Preparation method for implantable medical biological materials of animal origin

Abstract

The present invention provides a preparation method for implantable medical biological material of animal origin comprising the following procedures: Pre-processing, and washing of animal tissue materials; inactivation of virus; decellularizing cell; sodium chloride processing; molding and packaging sterilization. Cell-free ECM materials of animal origin produced by this method can achieve the goal of completely removing cell components of animal origin and composition of DNA, and at the same time, the natural ECM composition, three-dimensional structure and active growth factor which can induce and promote tissue regeneration retain. By using this process, endotoxin, organic solvents and toxic solvent residue are thus omitted and products with different sizes, thickness and mechanical strength can be formed.

Claims

1. A preparation method for an implantable medical biological material of animal origin, comprising the following steps: step 1, pre-processing, separating and washing of animal tissue material taking out fresh animal tissue and washing the flesh animal tissue with injection water for 3 times to obtain washed animal tissue; step 2; inactivating virus inactivating the virus in the washed animal tissue obtained in the step 1 by using ethanol solution with a low concentration of peracetic acid to obtain inactivated material; conducting a cleaning step in an oscillation ultrasonic cleaner at a constant temperature; wherein the peracetic acid takes up a percentage of 0.050.2% by volume, an inactivation time ranges from 1 h to 2 hours; wherein the constant temperature ranges from 4 C. to 40 C.; cleaning the inactivated material in a phosphate buffer solution for 25 times; wherein each time the cleaning lasts for 15 minutes; detecting a pH value of the phosphate buffer solution after each time of the cleaning: when a pH value reaches a range from 6.5 to 7.5, cleaning the inactivated material with flow injection water until a conductivity is lower than 1.5 um/s, such that a first cleaned material is obtained; step 3, decellularizing cell wherein the step 3 is conducted in an oscillation sink having an ultrasonic cleaner at a constant temperature; placing the first cleaned material obtained in the step 2 into the oscillation sink; injecting a sodium hydroxide solution into the oscillation sink, and turning on the ultrasonic cleaner; wherein a cleaning time by using the sodium hydroxide solution ranges from 5 to 30 minutes: wherein a concentration of the sodium hydroxide solution ranges from 5 mmol/L to 100 mmol/L; turning off the ultrasonic cleaner; pouring out the sodium hydroxide solution; injecting a phosphate buffer solution into the ultrasonic cleaner and turning on the ultrasonic cleaner; wherein a cleaning time by using the phosphate buffer solution ranges from 5 minutes to 20 minutes; repeating the cleaning with the sodium hydroxide solution for 25 times to obtain a second cleaned material; detecting a pH value of the phosphate buffer solution after each time of the cleaning; cleaning the second cleaned material by using a flowing injection water when the pH value of the phosphate buffer solution is in a range from 6.5 to 7.5; terminating the cleaning until a conductivity is lower than 1.5 um/s; step 4, sodium chloride treating wherein the step 4 is conducted in the oscillation sink having the ultrasonic cleaner at a constant temperature; injecting a sodium chloride solution into the oscillation sink, and turning on the ultrasonic cleaner to obtain a third cleaned material; wherein a cleaning time by using the sodium chloride solution ranges from 5-30 minutes; wherein a concentration of the sodium chloride solution is 0.015 mol/L or 2 mol/L, and a pH value of the sodium chloride solution is less than 7.8; washing the third cleaned material with the flowing injection water until a conductivity is lower than 1.5 um/s, such that a fourth cleaned material is obtained; step 5, molding step fixing the fourth cleaned material obtained in the step 4 to a plurality of devices, freezing and drying the fourth cleaned material, and drilling a plurality of micro holes on the fourth cleaned material using a laser; wherein sizes and shapes of the plurality of devices vary in accordance with different product requirements; securing the fourth cleaned material onto the plurality of devices and washing the fourth cleaned material by flowing injected water, such that a washed material is obtained; placing the washed material into a freeze drying machine and securing; performing a pre-designed lyophilization process to obtain a lyophilized material; wherein the pre-designed lyophilization process includes pre-cooling the washed material to a temperature of 2550 C., and keeping the temperature for 0.54 hours; rising the temperature to 15 C. and keeping the temperature for 412 hours; rising the temperature to 15 C. and keeping the temperature for 0.54 hours; rising the temperature to 25 C. and keeping the temperature for 4 hours; drilling a plurality of micro holes on the lyophilized material using a laser such that a drilled material is obtained: wherein a diameter of each of the plurality of micro holes of the drilled material ranges from 0.05 to 1 mm; wherein a distance between adjacent micro holes ranges from 0.1 cm to 2 cm: step 6, packaging sterilization performing a packaging step in a sterile condition; wherein a first layer of a packaging is a tyvek paper, a second layer of the packaging is a polyethylene plastic layer; performing an ethylene oxide sterilization after the packaging step.

2. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the animals is selected from the group consisting of pigs, cows and horses.

3. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the fresh animal tissue is selected from the group consisting of small intestinal submucosa, leather and pericardium.

4. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the percentage of the acetic acid peroxide in the step 2 is 0.1% by volume.

5. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein a inactivating time in the step 2 is 1 hour.

6. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein an oscillation frequency of the oscillation sink in the steps 3 and 4 is 100300 RPM.

7. The preparation method for the implantable medical biological material of animal origin of claim 6, wherein the oscillation frequency of the oscillation sink in the steps 3 and 4 is 200 RPM.

8. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein an ultrasonic frequency of the ultrasonic cleaner in the steps 3 and 4 is 2080 kHz.

9. The preparation method for the implantable medical biological material of animal origin of claim 8, wherein an ultrasonic frequency of the ultrasonic cleaner in the steps 3 and 4 is 45 kHz.

10. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the cleaning time by using the sodium chloride solution in the step 4 is 20 mins.

11. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the concentration of the sodium chloride solution in the step 4 is 5-20 mmol/L.

12. The preparation method for the implantable medical biological material of animal origin of claim 11, wherein the concentration of the sodium chloride solution in the step 4 is 10 mmol/L.

13. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the cleaning time for the material by using the phosphate buffer solution in the step 3 is 15 min.

14. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein a cleaning time for the material by using the sodium hydroxide solution in the step 3 is 20 min.

15. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the concentration of the sodium hydroxide solution in the step 3 is 0.015 mol/L.

16. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the plurality of devices in the step 5 are stainless steel devices.

17. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the pre-designed lyophilization process in the step 5 further comprises: pre-cooling the washed material to a temperature of 25 C. and keeping the temperature for 2 hours; rising the temperature to 15 C. and keeping the temperature for 8 hours; rising the temperature to 15 C. and keeping the temperature for 2 hours; rising the temperature to 25 C. and keeping the temperature for 4 hours.

18. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the diameter of each of the plurality of micro holes drilled by the laser in the step 5 ranges from 0.2 to 0.5 mm.

19. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the distance between adjacent micro holes drilled by the laser in the step 5 ranges from 0.5 to 1 cm.

20. The preparation method for the implantable medical biological material of animal origin of claim 1, wherein the fresh animal tissue is selected from the group consisting of small intestine tissue, dermal tissue, and pericardial tissue.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a clipping schematic view of the second embodiment;

(2) FIG. 2 is an optical microscope view of the second embodiment;

(3) FIG. 3 is an electron microscopy ultramicro structure of the second embodiment.

DETAILED DESCRIPTION OF THE INVENTION

(4) The present invention is described with the accompanying embodiments but not limited to the embodiment.

First Embodiment

(5) The preparation of substrate material of pig's small intestinal submucosa.

(6) 1. Pre-Processing, Separation and Washing of Animal Tissue Materials

(7) Taking out freshly slaughtered pig's small intestine tissue, cleaning and isolating small intestine submucosa, then washing the isolated small intestine submucosa with injection water for 3 times.

