 3.94% and 3.71%, all is lower than 5%.Show all haemolysis not of material that embodiment 1, embodiment 2 and embodiment 3 provided, the hemolytic test requirement of composite biological material.
5.4 hypersensitive test
The lixiviating solution of the material that embodiment 1, embodiment 2 and embodiment 3 are provided and normal saline treated animal dermoreaction all do not have erythema is not had edema, proves that bioavailability bracket material provided by the present invention does not have sensitization, meets the requirement of biomaterial hypersensitive test.
6. conclusion
According to the experimental technique that present embodiment provided, the correlation technique and the standard of the medical apparatus and instruments biological assessment of formulating with reference to international standard ISO10993-1, the biocompatibility and the immunogenicity of the material that embodiment 1, embodiment 2 and embodiment 3 are provided are tested and are estimated, the result confirms bioavailability bracket material avirulence provided by the present invention, nonirritant and immunogenicity, and the bio-compatible part is good.
Embodiment 5: the porosity of the pig intestinal mucosa lower floor substrate of medical bioavailability bracket material provided by the present invention and conventional method preparation and mechanical strength contrast
Adopt the material that scanning electron microscope provides embodiment 1 respectively and the pig intestinal mucosa lower floor stromal surface of conventional method preparation to scan, measure its surface apertures size.Place 10% (volume fraction) neutral formalin fixing respectively both, after dehydration, transparent, waxdip, embedding, section and dewaxing, HE dyeing.Carry out the detection of porosity by following method.Porosity=[weight (g) after weight (g)-lyophilization of infiltration D-Hanks]/volume (cm
3) * 100%.Every group is detected 10, averages.
With Tianjin, island AG20KNA Material Testing Machine the material that the pig intestinal mucosa lower floor substrate and the embodiment 1 of conventional method preparation provided is carried out tension test, the speed of tension test is 10mm/ minute.
The material surface that result: embodiment 1 provides is more smooth, and hole is even, softness, and good springiness, water absorption is strong, and putting into water behind the expressed water again can restore to the original state immediately.Its collagen fiber are interconnected to porous network structure, mesh size basically identical, about 30 ~ 60 μ m.HE dyeing, visible support is light red, and is wavy, often has branch also to interweave mutually.Porosity is (90 ± 5.8) % (accompanying drawing 1a).The pig intestinal mucosa lower floor stromal surface of conventional method preparation shows uneven, the fracture of part collagen fiber, and pore size is inhomogeneous, and 30 ~ 200 μ m do not wait.Porosity is (75 ± 6.3) % (accompanying drawing 1b).
Stretch test result shows: the yield stress of the pig intestinal mucosa lower floor substrate of conventional method preparation is 6.24 ± 0.72MPA, and strain is 36.23 ± 9.52; The yield stress of the material that embodiment 1 is provided is 10.31 ± 0.65MPA, and strain is 57.21 ± 7.55.
Conclusion: with the pig intestinal mucosa lower floor matrix phase ratio of conventional method preparation, the fiber alignment rule of material provided by the present invention, hole is more even, and porosity obviously increases, and has higher mechanical strength.
Embodiment 6: the mensuration of growth factor content in the medical bioavailability bracket material provided by the present invention
Get the lyophilized powder 10mg of the material that embodiment 1 provided, placing PBS1ml (pH=7.4) to shake for 37 ℃ hatches, in different time points sampling, press EL ISA test kit description bioassay standard product and sample neutral and alkali fibroblast growth factor (β-FGF), VEGF (VEGF), platelet derived growth factor (PDGF), the β transforming growth factor (protein content of β-TGF).
BFGF content is about (101.8 ± 2.163) ng/L in the PBS Incubating Solution, and VEGF content is about (89.8 ± 2.053) ng/L, and PDGF content is about (45.8 ± 4.834) ng/L, and β-TGF content is about (35.8 ± 3.065) ng/L.
Embodiment 7: use the active corium of medical bioavailability bracket material external structure provided by the present invention
The material that embodiment 1, embodiment 2 and embodiment 3 are provided is inserted in 6 orifice plates respectively, soak 48h with IMDM, cultivate going down to posterity the 3rd generation fibroblast plant on ADM with 1.0 * 106/cm2 density, IMDM adds 10% (volume fraction) calf serum, and the cell culture incubator of 37 ℃, 5% (volume fraction) CO2, saturated humidity is cultivated.Change liquid every other day.Observation of cell growth and metamorphosis situation under the inverted microscope.
