Novel Process for 3D Printing Decellularized Matrices

Summary

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

Abstract

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs^1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 °C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 °C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

Introduction

Current tissue engineering techniques for clinical applications such as bone, cartilage, tendon, and ligament reconstruction use auto- and allografts to repair damaged tissue. Each of these techniques is performed routinely as a "gold standard" in clinical practice by first harvesting the donor tissue either from the patient or a cadaveric match, and then placing the donor tissue into the defect site². However, these strategies are limited by donor site morbidity, donor site scarcity for large defects, risk of infection, and difficulty finding grafts that match the desired geometry. In addition, studies have shown that allografts used for reconstruction have reduced mechanical and biologic properties when compared with native tissue³. With these considerations in mind, tissue engineers have recently turned to three dimensional (3D) bioprinting to produce custom, complex geometries that are biologically active and designed to accommodate defect size and shape while providing sufficient mechanical properties until biologic remodeling is complete.

Ideally, a 3D-printed scaffold would consist of a polymeric backbone that can retain the required mechanical stability of native tissue while the incorporated biologics offer biochemical cues to surrounding cells, leading to their migration, proliferation, differentiation, and tissue production^2,5. Unfortunately, most constructs that contain biologic components are made with gels or polymers that are too weak to withstand in vivo forces experienced by the targeted tissues for auto/allograft reconstruction. Other polymers such as polylactic acid (PLA) are bioresorbable, 3D printable and structurally sound, but are printed at temperatures at or above 210 °C - making it impossible for biologics to be co-printed during fabrication. Polycaprolactone (PCL) is another FDA-cleared, bioresorbable polymer that can be 3D printed at a lower temperature (65 °C), which has become increasingly popular in fabricating patient-specific implants with complex morphologies^5,6,7,8,9. However, most bioprinters using pneumatic technology make it impossible to print PCL at lower temperatures where biological activities can remain unharmed. To date, the integration of these polymers with auto/allografts into a novel printable biomaterial has yet to be accomplished. In the absence of such a material, a true tissue engineered approach to tissue reconstruction is unlikely. Therefore, we have sought to combine PLA, PCL, and decellularized allograft matrices (DM) to utilize the advantages of each material in order to manufacture a viable construct capable of reconstructing complex tissues. This process would provide the initial mechanical strength necessary to resist in vivo forces and the thermal stability to accommodate additive manufacturing in a construct that induces formation of the desired tissue.

In a recent attempt to address the aforementioned hurdles, we showed that it is feasible to encapsulate decellularized cartilage extracellular matrix within a thermally protective PLA barrier that can be extruded within PCL filaments, maintaining the ability of DM to influence surrounding host cells². This has inspired us to seek clinically effective approaches for tissue reconstruction. In the current study, we utilize the platform technology to build all-in-one scaffolds that include PLA, DM, and PCL (PLA-DM/PCL).

Our goal is to improve the efficacy and utility of allografts using the proposed novel biofabrication technique to more accurately recapitulate native tissue, to ultimately use them in various applications.

Protocol

1. Obtaining and Preprocessing Microspheres

1. Produce microspheres with the desired matrix encapsulated (PLA-DM)².
   NOTE: It is imperative that the microspheres are of uniform size. For this reason, sieving the microspheres prior to use is essential. Although matrix decellularization and encapsulation have been detailed in previous publications², a brief summary of the process follows.
   1. First, harvest cartilage plugs from porcine hind limbs. Decellularize the cartilage in a series of washes with 0.05% trypsin/0.5 mm tetrasodium ethylenediaminetetraacetic acid (EDTA), Dulbecco's modified Eagle's medium (DMEM), and 1.5% peracetic acid and 2.0% Triton X-100 for 4 h each with distilled water washes before and after each step².
   2. Drain the decellularized matrix, freeze it, lyophilize, grind, and dissolve into pepsin solution. Following dissolution, mix the pepsin solution with PLA which has been dissolved in dichloromethane.
   3. Add the mixture dropwise into a 3% polyvinyl alcohol in water solution. Centrifuge the resulting microspheres, rinse, drain, and lyophilize again.
      NOTE: For full details on the process see the previously published protocol².
2. Sieve the microspheres.
   1. Ensure that all sieve plates have been thoroughly cleaned and are dry prior to use. If necessary, clean sieves using ultrasonic cleaner to ensure that all spheres are removed from the sieve.
   2. Assemble the sieve shaker with the 106 µm sieve tray at the top, the 53 µm tray after that, and the sieve pan at the bottom.
   3. Place dry microspheres in the topmost sieve tray and place the lid on the top tray. Turn on coarse sieving for 8 to 10 min. Repeat on fine for 8 to 10 min.
      NOTE: The sieve times may need to be increased or decreased depending on the batch.
   4. Carefully remove the sieve plates one by one and place them upside down on a large weigh paper. Tap the sides gently to ensure that most of the spheres have fallen out of the sieve and onto the paper.
   5. Discard the oversized spheres (>106 µm) and undersized spheres (<53 µm). Add spheres that are in the 53 to 106 µm size range to a labeled centrifuge tube with the type and batch number then place in a -20 °C freezer until further use.

