A 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 superior and inferior surface layers, forming a sandwich structure. The biological material integrates the advantages of UBM and SIS: {circle around (1)} High bioactivity with bionic structure; {circle around (2)} UBM isolates the immunogenicity of SIS and directly contacts host tissue; {circle around (3)} SIS can make up for the disadvantage of low mechanical strength of UBM, the preparation of SIS is easier, and its thickness is subject to change after composition; {circle around (4)} raw materials of same origin are feasible for industrial large-scale production. The biological material can be applied to filling, reinforcement, restoration or reconstruction of fascia, meninx, pleura, pelvic floor, derma, solid viscera and various soft tissue defect, possessing good clinical practicability.

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.

2. The biological material with composite extracellular matrix components of claim 1, wherein the SIS is obtained from mammalian small intestine that is delaminated mechanically to remove serosa, mucosa and muscularis layers and then decellularized.

3. The biological material with composite extracellular matrix components of claim 1, wherein the UBM is obtained from mammalian urinary bladder that is treated mechanically to remove serosa, muscularis external, submucosa, and muscularis mucosa layers and then decellularized.

4. The biological material with composite extracellular matrix components of claim 1, wherein the number of layers in the interlayer is 120.

5. The biological material with composite extracellular matrix components of claim 1, wherein the number of layers in the superior surface layer and the inferior surface layer is 110 respectively.

6. The 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 the interlayer, the superior surface layer and the inferior surface layer are bonded among them with the aforesaid method too.

7. The biological material with composite extracellular matrix components of claim 6, wherein said medical adhesive comprises one or several of chitosan, collagen, fibrin glue, hyaluronic acid, chondroitin sulfate, hydrogel, bone glue, gelatin, and pectin.

8. The biological material with composite extracellular matrix components of claim 6, wherein technical parameters of vacuum pressing are as follows: Vacuum pressure: 50760 mmHg, acting duration: 0.572 h.

9. The biological material with composite extracellular matrix components of claim 1, wherein the biological material comprises perforations that penetrate the biological material.

10. The biological material with composite extracellular matrix components of claim 9, wherein the perforations have a diameter of 1 mm to 5 mm, wherein the spacing between the perforations is 0.5 cm to 5 cm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The FIGURE 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

[0029] 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

[0030] Use Abraham method to prepare porcine decellularized urinary bladder matrix (UBM) and decellularized small intestinal submucosa (SIS): 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

[0031] Use Abraham method to prepare porcine decellularized urinary bladder matrix (UBM) and decellularized small intestinal submucosa (SIS): 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

[0032] According to GB/T528-2009, 3 samples were taken, each being 4 cm1 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 343 N/cm.

[0033] Three samples were taken and cut into 2 cm5 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 62 N/cm, and the force for maintaining peeling was 1.50.5 N/cm.

[0034] 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 01.

[0035] Cell migration: The said material was powered under low temperature and then degraded by protease, the concentration of enzymatic products being 50 g/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 205672, and that for positive control was 210535, that for negative control was 132865. No significant difference was detected between the said material and positive control regarding cell migration (P>0.05).

[0036] Endotoxin content of the said biological material was determined by the method claimed in GB/T14233.2. The water for endotoxin test was used as extracting agent, and the extraction was performed at 37 C. for 24 h. Endotoxin content was determined by kinetic turbidimetric limulus tests, and the endotoxin concentration of diluted extract was re-determined to rule out the interference. The endotoxin content of the said material was 5EU/device.

[0037] 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 g/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%.

[0038] 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.

[0039] 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.

[0040] The animal model with defects of rectus abdominis sheath and rectus abdominis was established in dogs, the defect area being 105 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.