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Beijing University of Chemical Technology: 3D Printed HA/PCL Tissue Engineering Scaffolds

3D printed bone scaffolds used for tissue engineering purposes need to have a good amount of mechanical strength, since the scaffold needs to be able to provide support for the tissue. As bone scaffolds also require the correct pore structure to help provide a good environment for the differentiation, proliferation, and repairing of damaged tissue cells, bioactive materials, such as polycaprolactone (PCL) and hydroxyapatite (HA), are needed.

Researchers Zhiwei Jiao, Bin Luo, Shengyi Xiang, Haopeng Ma, Yuan Yu, and Weimin Yang, from the Beijing University of Chemical Technology (BUCT), published a paper, titled “3D printing of HA / PCL composite tissue engineering scaffolds,” about their work constructing nano-HA/PCL and micro-HA/PCL tissue engineering scaffolds using the melt differential FDM 3D printer they developed.

The abstract reads, “Here, the internal structure and mechanical properties of the hydroxyapatite/polycaprolactone scaffolds, prepared by fused deposition modeling (FDM) technique, were explored. Using hydroxyapatite (HA) and polycaprolactone (PCL) as raw materials, nano-HA/PCL and micro-HA/PCL that composite with 20 wt% HA were prepared by melt blending technology, and HA/PCL composite tissue engineering scaffolds were prepared by self-developed melt differential FDM 3D printer. From the observation under microscope, it was found that the prepared nano-HA/PCL and micro-HA/PCL tissue engineering scaffolds have uniformly distributed and interconnected nearly rectangular pores. By observing the cross-sectional view of the nano-HA/PCL scaffold and the micro-HA/PCL scaffold, it is known that the HA particles in the nano-HA/PCL scaffold are evenly distributed and the HA particles in the micro-HA/PCL scaffold are agglomerated, which attribute nano-HA/PCL scaffolds with higher tensile strength and flexural strength than the micro-HA/PCL scaffolds. The tensile strength and flexural strength of the nano-HA/PCL specimens were 23.29 MPa and 21.39 MPa, respectively, which were 26.0% and 33.1% higher than those of the pure PCL specimens. Therefore, the bioactive nano-HA/PCL composite scaffolds prepared by melt differential FDM 3D printers should have broader application prospects in bone tissue engineering.”

Melt differential 3D printer.

PCL is biocompatible, biodegradable, and has shape retention properties, which is why it’s often used to fabricate stents. But on the other hand, due to an insufficient amount of bioactivity, the material is not great for use in bone tissue engineering. HA, which has been used successfully as a bone substitute material, has plenty of bioactivity, which is why combining it with PCL can work for bone tissue engineering scaffolds.

“On the whole, the existing tissue engineering scaffolds preparation process have problems of low HA content, easy agglomeration, low stent strength, and single printing material,” the researchers explained.

“The HA/PCL composite particles are used as printing materials, and the mechanical properties and structural characteristics of the two tissue engineering scaffolds are compared and analyzed. The raw material of the melt differential 3D printer is pellets, which eliminates the step of drawing compared to a conventional FDM type 3D printer. The 3D printer is melt-extruded with a screw, and a micro-screw is used for conveying and building pressure. At the same time, precise measurement is performed by a valve control system. This printing method shows advantages in simple preparation process of the composite material, higher degree of freedom in material selection, simple printing process, and shorter preparation cycle of tissue engineering scaffolds.”

The team mixed PCL particles and HA powder together to make the scaffolds. Their melt differential 3D printer uses pellets, and features a fixed nozzle with a platform that moves in three directions. A twin-screw extrusion granulator was used to prepare the PCL material, and the melt differential 3D printer fabricated the tissue engineering scaffolds out of the nano-HA/PCL and micro-HA/PCL composite particles.

The working principle diagram of the polymer melt differential 3D printer.

