*2.1. Sca*ff*old Design*

Autodesk® Fusion 360™ (Autodesk Inc., San Rafael, CA, USA) was used to design the 3D models. The basic 3D model was designed as a cylinder with a diameter of 15 mm and a height of 1 mm. The 3D

model (.stl file) was then loaded into Perfactory RP for slicing, with a layer height equal to 0.25 mm. The sliced file (.bpl file) was loaded into Visual Machine, and the infill patterns were selected. A linear pattern was selected for the orthogonal scaffolds (i.e., the control group), and a sinusoidal waveform was selected for the wavy scaffolds (Figure 1). For wavy scaffolds, the amplitude and the wavelength of the sinusoid were varied systematically to develop 5 distinct scaffold designs (Table 1).

**Figure 1.** Wavy scaffold design containing 4 layers (**left**) and schematic showing the strut design for wavy scaffolds (**right**).


**Table 1.** Design and printing parameters for the scaffolds.

1 Strut-to-strut distance. 2 Print temperature. 3 Young's modulus from compression tests. 4 Micro-CT results.

### *2.2. 3D Printing of Sca*ff*olds*

3D Bioplotter (EnvisionTEC, Gladbeck, Germany) was used to print the scaffolds using PCL pellets (MW = 55 kDa, Polysciences Inc., Warrington, PA, USA). The print temperature and pressure were set to 80 ◦C and 700 kPa (7 bar), whereas the print speed was varied from 4 to 6 mm/s for each design to achieve a similar strut size (see Table 1 for actual values for each design).

### *2.3. Characterization of the Sca*ff*olds*

3D printed scaffolds were imaged by using a scanning electron microscope (SEM, JSM-7900F, JEOL, Tokyo, Japan) and a micro-computed tomography scanner (micro-CT, SkyScan 1275, Bruker, Billerica, MA, USA). SEM images were used to measure the strut size and the strut-to-strut distance. Micro-CT was used to measure the porosity of the scaffolds. Compression tests were performed on 3D printed scaffolds using an Instron machine (model 3343) with a 1000 N load cell and a 0.5 mm/min displacement rate. Three samples for each scaffold group were tested.

### *2.4. Preparation of the Sca*ff*olds for Cell Culture*

Sca ffolds were sterilized by immersing them in a 75% ethanol solution for 30 min, followed by 1 h of ultraviolet (UV) light exposure (by a germicidal lamp) for each side of the sca ffold. Sca ffolds were then incubated in 300 μL of fibronectin solution (20 μg/mL, bovine fibronectin plasma, Invitrogen) overnight to enhance cell attachment. Fibronectin solution was removed, and sca ffolds were washed with Dulbecco's Phosphate Bu ffered Saline (DPBS, Gibco, New York, NY, USA). Sca ffolds were then moved into a new well and kept in growth media prior to cell seeding.

### *2.5. Cell Culture and Reagents*

Human mesenchymal stem cells (hMSCs, passage 4, Lonza, Walkersville, MD, USA) were cultured in growth media ( α-MEM (minimum essential medium) supplemented with 10% fetal bovine serum (FBS, Gibco, New York, NY, USA) and 1% penicillin-streptomycin (pen-strep, Gibco, New York, NY, USA)). Prior to seeding, each sca ffold was removed from the growth media and placed in a single well in a non-treated 24 well plate. The human mesenchymal stem cell (hMSC) suspension (133,000 cells/mL) was seeded from the top of the sca ffolds (300 μL per sca ffold, corresponding to approximately 5000 cells/cm2). Cells were incubated at 37 ◦C for 60 min to allow cell attachment. Sca ffolds were then flipped, and the same amount of cell suspension was seeded from the top, followed by 60 min incubation at 37 ◦C. The sca ffolds were then transferred to a new non-treated 24 well plate, and 1 mL of fresh growth media was added into each well. The sca ffolds were incubated for 7 days in growth media. For osteogenic di fferentiation studies, growth media was replaced with osteogenic induction media (hMSC osteogenic di fferentiation medium BulletKit ™, Lonza, Basel, Switzerland) at Day 7, and cells were cultured for an additional 14 days. The media was refreshed every 3 days in cell culture studies.

### *2.6. Cell Culture and Characterization*

For stem cell growth studies, the AlamarBlue assay (AlamarBlue ™ Cell Viability Reagent, Invitrogen) and PicoGreen assay (Quant-iT ™ PicoGreen ™ dsDNA Assay Kit, Invitrogen) were used to evaluate the cell proliferation at Days 1, 4, and 7, according to the manufacturer's protocol. A Tecan plate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland) was used to complete the assays for these studies. To visualize the attached cells on the sca ffolds, cells were washed with DPBS (3×), fixed with 4% formaldehyde for 15 min, followed by DPBS wash (3×), and permeabilization in 0.25% Triton-X DPBS solution for 1 h. Cells were stained for F-actin using rhodamine phalloidin (1:40 in DPBS, Invitrogen). Cell nuclei were stained with <sup>4</sup>,6-diamidino-2-phenylindole (DAPI, 1:2000 in DPBS, Sigma, St. Louis, MO, USA). At Day 7, cells were immunostained for vinculin using the anti-vinculin-FITC antibody (1:50, mouse monoclonal, Sigma). For this purpose, cells were incubated in 10% goa<sup>t</sup> serum (in PBS) for 30 min, washed with staining solution (3<sup>×</sup>, 3% bovine serum albumin + 0.1% Tween-20 +0.25% Triton-X), and incubated in vinculin antibody in staining solution overnight at 4 ◦C. Cells were imaged by using a confocal and multiphoton microscopy (TCS SP8 MP, Leica, Wetzlar, Germany).

For di fferentiation studies, calcium deposition was evaluated at Day 21 by using the alizarin red staining kit (AR, Sigma, St. Louis, MO, USA). After staining was completed, cells were washed with DPBS (3×), and incubated in 10% cetylpyridinium chloride (Sigma, St. Louis, MO, USA) in sodium phosphate bu ffer (10 mM, pH 7, Sigma) to remove the stain. This solution was then used to quantify calcium content by using a Tecan plate reader (scanned at 405 nm). Alkaline phosphatase activity was studied with the QuantiChrom ™ Alkaline Phosphatase Assay Kit (ALP assay Kit, BioAssay Systems, Hayward, NY, USA). Cells cultured within the sca ffolds were first lysed with 0.2% Triton-X followed by 3 freeze-thaw circles. The lysate was then reacted with p-nitrophenyl phosphate working solution and scanned at 405 nm using a plate reader (Infinite M200 Pro, Tecan). For osteocalcin (OC) staining, cells were fixed at Day 14 and Day 21. Cells were incubated in 10% goa<sup>t</sup> serum (in PBS) for 30 min, washed with staining solution (3<sup>×</sup>, 3% bovine serum albumin +0.1% Tween-20 +0.25% Triton-X), and

incubated with the OC primary antibody (1:200, monoclonal mouse, Invitrogen) in the staining solution overnight at 4 ◦C. After removing the antibody containing staining solution and washing the samples with fresh staining solution, cells were incubated in Alexa Fluor 488 rabbit anti-mouse secondary antibody (1:100, Invitrogen) in staining solution for 2 h. Samples were then stained with phalloidin (rhodamine phalloidin, Invitrogen) and DAPI to visualize F-actin and cell nuclei, respectively. Cells were imaged by using a confocal and a multiphoton microscopy (TCS SP8 MP, Leica). All of the collected images were processed using ImageJ (NIH, Bethesda, MD, USA) for further analysis.
