BIOLOGICAL MATERIAL WITH COMPOSITE EXTRACELLULAR MATRIX COMPONENTS

Abstract

A biological material with composite extracellular matrix component, in which decellularized small intestinal submucosa (SIS) is treated as the interlayer and decellularized urinary bladder matrix (UBM) is treated as superior and inferior surface layers. The interlayer is totally encapsulated by the mentioned superior and inferior surface layers, forming a sandwich structure with advantages of integrating UBM and SIS to have high bioactivity with bionic structure, UBM isolates the immunogenicity of SIS and direct contact with host tissue, and after implantation the basic type of inflammatory interaction in the host-implant marginal zone is the same as that of pure UBM, with high biocompatibility; effective endotoxin removal optimize the biosafety of the material after implantation; feasibility for industrial large-scale production; the stiffness of the material can be maintained even after hydration, with good handling feel and fit condition, beneficial for the suture fixation and also shorten the fixation or surgery time.

Claims

1. A biological material with composite extracellular matrix components, comprising: an interlayer containing a decellularized small intestinal submucosa (SIS); a superior surface layer containing a decellularized urinary bladder matrix (UBM); an inferior surface layer containing a UBM; wherein the superior surface layer and the inferior surface layer encapsulated completely the interlayer to form a sandwich structure; wherein the thickness of the superior surface layer is 0.05 mm-0.2 mm; wherein the thickness of the inferior surface layer is 0.05 mm-0.2 mm; wherein the SIS were processed for effective endotoxin removal; wherein the effective endotoxin removal process is as follows: the SIS is treated with lipid reduction process to reduce the lipid content to less than 2% and subsequently treated with an alkali solution.

2. A biological material with composite extracellular matrix components of claim 1, wherein the bending length of the biological material is reduced by no more than 50% after 10 min of hydration.

3. A biological material with composite extracellular matrix components of claim 1, wherein the endotoxin content of the biological material is less than 0.5 EU/g; preferably, the endotoxin content of the biological material is less than 0.1 EU/g.

4. A biological material with composite extracellular matrix components of claim 1, wherein the lipid reduction process comprises organic solvent treatment for 2-16 h.

5. A biological material with composite extracellular matrix components of claim 1, wherein alkali solution process comprises 0.1-2% (w/v) alkali solution treatment for 0.25-1 h, wherein said alkali comprises sodium hydroxide, potassium hydroxide.

6. A biological material with composite extracellular matrix components of claim 1, wherein the SIS is treated with lipid reduction process to reduce the lipid content to less than 1%, and followed by alkali treatment, and then detergent treatment.

7. A biological material with composite extracellular matrix components of claim 1, wherein the number of layers in the interlayer is 1˜20; wherein the number of layers in the superior surface layer and the inferior surface layer is 1˜10 respectively.

8. A biological material with composite extracellular matrix components of claim 4, wherein the said interlayer is bonded to the superior surface layer and the inferior surface layer by one or several of such methods as medical adhesive, suturing and tying, and vacuum pressing; wherein the layers in interlayer, superior surface layer and inferior surface layer are bonded among them with the aforesaid method too.

9. A biological material with composite extracellular matrix components of claim 6, wherein the technical parameters of the vacuum pressing are as follows: Vacuum pressure: −50˜−760 mmHg, acting duration: 0.5˜72 h.

10. A biological material with composite extracellular matrix components of claim 1, wherein the biological material is applied to the laparoscopic repair of inguinal hernia, femoral hernia, and abdominal wall hernia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 shows the structure of the present invention, wherein digit 1 indicates decellularized small intestinal submucosa (SIS), and digit 2 indicates decellularized urinary bladder matrix (UBM)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0052] Following specific embodiments are used to further expound the present invention. Those embodiments are only for explaining the present invention, and shall not limit the scope of the present invention. It shall be understood that technicians in the same field can make various changes or modifications after they read the content of the present invention, and all those changes and modifications are equivalent to the present invention and therefore fall within the scope of the present invention.

Example 1

[0053] Porcine decellularized urinary bladder matrix (UBM) and decellularized small intestinal submucosa (SIS) were prepared. Spread a monolayered UBM out smoothly (the smooth surface being downward), composite SIS into an independent layer, each SIS being overlapped by 50%. Then place 4 aforesaid independent layers on the aforesaid UBM, each layer being interlaced by 90°. Place a monolayered UBM on the surface of the aforesaid layers (the smooth surface being upward). Dissipate bubbles, bind all interlayers with medical chitosan as adhesive, then press the above layers under −250 mm Hg for 24 h to make it become a whole material. The aforesaid material is perforated through all layers, the hole spacing being 5 mm, and the diameter of hole being 1 mm.