Seeing under the inverted microscope in material periphery that embodiment 1, embodiment 2 and embodiment 3 are provided and the mesh all has fibroblast adherent, and cell begins propagation behind the 3d, growth (accompanying drawing 2a) in SCM surface and mesh.Be cultured to 14d, filled by fibroblast and secreted substrate thereof in the visible mesh under the mirror, material is not seen obvious contraction (accompanying drawing 2b).Be cultured to 21 days, material does not have degraded.
Conclusion: present embodiment is planted fibroblast on biologic bracket material provided by the present invention, fibroblast forms a monolayer at material surface, be full of by fibroblast and excretory substrate thereof in the mesh gap of material, material provided by the present invention does not shrink, and being cultured to 21 days does not have degraded.Show that it provides the place of adhering to for the fibroblast of newborn corium,, can prepare the organization engineering skin and the wound dressing that are used for the treatment of skin injury, burn, wound for cell attachment propagation and differentiation provide solid space.
Implement 8: medical bioavailability bracket material provided by the present invention is used for the treatment of hernia and abdominal-wall defect
Case is selected: oblique hernia patient 10 examples, straight hernia patient 5 examples.
Operation method: turn in the high-order separation also of conventional row hernical sac, get patch size and be generally 6cm * 8cm, sticking patch should be cut spoonful shape and pass through to hold spermatic cord in the internal ring corresponding position.Sticking patch sutured, inboard are stitched to the pubic tubercle place, should surpass and covering tuberosity 115～210cm, and sew up with conjoined tendon.The sticking patch lower edge should be sewed up with inguinal ligament and iliopubic tract, the outside surpasses internal ring top 2.0～3.0cm, and upper limb and obliquus externus abdominis m. backing layer are sewed up, and makes sticking patch cover inguinal whole bottom and also surpasses bottom boundary, made sufficient tissue contact surface and kept suitably lax, should avoid curling.
Curative effect: but postoperative out-of-bed activity after 4～6 hours; Complication does not take place in postoperative; All the case postoperative was left hospital in 3～7 days.All follow up a case by regular visits to, the shortest 6 months, the longest 3 years, there is not recurrence.
Application Example 2 and embodiment 3 prepared materials can reach same effect.
Conclusion: material provided by the present invention can prepare the embedded material that is used for the treatment of abdominal external hernia and other abdominal-wall defect diseases.
Embodiment 9: medical bioavailability bracket material provided by the present invention is used for the treatment of anal fistula
Case is selected: selects in June, 2006 ~ 2007 anal fistula patient in year June totally 30 examples, the wherein simple property of low level anal fistula 10 examples, low level complexity anal fistula 4 examples, high-order property anal fistula 6 examples, high-order complexity flaccid paralysis 3 examples merely.
Operation method: seek internal orifice; Carry out debridement then, guarantee debridement and haemostatic effect.According to fistula road situation, the material that embodiment 1 is provided is cut into suitable size and dimension in advance, inserts the anal fistula probe; The material that traction embodiment 1 is provided enters the fistula road through internal orifice, draws through collar extension.The per rectum mucosa is with material bottom " 8 " word sutured.Remove the most advanced and sophisticated redundance of material, make most advanced and sophisticated parallel with anal skin.Collar extension covers aseptic dressing and changes dressings until healing every day.
Curative effect: through following up a case by regular visits to result such as following table:
| The example number | Healing NO/% | Recurrence NO/% | Infect NO/% | Patient satisfaction | |
| The simple property of low level anal fistula | 10 examples | 9/90％ | 0/0 | 0％ | Satisfied |
| Low level complexity anal fistula | 4 examples | 3/75％ | 1/25％ | 0％ | Satisfied |
| High-order simple property anal fistula | 6 examples | 5/67％ | 1/17％ | 0％ | Satisfied |
| High-order complexity flaccid paralysis | 3 examples | 2/67％ | 1/33％ | 0％ | Satisfied |
Application Example 2 and embodiment 3 prepared materials can reach same effect.
Conclusion: material provided by the present invention can prepare the embedded material that is used for the treatment of anal fistula.
