Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Summary

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Abstract

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

Introduction

In the tissue engineering field the ability to fabricate tissue scaffolds is vital. A suitable tissue scaffold has a 3D structure, is composed of biocompatible materials, and mimics in vivo tissue architecture to facilitate cell and tissue growth and remodeling. This scaffold must allow the transport of nutrients and the removal of wastes^1-4. One of the main obstacles in the production of these scaffolds is the ability to recapitulate specific geometrical features into a biocompatible material. Several biofabrication techniques have been reported to control the geometrical features of these scaffolds, examples are electrospinning^5-8, solvent-casting⁹, stereolithography¹⁰, and 3D-printing¹¹, among others. These techniques fall short in providing a relatively easy transfer of controllable internal and external architectural features, are expensive, are limited by their resolution and printability (e.g., nozzle gauge, material restriction), or require post-fabrication techniques which demands a long period of time to produce viable scaffolds¹².

In many commercial fabrication systems, the creation of internal voids, channels, and features is achieved using sand or other suitable removable or sacrificial materials. The metal or plastic part is formed around the sand mold, and once it is solidified, the sand is removed. In much the same manner, the next generation of biomaterials needs the biosand equivalent. Therefore, the BSA rubber was developed as a substitute for biosand. The BSA rubber is a newly formulated material that consists of bovine serum albumin crosslinked with glutaraldehyde. The ultimate goal is to recreate specific architectural features into a biodegradable collagen scaffold. The characteristics of the sacrificial biorubber that maintains dimensional fidelity with the mold of the original tissue are described.

Several combinations of BSA and glutaraldehyde concentrations were tested using a variety of solvents. This material was created by the reaction between BSA and glutaraldehyde. BSA rubber can be reaction injected into the intricate geometries of the tissue molds. Crosslinked BSA is trypsin labile and readily digested by the enzyme at mild pH and temperature conditions. Conversely, intact type I collagen is very resistant to trypsin digestion. These features were capitalized to selectively remove the BSA rubber leaving the collagen behind. The present work consisted of determining the ideal parameters needed to obtain a labile mold that can deliver specific architectural features to a biocompatible scaffold. The specific features that were evaluated included mixability, enzyme digestion, load bearing, and ability to be reaction injected into a negative mold. The combination of 30% BSA and 3% glutaraldehyde fulfills these requirements. This protocol provides the necessary guidelines to create these three-dimensional scaffolds. The prototype consists of a collagen scaffold that represents a branched architecture with one inflow and two outflow channel with diameters of 4- and 3-mm, respectively. This technique has the potential to mimic macro- and micro-environments of the tissue of interest. This technology provides a viable technique to deliver a specific geometrical instructive to a biodegradable material in a relatively easy and timely matter with high fidelity, which can be tuned to mimic the in vivo tissue elasticity and other characteristics of the tissue of interest.

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Protocol

1. Determine the Percentage of Solids in the Collagen Batch

1. Extract the collagen following a previously published procedure¹³. Thaw a minimum of 20 ml of collagen. Determine the initial percentage of collagen solids in the batch in order to manipulate the collagen concentration in the formed hydrogels.
   1. Cut three pieces of aluminum foil (about 6 x 6 cm) and shape each one as a pan by using the bottom of a 25 ml beaker. Record the weight of each pan.
   2. Add a small amount of collagen to each pan and record the total weight of the aluminum pan and collagen. Add 0.5 - 0.8 g of collagen on each aluminum pan.
      Note: After this step, there are three aluminum foil pans and each one should have a small amount of collagen. Make sure to record the weight of each (total 3) empty aluminum pan and the weight of the pan after the addition of collagen. 
   3. Calculate the weight of the collagen in each pan using the following formula:
      Collagen weight = Pan and collagen weight - Pan weight
   4. Place the three aluminum pans that hold the collagen in the oven at 100 °C for 24 hr.
   5. After 24 hr, record the weight of each aluminum pan and the dehydrated collagen.
   6. Calculate the weight of the dehydrated collagen using the following equation:
      Dehydrated collagen weight = Pan and dehydrated collagen weight - Pan weight
   7. Calculate the percentage of solid for the three samples to determine the collagen solid concentration using the formula below:
      
