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 f....

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 Eag....

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