Embodiment 10: medical bioavailability bracket material provided by the present invention is used for the treatment of recto-vaginal fistula
Case is selected: select in June, 2005 ~ 2007 recto-vaginal fistula patient in year October totally 5 examples, wherein, anal orifice and rectal intestine peripheral abscess 2 examples, postoperative rectal cancer 1 example, 2 examples behind the obstetrics and gynecology operation.
Operation: as the case may be, take through the perineum curved incision, separate the rectum vagina at interval, separating ranges surpasses fistula mouth 1cm on every side, prunes away around the fistula mouth and the scar tissue in fistula road.The degree of depth according to fistula mouth size and shape and fistula road is cut into suitable size and dimension with embodiment 1 prepared biology filling drain in advance with shears, as circular, cigarette shape, strip etc.The circular biological filling drain that cuts is clogged in rectum vaginal septa gap around the fistula mouth, noted sprawling neatly, the filling scope should surpass more than the fistula mouth 1cm; With the biological filling of strip or cigarette shape drain through the middle heart tamponade of two fistula mouths and circular biological filling drain in the fistula road, biology is clogged drain both sides and levator ani m. edge, rectal wall and vaginal wall sutured.Mucosal tissue is closed the rectal fistula mouth around sewing up the rectal fistula mouth, notes simultaneously the rectum end of strip or the biological filling of cigarette shape drain being fixed.Strip or the biological filling of cigarette shape drain vagina end transvaginal fistula mouth place are drawn.Remove biological filling drain outer end redundance, make biological filling drain vagina end parallel with vaginal mucosa.Intravaginal covers aseptic dressing and changes dressings until healing every day.
Curative effect: through following up a case by regular visits to, the result is as described below:
Patient's average time in hospital day is 8.2 days, behind the 10 routine corrective surgeries complication does not take place all, the equal first phase healing of otch.Through following up a case by regular visits to 1～9 year, there is not 1 example recurrence.
Application Example 2 and embodiment 3 prepared materials can reach same effect.
Conclusion: material provided by the present invention can prepare the embedded material that is used for the treatment of recto-vaginal fistula.
Embodiment 11: medical bioavailability bracket material provided by the present invention is used for the treatment of encysted rectum
Case is selected: select in June, 2003 ~ 2007 encysted rectum patient in year October totally 15 examples, it is middle and aged women.
Operation: get the about 3cm of the nearly vagina side of perineal position curved incision, cut skin, subcutaneous tissue, the passivity separation is walked around the external anal sphincter leading edge and is reached, and separates the open texture of recto-vaginal septum, separating ranges reaches 1cm on the weak area, reaches preceding intersection of rectum of isolating perineal body and levator ani m..Be cut into suitable size according to the big young pathbreaker embodiment 1 prepared biology filling drain of weak area in advance with shears.The biology filling drain that cuts is clogged in rectum vaginal septa weak area, noted sprawling neatly sutured.Biology is clogged the drain outer end draws through the perineal incision place.Sew up subcutaneous tissue and skin.Remove biological filling drain outer end redundance, make biological filling drain outer end parallel with otch skin.Otch covers aseptic dressing and changes dressings until healing every day.
Curative effect: through following up a case by regular visits to, the result is as described below: 15 routine patient's postoperative proctography checks all point out encysted rectum to disappear, and do not have stool and are detained.Follow up a case by regular visits to and do not see recurrence, cure rate 100% in 6 months～2 years.3～6 days postoperative hospital stayss, average 4.8 days.Postoperative main suit pain 2 examples all can tolerate, and need not to handle.It is normal that patient's anus profile is recovered, and no blood stasis is deviate from, and defecation is smooth and easy, and nothing is had blood in stool, anal stenosis, infection, fecal incontinence.
Application Example 2 and embodiment 3 prepared materials can reach same effect.
Conclusion: material provided by the present invention can prepare the embedded material that is used for the treatment of encysted rectum.
Embodiment 12: medical bioavailability bracket material provided by the present invention is used for the treatment of hemorrhoid
Case is selected: select in June, 2006 ~ 2007 hemorrhoid patient in year October totally 20 examples, wherein III degree internal hemorrhoid 10 examples, IV degree internal hemorrhoid 5 examples.