   8. Calculate the average collagen solid content of the batch using the percentage of collagen solids for each of the three samples.
      Note: The collagen that will be used is what is left of the hydrated collagen. None of the dehydrated collagen will be used.
   9. After determining the percentage of collagen solid (initial collagen concentration) of the batch, continue using the remaining hydrated collagen. Use a calibrated pH meter to adjust the pH of the collagen batch to 3.
      1. Add small amounts (2-5 µl at a time) of 12 N Hydrochloric Acid (HCl). Keep on ice at all times. Do not add the hydrochloric acid directly to the collagen — add the acid to the side of the tube. After adding the acid, use the spatula to push the acid to the collagen and quickly stir the mixture.
   10. Once it reaches a pH of 3, let the collagen sit O/N at 4 °C.

2. Preparation of the BSA Rubber

1. Prepare the Bovine Serum Albumin (BSA) solution following the procedure listed below.
   1. Prepare 2x Phosphate Buffered Saline solution (PBS). Add two PBS tablets to 100 ml of water to make 0.02 M PBS solution.
   2. Combine BSA with 2x PBS to create a 30% BSA solution using the procedure listed below.
      1. For example, to make a 30% BSA with 30 ml of 2x PBS, use 12.9 g of BSA. Add 1/3 of the 2x PBS (e.g., 10 ml) to a flask with a stir bar. Add 1/2 of the BSA (e.g., 6.45 g BSA) to the flask and using a spatula, wet the dry solute.
      2. Repeat this process of adding PBS and then BSA until all the solute and solvent is in the flask. Use the spatula to wet all the solute. It will look like a mixture of some liquid and clumps. Let it sit for approximately 30 min.
      3. Then, turn the stirrer on a low cycle and make sure that there are no clumps around the stir bar. It should take 90 min to get everything in solution or it can be left stirring O/N at 4 °C. After the solute is all dissolved, place the solution in a 20 ml syringe and capped with a 0.20 µm syringe filter. Press the plunger to expel liquid through filter and collect the sterilized solution in a new tube. Store at 4 °C.
   3. Prepare 3% glutaraldehyde solution by diluting 25% glutaraldehyde solution with sterile filtered 2x PBS. For example, for 10 ml of 3% Glutaraldehyde solution, use 2 ml of 25% glutaraldehyde and 8 ml of 2x PBS. Store at 4 °C.

3. Molds Treatment

Note: The prototype described in this paper uses a custom made stainless steel Y mold piece. The mold contains an inflow and two outflow channels of 4 and 3 mm, respectively. First, clean molds, spray them with unsaturated lard, and sterilize them. Prepare the molds following the procedure described below.

1. Clean stainless steel molds using sonicator at a frequency of 35 kHz. Place the molds in the sonicator and submerge them with water and ice. Keep the molds cold at all times while the sonicator is running. Run the sonicator for 2 periods of 90 min.
   1. After each period, use a needle to make sure that there is no material in the Luer lock stainless steel or brass connector. Use soap and water to clean the entire surface of the two sides of the molds. Verify that there is no obstruction in the channels.
2. Place the molds, screws, and Luer lock connector in an autoclave bag and autoclave it.
3. Fill the bottle that attaches to an air sprayer half way with commercially available lard (mixed fatty acid release agent). Replace the cap with a regular cap bottle. Place it in an autoclave bag and autoclave it.
   Note: Lard is used to facilitate the release of the material that will be reaction injected later on (BSA rubber). Do not place the sprayer bottle lid in the autoclave- it can damage the internal seal.
4. Warm up the lard for 45 sec or until its clear and liquid in a microwave. Screw the air sprayer lid to the lard bottle. Connect the lid with the sprayer. Attach the sprayer to the air source at the lab bench. Open the air valve, and open the nozzle of the sprayer until it starts wetting the surface of a paper towel.
5. Spray lard perpendicular to the mold surface until the surface is fully covered. After each piece has been sprayed, place them in a Petri dish and seal it. Place the molds at 4 °C for 2 hr.
6. Proceed to sterilize the molds by exposing the surface to UV light for 30 min. Place them back at 4 °C until they are ready to be reaction injected.

