Effect of Poly(sophorolipid) Functionalization on Human

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Effect of Poly(Sophorolipid) Functionalization on Human Mesenchymal Stem Cell Osteogenesis and Immunomodulation Ahmad S. Arabiyat, Patricia Diaz-Rodriguez, Josh D. Erndt-Marino, Filbert Totsingan, Shekar Mekala, Richard A Gross, and Mariah S. Hahn ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00434 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018

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ACS Applied Bio Materials

Full Title: Effect of Poly(Sophorolipid) Functionalization on Human Mesenchymal Stem Cell Osteogenesis and Immunomodulation.

Running Title: pLSL functionalization directs MSC behavior

Authors: Ahmad S. Arabiyata,c†, Patricia Diaz-Rodrigueza,c†, Josh D. Erndt-Marinoa,c, Filbert Totsinganb,c, Shekar Mekalab,c, Richard A. Grossb, c and Mariah S. Hahna,c *.

†A.S.A. and P.D.R. contributed equally to this work.

Affiliations: aDepartment

of Biomedical Engineering, bDepartment of Chemistry and Chemical Biology,

and cCenter

for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute

(RPI), 110 8th street, Troy, New York 12180, United States.

*Contact author Mariah S. Hahn, Ph.D. Professor, Biomedical Engineering

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Rensselaer Polytechnic Institute Tel: 518-276-2236 Email: [email protected]

ABSTRACT Sophorolipids are a class of glycolipids that can be polymerized via ring-opening metathesis polymerization giving rise to bioresorbable biomaterials. The surface chemistry of the resulting poly(sophorolipids) (pLSLs) can be modified using a combination of enzymatic and “click” chemistries to insert bioactive groups that influence cellular behavior. Mesenchymal stem cells (MSCs) are being actively investigated for engineered bone grafts for fracture repair due to their osteogenic potential, and more recently, due to their immunomodulatory capacity. The long-term goal of this work is to utilize functionalized pLSL foams loaded with MSCs as a bioresorbable scaffolds for bone fracture healing. Towards this goal, the present study evaluated the effect of various pLSL chemistries on the osteogenic and immunomodulatory behavior of MSCs. pLSLs functionalized with PO4, NH2 or COOH small functional groups, were fabricated into open porous foams and then cultured with MSCs in the presence of osteogenic medium for 72 h. Protein level assessments demonstrated that the PO4functionalized pLSL foams supported the highest degree of MSC osteogenesis as well as the highest levels of immunomodulatory factors pertinent to improved bone fracture healing.


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Cumulatively, these results suggest that further investigation of the long-term osteogenic commitment of MSCs in PO4-functionalized pLSL foams is warranted.

Keywords: poly(sophorolipids), mesenchymal stem cells, osteogenesis, immunomodulation for bone fracture repair

1. INTRODUCTION The market for bone grafts in the US is estimated at 1.6 million grafts annually, with an associated cost of $2.5 billion1,2. Autografts are considered the ‘gold standard’ of care for bone defects. However, their use is limited by supply, donor site morbidity, infection and the size of the defect1–3. Tissue engineered bone grafts may provide a suitable environment for osteogenic cells to proliferate and differentiate and thus may present an alternate approach for the treatment of bone defects4–6. Engineered bone grafts are typically comprised of an osteoconductive material with seeded or recruited osteogenic cell populations4,5. Mesenchymal stem cells (MSCs) have been a primary cell type utilized for engineered bone grafts not only due to their ability to undergo osteogenic differentiation4,7,8 but also more recently due to their capacity to promote regeneration through production of several immunomodulatory factors9,10. 3 ACS Paragon Plus Environment


