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Lacunaris bladder acellular matrix preserving biological activity factor and preparation

A bioactive factor, acellular matrix technology, applied in medical science, prosthesis and other directions, can solve the problems of poor bladder smooth muscle cell regeneration, destruction of bioactive factors, disordered and irregular arrangement, etc. Compatibility and easy access to materials

Inactive Publication Date: 2012-07-25
THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous studies, after bladder replacement with these prepared BAMs, bladder smooth muscle cells regenerated poorly. In the central area of ​​BAM replacements, smooth muscle cells were rare and irregularly arranged, which may be due to the destruction of endogenous bioactive factors

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of porcine bladder cell-free matrix, the preparation steps are as follows:

[0057] a) Place the bladder and part of the urethra of a pig weighing 20 kg in pre-cooled 10mM phosphate buffer at 4°C, pH 7.2-7.4, quickly bring it to the laboratory, and carefully remove the fat and serosa tissue on the outer surface , insert the infusion pimp catheter through the urethra and fix it on the urethra with silk thread, then wash the bladder with 10mM phosphate buffer saline, PH7. flush with urine;

[0058] b) Pour 100ml of digestive juice containing 0.25% / 0.038% trypsin / tetrasodium edetate into the bladder cavity through the infusion thong catheter, the pH is 7.2-7.4, clamp the infusion thong catheter, and then place the entire bladder Completely immerse in the digestion solution, shake and digest at room temperature for 2 hours, then pour out the digestion solution;

[0059] c) Rinse 3 times with 10mM phosphate buffer solution pre-cooled at 4°C, pH7.2-7.4...

Embodiment 2

[0072] Example 2 Preparation of rabbit bladder cell-free matrix, the preparation steps are as follows:

[0073] a) Place the bladder and part of the urethra of a New Zealand white rabbit with a body weight of 4 kg in pre-cooled 10mM phosphate buffer at 4°C, pH 7.2-7.4, quickly bring it to the laboratory, and carefully remove the fat and slurry on the outer surface Membranous tissue, insert the transurethral infusion pimp catheter and fix it on the urethra with silk thread, then wash the bladder with 10mM phosphate buffer solution, PH7. rinse with urine;

[0074] b) Pour 40ml of digestive juice containing 0.25% / 0.038% trypsin / tetrasodium edetate into the bladder cavity through the infusion thong catheter, the pH is 7.2-7.4, clamp the infusion thong catheter, and then place the entire bladder Completely immerse in the digestion solution, shake and digest at room temperature for 0.5 hours, then pour out the digestion solution;

[0075] c) Rinse 3 times with 10mM phosphate buffe...

Embodiment 3

[0088] Example 3. Isolation, cultivation and identification of seed cells

[0089] 3.1 Isolation, culture and identification of HBSMC

[0090] For a small piece of bladder tissue without obvious tumor growth, the urothelial layer was completely removed by mechanical dissection, and sequentially treated with 0.25% / 0.038% trypsin / tetrasodium edetate and 0.1% type I collagenase After digestion, the resulting cell suspension was inoculated in DMEM / F-12 full medium containing 10% FBS for primary culture and subculture. Inverted microscope was used for morphological observation, and the expression of smooth muscle actin and desmin was observed by immunostaining of cell slides.

[0091] Observed under an inverted microscope, the human bladder smooth muscle cells cultured in vitro were long-spindle-shaped, and showed a typical "peak-valley" shape after fusion; both smooth muscle actin and desmin staining were positive, and the positive rate was greater than 98%. The present inventio...

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PUM

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Abstract

The invention discloses a porous bladder acellular matrix retaining bioactive factors and a preparation method thereof. A bladder of a pig, a rabbit, a dog or cattle is handled by digestive juice, hypotonic buffer solution, hypertonic buffer solution with surfactants, buffer solution with nuclease, and deionized water. An abacterial bladder acellular matrix is obtained by cooling, freezing out and sterilizing. The bladder acellular matrix is not provided with cells, an urinary tract epithelial layer, a submucous layer or a blood circulatory system, thus having good biocompatibility and appropriate ultrastructural structure. Bioactive factors are retained, including cell adhesive factors, growth factors and chemokines. Therefore, the bladder acellular matrix is an ideal scaffold material for a bladder in tissue engineering.

Description

technical field [0001] The invention relates to a biological material and a preparation method thereof, in particular to a porous bladder cell-free matrix retaining biologically active factors and a preparation method thereof. Background technique [0002] Congenital malformations, infections, traumas, tumors and other lesions of the bladder can lead to structural damage and dysfunction of the bladder and require bladder replacement surgery. The gastrointestinal tract is currently the main replacement for bladder replacement. However, since the gastrointestinal tract is in the environment of the urinary system, it may cause a series of complications such as metabolic disorders, infection, stone formation and even malignant transformation. Therefore, finding a reasonable bladder substitute is a challenge before us, and tissue engineering technology undoubtedly provides us with a powerful tool. [0003] The basic principle of tissue engineering bladder is to inoculate the bl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/36A61L27/56
Inventor 杨斌孙则禹周六化戴玉田
Owner THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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