4. Reaction Injection of the BSA Rubber

Note: All the materials and solution should be keep cold until ready to use to prevent premature setting of the BSA rubber in the next steps.

1. Prepare the dispenser for delivering the BSA rubber to the molds following these steps.
   1. Sterilize all of the mixing components (two O rings, syringe cap, double syringe, mix tip, and 4:1 dispenser) by exposing them to UV light for 30 min in the polymerase chain reaction (PCR) hood.
      Note: A PCR hood was used because this procedure involves fixatives. These chemicals cannot be used in the cell/tissue culture hood due to the risk of exposure and toxicity to the cells. Any other hood that contains a UV light will be suitable. 
   2. Place the tip cap on the solution holder.
   3. Perform the mixing and injection at a 4:1 ratio of BSA:Glutaraldehyde. Add the 30% BSA to the double syringe chamber that will deliver the highest amount of solution (It will take approximately 4 ml to fill). Make sure to leave enough space to place the O ring in order to prevent overflowing and contamination of the adjacent chamber.
   4. Add 3% glutaraldehyde solution to the other chamber (it will take about 1 ml to fill). Make sure to leave enough space to place the O ring to prevent overflowing and contamination of the adjacent chamber.
   5. Place the double syringe on the dispenser. Tilt the assembly vertically so that the syringe cap is on top. Replace the cap with the mixing tip.
   6. Screw the two stainless steel mold pieces together.
   7. Place the assembly inside of an autoclave bag.
   8. To remove any air in the dispenser, hold it in the upright position and quickly squeeze the handle one time to release a small amount of the BSA mixed with the glutaraldehyde. Then quickly attach the stainless steel Y mold's Luer lock connector to the syringe tip.
   9. Hold the stainless steel Y mold with the left hand and the BSA-Glutaraldehyde mixture dispenser on the right. Alternate covering each of left and right exhaust of the outflow channels by pressing the exhaust to the sides of the autoclave bag to make sure the inside voids are filled with solution. Then, place the molds horizontally and inject again.
   10. Detach the molds from dispenser and place in a 25 mm Petri dish.
   11. Place Parafilm around the Petri dish to prevent dehydration of the rubber.
   12. Place the mold in the 4 °C fridge O/N.

5. Adjusting the Collagen Concentration

Note: The collagen should be kept on ice at all times during the process.

1. Modify the collagen concentration using the percentage of collagen solids.
   1. Make 10 ml of 1.75% collagen by adjusting the initial collagen concentration with cold water.
   2. Using a calibrated pH meter, adjust the pH to 3 using 12 N Hydrochloric Acid. Do not add the hydrochloric acid directly to the collagen- add the acid to the side of the tube. After adding the acid, use the spatula to push the acid into the collagen and quickly stir the mixture.
   3. Weigh 4 g of collagen in a separate conical tube.
   4. Centrifuge the collagen to remove air at 4 °C and 9,343 x g for 20-30 min.
   5. UV sterilize the cell culture hood for 30 min, and add 14 µl of laminin to the 4 ml collagen. This will result in a final laminin concentration of 10 µg/µl. Note: Laminin provides structural integrity, adhesion, and promotes various cellular responses.
   6. Turn the PCR UV light on for 20-30 min prior to using the hood for sterilization.
   7. Place the Luer lock cap, to attach to a 20 ml syringe, in ethanol for a 2 hr. Then, allow it to dry and place it in UV light.
   8. Gamma irradiate the collagen for 8.6 min to reach 1,200 cGy.
      Note: The time will depend on the decay of the Cesium source. Adjust the time to reach the same dosage.