A microcomputer-controlled electronic universal testing machine was used to test the scaffolds’ bending and tensile properties. A scanning electron microscope was used to observe the micro-HA particle size, as well as the scaffolds’ cross section, while an optical microscope was used to observe their surface structure and a transmission microscope was used to look at the nano-HA particles’ particle diameter and morphology. The scaffold material’s crystallization properties were analyzed using a differential thermal analyzer.

3D printing tissue engineering scaffolds.

Testing showed that the micro-HA was spherical, with a 5–40 μm diameter, and contained some irregularly-shaped debris. The nano-HA was rod-shaped, with a 20–150 nm length.

The crystallization peak temperature of the HA/PCL composites was higher than pure PCL material, because adding HA caused its molecular chain to form a nucleate after absorbing on the HA’s surface. Additionally, adding HA to pure PCL increased the material’s melting temperature, as the latter material had crystals “of varying degrees of perfection.”

The nano-HA/PCL and micro-HA/PCL tissue engineering scaffolds “could form a pre-designed pore structure and the pores were connected to each other,” which is seen in the image below.

“…the micro-HA/PCL and the nano-HA/PCL composite tissue engineering scaffolds can form a three-dimensional pore structure with uniform distribution and approximately rectangular shape.”

External views of micro-HA/PCL and nano-HA/PCL composite tissue engineering scaffolds.

These rectangular pores, with a 100-500 μm length and width, are good news for cell adhesion and proliferation, and the fact that they’re interconnected is positive for nutrient supply.

As for mechanical properties, the nano-HA/PCL specimens had the highest tensile and bending strengths – between 25 and 35% higher than the pure PCL. The micro-HA/PCL specimens had higher tensile and flexural strengths than the PCL, but the nano-HA/PCL was stronger than the micro-HA/PCL, because the HA’s modulus is higher than the PCL’s.

“In addition, nano-HA was more evenly distributed in the composite, while micro-HA had obvious agglomeration in the composite, so the tensile strength and flexural strength of nano-HA/PCL specimens were higher than that of micro-HA/PCL specimens,” the researchers wrote.

Finally, the pore structure of the nano-HA/PCL and micro-HA/PCL tissue engineering scaffolds offered a favorable environment for the discharge of cellular metabolic waste, in addition to facilitating nutrient transport and blood vessel growth. The researchers concluded that their 3D printed composite scaffolds had more potential applications in bone tissue engineering.

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Singapore: 3D Bioprinting with Magnesium Alloys to Create Bone Scaffolds

SEM micrographs of samples sintered at different temperatures in the regime of super solidus liquid phase sintering for 5 h, a) 535 °C, b) 550 °C, c) 565 °C, d) 580 °C, e) 595 °C, and f) 610 °C.

Strides in the medical field today via 3D printing have been staggering, and especially in bioprinting, with many different technologies and materials being created. Now, researchers in Singapore are exploring the use of alloys like magnesium in fabricating scaffolding, with their findings detailed in ‘Additive manufacturing of magnesium–zinc–zirconium (ZK) alloys via capillary-mediated binderless three-dimensional printing.’

Magnesium is an alloy that can be used in 3D printing and additive manufacturing, as a third-generation biomaterial useful in tissue engineering; however, as the researchers point out, there are myriad challenges. High affinity to oxygen and a low boiling temperature are issues, along with careful consideration that must be applied when disposing of magnesium powders due to the possibility of reactions with other chemicals.

High vapor pressure can be a major obstacle in using magnesium too, leading the researchers to explore AM processes with ambient temperature. This can allow for all the benefits of powder-bed inkjet 3D printing to be enjoyed, as it can be employed at ambient temperatures, no supports are required, and powder can be fully recycled. Here, the researchers have created a new 3D printing technique including a sintering process which transforms magnesium powder and green objects into functional parts that can be used in scaffolding, producing parts with mechanical properties as strong as human bone.