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The inflammatory immune response involved in bone fracture healing plays a vital role in bone formation and remodeling11–14. The fracture hematoma is critical to the initial inflammatory response of bone healing, as shown by upregulation of chemokines and infiltration of neutrophils and monocytes/macrophages15,16. Interestingly, disturbance of hematoma formation has been previously shown to impair fracture healing and lead to a significant decrease in bone mechanical properties14,17. The processes mediated by cytokine and chemokine release highlight the essential role that modulation of inflammation plays in bone healing. For example, the recruitment of inflammatory cells in the fracture hematoma is mediated by the release of chemokines during the initial inflammatory immune response. Interleukin-6 (IL-6), secreted by macrophages, inflammatory cells and cells of mesenchymal origin, acts as an initiator of the repair cascade11,18,19. Monocyte Chemotactic Protein-1 (MCP1, also known as CCL2), a chemokine upregulated in the fracture hematoma, plays a role in the infiltration of macrophages20 and osteoclastogenesis21, which are essential in stimulating MSC osteogenesis and bone remodeling, respectively14. Furthermore, the upregulation of interleukin-8 (IL-8, also known as CXCL8), evident in the initial stages of bone healing, stimulates the release of vascular endothelial growth factor (VEGF), thus promoting angiogenesis22,23. Despite playing essential roles in bone regeneration, the production of many of these cytokines and chemokines, including MCP-1 and IL-8, are often not considered or measured in MSCs utilized for bone regeneration applications. For example, the immunomodulatory capacity of MSCs is typically assessed through production of enzymes such as prostaglandin E2 synthase (PTGES-2), indoleamine 2,3-dioxygenase (IDO), 4 ACS Paragon Plus Environment

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cyclooxygenase-2 (COX-2) and of cytokines such as hepatocyte growth factor (HGF)24–28. The upregulation of the enzymes IDO and PTGES-2 function as immunomodulatory elements through minimizing immune-mediated tissue injury by suppression of T cell function29,30, and through the synthesis of the immunomodulatory factor prostaglandin E2 (PGE2), respectively31,32. Moreover, the activity of the enzyme COX-2 is associated with the production of PGE2 and the regulation of MSC osteogenic differentiation33,34. On the other hand, HGF secreted by MSCs has been shown to exhibit immunomodulatory effects through reducing the ability of dendritic cells to induce inflammatory Th1 cells and modulating the production of the immune suppressive cytokine interleukin-10 from monocytes28,35. Sophorolipids are a class of extracellular glycolipids that are synthesized in large quantities by the yeast Candida bombicola when grown on carbohydrates, fatty acids or their mixtures36,37. Biological applications of sophorolipids have been reported for their therapeutic roles in modulating inflammation38. The development of poly(lactonic sophorolipids), pLSLs, via ring-opening metathesis polymerization (ROMP) was previously described for diacetylated lactonic forms of sophorolipids39,40. The resulting pLSLs are amphiphilic, containing both hydrophobic and hydrophilic segments40,41. Structurally, pLSLs present biodegradable ester and acetal linkages that produce natural structures upon hydrolytic degradation, an attractive feature of potential bioresorbable biomaterials40. We previously reported chemo-enzymatic routes to introduce clickable methacrylate moieties into lactonic sophorolids (LSLs), which subsequently transfer to the polymer chains following ROMP41. The addition of these clickable



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moieties into sophorolipids can be used to alter the chemistry of pLSLs through the introduction of bioactive groups which regulate cellular differentiation and function. Altering the surface chemistry of biomaterials through the addition of small functional groups has been previously established to impact stem cell adhesion and differentiation through changes in the hydrophilicity and charge of the material42,43. For example, Benoit et al. demonstrated that MSCs grown on poly(ethylene glycol) (PEG) gels presenting were induced to preferentially undergo osteogenic, chondrogenic or adipogenic differentiation depending on the presented functional group42. Specifically, phosphate (PO4) groups have been linked to directing osteogenic differentiation42,43 due to their role in mineralization and calcium deposition44. Similarly, carboxylic acid (COOH) groups, which are present on the surfaces of glycosaminoglycans42, have been shown to play a role in promoting chondrogenesis42,45. We previously demonstrated that methacrylate (MA) moieties on pLSLs can be functionalized via thiol-ene click chemistry to add small functional groups41. Additionally, pLSL films have been previously examined for their ability to promote MSC osteogenesis and to support robust cellular attachment41. However, the ability of functionalized pLSLs to be formed into 3D scaffolds and to effect changes in MSC osteogenic and immunomodulatory responses remains to be elucidated. The goal of the present study is to evaluate how different pLSL chemistries, presented when fabricated into porous foams, influence the osteogenesis and immunomodulatory capacity of MSCs. We first fabricated various pLSLs, functionalized with either PO4, COOH or NH2 groups, into biologically relevant, 3D porous foams. We subsequently examined their 6 ACS Paragon Plus Environment

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potential utility as bone regeneration scaffolds through phenotypically profiling seeded MSCs with protein level measures of osteogenic markers, inflammatory cytokine/chemokines associated with bone regeneration, and more traditional MSC immunomodulatory factors following 72 h culture in osteogenic medium.