6.Casting Collagen on BSA Rubber

1. To polymerize the collagen, use an 8:1:1 ratio (collagen:HEPES:MEM). The following procedure is based on an initial 4 g of acidified collagen (from Step 5.1.8).
   1. Make a 0.2 N HEPES solution in water, and adjust the pH to 9 by adding small amounts (1-5 µl) of 1-5 M sodium hydroxide (NaOH) solution. Using a calibrated pH meter, monitor the pH of the solution after each addition. Store at 4 °C or keep on ice.
   2. Turn on the UV light of the tissue culture hood for 20-30 min to sterilize the hood.
   3. Autoclave forceps, spatula, and scalpel for sterilization.
   4. Mix 1.5 ml of 0.2 N HEPES (pH 9) and 1.5 ml of 10x MEM using the tissue culture hood. Make sure to keep it on ice and vortex it for 5 sec prior to use. Store at 4 °C or keep on ice.
   5. To sterilize the PCR hood, turn on the UV light for 30 min.
   6. Place a 12 well plate and a 20 ml syringe in -20 °C for 10 min, or until ready to continue. Note: Keep all materials cold until ready to use. The increase in temperature induces premature collagen fibrillogenesis.
   7. Spray all tubes and well plates that are going to be in the hood with 70% ethanol and let them dry for sterilization. Place a 20 ml syringe on ice to cool for later use.
   8. In the PCR hood, open the stainless steel molds to release the BSA rubber and using a scalpel, cut the exhaust channels of the BSA rubber mold.
   9. Under the PCR hood, open the sterile collagen tubes and add 1 ml of the HEPES-MEM solution (make sure that before extracting the HEPES-MEM solution, that it is well mixed and there are no solid deposits).
   10. Using the sterile spatula, thoroughly mix the collagen and buffer solution.
   11. Close and vortex it quickly to ensure a well-mixed hydrogel.
   12. Transfer to a cold 20 ml syringe.
   13. With one hand, hold the BSA Rubber inside of the well and, using the other, dispense half of the collagen hydrogel solution onto the bottom of the well.
   14. Using the sterile tweezers, ensure that the rubber inflow and outflow ends are touching the sides of the well.
   15. Pour collagen solution on top of the rubber until is completely covered.
   16. Ensure that the BSA rubber is suspended within the collagen and that there are no bubbles, especially near the ends of the rubber.
   17. Place the cover and wrap Parafilm around the circumference of the well.
   18. Put in the incubator for 1 hr at 37 °C. Keep the PCR UV light on.
2. After the polymerization of the collagen, UV crosslink the hydrogel via the following procedure.
   Note: The crosslinking of the collagen will be done using a UV crosslinker apparatus in which the amount of energy can be controlled.
   1. Turn on the UV crosslinker and use the energy setting to irradiate the empty chamber with 630,000 µJ/cm².
   2. Remove the gels from the incubator.
   3. Spray hands with ethanol, and, inside the chamber, remove the lid as quickly as possible.
   4. Close the chamber and UV crosslink the hydrogels by selecting the energy setting and irradiating 630,000 µJ/cm².
   5. After the crosslinking cycle, turn off the UV light on the PCR hood
   6. Spray hands with ethanol and open the chamber, quickly placing the lid back onto the well plate. Move the well plate to the PCR hood.
   7. Using the sterile spatula, gently loosen and remove the gel from the well. Flip the gels under the hood to crosslink the bottom of the hydrogel. Repeat step 6.2.3 and 6.2.4.

7. Enzyme Digestion of the BSA Rubber

1. In order to have a hollow collagen scaffold, remove BSA Rubber in a way that does not affect the dimensions embedded in the hydrogel. The procedure is described below.
   1. Turn on the UV light for 20-30 min to sterilize the tissue culture hood.
   2. Make 0.25% trypsin solution pH 7.8. For example, for 15 ml of water, add 0.0376 g of trypsin in a 50 ml conical tube. Adjust the pH to 7.8 by adding small amounts (2-5 µl) of 1 M NaCl. Place the solution in a 20 ml syringe and capped with a 0.20 µm syringe filter. Press the plunger to expel liquid through filter and collect the sterile solution in a new tube.
   3. Turn on the water bath and set the temperature to 30 °C.
   4. After 30 min, turn off the UV light. Spray ethanol on all the tubes and materials that will be used in the hood.
   5. Transfer the collagen hydrogel under the hood and place in separate conical tube.
   6. Add around 3-5 ml (just enough to cover the gels) of 0.25% trypsin solution with a pH of 7.8 to each tube.
   7. Seal the tubes with Parafilm and vortex lightly for approximately 1 min.
   8. Place in the 30 °C water bath for 15-24 hr. While in the water bath, lightly vortex the gels frequently until the BSA rubber has been digested or removed from the hydrogel.
      Note: In order to determine if the BSA rubber has been removed, either the rubber is floating in the trypsin solution or there are broken-down pieces. There must be no visual dark areas within the hydrogel.
2. To ensure that all the BSA rubber and trypsin has been removed from the hydrogels, rinse it as described below.
   1. Turn on the PCR hood UV light for 20-30 min.
   2. Prepare Mosconas solution. Combine potassium chloride (KCl, 28.6 mM), (NaHCO₃, 11.9 mM), glucose (9.4 mM) and (NaH₂PO_4, 0.08 mM) in water. Adjust pH to 7.4 with 1 M NaOH or 12 M HCl solution. Place the solution in a 20 ml syringe and use a syringe filter of 0.20 µm to sterilize the solution.
   3. Spray everything with ethanol prior placing them on the hood and allow the ethanol to dry.
   4. Open the tube and transfer 5-10 ml of sterile Mosconas solution to a new 50 ml conical tube.
   5. Transfer the collagen hydrogel to the conical tube that contains the enzyme solution. Make sure that the hydrogel is completely covered with the solution. Leave the tube in the shaker in the fridge at 4 °C for 30 min.
   6. Aspirate the Mosconas solution and repeat step 7.2.5 twice.
   7. Store at 4 °C.