2. Microsphere Quality Control Assessments

Note: See Figure 1.

1. Perform macroscopic/visual assessment to check that the microspheres are uniform and spherical, with no aggregates present.
2. Assess the microspheres using a scanning electron micrograph (SEM).
   1. For this, place microspheres onto an SEM chuck and sputter coat with gold-palladium in argon atmosphere using a sputter coater to a thickness of 4 nm.
   2. Observe surface features, morphology, and diameters of the microspheres using a 10 kV accelerating voltage and a 10 mm working distance to ensure that production and sieving of the microspheres was successful.

3. Filament Creation for 3D Printing

1. Measure and record the mass of the microspheres obtained from steps 2 and 3; at least 25 g is needed.
2. Add polycaprolactone (PCL) powder to the microspheres for a 1:4 weight ratio of microspheres to PCL.
3. Mix the powder mixture on a miniature rolling mixer at 20 rpm for 5 min then flip the container and mix at 20 rpm for an additional 5 min (see Figure 2).
4. Many commercially available extruders (see the Table of Materials) have insulating jackets because their intended working temperatures are for traditional fused deposition modeling (FDM) filaments. Modify the extruder (if necessary) by removing the insulating material and use it in combination with desktop fans (which blow ambient air onto the extruder and extruded filament) to use of the extruder at lower temperatures.
   NOTE: Desktop fans which blow ambient air to cool the extruder and filament are useful for this procedure.
5. Setup the equipment setup for extrusion. See Figure 3.
   1. Setup the extruder so that its outlet is ~60 cm from the inlet to the spooler, with a direct path from the extrusion outlet to the spooler inlet.
      NOTE: The spooler can optionally be raised 3-4 inches from the bench if it is found that the filament is drooping to the point of touching the benchtop.
   2. Place a desktop fan ~15 cm from the heating jacket and direct it towards the heating jacket to offer cooling with ambient air throughout filament production. Place a second cooling fan approximately halfway between the extruder and spooler and direct it towards the extrudate to assist in cooling the filament with ambient air.
   3. Adjust the positioning as needed throughout the process.
6. Set the modified extruder heating element to 52 °C, turn on the desktop cooling fans, and allow the instrument to come to equilibrium for 20 to 30 min. Ensure that the proper nozzle is attached to the extruder.
7. Just before beginning, fill the extruder hopper with the microsphere/PCL mixture from step 3.3. Turn on the spooler and the extruder auger to initiate extrusion of filament.
8. When the initial filament is extruded, manually pull the extrudate from the extrusion outlet with forceps and feed it to the filament spooler.
9. The desired filament will take some time to come out of the spooler. Using separate spools or tape, clearly mark when the filament composition visually appears uniform.
10. Monitor the process closely and modify parameters as necessary. Adjust the extruder temperature, extrusion auger speed, and spooler speed to obtain a 1.75 mm diameter filament as measured by calipers. Adjust the fans as needed to cool the filament properly to avoid non-circular filament cross-sections. Mix and refill the hopper as necessary.
    NOTE: Close attention is required during this process to obtain adequate filament for subsequent 3D printing. The above parameters will change depending on the ambient conditions, the fill level and uniformity of the mixture in the hopper, and the thermodynamics and flow dynamics of the specific batches of PCL and microspheres.
11. Continue extruding until all of the powder has been used and the hopper is almost empty. Add PCL powder (without microspheres) to the hopper to flush out the microsphere mixture that is currently in the extruder. Continue adding PCL powder to the hopper until no more microspheres are visible in the extrudate.
12. Be sure to label and separate the filament which contains the microspheres in the desired concentration, as after the filament is cooled it is harder to distinguish the uniform filament from non-uniform filament.
13. Continue extruding until there is minimal powder left in the hopper, then turn off the spooler, extruder auger, extruder heating element, and fans.