(8) 2. Inactivation of Virus

(9) Inactivating the virus by using the method of ethanol solution with low concentration of peracetic acid. This cleaning step is conducted in the oscillation ultrasonic cleaners with constant temperature. Peracetic acid takes up the percentage of 0.050.2% by volume (preferred for 0.1%) and the inactivation time is 12 h (preferably for 1 h). The oscillation frequency of cleaners ranges from 30 to 600 RPM (preferably ranges from 100 to 300 RPM, 200 RPM is considered to be the best). The ultrasonic frequency ranges from 20 to 80 KHZ (preferably ranges from 20 to 50 KHZ, further preferably ranges from 35 to 50 KHZ, most preferably for 45 KHZ). The temperature ranges from 4 to 40 C. Inactivated materials are cleaned for 25 times in phosphate buffer, each of the cleaning process being 15 minutes. Detecting the pH value of the phosphate buffer after the cleaning process, when pH reaches 6.5 to 7.5, the above processed material is cleaned with flow injection water until conductivity is detected as being lower than 1.5 um/s. Preparation of phosphate buffer is achieved by dissolving 7.9 g NaCl, 0.2 g KCl, 0.24 g KH.sub.2PO.sub.4 and 1.8 g K.sub.2HPO.sub.4 into 800 ml distilled water, adjusting the pH value by adding HCl solution to achieve a pH value of 7.4, then adding distilled water until the constant volume reaches 1 L.

(10) 3. Decellularized Cell

(11) The cleaning steps are conducted in the oscillation sink having ultrasonic cleaners with constant temperature. First of all, the materials are placed into the oscillation sink for cleaning, and then sodium hydroxide solution is injected into the oscillation sink, turning on the cleaners. The cleaning time is 530 min (preferably for 20 min). The sodium hydroxide solution concentration is 5100 mmol/L (preferably for 520 mmol/L, further preferably for 10 mmol/L). Then the cleaners are turned off and sodium hydroxide solution is poured out. Phosphate buffer solution is injected into the cleaner and turning on the cleaner. The cleaning time is 520 minutes (preferably for 15 min), repeating the phosphate buffer cleaning process for about 25 times, detecting the pH value of the phosphate buffer solution after cleaning. When the pH value of the detected phosphate buffer solution is in the range from 6.5 to 7.5, the processed material by is cleaned by the flowing water for injection. The flowing water cleaning process is terminated when a conductivity lower than 1.5 um/s is detected. The oscillation frequency of the cleaning step ranges from 100 to 300 RPM (preferably for 200 RPM), ultrasonic frequency ranges from 20 to 80 KHZ (preferably 2050 KHZ, further preferably of 3550 KHZ, most preferably for 45 KHZ). The preparation of phosphate buffer solution can be obtained as in step 2.

(12) 4. Sodium Chloride Treating

(13) This step is conducted in an oscillation sink having ultrasonic cleaners with constant temperature. Materials are placed into the oscillation sink for cleaning, and sodium chloride solution is injected into the oscillation sink, turning on the cleaners. The cleaning time is 530 min (preferably for 20 minutes). The concentration of sodium chloride solution is 0.015 mol/L or 2 mol/L (preferably 0.015 mol/L). The pH value is less than 7.8. The materials are washed with flowing injection water and conductivity of the cleaning water is detected. The washing step is terminated when the conductivity of the detected cleaning water is lower than 1.5 um/s. The oscillation frequency of the cleaning sink in this cleaning step ranges from 100 to 300 RPM (preferably for 200 RPM). The ultrasonic frequency ranges from 20 to 80 KHZ (preferably 2050 KHZ, further preferably 3550 KHZ, the most preferably is 45 KHZ).

(14) 5. The Molding Step

(15) The sizes and shapes of plurality of stainless steel devices vary in accordance with different product requirements. The material is fixed on the stainless steel device. Layers of the material may overlap according to different product requirements. The material which has been washed by flowing injected water is placed and fixed on the device into freeze drying machine. The lyophilization process is performed as pre-designed. The pre-designed lyophilization process is carried out as following: pre-cooling the material to 2550 C. (preferably for 25 C.) and conserving for 0.54 hours (preferably for 2 h); rising a temperature of 15 C. and conserving for 412 hours (preferably for 8 h); rising a temperature of 15 C. and conserving for 0.54 hours (preferably for 2 h), rising the temperature up to 25 C. and conserving for 4 hours. After the lyophilization process, laser is used to punch micro holes on the lyophilizated material. Aperture of the lyophilizated material ranges from 0.05 to 1 mm (preferably 0.20.5 mm). The distance between the adjacent two holes ranges from 0.1 to 2 cm (preferably 0.51 cm).

(16) 6. Packaging Sterilization

(17) The packaging step is performed in sterile conditions. One layer of the packaging is tyvek paper, the other layer is polyethylene plastic. After packaging, an ethylene oxide sterilization is performed.

The Second Embodiment

(18) Testing the physical and chemical properties, histology, growth factors and biological performance of the decellularized small intestinal submucosa matrix material obtained from the first embodiment.