Example 2

[0054] Porcine decellularized urinary bladder matrix (UBM) and decellularized small intestinal submucosa (SIS) were prepared. Spread 2 layers of UBM out smoothly (the smooth surface being downward), composite SIS into an independent layer, each SIS being overlapped by 50%, then place 6 aforesaid independent layers on the surface of UBM, each layers being interlaced by 90°. Spread 2 layers of UBM on the surface of the aforesaid layers (the smooth surface being upward). Dissipate bubbles, bind all interlayers with medical collagen as adhesive, then press the above layers under −300 mm Hg for 36 h to make it become a whole material. The aforesaid material is perforated through all layers, the hole spacing being 8 mm, and the diameter of hole being 2 mm.

Example 3

[0055] According to GB/T528-2009, 3 samples were taken, each being 4 cm×1 cm in size and dumbbell-like in shape; aforesaid 3 samples were hydrated and then their two ends were fixed to a mechanical tester and pulled at the speed of 10 mm/min, and the tensile strength of those samples was 34±3 N/cm.

[0056] Three samples were taken and cut into 2 cm×5 cm in size; two ends of those samples were fixed to upper and lower clips of a tensile machine respectively, and those samples were peeled continuously at the speed of 10 mm/min till the overlapped part of them laminated; the force at stratification was recorded. The peeling strength of SIS-SIS and UBM-SIS was 6±2 N/cm, and the force for maintaining peeling was 1.5±0.5 N/cm.

[0057] The cytotoxicity of the said material was evaluated by the method claimed in GB/T 16886.5. NIH3T3 cells and L929 cells were used, and cell culture medium was used as extracting agent, extracts of gradient concentrations were used as cell culture media, and MTT method was used to determine the cell viability. The cytotoxicity of the said biological material was graded to be 0˜1.

[0058] Cell migration: The said material was powered under low temperature and then degraded by protease, the concentration of enzymatic products being 50 μg/mL. Cells were starved for 24 h, and Boyden chamber method was used to determine 6 h-migration of cells, medium with and without 10% fetal bovine serum being used as positive and negative control respectively. Migrated cells for the said material was 2056±72, and that for positive control was 2105±35, that for negative control was 1328±65. No significant difference was detected between the said material and positive control regarding cell migration (P>0.05).

[0059] The hemocompatibility of the said material was determined by the method claimed in GB/T14233.2. Contact group: The back of rats was dehaired, applied with 50 μg/mL enzymatic products for once a day, consecutive 20 days. Oral administration group: 1 ml of extract was administered orally every other day within 7 days, 4 administrations in total; intramuscular injection and intravenous injection group: 0.15 mL of extract was injected every other day within 7 days, 4 injections in total. Rats in aforesaid 4 groups were killed 30 days and 90 days respectively after the administration, and the venous blood was collected for detection. The hemolytic ratio was calculated by the following formula: Hemolytic rate (%)=(absorbance of sample minus absorbance of negative control) divided by (absorbance of positive control minus absorbance of negative control)×100%. The hemolytic rate of the said material was ≤5%.

[0060] The intradermal stimulation of the said material was evaluated by the method claimed in GB/T 16886.10. Rabbits were subject to the intradermal injection of 0.2 mL of extract and 0.2 mL of control (PBS) respectively, and the skin reaction of the injected region was observed 15 min, 1 h, 2 d, and 3 d after the injection; the stimulation grades was scored as erythema and edema. The said material showed no intradermal stimulation.

[0061] The sensitization of the said material was evaluated by the maximal dose method claimed in GB/T 16886.10. The solution of pure starch was used as negative control. Extracts of the said material and control were oral administered consecutively for one week. Rats were observed for one week after oral administration. Weight, clinical toxic symptoms and grade of toxicity were daily recorded. All rats were killed after the test, and the pathological examination demonstrated that the said material showed no delayed super-sensitivity.

[0062] The animal model with defects of rectus abdominis sheath and rectus abdominis was established in dogs, the defect area being 10×5 cm.sup.2; the said material was cut into suitable size for defect repair, pure SIS and pure UBM being used as controls. The incidence of seroma in the repair region was 33% for pure SIS, and no seroma occurred for pure UBM and the said material. Repair region was harvested 2 weeks, 1 month, 2 months, and 4 months respectively after the aforesaid repair and subject to staining of CD68, CCR7, and CD163 to observe the classification and density of infiltrated cells and the ratio of M1 macrophages to M2 macrophages. It was confirmed that the basic type of inflammatory interaction of said material in the host-implant marginal zone was the same as that of pure UBM, and repair efficiency was close to that achieved by pure UBM.

