Albumin tissue scaffold

A tissue scaffold that made of albumin having continuous solid network and void are disclosed. Methods for preparing albumin tissue scaffolds from animal albumins are also disclosed.

DESCRIPTION OF PREFERRED EMBODIMENTS

The tissue scaffold having a continuous solid network. The solid network of tissue scaffold consists of a polymer of albumin protein prepared from polymerization reaction. There are two preparative methods, chemical crosslinker-catalyzed polymerization reaction and transglutaminase-catalyzed polymerization reaction, both can generate polymeric albumin. The preferred animal albumin is selected from the group consisting of bovine albumin, human albumin, and porcine albumin. The polymerization reactions preferably have mild conditions in which no organic solvents, 100% aqueous phase, neutral pH value, mild buffer and salt strengths, no excess heat generation during polymerization reaction, no heating requirement, and no chaotropic agent.

Commercial available albumins from animals are provided in dried and lyophilized powders. These powders were dissolved in a suitable reaction buffer to make an albumin solution. The preferred buffer substance is selected from the group consisting of BICINE, HEPES, MOPS, and TRIS. In a chemically cross linking reaction, a di-aldehyde was added to the albumin solution. In an enzymatically cross linking reaction, a transglutaminase was added to the albumin solution.

The polymerization reaction was carried out at the temperature of 37° C. Extensive cross links among individual albumin molecules occurred during incubation. The proceeding of polymerization can be traced using stirring. The reaction, at first, became high viscous, and then it turned into a solid form. The time required for curing solution is vary, which greatly depend on the amounts of cross linkers and albumin that are used. The preferred time for reaction incubation is between 0.5 to 24 hours.

In the present invention, it was found that not all albumins will be incorporated into high molecular weight polymers after polymerization reaction. Some albumins have shown to un-polymerization or low degree of polymerization. The components of polymeric albumin is typically assay by using SDS-PAGE analysis. A denaturing solution and a mechanical homogenizer are applied for disrupting noncovalent protein-protein interactions among albumin polymers. The preferred denatured agents are urea and guanidine. The preferred mechanical homogenization method is selected from the group consisting of pipetting, chopping and mincing, French press, pestle homogenizer, motor-driven tissue homogenizer, and warning blender.

In the present invention, it has found that centrifugation can effectively recover high molecular weight albumin polymers from the polymerization reaction. High molecular weight albumin polymers are insoluble, can be pelleted by centrifugation at about 2,330 g force for about 5 min. Albumin oligomers and low molecular weight albumin polymers remain in the supernatant.

In the present invention, albumin polymers comprise high molecular weight albumin polymers which is essential free of low molecular weight albumin polymer and albumin oligomers. The albumin polymer can be prepared from an enzymatic or a chemical polymerization reaction.

In the present invention, the albumin polymer is subject to wash by a diluted solution before freeze-drying. The preferred substance is pure water or a diluted acid solution which selected from the group consisting of formic acid, acetic acid, lactic acid and citric acid. The washed albumin polymer was transferred into a casting mold, frozen in low temperature, and then freeze-drying. A freeze-dryer can maintain the vacuum under less than 100 mtorr of pressure is used.

In the present invention, vaporous formaldehyde was used to cross link among the albumin polymers. Formaldehyde treatment fixes the shape and the size of albumin tissue scaffold.

2 g bovine serum albumin (purity>98%; Sigma) was dissolved in 19 mL buffer of 50 mM BICINE, pH 8.3. The solution was concentrated by using a spin concentrator (GE Healthcare) to the final volume of 10 mL. Albumin solution was stored in 4° C. refrigerator. Diluted glutaraldehyde regents at the concentrations of 25%, 12.5%, 6.25%, 3.13%, 1.56, and 0.78% were fresh made from 50% glutaraldehyde solution (Sigma) and pure water (Millipore). The reagents were kept on ice to prevent the spontaneous degradation of very diluted glutaraldehyde solution. 0.020 mL of various concentrations of glutaraldehyde was combined with 0.180 mL of bovine serum albumin solution in fresh plastic tube, mixed up immediately by a vortex mixer at top speed. Samples were incubated at 37° C. The following observations were noted after 30 min incubation:

Samples in EXAMPLE 1 were return to the incubator, and an additional incubation of 11.5 hours was performed. The state of each sample was the same as before. 3.4 mL of 8 M urea solution were added to every sample. For those solid state samples, the content was transferred to a tissue grinder (Kontes), and then homogenized by a homogenizer (IKA) at the rotational speed of 2000 rpm for several strokes. The homogenization was keep on ice during processing to prevent sample overheat. For those liquid state samples, content was mixed by a vortex mixer. The resulted homogenates were analyzed by SDS-PAGE analysis. NuPAGE LDS sample buffer (Life Technologies) included reducing agent was added, and then loaded to NuPAGE Bis-Tris Mini gel (Life Technologies). After electrophoresis, gel was stained with Instant blue (Novexin) to reveal protein bands. Following observation were noted after gel stain:

Preparation of the albumin tissue scaffold was done as follows. 2 g bovine serum albumin, purity>98% purchased from Sigma, was dissolved in 8.8 mL buffer of 50 mM BICINE, pH 8.3. The albumin solution was kept in 4° C. refrigerator. 0.026, 0.020, 0.016, and 0.013 mL of 50% glutaraldehyde solution were combined with 1 mL of albumin solution in tubes which correspond to 1:15, 1:20, 1:25, and 1:30 weight ratio of glutaraldehyde to albumin, respectively. Samples were incubated at 37° C. for 2 hours. 40 mL of the ice-cold solution of 6 M urea, 0.1 M sodium acetate, pH 5.0 was added to each sample, and then homogenized. The resulted homogenate was centrifuged at 2,330 g for 5 min. The pellets, which containing high molecular weight albumin polymers, were recovered in every sample. 40 mL of 0.1% lactic acid (Sigma) was added to suspend the albumin polymers, incubated on room temperature for 5 min, and then pelleted by centrifugation 2,330 g for 5 min. The lactic acid washing step was repeated more twice to remove urea from albumin polymers. A volume of 0.1 mL of albumin polymer was transferred to 96-well culture plate (Falcon) using a positive-displacement pipette (Gilson). The plate was kept in a −80° C. deep freezer (Thermo) for 1 hour, then moved to a freeze dryer (VirTis) for 24 hours. The porous scaffold was obtained after freeze-drying. The plate was placed in a 2.5-L container included 250 mL of 4% paraformaldehyde (Sigma) in the bottom of container. The vaporous cross linking treatment was performed at room temperature for 1 hour. Prepared tissue scaffold was then stored in a dry box.

Scanning electron microscopes. Albumin tissue scaffolds were mounted onto sample holder using a conductive tape (EMS). Samples were coated by gold and observed under SEM (JEOL). For observing inner structure, used albumin tissue scaffolds were saved after surface examination, horizontally cut through the center by a blade (Leica) into the half. Surface pore diameters were estimated as followings:

Water binding. Albumin tissue scaffold was soaked in pure water (Millipore), and then determined the wet weight. A filter paper (Whatman) was used to blot off the water from wet albumin tissue scaffold to semi-dry, and then placed the samples in a 60° C. oven for 2 hours. The dried weight of dehydrated sample was then determined. The water binding was calculated as the weight ratio that divided the wet weight by the dried weight. The following results were obtained:

Cyclic compressive test. Sample was rinse by Milli Q water. Sample was placed in a 3-cm tissue culture dish contained 1 mL of the Milli Q water. A cyclic compressive testing was setup and performed at ambient by a testing machine (Instron).

Cell adhesion. Albumin tissue scaffold was soaked in pure water (Millipore), washed by Dulbecco's PBS (Invitrogen) three changes, and then culture medium three changes (Invitrogen). A cell suspension of MSC (Cambrex) was prepared in the culture medium at the density of 1e6 cells per mL. 10 μL of cell suspension was transferred onto the prepared albumin tissue scaffold. After 24 hour incubation, sample was washed by Dulbecco's PBS three times, and then fixed by 4% paraformaldehyde/PBS for 1 hour at room temperature. Sample was soak in 6.8% sucrose/PBS overnight, dehydrated by acetone, and the dried by critical point dryer (Tousimis). Samples were coated by gold and observed under SEM (JEOL).

Preparation of the albumin polymer was done as follows. 0.05 g human, bovine, or porcine serum albumin (purity>98%, all from Sigma) was dissolved in 0.475 mL of 50 mL BICINE, pH 8.3 buffer. Polymerization reaction was carried out by adding 0.5 mL of 1 mg/mL microbial transglutaminase (AJINOMATO) and 0.025 mL of 0.5 M DTT (Sigma) into albumin solution. The reaction was incubated at 37° C. for 18 hr. The resulted albumin solid was homogenized in 9 mL of 6 M urea, 0.1 M sodium acetate, pH 5.0. The homogenate was spin down at 2,330 g for 5 min, and the supernatant was discarded. 9 mL of 0.1% lactic acid was added to suspend the pelleted albumin polymers. The suspension was spin down at 2330 g for 5 min. The lactic acid washing step was repeated more twice. A volume of 0.1 mL of albumin polymer was transferred to 96-well culture dish. The plate was frozen at −80° C. for 1 hour, subsequently moved to freeze dryer for 24 hours. After freeze-drying, porous tissue scaffolds was generated. The plate was then placed in a 2.5-L sealed container included 250 mL of 4% paraformaldehyde. The cross linking treatment was performed at room temperature about 25° C. for 1 hour. Prepared tissue scaffold was then stored in dry box. The examinations revealed that the sponge have following characterizations: pore diameter between about 54 μm to about 124 μm, water binding of about 43.4±1.5, and having resilient property in water.