Operation method: prick art or whitehead operation excision hemorrhoidal tissue in the outer stripping of tradition; According to concrete condition in the operation material that embodiment 1 is provided is cut into suitable size in advance with shears, on one side and the fillet that will be wherein cut out wide a 0.2 ~ 1cm be used as drainage strip, be filled under the mucosa in the wound surface, note sprawling neatly.Drainage strip through mucous membrane otch is drawn; Sew up the mucosal tissue that cuts; Remove the most advanced and sophisticated redundance of drainage strip, make hemorrhoid wound surface implant most advanced and sophisticated parallel with mucosa.One piece of one of anal placement povidone iodine cotton balls and ZHICHUAN SHUAN, and change dressings until healing every day.
Curative effect: 10 routine postoperatives are when average daily no pain, archorrhagia, and average hospital days is (3.62 ± 1.23) sky, and the time of resuming work is 7.03 ± 1.83 days (4～10 days), follow up a case by regular visits to 3 months to 1 average annual nothing recurrence.
Application Example 2 and embodiment 3 prepared materials can reach same effect.
Conclusion: material provided by the present invention can prepare the embedded material that is used for the treatment of hemorrhoid.
Embodiment 13: medical bioavailability bracket material provided by the present invention is used for the treatment of the damaged zoopery of vascular tissue
The material webs that embodiment 1 is provided does not have the wound prolene suture with 8-0 and is stitched into pipe continuously along the longitudinal axis to the polyethylene axle.
Get 24 of bull new zealand white rabbits, adopting the random digit method to divide equally is 2 groups, and the matched group graft materials is for making pig intestinal mucosa lower floor pipe; The experimental group graft materials is the pipe of material provided by the present invention.Length is 15mm.Operating procedure is as follows: behind the rabbit anesthesia, make the outside, throat longitudinal incision, the free common carotid artery that exposes.Wipe out the part tremulous pulse betwixt, cause about 15mm vascular defects.The graft materials two ends are not had the capable end to end anastomosis bridge joint of wound suture interrupted suture with blood vessel two broken ends of fractured bone with 8-0 respectively repair that this is damaged.Check and confirm the unobstructed property of blood flow that observe 30min, it is good to keep clear.Postoperative subcutaneous injection heparin 1250U/kg 2 every day in week.In postoperative 1,2 week with did the blood vessel Color doppler ultrasound in 1,2 month; Draw materials in the time of 1,2 month respectively at postoperative in two batches for every group and do histology observing tube wall construction and tissue reaction's (hematoxylin-eosin staining and Masson dyeing).
Main observation index: 1. vitro detection support burst pressure, hemolysis rate, cytotoxicity.2. the back observation unobstructed property of support that implants and histology's performance.
The result: tube wall does not all take place and breaks, reveals phenomenons such as blood in all supports in implantation.1 all ultrasonic examinations showed 8 generation obturations after 12 rabbits were made small intestinal submucosa intravascular stent implantation, and tube chamber built-in wall thromboembolism is seen in the capable histological examination of drawing materials; 4 in 2 months detection, keep clear (patency rate 33.3%) in addition, but all have aneurysmal to change (accompanying drawing 3c).After the pipe implantation of 12 material provided by the present invention, 1 obturation when 2 weeks; All the other 11 keep clear always, patency rate 91.6% (accompanying drawing 3b) is significantly higher than matched group (P＜0.05), and wherein 2 aneurysmal the time occurs since 2 weeks and change (18.1%), be lower than matched group (P＜0.05), 8 then keep smooth tubular structure in addition.Histology finds that the support that material group provided by the present invention keeps clear is existing complete endothelium covering in the time of 1 month after surgery, a small amount of vascular smooth muscle cell of having grown in the collagen fiber of outer small intestinal submucosa, NIP infiltration phenomenon (accompanying drawing 3a); And the small intestinal submucosa group in the time of 1 month after surgery the collagen composition obviously reduce, do not see neointimal hyperplasia.
Application Example 2 and embodiment 3 prepared materials can reach same result.
Conclusion: material provided by the present invention can be prepared into small-caliber vascular stent, blood compatibility is good, mechanical property is fit to, can be used for repairing vascular defects, can keep long-term unobstructed and complete vascular wall tissue to reinvent, and form or thrombotic sign without any graft infection, aneurysm, obviously be better than the pig intestinal mucosa subsurface material of conventional method preparation.Material provided by the present invention can be used for preparing the damaged embedded material of reparation vascular tissue.