(19) 2. Testing the physical properties of the obtained materials having eight layers. The testing items include the following: suture retention, tensile strength, bursting strength and porosity.

(20) 1). Suture retention test: 2-0 surgical suture or stainless steel wire of the same diameter is used to stitch on the edge of one end of the eight-layer material and located about 2 mm off the edge, the other end of the suture or stainless steel wire of the eight-layer material is fixed on the tension meter. The wire is being stretched at a speed of 20 mm/min until the suture point is torn. Pulling force is recorded when suture point is torn. 3 batches of samples are tested according to the above method under the same condition. Results show that the suture tensile strength is greater than or equal to 50.5 N.

(21) 2). The tensile strength testing method: tensile (compressed) tester is used, as shown in FIG. 1. Eight-layer materials are cut into samples. The humidity of the materials after cutting ranges from 40% to 60%. Samples are placed under the temperature of 22 C.2 C. for 2 hours and tested immediately thereafter. Two ends of the sample are fixed onto clamp heads of tensile testing machine and the sample is tested at a speed of 100 mm/min until the sample fractures. The samples are tested transversely and longitudinally respectively. The force when the sample is broken is recorded with a unit of N. 3 batches of the above samples are tested under the same condition. Results show that longitudinal force is 15 N and the transverse force is 8 N.

(22) 3). Bursting strength testing:

(23) using the tensile (compressed) tester, the eight-layer materials are cut into 23*23 mm square samples for spare use. Cut materials are placed under the condition where the relative humidity of cut material ranges from 40% to 60% and the temperature is 24 C.2 C. for 2 hours. Experiment is conducted immediately thereafter. Samples are secured to the stretcher workbench with ring clamps, making the spherical probe pass through the samples at a speed of 750 mm/min. Force when the samples are worn out is recored. Three batches of samples are tested according to the above method. Results show that the bursting strength is greater than 120 N.

(24) 4). Porosity determination: determining the porosity of the material by mercury injection method. Results show that the porosity is not less than 85%.

(25) 2. Testing the chemical performance of the eight-layer materials obtained from above process, wherein the testing items include viruses, pH value, residues of endotoxin and DNA.

(26) 1) Preparation of testing liquids: homogeneous thickness parts of the sample are taken; the sample is cut into debris with an average area of 1 cm.sup.2; cut samples are washed with water and sample is dried. Then sample is placed into a glass container, water is added into the glass container, wherein the ratio between the total surface area of the sample (cm2) and the volume of water (mL) is 5:1. Glass container is covered and the glass container is placed into a pressure steam sterilizer. Above sample is heated at a temperature of 121 C.1 C. for 30 minutes. Sample and liquid are separated after heating. Heated liquid is cooled to room temperature as test fluid. The same volume of water is taken as a blank comparison.

(27) 2) Virus detection: Pseudorabies are taken as the indicate virus; detecting the DNA copy number of viruses through real-time and quantitative PCR method. Three batches of samples are detected. Results show that the viral DNA copy number is 0.

(28) 3). pH value testing: experiment is conducted according to the regulations published in GB/T14233.1 (the medical infusion, transfusion, injection equipment checking method 1th part: methods of chemical analysis) method specified in 5.4.1. Results show that the pH value difference between the test solution and control solution is less than 1.5.

(29) 4). Endotoxin: Every 6 cm.sup.2 of samples should mix with 1 ml extraction medium. The experiment is conducted under the temperature of 371 C. for 722 hours. The extraction medium is saline solution. The experiment is conducted following the regulations published in GB/t 14233, which are the medical infusion, transfusion, injection equipment checking method 2nd part: biological test methods), and testing three batches of samples. Results show that the endotoxin content is less than 5 EU/g.

(30) 5) Detection of DNA residual: Samples obtained from the first embodiment are detected based on biological agents' residual DNA detection method (the Chinese Pharmacopoeia 2010, Appendix method for determination of IX-B residues of exogenous DNA) by a fluorescence staining method. Results show that the residues of DNA materials do not exceed 150 pg/g.

(31) 3. Histological Detection

(32) 1) Observation through optical microscopy: the material is coated with paraffin and the material is colored with hematoxylin-eosin staining. The coated and colored material is observed in the optical microscopy through inverted phase contrast microscope. As shown in FIG. 2, there is no residue of cell-free and cell debris, collagen is in a row under the microscopy.