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Results

The results demonstrate that this biofabrication technique is efficient in generating 3D scaffolds that can mimic the spatial arrangement seen in in vivo tissue. The architectural features are vital parameters for tissue engineering application, playing a crucial role in the in vivo cell interaction and functionality of the tissue.

The consistency and mixability of the BSA rubber was an important parameter in p...

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Discussion

Biofabrication is a highly multidisciplinary field in which biology and engineering principles merge to generate complex materials that mimic native tissue. In order to achieve this, there is a need to develop techniques that use the information gathered from in vivo tissue and translate it into an in vitro scaffold. In this way, a platform can be engineered that closely resembles the architectural, functional, and mechanical properties of the in vivo tissue. The optimal scaffolding material sh...

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Materials

Name	Company	Catalog Number	Comments
Collagen type I			Collagen extracted from calf hide
Hydrocloric Acid (HCl)	Sigma-Aldrich	7647-01-0
Phosphate Buffer Solution (PBS Tablets)	MP Biomedical	U5378	1 tablet per 100 ml makes 1x PBS
Albumium from bovine serum (BSA)	Sigma-Aldrich	A9647
Glutaraldehyde	Sigma -Aldrich	G5882	Toxic
Lard	Fields	3090
Stainless Steel Molds			Milled using Microlution Machine
Air Brush Kit	Central Pneumatic	47791
Mixing Tip for double syringe	Medmix	ML2.5-16-LLM	Mixer, DN2,5X16, 4:1 brown, med
Small O ring for double syringe	Medmix	PPB-X05-04-02SM	Piston B, 5 ml, 4:1, PE natural
Double Syringe cap	Medmix	VLX002-SM	Cap, 4:1/10:1, PE brown, med
Big O ring for double syringe	Medmix	PPA-X05-04-02SM	Piston A, 5 ml, 4:1
Double Syringe	Medmix	SDL X05-04-50M	Double syringe, 5 ml, 4:1
Double Syringe Dispenser	Medmix	DL05-0400M	Dispenser, 5 ml, 4:1, med , plain
Laminim			3.6 mg/ml - extracted USC lab
20 ml Syringe Luer Lock Tip	BD	302830
Luer Lock Caps	Fisher	JGTCBLLX
HEPES	Sigma -Aldrich	H4034
Gibco Minimum Essential Media 10x (MEM)	Life Technologies	1143-030
Trypsin	Life Technologies	27250-018
UV Crosslinker	Spectroline UV	XLE1000
Sodium Cloride (NaCl)	Fisher	S271-10	To prepare Mosconas
Potassium chloride (KCl)	Sigma -Aldrich	P5405-250	To prepare Mosconas
Sodium Bicarbonate (NaHCO3)	Fisher	BP328-500	To prepare Mosconas
Glucose	Sigma -Aldrich	G-8270	To prepare Mosconas
Sodium Phosphate didasic (NaH2PO4)	Sigma-Aldrich	S-7907	To prepare Mosconas
Sterile Filter for syringes	Corning	431224