Example 4

Bending Length and Flexural Rigidity Measurement:

[0063] Control: 8-layer SIS

[0064] Sample A: 6-layer SIS+UBM as the upper and lower surface layers, the thickness of surface layer is 0.05 mm

[0065] Sample B: 6-layer SIS+UBM as the upper and lower layers, the thickness of the surface layer is 0.1 mm

[0066] Sample C: 6-layer SIS+UBM as the upper and lower layers, the thickness of the surface layer is 0.2 mm

[0067] Vacuum lamination is used to prepare the sample to be tested.

[0068] Spiral thickness meter was used to test the prepared controls and samples, and 5 points for each sample were tested randomly. Average values are shown in Table 1. No significant difference in the overall thickness of materials was observed in each group

[0069] An analytical balance was used to test the mass per unit area of the controls and samples. No significant difference was found in the mass per unit area in each group of materials, and the results are shown in Table 1.

TABLE-US-00001 TABLE 1 Thickness and mass per unit area of test samples Sample thickness (mm) Mass per unit area (g/m.sup.2) Control 0.25 143 Sample A 0.22 132 Sample B 0.30 147 Sample C 0.25 135

[0070] Methods: The bending length and flexural rigidity of unhydrated materials with a width of 1 cm and a length of 20 cm were measured by the section method. The material was immersed in saline at room temperature for 2 or 10 min After taking the material out, both the bending length and flexural rigidity were measured. Each sample was measured 5 times and the average value was calculated. The results are shown in Table 2. Bending length is defined as the length of a rectangular strip of fabric, fixed at one end and free at the other, that will bend under its own weight to an angle of 7.1°; flexural rigidity is defined as the ratio of the small changes in bending moment per unit width of the material to its corresponding small changes in curvature.

TABLE-US-00002 TABLE 2 Bending length and flexural rigidity Bending length Bending length Bending length flexural rigidity flexural rigidity flexural rigidity (before (2 min after (10 min after (before (2 min after (10 min after hydration, cm) hydration, cm) hydration, cm) hydration, mN .Math. cm) hydration, mN .Math. cm) hydration, cm) Control 6.5 1.3 1.0 39.3 0.7 0.4 Sample A 6.2 4.0 3.4 31.5 16.9 11.4 Sample B 6 4.5 3.7 31.3 26.8 15.8 Sample C 6 4.8 4.2 29.2 27.9 21.0

[0071] According to Table 2, the bending length of the samples A, B and C did not exceed 50% of the values, when “10 min after hydration sample” was compared to “before hydration sample”. Thus, the structure of UBM as the upper and lower layers and SIS as the middle layer and the thickness of UBM is in the range of 0.05-0.2 mm, can reduce the bending length of the whole material by no more than 50%, after hydration for 10 min. When the thickness of the overall material remains unchanged, as the thickness of the UBM increases, the reduction of the bending length after hydration become less, leading to better operation performance.

[0072] Clinical research summary: A randomized, single-blind, parallel-controlled, multi-center clinical trial was used to evaluate the effectiveness and safety of the biological material for the treatment of inguinal hernia. There are 150 patients of open surgery, including 75 cases in the control group and 75 cases in the experimental group. The test device was sample A shown in the example 4, and the control device was the control shown in example 4 (porcine SIS products). The main observation index was the recurrence rate of inguinal hernia at 6 months after operation. The secondary safety evaluation indexes were postoperative fever, hematoma/seroma in the operation area, evaluation of infection in the incision area, pain scale, foreign body sensation in the groin area, and allergic reactions.

[0073] Results:

[0074] Recurrence rates of hernia at 6 months after operation

[0075] Experimental group: None.

[0076] Control group: 2 cases.

[0077] Recurrence rates after 18 months

[0078] Experimental group: None.

[0079] Control group: 4 cases.

[0080] Other indexes:

[0081] The material operation feeling of the experimental group was better than that of control group. The material fixation time in the experimental group was shorter than the control group, making the operation time significantly shorter than the control group.

TABLE-US-00003 TABLE 3 Patch operation time Item Control group Experimental group Patch fixation time 12.02 ± 4.83  9.89 ± 4.12 Operation duration 68.06 ± 18.10 63.06 ± 18.00 (min)

[0082] Based on the above clinical results, the biomaterial of the present invention has a good operating feel. The biomaterial can maintain the stiffness of the material after hydration and had a good fit, which were conducive to surgical suture operations and shorten the material fixation time.