2. MATERIALS AND METHODS

2.1 pLSL synthesis and functionalization All chemicals and solvents were purchased in the highest available purity and were used without further purification. Crude lactonic sophorolipid 6',6"-diacetate (LSL[6' Ac, 6" Ac]) was produced by fermentation of Starmerella (Candida) bombicola following the previously reported protocol36,46. 1H NMR data were collected on a Bruker 600 MHz or Varian 500 MHz spectrometer with deuterated dimethyl sulfoxide (DMSO-d6) as solvent and processed using MestReNova software. Gel permeation chromatography (GPC) analysis was carried out on a Waters HPLC system equipped with two columns in series (Agilent RESIPORE 300x7.5 mm and PLgel 5 µm, 103 Å, 300x7.5 mm). Tetrahydrofuran (THF) was the eluent at flow rate of 1 mL/min and calibration was performed using polystyrene standards. Electrospray ionization mass spectrometry (ESI-MS data; positive mode) was performed using a ThermoScientific LTQ ORBITRAP XL Mass Spectrometer.

2.1.1 Synthesis of LSL[6'Ac, 6"Ac] monomer



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LSL[6' Ac, 6" Ac] monomer was prepared by microbial fermentation according to the method reported in literature46. Pure LSL[6' Ac, 6" Ac] was separated from crude sophorolipids (extracted from fermentation mixture) by flash chromatography using silica gel and chloroform and methanol (10:1) as the eluent. This was followed by recrystallization from a 1:1 (v/v) mixture of ethyl acetate and hexane (yield = 85%). The resulting product was characterized by 1H NMR and ESI-MS, which gave data consistent with those reported previously41,47.

2.1.2 Synthesis of LSL[6'MA, 6"Ac] monomer Lactonic sophorolipid 6'-methacrylate 6"-acetate (LSL[6' MA, 6" Ac]) monomer was synthesized from LSL[6' Ac, 6" Ac] by lipase-catalyzed selective 6'-deacetylation followed by selective 6'-methacrylation catalyzed by immobilized lipase PS (Supplemental Figure S1)41. Briefly, 5 mL of anhydrous THF, 960 µl (8 mmol) of vinyl methacrylate, and 80 mg of Amano lipase PS-IM (immobilized on diatomaceous earth) were added to Lactonic sophorolipid 6” – acetate (LSL[6'OH, 6"Ac], 0.65 g; 1 mmol) synthesized from LSL[6' Ac, 6" Ac] by lipasecatalyzed selective 6'-de-acetylation in phosphate buffered saline (PBS). The reaction mixture was stirred at 45 °C under nitrogen until all LSL starting material was consumed (typically 812 h). Upon completion, the solvent and unreacted vinyl methacrylate were removed by rotoevaporation. The crude product (yellowish solid) was purified by flash chromatography using chloroform/methanol as the eluent (gradient: 0-20% (v/v) methanol). The final product was recovered as a white powder (mp 131-143 °C, yield = 80%).


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2.1.3 Synthesis of copolymer methacrylate derivatized poly(LSL[6' Ac, 6" Ac]95%-co-LSL[6' MA, 6" Ac] 5%) (pLSL-MA) by ROMP The copolymer poly(LSL[6' Ac, 6" Ac]95%-co-LSL[6' MA, 6" Ac]

5%)