Embodiment 14: medical bioavailability bracket material provided by the present invention is used for the treatment of the damaged zoopery of nervous tissue
Choose 24 of healthy SD rats, be divided into three groups by the principle of completely randomization, i.e. the neural group of embodiment 1 material bridge joint, nerve autograft matched group and blank group.Every group each 8, all adopt right sciatic nerves as repairing model.Postoperative 6 week and got in 10 weeks and cut, respectively 4.
Separate and to appear the rat right sciatic nerves, cut the sciatic nerve of 10mm length with double-edged razor blade, cause the neurologic defect model apart from sciatic nerve outlet 1cm.The neural group of embodiment 1 material bridge joint is sewed up the damaged two ends of bridge joint with the material that ready embodiment 1 is provided under operating microscope.Sciatic nerve section under the nerve autograft matched group will cut is near, far-end is put upside down and is sutured in that sciatic nerve is near, far-end.The blank group is spacious puts damagedly, does not do any processing.: the far-end of sewing up mouthful respectively at near-end of sewing up mouthful from near-end in 6 weeks of postoperative, 10 weeks and far-end cuts the sciatic nerve of rat, carries out histological observation; And carry out electric physiological detection with viso-scope, measure sciatic incubation period of rat both sides and bring out the wave amplitude of current potential.
The result shows: as seen neural group of embodiment 1 material bridge joint and nerve autograft group all have nervous tissue's regeneration.Compare with the nerve autograft group, the preclinical retardation rate of the neural group of embodiment 1 material bridge joint increases to some extent, but there was no significant difference (P＞0.05).
Application Example 2 and embodiment 3 prepared materials can reach same result.
Conclusion: material provided by the present invention has the effect that promotes the peripheral nerve axon growth, repairs peripheral nerve defection, can be used for preparing the damaged embedded material of reparation nervous tissue.
Embodiment 15: medical bioavailability bracket material provided by the present invention is used for the treatment of the damaged zoopery of tendon tissue
Get 20 of healthy adult new zealand rabbits, excision posterior cord heel string, resection length is 1.5cm.Substituting of long embodiment 1 material that provides of 1.5cm is provided.1,4,8,12,16 weeks of postoperative are respectively put to death 4 animals, draw materials to comprise experimental side and normal control side, carry out histological examination and mechanical test respectively.
Histological observation: 1 week of postoperative, a large amount of ellipses of experimental side or round-formed fibrocyte, the collagen integral frame is still complete, and is fine and close; In 4 weeks of postoperative, a large amount of ellipses of experimental side or round-formed fibrocyte are assembled agglomerating, and the cell peripheral in cell aggregation district has newborn collagen to form, and focal streak ripe collagen is island and distributes, and forms " collagen island " (accompanying drawing 4a); In 12 weeks of postoperative, experimental side collagen band connects in flakes, and immature collagenous region is obviously dwindled, and forms the focal zone that is dispersed in distribution; In 16 weeks of postoperative, the performance of experimental side and normal tendon is very near (accompanying drawing 4b).
Mechanical test: 1 week of postoperative, experimental side maximum load 54N ± 6N; 4 weeks of postoperative, experimental side maximum load 59N ± 5N; 8 weeks of postoperative, experimental side maximum load 98N ± 9N; 12 weeks of postoperative, experimental side maximum load 154N ± 14N, 16 weeks of postoperative, experimental side maximum load 169N ± 10N; Normal tendon: maximum load 172N ± 7N.In prompting 1 to 16 week of postoperative, the mechanical strength of implant strengthens and approaching normal tendon tissue gradually.
Application Example 2 and embodiment 3 prepared materials can reach same result.
Conclusion: material provided by the present invention has that to repair tendon damaged, and reaches the effect of tendon mechanical strength, can be used for preparing repairing the damaged embedded material of tendon tissue.
