Synthesis and Biological Evaluation of Well-Defined Poly(propylene fumarate) Oligomers and Their Use in 3D Printed Scaffolds

A ring opening polymerization method for synthesizing oligomeric poly­(propylene fumarate) (PPF) provides a rapid, and scalable method of synthesizing PPF with well-defined molecular mass, molecular mass distribution (<i>Đ</i>_m), and viscosity properties suitable for 3D printing. These properties will also reduce the amount of solvent necessary to ensure sufficient flow of material during 3D printing. MALDI mass spectrometry precisely shows the end group fidelity, and size exclusion chromatography (SEC) demonstrates narrow mass distributions (<1.6) of a series of low molecular mass oligomers (700–3000 Da). The corresponding intrinsic viscosities range from 0.0288 ± 0.0009 dL/g to 0.0780 ± 0.0022 dL/g. The oligomers were printed into scaffolds via established photochemical methods and standardized ISO 10993-5 testing shows that the 3D printed materials are nontoxic to both L929 mouse fibroblasts and human mesenchymal stem cells

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Supplementary Material

Effect of Chemical and Physical Properties on the In Vitro Degradation of 3D Printed High Resolution Poly(propylene fumarate) Scaffolds

Two distinct molecular masses of poly­(propylene fumarate) (PPF) are combined with an additive manufacturing process to fabricate highly complex scaffolds possessing controlled chemical properties and porous architecture. Scaffolds were manufactured with two polymer molecular masses and two architecture styles. Degradation was assessed in an accelerated in vitro environment. The purpose of the degradation study is not to model or mimic in vivo degradation, but to efficiently compare the effect of modulating scaffold properties. This is the first study addressing degradation of chain-growth synthesized PPF, a process that allows for considerably more control over molecular mass distribution. It demonstrates that, with greater process control, not only is scaffold fabrication reproducible, but the mechanical properties and degradation kinetics can be tailored by altering the physical properties of the scaffold. This is a clear step forward in using PPF to address unmet medical needs while meeting regulatory demands and ultimately obtaining clinical relevancy

Cell viability by CCK-8 assay under LUT pretreatment and the morphology of H9c2 cells.

<p>A. CCK-8 assay detected the toxicity of LUT on H9c2 myocardiocytes. Compared to DMSO group, 32 and 64μM of LUT reduced the cell viability (**<i>p</i><0.01, ***<i>p</i><0.001), 16μM of LUT was chosen for the further experiments. B. The morphology of H9c2 cells in control, AR and LUT+AR group.</p

AV/PI detected the early apoptotic cell percentage of H9c2 cells.

<p><b>A.</b> AR significantly increased the early apoptotic cell percentage of H9c2 cells compared with control group (**<i>p</i><0.01). However, LUT pretreatment significantly decreased the apoptosis rate compared to AR group (#<i>p</i><0.05). B. Overexpression of miR-208b-3p under AR significantly increased the early apoptotic cell percentage of H9c2 cells compared with AR group (***p<0.001). However, LUT pretreatment had no statistical significance compared to 208m+AR group (<i>p</i>>0.05). C. Knocking down miR-208b-3p expression under AR decreased the early apoptotic cell percentage of H9c2 cells compared with Ni+AR group (*<i>p</i><0.05). D. Knocking down Ets1 expression under AR increased the early apoptotic cell percentage of H9c2 cells compared with si-Nc+AR group (*<i>p</i><0.05).</p

Hemodynamic parameters in different groups.

<p>Hemodynamic parameters in different groups.</p

Photographs of various stages of the new method.

<p>a, b. The mouse was located on its right side and connected with ventilation and a Data Analysis System. The skin was depilated and disinfected. c. The skin was covered with aseptic hole-towel. d. A 1 cm skin incision and a purse suture were made. e. The pectoralis muscles were separated and the intercostal muscles were exhibited. f. A small hole was made at the fourth intercostal muscle. g. A small wet sterile gauze was used to protect the lungs. The LAD (↑) and left auricle (◀) were displayed. h. One hand held the microneedle holder with a surgical suture needle to pass underneath the LAD and another hand held a smooth tying forceps to widen the hole. i. An approximate 2-mm diameter loop was made. j. A 4–0 suture was passed through the third and fifth intercostal muscle. k. A deflated balloon was placed in the loop. l. The balloon was inflated. m. The chest cavity was closed by straining the purse suture. n. The wound was closed. o. In the classic method, a PE-10 tube was used and a slipknot was made.</p

Myocardial injury in different groups.

<p>a. Representative photographs of Evans blue/TTC-stained heart sections in different groups. b. Percentage of area at risk over total cross-sectional areas of the heart in different groups (n = 6). c. Percentage of infarct size over area at risk in different groups (n = 6). ###<i>p</i><0.001 vs. sham group.</p

Ets1 was a target of miR-208b-3p.

<p>A. MiR-208b-3p was detected to be up-regulated under A/R injury, LUT pretreatment could down-regulate it in H9c2 cells, **<i>p</i><0.01, *<i>p</i><0.05 as compared with control group; ##<i>p</i><0.01 as compared with AR group. Ets1 mRNA was detected to have no statistical significance under A/R injury and LUT pretreatment (<i>p</i>>0.05). Ets1 mRNA was detected to have no statistical significance under miR-208b-3p mimic/inhibitor transfecting (<i>p</i>>0.05). B. Ets1 was decreased after A/R injury, but increased by LUT pretreatment in H9c2 cells, ***<i>p</i><0.001 as compared with control group, ##<i>p</i><0.01 as compared with AR group; Over-expressing miR-208b-3p decreased Ets1, #<i>p</i><0.05 as compared with AR group, and abolished the effects of LUT on Ets1 (<i>p</i>>0.05). Knocking down miR-208b-3p increased Ets1, #<i>p</i><0.05 as compared with AR group. And Ets1 was decreased after transfecting Ets1-siRNA, ###<i>p</i><0.001 as compared with AR group, and abolished the effects of LUT on Ets1 (<i>p</i>>0.05). C. By transfecting of the reporter plasmid with (WT group) or without (MUT group) the putative miR-208b-3p binding site, the luciferase activity decreased after miR-208b-3p overexpression and increased after miR-208b-3p knock-down in H9c2 cells; ***<i>p</i><0.001 as compared with WT+Nm group, **<i>p</i><0.01 as compared with WT+Ni group. In addition, mutating the predicted binding site of miR-208b-3p (MUT+208m, MUT+208i group) abolished the process mentioned above (<i>p</i>>0.05).</p

Representative electrocardiogram during different period and a schematic diagram of surgical procedure.

<p>a. Electrocardiogram changes during I/R. Before ligation of the LAD, the ST segment was located at the baseline. The ST segment elevated during ischemia and it declined in reperfusion period. b. The schematic diagram of surgical procedure, recovery time, ischemia operating time and reperfusion operating time.</p