was prepared

according to the method reported previously (Supplementary Figure S2)41. In a 5 mL coneshaped micro reaction vessel were dissolved LSL[6' Ac, 6" Ac] (655 mg; 0.95 mmol) and LSL[6' MA, 6" Ac] (36 mg; 0.05 mmol) in 1.35 mL of anhydrous THF. The vessel was capped with a rubber septum and equilibrated to 60 °C in an external heating block. A stock solution of Grubbs 2nd generation (G2) catalyst was prepared separately by dissolving 42.5 mg G2 into 250 µl anhydrous THF in a vial and preheated to 60 °C. Thereafter, 50 µl of G2 stock solution was transferred into the vessel containing the monomer solution, so that the catalyst ratio was 1 mol% relative to total moles of monomer. The vessel was resealed with the rubber septum. The polymerization mixture was maintained at 60 °C with magnetic stirring for 30 min without protection of inert gas. Subsequently the polymerization vessel was cooled down to room temperature with an external cold water bath, and 3 mL of 30 % (v/v) ethyl vinyl ether in THF was added to quench the reaction. After vigorous mixing for 15 min, the polymer product was isolated with removal of unreacted monomer by precipitation (twice) in 100 mL of ethyl acetate. The final polymer (pLSL-MA) was dried under vacuum at 20 mmHg, 45 °C for 48 h (yield = 60%, GPC: Mn = 81670 g/mol and Ɖ = Mw/Mn = 2.06). Characterization by 1HNMR is provided in Supplemental Figure S3. This functionalization level was selected to be



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intermediate within the range presented in Benoit et al.42, in which small functional groups such as those examined herein were found to have marked effects on MSC differentiation.

2.1.4 Functionalization of methacrylate-derivatized copolymer (pLSL-MA) via thiol-ene click reaction Functionalization of pLSL-MA with the following thiol compounds was conducted via thiol-ene click chemistry41: 1) HSCH2CH2NH2, 2) HSCH2CH2COOH, 3) HSCH2CH2PO4. In brief, the thiol compound (4 eq, relative to methacrylate units) and DMPA photoinitiator (0.2 eq) were added to the pLSL-MA (200 mg) copolymer in DMSO (5 mL). The resulting mixture was stirred at room temperature under 365 nm UV irradiation for 24 h. Upon completion, the mixture was added dropwise to 25 mL of distilled water, and the precipitate was collected by centrifugation, washed twice with distilled water and dried under vacuum for 48 h. The resulting pLSL products were characterized by 1H-NMR (Supplemental Figures S4 and S5).

2.2 Fabrication of polymeric foams pLSL foams were obtained using a salt-fusion/salt-leaching technique48. In brief, NaCl (Acros Organics #207790010) particles were separated using a 300 µm sieve to select for particles > 300 µm in diameter. The harvested salt population displayed an average transverse diameter of 372 ± 7 µm (max: 650 µm). Then, 2 g of the sieved salt was transferred to a 2.0 cm diameter glass vial. Water was added at 6.9% (w/w), after which pressure was applied to force


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close contact between the wetted salt crystals. The resulting salt disc was then fully dried under vacuum to form a fused salt template of diameter 1.7 cm and thickness of 0.5 cm. Polymeric solutions were prepared by dissolving the different pLSLs in THF at 10% polymer concentration. Salt templates were wetted with a gradient solution of ethanol (EtOH) and THF, starting with 100% EtOH and ending with 100% THF, after which 1 mL of polymer solution was applied to each salt template. The solubilized polymer was allowed to diffuse into the salt matrix for 24 h, and the salt template was flipped after 12 h to facilitate even polymer penetration. The solvent was slowly evaporated for 24 h under ambient conditions and the dried polymer scaffolds were wetted with gradient solutions of THF and EtOH followed by gradient solutions of EtOH and Dulbecco’s phosphate buffered saline (DPBS; Lonza). Then, the salt was dissolved into DPBS with changes to fresh DPBS every 15 min for 3 h, and then overnight. Thereafter, pLSL foams were dried at 30 ºC for 48 h in an oven, and then sterilized with ethylene oxide using a 12 h cycle. Sterilized pLSL foams were wetted with gradient solutions of EtOH and DPBS, starting with absolute EtOH and ending with 100% DPBS and separated into 4 equally sized discs. Negligible swelling of the foams was observed following aqueous immersion, although the surfaces of all of the pLSL foams were wettable. Prior to cell seeding, the discs (n = 4 per pLSL formulation) were placed into 24 well-plates, DPBS was removed and osteogenic induction cell culture medium was added.