(33) 2) Ultrastructural observation. Results: the material is porous, fibre without fracture, pore size, average pore size of 200 um, the porosity is greater than 85%, as shown in FIG. 3.

(34) 4. Growth Factor

(35) Every 6 cm.sup.2 samples should mix up with 1 ml extraction medium; the preparation of the testing liquids is conducted under the temperature of 371 C. for 722 hours. The extraction medium is saline solution. Growth factor (bFGF) and vascular endothelial growth factor (VEGF) levels in the extraction medium are detected by ELLISA method. Results show that the level of bFGF is 121.82.683 ng/L and VEGF is 93.83.033 ng/L.

(36) 5. Biological property testing is conducted, wherein the testing items include cytotoxicity, delayed-type hypersensitivity, and skin reaction.

(37) 1) Cytotoxicity: method: every 6 cm.sup.2 samples should mix up with 1 ml extraction medium. The preparation of the testing liquids is conducted under the temperature of 371 C. for 242 hours. The extraction medium is MEM serum-containing medium. The testing experiment is conducted following the regulation published in GB/T 16886.5-2003, such as the biological evaluation of medical devices part 5: in vitro cytotoxicity test. Result shows that the toxicity of cell is less than or equal to the grade 1.

(38) 2) Delayed-Type Hypersensitivity:

(39) Every 6 cm.sup.2 samples should mix up with 1 ml extraction medium. The preparation of the testing liquids is conducted under the temperature of 371 C. for 722 hours. The extraction medium is saline extract and cottonseed oil. The testing experiment is conducted following the regulation published in GB/T 16886.10-2005 (the biological evaluation of medical devices part 10th: stimulating and delayed-type hypersensitivity test). Results show that there is no delayed-type hypersensitivity reaction.

(40) 3). Intradermal reaction: the corresponding proportion between the leaching medium and the samples is that every 6 cm samples should be mixed with 1 ml leaching medium. The experiment is conducted under the temperature of 371 C. It takes 722 hours to prepare the leaching medium. The leaching medium is saline or cottonseed oil. The experiment is conducted following the regulations of GB/T 16886.10 2005 (the biological evaluation of medical devices part 10: stimulation and delayed-type hypersensitivity test). Results: the average comparison score between the tested sample and the solvent is less than 1.0.

The Third Embodiment

(41) The preparation of cell-free pig dermal matrix material. Freshly slaughtered pig dermal tissue is taken as raw material. The preparation method is the same as the first embodiment.

The Fourth Embodiment

(42) Testing the performance (physical and chemical properties, histology, growth factors and biology) of the cell-free pig dermal matrix material obtained from the third embodiment. The testing method is the same as stated in the second embodiment. Results show that the suture tensile strength of the decellularized dermal matrix material obtained from the third embodiment is greater than 5 N. The transverse and longitudinal tensile strength are greater than 20 N. The bursting strength is greater than 120N and the porosity is larger than 80%. The copy number of the viral DNA is 0. The endotoxin content is less than 5 eu/g and the amount of DNA residual does not exceed 150 pg/g. There is no delayed-type hypersensitivity or intradermal reaction occurring.

The Fifth Embodiment

(43) Preparation of the decellularized pig pericardium substrate materials.

(44) Freshly slaughtered pig's pericardial tissue is taken as raw material. Preparation method is the same as stated in the first embodiment.

The Sixth Embodiment

(45) Testing the performance (physical and chemical properties, histology, growth factors and biological) of the decellularized pig pericardium substrate materials obtained from the fifth embodiment. The testing method is the same as stated in the second embodiment.

(46) Results show that the suture tensile strength of the decellularized pig pericardium substrate materials obtained from the fifth embodiment is greater than 5 N. The transverse and longitudinal tensile strength are greater than 20 N. The bursting strength is greater than 120N and the porosity is larger than 85%. The copy number of the viral DNA is 0. The endotoxin content is less than 5 EU/g and the amount of DNA residual does not exceed 150 pg/g, there is no delayed-type hypersensitivity or intradermal reaction occurring.

(47) The above embodiments are the descriptions of this invention. This invention should cover all equivalent modifications and combinations of these embodiments, and is not limited to the above embodiments.