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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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/organs1,2. 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. Decel.......

Representative 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 repair10,11,12. The use of decellularized matrix in engineering approaches to tissue repair has been a subject of much interest and success in the recent past2,3,14<.......

Acknowledgements

This project was partially funded by a grant from the Pediatric Orthopaedic Society of North America (POSNA) and the National Institutes of Health grant NIBIB R21EB025378-01 (Exploratory Bioengineering Research Grant).

....

Materials

NameCompanyCatalog NumberComments
Sieve machineHaver & Boecker TylerRo-Tap RX 29-E Pure
Sieve 90 umFisherbrand170328156No. 170
Sieve 53 umFisherbrand162513588No. 270
Sieve 106 umFisherbrand162018121No. 140
Sputter coaterLeican/a
Scanning Electron MicroscopeHitachi, USAn/a
Filabot EX2Filabot.comFB00061
Filabot SpoolerFilabot.comFB00073
CAPA 6506Perstorp24980-41-4
Phosphate buffered saline, PBSGibco10010023
6" FanComfort Zone, Amazonn/a
Ultrasonic Water BathCole ParmerSK-08895-13
DreamerFlashForgen/a
Drum MixerCustom maden/aSimilar 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 BalanceMettler Toledo, Fisher Scientific01-913-851
Simplify3DSimplify3Dn/a
SolidWorksSolidWorksn/a
MicrospheresProduced in-house, see concurrently submitted JoVE submission
p-nitrophenyl phosphate, disodium salt, hexahydrateMillipore4876-5GM
Phosphatase, alkalineRoche Diagnostics GmbH10 713 023 001
Absorbance ReaderTecanSunrise
Tris-HCl BufferSigma-AldrichT6455-100ML
Heated shakerNew Brunswick ScientificExcella E24

References

  1. Hutchmaker, D., Teoh, S., Zein, I., Ng, K. W., Schantz, J. -. T., Leahy, J. C. Design and Fabrication of a 3D Scaffold for Tissue Engineering Bone. Synthetic Bioabsorbable Polymers and Implants. 15 (2), 845-847 (1988).
  2. Ghosh, P., Gruber, S. M. S., Lin, C. -. Y., Whitlock, P.

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3D PrintingDecellularized MatricesTissue engineered ScaffoldsMicrospheresSievePolycaprolactoneExtruderFilament MaterialTissue Engineering

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