2.3 Pore size, porosity and morphology



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Scanning electron microscopy (SEM) was used to evaluate pore morphology and interconnectivity in pLSL foams. The scaffolds were freeze-fractured in liquid N2, and the cross sections were sputter-coated with platinum (Denton Vacuum). SEM images were obtained at 20 kV using VERSA 3D Dual Beam. Obtained SEM images were used to measure pore size via assessment of the straight line distance across complete pores using ImageJ software 1.52a. At least 46 pores were measured to obtain the average pore size of the pLSL foams. Porosity was evaluated by two approaches. As a first estimate, we measured the radius (r), thickness (t) and mass (m) of three salt templates. The mass measures were then converted to an equivalent volume of salt by dividing by the density of NaCl (2.16 g/cm3). Porosity was subsequently estimated at 75 ± 2% by dividing the volume of salt by the volume of the template (r2t). Given the autofluorescence of the pLSL scaffolds, further assessment of porosity was based on void area in cross-sectional images of wetted scaffolds obtained via confocal microscopy. Resulting quantification indicated an average porosity of 70 ± 2%.

2.4 Cell culture Cryopreserved bone marrow derived MSCs (Texas A&M Institute for Regenerative Medicine) were thawed and expanded in Minimum Essential Medium-α (α-MEM, Gibco) supplemented with 16.5% MSC-qualified fetal bovine serum (FBS; Atlanta Biologicals), 1% antibiotic solution (10,000 IU/mL penicillin, 10,000 µg/mL streptomycin; Life Technologies) and 1% glutamine (Glutamax; Life Technologies) in a 37 °C-5% CO2 jacketed incubator. MSCs were harvested at passage 5 and seeded into the pLSL foams at 3.5 x106 cells/mL in osteogenic 12 ACS Paragon Plus Environment

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medium: Dulbecco’s Modified Essential Medium (DMEM; Life Technologies) supplemented with 10% FBS, 50 μg/mL L-ascorbic acid 2-phosphate (Sigma), 0.1 μM dexamethasone (Sigma), 10 mM β-glycerophosphate (Sigma), and 1% antibiotic solution (10,000 IU/mL penicillin, 10,000 µg/mL streptomycin; Life Technologies). Cell suspensions were pipetted three times on each side of the pLSL discs to promote homogeneous cell seeding. After 24 h, pLSL foams were moved to a new 24 well-plate with fresh osteogenic medium and cultured for an additional 48 h. Culture in osteogenic medium was conducted to reflect the osteogenic environment that the cells experience during fracture healing. A 72 h endpoint was selected based on literature indicating this as a relevant time point for initial screening of biomaterial osteogenic and/or immunomodulatory effects on MSCs49–52. The homogeneity of MSC seeding through the interconnected porous structure of the pLSL foams is shown in Supplemental Figure S6.

2.5 MAGPIX immunoassay multiplexing Following 72 h of culture, culture medium was collected for analysis of secreted cell products. In addition, protein was extracted from each of the foams using a modified version of a previously described extraction protocol53. Briefly, pLSL foams were rinsed with DPBS for 5 min, transferred into 1.7 mL microcentrifuge tubes containing 200 µl of lysis buffer (100 mM Tris, 500 mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM dithiothreitol, pH~7.8), homogenized using plastic pestles (Kimble Chase) and then incubated for 20 min at room temperature with mixing every 5 min. Homogenized samples were centrifuged for 5 min at 10,000 rpm to pellet foam



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particulates, and the supernatant (homogenate) was collected and stored at -80 ºC until the time of analysis. Selected cytokines and growth factors from foam homogenates and culture medium were quantitatively characterized using multiplex protein analyses. Specifically, MILLIPLEX® MAP assays for panels of human cytokines/chemokines (Millipore) were performed using a MAGPIX® immunoassay system (Luminex) according to the manufacturer’s protocol. The concentrations of proteins of interest were obtained from measured median fluorescence intensities (MFI) and associated standard curves. Markers assessed from the harvested culture medium included soluble factors IL-8, MCP-1, IL-6, osteoprotegerin, osteoactivin and HGF. Markers assessed in foam homogenates included bone morphogenetic protein-2 (BMP-2) and collagen type 1 α1 (COL1A1). Results were normalized to the DNA concentration of each sample as assessed via the PicoGreen assay (Life Technologies) according to the manufacturer’s instructions.

