Supporting Information. Templated Repair of Long Bone Defects in Rats. with Bioactive Spiral-Wrapped Electrospun

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1 Supporting Information Templated Repair of Long Bone Defects in Rats with Bioactive Spiral-Wrapped Electrospun Amphiphilic Polymer/Hydroxyapatite Scaffolds Artem B. Kutikov,, Jordan D. Skelly, David C. Ayers, and Jie Song,, * Department of Orthopedics and Physical Rehabilitation. University of Massachusetts Medical School. 55 Lake Ave North, Worcester, MA 01655, USA Department of Cell and Developmental Biology. University of Massachusetts Medical School. 55 Lake Ave North, Worcester, MA 01655, USA Corresponding Author * address: Jie.Song@umassmed.edu; phone: ; fax:

2 1. MATERIALS and METHODS: 1.1 MSC viability and proliferation following lentiviral GFP transduction MTT cell viability assay (Roche, Indianapolis, IN) was performed to quantify cell viability 24 h after lentiviral transduction. Percent cell viability was normalized to untreated MSC controls. Cell proliferation over 72 h was quantified by MTT (n=3) and presented as fold change from initial cell viability (24 h following lentiviral transduction). 1.2 Pathology of vital/scavenger organs To identify any potential negative systemic effects of HA-PELA and its degradation products, heart, kidney, liver, lung, spleen, and pancreas tissues were retrieved from the sacrificed rats at 12 weeks and 24 weeks after receiving the implants. The organs were fixed in periodate-lysine-paraformaldehyde fixative at 4 ºC for 2 days then paraffin embedded, sectioned, and stained with hematoxylin and eosin for pathological evaluation. 1.3 Osteogenic and adipogenic differentiation of MSCs Osteogenic and adipogenic differentiations were performed by incubating the untreated MSCs or GFP-MSCs in MSC expansion media containing osteogenic (10 nm dexamethasone, 20 mm β-glycerol phosphate, 50 µm L-ascorbic acid 2-phosphate) or adipogenic (0.5 µm dexamethasone, 0.5 µm isobutylmethylxanthine, 50 µm indomethacin) supplements. Alizarin red S and oil red O staining was performed following 2 weeks of incubation to assess the potency of osteogenic and adipogenic differentiation, respectively. For quantification, alizarin red S stain was quantified by extraction with acetic acid, neutralization with ammonium hydroxide, and colorimetric detection at 405 nm, as previously described 1. Oil red O staining was quantified by extraction with isopropanol and colorimetric detection at 450 nm, as previously described. 2 2

3 1.4 Biomechanical testing Using a Dremel drill, the center of the PEEK plates fixating the explants were thinned to minimal attachment to facilitate the handling of those explants that were not fully bridged with calcified callus during potting. The explants were then potted in zinc-coated aluminum hex nuts with bone cement following literature protocol. 3 After both ends were potted, the thinned center of the PEEK plate was carefully trimmed with scissors without damaging the underlying graft/callus. Using a mini-torsion tester equipped with an ep2 controller (Admet, Norwood, MA), the explants were torqued to failure at 1 /s. Maximum torque recorded was reported as the failure torque, torsional stiffness was determined from the linear region of the torque/displacement curve, and torsional energy was determined form the area under the torque/displacement curve. 3

4 2. RESULTS Figure S1. Lentiviral GFP labeling of MSCs. (A) Fluorescence microscopy of MSCs transduced with lentiviral vectors driving GFP expression by CMV, UBC, or EF1α promoters with increasing MOI s. Scale bar = 150 µm. (B) Viability of MSCs upon 24-h exposure to various MOI s of EF1α -GFP lentivirus relative to untreated controls. (C) Fold change in cell viability of GFP-MSCs (transduced with LV-EF1α-GFP at MOI = 25) vs. untreated MSCs over 72 h. (D) Alizarin red S staining of untreated MSCs and GFP-labeled MSCs following 14-day culture in osteogenic media; Scale bar = 250 µm. (E) Oil red O staining of untreated MSCs and GFPlabeled MSCs following 14-day culture in adipogenic media; Scale bar = 250 µm. (F) 4

5 Quantification of alizarin red stains in the MSC and GFP-MSCs cultures after 14 days in expansion (negative control) and osteogenic media. The stains were released by acid treatment for spectroscopic quantifications. (G) Quantification of Oil red stains in the MSC and GFP- MSCs cultures after 14 days in expansion (negative control) and adipogenic media. The stains were released by isopropanol for spectroscopic quantifications. 5

6 Figure S2. Hematoxylin and eosin staining of the center of the scaffolds showing bone formation, blood vessel formation, and immune response at 12 weeks post-op. S = scaffold, NB = new bone, BM = bone marrow. Arrowheads indicate blood vessels and * indicates macrophages/foreign body giant cells. Figure S3. Hematoxylin and eosin staining of vital/scavenger organs (heart, liver, kidney, lung, spleen and pancreas) collected at (A) 12 weeks and (B) 24 weeks after implantation of HA- PELA. No abnormality was detected compared with un-operated healthy controls. 4 Figure S4. Torsional testing of 12-week explants as a function of treatment (HA-PELA alone, MSCs, 500 ng rhbmp-2). Data are presented as scatter plots with mean ± standard deviation. 6

7 REFERENCES (1) Gregory, C. A; Gunn, W. G.; Peister, A.; Prockop, D. J. An Alizarin Red-Based Assay of Mineralization by Adherent Cells in Culture: Comparison with Cetylpyridinium Chloride Extraction. Anal. Biochem. 2004, 329, (2) Laughto, C. Measurement Attached of the Specific Lipid Content of Cells in Microtiter Cultures. Anal. Biochem. 1986, 314, (3) Nussbaum, D. a.; Gailloud, P.; Murphy, K. The Chemistry of Acrylic Bone Cements and Implications for Clinical Use in Image-Guided Therapy. J. Vasc. Interv. Radiol. 2004, 15, (4) Filion, T. M.; Li, X.; Mason-Savas, A.; Kreider, J. M.; Goldstein, S. A.; Ayers, D. C.; Song, J. Elastomeric Osteoconductive Synthetic Scaffolds with Acquired Osteoinductivity Expedite the Repair of Critical Femoral Defects in Rats. Tissue Eng. Part A 2011, 17,