Embodiment 16: medical bioavailability bracket material provided by the present invention is used for the treatment of bone and the damaged zoopery of cartilaginous tissue
After the healthy new zealand white rabbit anesthesia, in the about 1cm of patella lower edge, the bone that vertical condyle of femur bores a diameter 6mm, dark 10mm is damaged, the damaged model of preparation rabbit femoral condyle.It is damaged that the material that adopts embodiment 1, embodiment 2 and embodiment 3 to be provided is respectively repaired condyle of femur.Clinical follow animal diet followed, activity and wound change.And put to death animals in postoperative 2,4,8 and 12 weeks, draw materials, observe material and bone damaged be connected degree and skeletonization situation.2 weeks of postoperative are taken out material, transmission electron microscope observing osteoblast form.HE dyeing, microscopically is observed skeletonization and material degradation situation.
The result: each treated animal postoperative is suffered from the limb limping, can normally take action after about 1 week, and activity freely.The postoperative animal all can normally be looked for food, and wound does not have infection, average 10d wound healing.Wound healing is good when drawing materials, and no material exposes and discharges.Gross examination of skeletal muscle: in 2 weeks of postoperative, material is connected with the defective region surface of bone; In 4 weeks, it is harder that material touches, not movable, firmly is connected with the defective region surface of bone; In 8 weeks, material is at bone defective region skeletonization; In 12 weeks, material touches hard, and color and luster is replaced by osseous tissue substantially near host bone.Electron microscopic observation: 2 weeks of postoperative, histological observation: 2 weeks of postoperative, the visible osteoblast of edge of materials is assembled, and cell is active, the visible projection in surface, karyon is big, contain and enrich kernel, see in the endochylema and enrich endoplasmic reticulum, the also visible many active osteoblast of material internal, there is area of new bone to form, visible a large amount of new vesselses in the material; In 4 weeks, area of new bone further increases, and the part area of new bone merges, and forms bone trabecula, accidental little pulp cavity; Still see a large amount of new vesselses (accompanying drawing 5a) in the material.In 8 weeks, material has degraded in various degree, and a large amount of area of new bone form and are fused into bone trabecula, and little pulp cavity exists in a large number; In 12 weeks, material is degraded substantially, and bone trabecula merges, and forms the spongy bone pulp cavity, and (accompanying drawing 5b) appears in visible fat drop in the pulp cavity.
Conclusion: material provided by the present invention can repair bone and cartilaginous tissue is damaged, reinvents bone and cartilaginous tissue, can be used for preparing reparation bone and the damaged embedded material of cartilaginous tissue.
Embodiment 17: it is damaged that medical bioavailability bracket material provided by the present invention is used for the treatment of oral mucosas tissue
Select extension tooth plantation mucomembranous defect patient 3 examples, at once tooth plantation mucomembranous defect patient 2 examples, carcinoma of maxillary sinus excision mucomembranous defect patient 3 examples, the swollen thing excision of palatine mucomembranous defect patient 1 example, damaged patient's 2 examples of wound Mucosa of lip, white macula and lichen planus excision mucomembranous defect patient 1 example, operation method is with thoroughly excision of pathological change of oral cavity, soft tissue release or after implanting the plantation nail routinely, wound surface thoroughly stops blooding, the normal saline flushing wound surface; The size damaged according to oral mucosas tissue, the material that embodiment 1 is provided is cut into suitable size and dimension in advance, spreads on the damaged wound surface, sews up, fixes.Postoperative was removed dressing in 8 days.
The equal I phase of 12 routine patient's wound surface heals, and the graft color and luster was a magnetic white when postoperative was taken out stitches in 8 days, combined well with surrounding tissue, did not have obvious hyperemia and edema.Postoperative January, do not find rejection, acellular dermal matrix is white powder, and wound surface epithelization degree height is good with wound surface surrounding edge tissue bond, attaches closely with basilar part, and it is unclear to demarcate.
Application Example 2 and embodiment 3 prepared materials can reach same result.
Conclusion: material provided by the present invention can be repaired reconstruction of oral defect, can be used for preparing the embedded material of repairing reconstruction of oral defect.
Embodiment 18: medical bioavailability bracket material provided by the present invention is used for the urinary tract reparation
After male Wistar rat is anaesthetized successfully, get the median abdominal incision incision and appear bladder, about 1 * 1cm is excised at the bladder top of dissociating, and the material that Application Example 1 is provided is cut into suitable shape and size in advance, is fit to the bladder defective region with the absorbable thread holostrome.1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks are got the material of implantation after surgery respectively, and hematoxylin-eosin staining is observed rat bladder and repaired situation.