4. Printing with the Filament

1. Design a geometry of the desired shape and form using a computer aided design software. Then slice the model and dictate the toolpath using slicing software that is compatible with the 3D printing machine being used.
2. Load the filament from step 3 onto any standard FDM printer, fitted with standard nozzles of the desired diameter (typically 0.4 mm). Begin the print (typically at 65-70 °C and 300 mm/min linear speed) as the custom filament is deposited layer-by-layer by the machine.
3. Make sure to pay special attention to the first layer and adjust settings as needed to get a good quality print.
   NOTE: Adjustments may be made to the print speed, print temperature, platform temperature, extrusion multiplier, and other parameters. Refer to the printer and slicing manufacturer's troubleshooting guide for further assistance.

5. Quality Control Assessment

1. Place the printed constructs on SEM chucks and sputter coat with gold-palladium in argon atmosphere using a sputter coater to a thickness of 4 nm.
2. Observe under the microscope using a 10 kV accelerating voltage and a 10 mm working distance to check surface features and for the presence or absence of microspheres if applicable.

6. Functional Testing of the Printed Constructs

NOTE: Alkaline phosphatase (ALP) can be used as a surrogate for decellularized matrix to determine if encapsulated proteins are biologically active after the filament production process. ALP is used because it catalyzes a reaction from a substrate, p-nitrophenyl phosphate, to change from colorless to yellow byproducts, p-nitrophenol and inorganic phosphate, but only if ALP is in the functional conformation.

1. Print a geometry (n = 3) that has an end mass of at least 400 mg with the ALP microsphere filament (PLA-ALP/PCL) using identical print parameters as the PLA-DM/PCL scaffolds. Also print PCL-only (PCL(-)) scaffolds of the same geometry as the PLA-ALP/PCL scaffolds. Submerge them in 1 mL Tris-HCl buffer and incubate for 24 h at 37 °C and 110 rpm rotation to allow enzyme diffusion.
2. Add 1 mL of 1 mg/mL p-nitrophenyl phosphate, disodium hexahydrate in Tris-HCl. Incubate at 37 °C, 110 rpm for an additional 10 h. Read the supernatant absorbance at 415 nm.

Results

After sieving, microspheres should appear uniform and be free from aggregates. Under SEM, the sieved microspheres may have small pores on their surface, but will otherwise be spherical and smooth, as shown in Figure 1. All extruded filaments should be of uniform diameter and circular cross-section. A filament that contains microspheres (PLA-DM/PCL) will have a slightly more matte finish while a PCL-only (PCL(-)) filament would look more glossy. The PLA-DM/PCL...

Discussion

Both decellularized matrices and 3D printed PCL scaffolds have independently been shown to allow adhesion and proliferation of cells, validating their use for osteochondral repair^10,11,12. The use of decellularized matrix in engineering approaches to tissue repair has been a subject of much interest and success in the recent past^2,3,14<...

Materials

Name	Company	Catalog Number	Comments
Sieve machine	Haver & Boecker Tyler	Ro-Tap RX 29-E Pure
Sieve 90 um	Fisherbrand	170328156	No. 170
Sieve 53 um	Fisherbrand	162513588	No. 270
Sieve 106 um	Fisherbrand	162018121	No. 140
Sputter coater	Leica	n/a
Scanning Electron Microscope	Hitachi, USA	n/a
Filabot EX2	Filabot.com	FB00061
Filabot Spooler	Filabot.com	FB00073
CAPA 6506	Perstorp	24980-41-4
Phosphate buffered saline, PBS	Gibco	10010023
6" Fan	Comfort Zone, Amazon	n/a
Ultrasonic Water Bath	Cole Parmer	SK-08895-13
Dreamer	FlashForge	n/a
Drum Mixer	Custom made	n/a	Similar piece of equipment: https://www.coleparmer.com/i/argos-technologies-flexiroll-digital-tube-roller-shaker-120-vac/0439744?PubID=UX&persist=true&ip= no&gclid=CjwKCAjw- dXaBRAEEiwAbwCi5khGDMz0 dTjsraEsBGfhMEH7ytx LQWGUPNgUJYQ1p3vj_yxkYoI_ ixoC9GwQAvD_BwE
Micro Balance	Mettler Toledo, Fisher Scientific	01-913-851
Simplify3D	Simplify3D	n/a
SolidWorks	SolidWorks	n/a
Microspheres	Produced in-house, see concurrently submitted JoVE submission
p-nitrophenyl phosphate, disodium salt, hexahydrate	Millipore	4876-5GM
Phosphatase, alkaline	Roche Diagnostics GmbH	10 713 023 001
Absorbance Reader	Tecan	Sunrise
Tris-HCl Buffer	Sigma-Aldrich	T6455-100ML
Heated shaker	New Brunswick Scientific	Excella E24