Fish skin-derived medical collagen membrane with compact outer layer and loose inner layer and preparation method of medical collagen membrane
A collagen and fish skin technology, applied in the field of medical materials, can solve the problems of cytotoxicity, inability to effectively block growth, and inhibit cell adhesion and proliferation in cross-linked collagen membranes.
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Embodiment 1
[0044] Example 1: Preparation of non-denatured collagen derived from fish skin
[0045]Weigh 100g of fish skin, add 20 times the volume of 0.1mol / L NaOH solution and magnetically stir for 24 hours to remove non-collagen components and impurities, and eliminate the influence of endogenous proteases on the collagen structure. During this period, 0.1mol / L NaOH solution Replace 2 times. After the fish skin is swollen with alkali, the fish scales are removed with a knife and cut into small square pieces of about 5×5 mm. Wash the fish skin repeatedly with pre-cooled deionized water until neutral, then add 0.5mol / L acetic acid solution and 0.5% (w:w) pepsin to carry out enzymatic extraction at a material-liquid ratio of 1:60, and stir with medium speed magnetic force for 48h . After the enzymolysis extract was refrigerated and centrifuged at 9000r / min for 30min, the supernatant was collected and salted out with finely ground NaCl. The final actual concentration of NaCl was controll...
Embodiment 2
[0048] Example 2: Preparation of a medical graded collagen membrane with a dense outer layer and a loose inner layer from fish skin
[0049] The non-denatured collagen derived from fish skin obtained according to Example 1 was dissolved in 0.05M acetic acid to prepare 2.5%, 1.5%, and 0.5% collagen suspensions, stood at 4°C for 48 hours, and then stirred until homogenized. After the dissolution is complete, put the 2.5% collagen solution into a stainless steel dish, spread a thin layer, and stand at a low temperature to obtain a glue block. Then, according to the gradient, 1.5% collagen solution was covered on the lower layer of 2.5% collagen gel block, and the uppermost layer was 0.5% collagen solution, which was pre-cooled at 40°C for 12 hours and then freeze-dried. 60mM EDC / NHS (nEDC / nNHS=2.5:1, pH 5.5), soaked in 50mM MES in 40% ethanol solution for 4h, with 0.1M Na 2 HPO 4 The solution was washed for 4 hours, and then washed repeatedly with deionized water. After freeze...
Embodiment 3
[0050] Example 3: Preparation of a medical gradient collagen film with a dense outer layer and a loose inner layer from fish skin
[0051] The non-denatured collagen derived from fish skin obtained according to Example 1 was dissolved in 0.05M acetic acid to prepare 3%, 2%, and 1% collagen suspensions, left standing at 4°C for 48 hours, and then stirred until homogenized. After the dissolution is complete, put the 3% collagen solution into a stainless steel dish, spread a thin layer, and stand at a low temperature to obtain a glue block. Then, according to the gradient, 2% collagen solution was covered on the lower layer of 3% collagen gel block, and the uppermost layer was 1% collagen solution, which was pre-cooled at 40°C for 12 hours and then freeze-dried. 60mM EDC / NHS (nEDC / nNHS=2.5:1, pH 5.5), soaked in 40% ethanol solution containing 50mM MES for 4h, with 0.1M Na 2 HPO 4 The solution was washed for 4 hours, and then washed repeatedly with deionized water. After freeze...
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