The result: visible urinary bladder transitional epithelium forms on postoperative 1 all materials, has inflammatory cell to invade profit (accompanying drawing 6a) simultaneously; Visible new capillary vessel infiltrates on the 2nd all materials; The 4th week, existing complete urinary bladder transitional epithelium formed, simultaneously visible a small amount of immature contractile fiber cells; The 10th all visible significantly smooth muscle muscle bundles (accompanying drawing 6b).
Application Example 2 and embodiment 3 prepared materials can reach same result.
Conclusion: material provided by the present invention migrates to the damaged place of urinary tract urinary system epithelium, smooth muscle and serous coat regeneration, can repair urethra and bladder body is damaged, reinvents urinary tract, can prepare and be used to repair the damaged embedded material of urinary tract.
Embodiment 19: medical bioavailability bracket material provided by the present invention is used for rebuilding treatment female incontinence and uterine prolapse at the bottom of the basin
Case is selected: select in June, 2005 ~ 2007 female incontinence patient in year October totally 5 examples, the uterine prolapse patient is totally 5 examples.
Operation: adopt hypomere suspension and bilateral jarjavay's ligaments suspension in the urethra respectively, respectively that lower urinary tract and uterus suspention is fixing with the material that embodiment 1 is provided.
Curative effect: followed up a case by regular visits to 3 months to 1 year, 10 routine patients are all respond well, and complication such as urinary tract obstruction, infection do not take place in recurrence.
Application Example 2 and embodiment 3 prepared materials can reach same result.
Conclusion: material provided by the present invention can prepare and be used for rebuilding the implantation equipment of relaxed pelvic floor diseases such as treatment urinary incontinence and uterine prolapse at the bottom of the basin.
Claims (25)
1. the preparation method of a medical bioavailability bracket material is characterized in that, described preparation method may further comprise the steps:
A. prepare submucous layer of small intestine substrate step, described step is the submucous layer of small intestine tissue of separation of human or animal, soaks 16h in the solution that contains 100mmol/L ethylenediaminetetraacetic acid and 10mmol/L sodium hydroxide; Rinse well with deionized water then, place the solution that contains 1mmol/L hydrochloric acid and 1mmol/L sodium chloride to soak 6～8h; With in 1mmol/L sodium chloride phosphate buffer, soaking 16h behind the deionized water rinsing; Then with in PBS solution, soaking 2h behind the deionized water rinsing; Reuse deionized water rinsing 2h; Each step of said process all at room temperature carries out, and the ratio of material and liquor capacity all remained on 1: 100;
B. lyophilization, pulverising step, described step are to use vacuum freeze mixed solution is carried out lyophilization, use pulverizing homogenate equipment then and carry out pulverization process, and described step can repeat 2-3 time;
C. dissolving step, described step are to use dilute acid soln submucous layer of small intestine stromatolysis after handling is pulverized in lyophilization;
D. prepare collagen stroma solution step, described step is the tendon of people or animal or cartilage, skin histology to be shredded the back immerse 0.05mol/L acetic acid, puts in 4 ℃ of refrigerators, shakes, and behind the 48h, moves in the sterilization centrifuge tube; The centrifugal 30min of 4000r/min, draw supernatant, the ratio that adds 5g NaCl in the 100mL supernatant adds the NaCl crystal, centrifugal, abandoning supernatant stays the collagen stroma precipitation, use vacuum freeze liquid and carry out being dissolved in the 0.05mol/L acetic acid behind the lyophilization 48h, make collagen stroma solution;
E. blend step, described step is mixed in proportion submucous layer of small intestine matrix solution and collagen stroma solution;
F. add chondroitin sulfate C solution step, described step is that the chondroitin sulfate C drips of solution is added in the above-mentioned mixed solution, and constantly stirs, and the whole mass concentration that makes chondroitin sulfate C is 10-80g/L;
G. forming step, described step is that mixed solution is poured in ready-formed glass or the stainless steel mould, rolls flatly, the refrigerator pre-freeze of putting into-80 ℃ is more than 24 hours;
H. lyophilization step, described step are to use vacuum freeze mixed solution is carried out lyophilization;
I. cross-linking step, described step are to use the composite material of methods such as cross-linking agent, ultraviolet radiation after to lyophilization to carry out crosslinking Treatment;
J. the step of washing and eliminating residual cross-linking agent and regulate pH value, described step are to use the material of neutral buffered liquid after to crosslinking Treatment to wash, and use Freamine hatch to pH value be 7.35 ~ 7.55;
K. introduce the step of antibiotic coating;
L. sterilization steps.
2. preparation method according to claim 1, it is characterized in that, the step of described introducing antibiotic coating is the mode by spraying, soaking or instil, antibiotic homogenizing aqueous solution is incorporated in the tissue, through evaporation and/or after using desiccant and removing water basically, add at least a amphiphatic molecule solution soluble in water by same method again, obtain antibiotic coating through evaporation and/or after using the desiccant drying again.