Example 5

[0083] Porcine SIS was the experimental material. SIS was separated from the freshly obtained porcine small intestine, and it was cleaned briefly with peracetic acid and phosphate buffer, disinfected, freeze-dried and used as sample 1.

[0084] After Sample 1 was cleaned with a mixture of chloroform/ethanol (3:1, v/v) for 10 times as degreasing treatment, the lipid content was detected to be 1.2%, which was used as sample 2. Next, sample 2 was rehydrated step by step to remove organic solvent residues, treated 0.2% sodium hydroxide solution for 60 min, rinsed with injection water, freeze-dried and then used as sample 3.

[0085] Sample 1 was treated 0.2% sodium hydroxide solution for 60 min, rinsed with injection water, freeze-dried and used as sample 4.

[0086] For preparation of sample 5, methods described in U.S. Pat. No. 6,206,931 were used. Adult porcine small intestine was washed with water and treated with 0.2% peracetic acid/5% ethanol aqueous solution for 2 h. SIS was separated and freeze-dried.

[0087] The samples were layered and made into materials for the tests. Each material was cut into small pieces to detect the endotoxin content: Endotoxin-free water was used for 2 h extraction procedure. Dynamic spectrophotometry was used to detect the endotoxin content of the material. Whenever needed, a gradient concentration dilution was done before measuring the endotoxin content. The test results are shown in Table 4:

TABLE-US-00004 TABLE 4 The endotoxin content of the samples Endotoxin content (EU/g) Sample 1 786.98 Sample 2 4.98 Sample 3 0.05 Sample 4 467.32 Sample 5 1.37

[0088] The endotoxin detection has the problem of false negative or false positive result. So, in order to further clarify the endotoxin removal effect, macrophage activation method was used as a semi quantitative analysis to detect the activity of endotoxin residue. If the secretion of TNF-α is higher, more severe inflammatory reactions are expected after implantation, which reflects a higher endotoxin content present in the implant.

[0089] THP-1 mononuclear macrophages in the logarithmic phase were seeded into a 24-well culture plate at a density of 1×10.sup.4 cells/well, and cultured overnight at 37° C. After overnight culture, the cells were replenished with fresh media. The samples from each group were cut into 1×1 cm.sup.2 and placed in the upper chamber of the transwell, which was placed in the 24-well culture plate with an addition of 0.5 mL culture medium to the upper chamber. After co-culturing for 24 h, the cell culture supernatant was collected, centrifuged and used for the detection of TNF-α level by ELISA. Alamar blue reagent was added to the cells for staining and counting the live cells. LPS was used as the positive control and the complete medium served as the negative control. The TNF-α content of each test sample was shown in Table 5.

[0090] The results in Table 5 showed that the method of degreasing and alkali treatment, as disclosed in the present invention, is effective for endotoxin removal. This method thoroughly removes endotoxins from raw materials.

TABLE-US-00005 TABLE 5 TNF-α content in the samples TNF-α content (pg/50000 cells) Sample 1 674.2 Sample 2 217.6 Sample 3 4.9 Sample 4 441.3 Sample 5 95.4 Positive control 2,049.5 Negative control 2.3

Example 6

[0091] Porcine SIS was the experimental material. SIS was separated from the freshly obtained porcine small intestine, and it was cleaned briefly with peracetic acid and phosphate buffer, disinfected, freeze-dried and used as sample 6.

[0092] After Sample 1 was cleaned with lipase for 12 h at 4° C., and then freeze dried, followed by treatment of chloroform/ethanol (1:1, v/v) for 2 times. The lipid content was detected to be 1.2%, which was used as sample 7. Next, sample 7 was rehydrated step by step to remove organic solvent residues, treated 1.5% sodium hydroxide solution for 60 min, rinsed with injection water, freeze-dried and then used as sample 8.

[0093] Sample 6 was treated 1.5% sodium hydroxide solution for 60 min, rinsed with injection water, freeze-dried and used as sample 9.

[0094] The samples were layered and made into materials for the tests. Each material was cut into small pieces to detect the endotoxin content: Endotoxin-free water was used for 2 h extraction procedure. Dynamic spectrophotometry was used to detect the endotoxin content of the material. Whenever needed, a gradient concentration dilution was done before measuring the endotoxin content. The test results are shown in Table 6:

TABLE-US-00006 TABLE 6 The endotoxin content of the samples Endotoxin content (EU/g) Sample 6 786.98 Sample 7 5.30 Sample 8 0.38 Sample 9 345.61