The research team customized their own ink-jet 3D printer for this study, working to overcome previous challenges with the use of magnesium. Maintaining oxygen percentages at the lowest levels possible was of ‘paramount importance’:

“Conserving oxygen in green objects in low level indicates the promise of formulated solvent for AM of Mg-based alloys,” stated the researchers.

3D printed green samples showed no change at all in composition after the sintering process, leaving the team to point out that this means it is a ‘compositionally zero-sum process.’ With temperature variations, both density and stability were affected. The researchers state that dimensional precision is another element of paramount importance and is influenced when deviations occur in printing. Swelling may cause substantial problems too, resulting in shape loss of printed objects, noted at an increased sintering temperature from to 595 °C and 610 °C. Swelling can also interfere with functionality of components.

Samples after 5 h sintering at different temperature in the range of 535 °C to 610 °C.

In continuing to examine other features, the researchers found that density increases with temperature. In studying the effects of holding time on physical and mechanical properties, they also found that strength may be low even though density has become high. Overall though, for overcoming the challenges required in creating scaffolds, mechanical integrity must be present, along with balanced stiffness and strength:

“Mechanical properties of scaffolds could significantly affect cells behavior and the osteointegration between host tissues and the scaffold; premature collapse of subchondral bonearound bone defects may happen if the scaffold provides more than enough mechanical support,” said the researchers. “Thus, stiffness and strength of scaffolds should be modulated to match with those of host tissues in order to avoid post-surgery stress shielding effects and promote tissue regeneration.”

Healthy scaffolds exhibit good pore percentage, size, and shape, offering osteointegration, nutrients transportation, tissue in-growth, and waste products removal. With all those quotients in order, bone tissue regeneration is possible.

“Mg based alloys classify as a third generation of biomaterials when it comes to clinical outcomes, and capillary-mediated binderless 3D printed Mg part after sintering can provide comparable properties with bone,” stated the researchers.

In their paper, the researchers explain more about the structure of human cortical bone, a hierarchical ‘organization’ of three sizes to include:

  1. Haversian and Volkmann vascular canals having diameter in the range of 40 to 100 μm
  2. Osteocyte lacunae with size ranging from 10 to 30 μm
  3. Canaliculi having diameters on an order of a few tens of nanometers

Issues in porosity can be dealt with as larger pores are created in 3D to compensate for a required percentage, thus refining scaffold for better tissue engineering with bone.

“Increasing holding time from 5 h to 20, 40, and 60 h at optimum sintering temperature of 573 °C allowed steady improvements in microstructural, physical, and mechanical properties for each additional hold time while avoiding the undesirable dimensional loss. Interconnected open-porous structures with apparent porosity of 29%, average pore size of 15 μm, compressive strength of 174 MPa, and elastic modulus of 18 GPa were achieved,” concluded the scientists. “These values are well comparable with those seen for human cortical bone types.”

There is a huge momentum between 3D printing and the medical field today, and it just keeps growing as scientists and researchers continue to work toward the holy grail of fabricating human organs. Along with that, many different types of medical implants have been created and are now improving the quality of patients lives, from facial implants to those meant to facilitate knee replacements. Tissue engineering continues to be at the forefront of 3D printing also with the range of bioinks continuing to expand.

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Schematic illustrations of super-solidus liquid phase sintering process of 3D printed parts, a) total decomposition of interparticle bridges in a green sample after reaching 400 °C, b) nucleation of liquid phase along the grain boundaries and within the discrete islands throughout the grains at the temperature above the solidus, c) breaking MgO film for several particles with increasing temperature, leaking the liquid phase, forming liquid bridges among particles, and d) break down of MgO film, formation of liquid bridges between adjacent Mg particles, and growth of sinter necks diameter in the sample sintered at 573 °C for 40 h.

[Source / Images: Additive manufacturing of magnesium–zinc–zirconium (ZK) alloys via capillary-mediated binderless three-dimensional printing]