3. preparation method according to claim 1; it is characterized in that; described cross-linking agent is selected from glutaraldehyde, formaldehyde, the carbodiimides, 1 of suitable concentration; hexamethylene-diisocyanate, genipin, 1; 4-two (3; the 4-hydroxy benzenes)-2,3 dimethylbutane, epoxide, the dicarboxylic acid compound that contains disulphide functional group, acyl azide and diphenylphosphine hydrochlorate and glyoxalic acid.
4. preparation method according to claim 1 is characterized in that described ratio is 1: 1-1: 5.
5. preparation method according to claim 1 is characterized in that, described crosslinking Treatment is to use ultraviolet radiation to handle.
6. preparation method according to claim 1 is characterized in that, described crosslinking Treatment is to be immersed in 0.005% glutaraldehyde solution, puts 4 ℃ of refrigerator 24h hours.
7. preparation method according to claim 1 is characterized in that, described dilute acid soln is 1 ~ 6% acetic acid solution.
8. according to claim 1 or 3 described preparation methoies, it is characterized in that described cross-linking agent is 0.005% glutaraldehyde.
9. preparation method according to claim 2, it is characterized in that described amphiphatic molecule is selected from alkyl sulfate, alkylsulfonate, alkyl aryl sulfate, di alkylaryl sulfate, alkylaryl sulfonates, di alkylaryl sulfonate, cycloalkyl sulfate, cycloalkyl sulfonate, alkyl-cycloalkyl sulfate.
10. preparation method according to claim 2, it is characterized in that described antibiotics salt is selected from aminoglycoside antibiotics, tetracycline antibiotics, lincosamides, quinolone antibiotic, nitro glyoxaline antibiotic, sulfonamides antibiotic, methylfuran class antibiotic, itrofurans antibiotic or above-mentioned antibiotic compositions.
11. preparation method according to claim 10 is characterized in that, described antibiotic is the compositions of sulmycin and metronidazole.
12. a medical bioavailability bracket material is characterized in that, the application of described material in the preparation tissue engineering material.
13. application according to claim 12 is characterized in that, described application is organization engineering skin and the wound dressing that preparation is used for the treatment of skin injury, burn, wound.
14. the application according to claim 12 is characterized in that, described application is the implantation equipment that preparation is used for the treatment of abdominal external hernia and abdominal-wall defect.
15. the application according to claim 12 is characterized in that, described application is the implantation equipment that preparation is used for the treatment of anal fistula.
16. the application according to claim 12 is characterized in that, described application is the implantation equipment that preparation is used for the treatment of the recto-vaginal fistula prosthesis.
17. the application according to claim 12 is characterized in that, described application is the implantation equipment that preparation is used for the treatment of encysted rectum.
18. the application according to claim 12 is characterized in that, described application is the implantation equipment that preparation is used for the treatment of hemorrhoid.
19. the application according to claim 12 is characterized in that, described application is that preparation is used to repair the damaged implantation equipment of vascular tissue.
20. the application according to claim 12 is characterized in that, described application is that preparation is used to repair the damaged implantation equipment of nervous tissue.
21. the application according to claim 12 is characterized in that, described application is that preparation is used to repair the damaged implantation equipment of tendon tissue.
22. the application according to claim 12 is characterized in that, described application is that preparation is used to repair bone and the damaged implantation equipment of cartilaginous tissue.
23. the application according to claim 12 is characterized in that, described application is that preparation is used to repair the implantation equipment of reconstruction of oral defect.
24. the application according to claim 12 is characterized in that, described application is the implantation equipment that preparation is used to repair the urinary tract damage.
25. the application according to claim 12 is characterized in that, described application is that preparation is used for the implantation equipment rebuild at the bottom of the basin.
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