2.6 Western blot analysis Western blots were used to semi-quantitively compare protein expression of inflammatory markers by MSCs cultured within the various pLSL foams following a previously utilized protocol54. Briefly, sample homogenates containing equal amounts of DNA (200 ng) were denatured by the addition of β-mercaptoethanol, heated at 95 ºC for 10 min and then loaded into a 13% polyacrylamide gel. Proteins were separated by electrophoresis and subsequently transferred to a nitrocellulose membrane (Thermo Scientific). Thereafter, the 14 ACS Paragon Plus Environment

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membrane was rinsed twice with distilled-deionized H2O and then blocked with a 5% bovine serum albumin solution (BSA; Fraction V, Fisher Scientific) in TBST/NaN3 (25 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween 20, 0.05% NaN3) for 1 h at room temperature. Primary antibodies against PTGES-2 (Abcam, AB96189), IDO (Santa Cruz Biotechnology, sc-365086), COX-2

(Santa

Cruz

Biotechnology,

sc-166475)

and

glyceraldehyde

3-phosphate

dehydrogenase (GAPDH; Santa Cruz Biotechnology, sc-32233) were diluted in a solution of 5% BSA in TBST/NaN3 and applied to the blots overnight at 4 ºC with constant shaking. Bound primary antibodies were detected by applying the appropriate alkaline phosphatase-conjugated or horseradish peroxidase-conjugated secondary antibody (Jackson Immunochemicals) for 1 h at room temperature, followed by the application of Novex chemiluminescent substrate (Life Technologies) or Luminol chemiluminescent reagent (Santa Cruz Biotechnology), respectively. Chemiluminescence was detected using a ChemiDoc™ XRS+ System equipped with ImageLab™ software (Bio-Rad Laboratories). Exposure times were set manually to prevent signal saturation. Relative protein levels were quantified using integrated band densiometry with Adobe Photoshop CS2 (Version 9.0) and then normalized to GAPDH. Representative blot images are shown in Supplemental Figure S7.

2.7 Statistical analysis All data are reported as the mean ± standard error of mean. Sample means were compared using one-way ANOVA (SPSS Version 24.0) followed by a Tukey post-hoc test. Statistical significance was determined at p-value < 0.05. 15 ACS Paragon Plus Environment


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3. RESULTS

3.1 pLSL synthesis and porous scaffold fabrication Methacrylate-derivatized pLSL (pLSL-MA) as well as functionalized derivatives were synthesized per established methods, characterized and then formed into porous scaffolds. pLSL-MA was first prepared by ring opening metathesis polymerization (ROMP) of a mixture of monomers consisting of 95 mol% LSL[6'Ac, 6"Ac] and 5 mol% LSL[6'MA, 6"Ac] using 1 mol% Grubbs 2nd generation catalyst (Supplemental Figure S2)41,47. The resulting copolymer displayed a number average molecular weight (Mn) of 81,670 g/mol with a polydispersity of 2.06 by GPC. 1H NMR analysis revealed a fractional methacrylate-derivatization of ~5.5% through relative integration of the methacrylate double bond proton signals at 6.04 ppm (a) or 5.68 ppm (b) and the sugar proton signal at 4.71 ppm (H-4") (Supplemental Figure S3). Subsequent functionalization of pLSL-MA with three distinct thiol compounds was performed via thiol-ene click reaction under UV irradiation at 365 nm using 2,2-dimethoxy2-phenylacetophenone (DMPA) as photoinitiator (Supplemental Figure S4A)55. The reaction was monitored by 1H NMR. Disappearance of the two double bond proton signals (at 6.04 ppm and at 5.68 ppm) of the methacrylate unit was observed upon completion of each reaction, as shown in Supplemental Figure S4B. The structures of resulting products (pLSL-MA, pLSL-


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NH2, pLSL-PO4 and pLSL-COOH) are shown in Figure 1, and full 1H NMR spectra are provided in Supplementary Figure S5. The functionalized pLSLs were formed into porous foams by a salt-fusion/salt-leaching method outlined schematically in Figure 2A. Sieved NaCl particles were utilized in forming the salt template with a diameter of 1.7 cm and thickness of 0.5 cm. Due to the fused nature of the salt within the template mold, the resulting pLSL foams exhibited an interconnected porous structure (Figure 2B), with an average pore size of 337 ± 7 µm and a porosity of 70-75%. Representative macroscopic images of the pLSL foams are shown in Figure 2C.

3.2 Lineage progression of MSCs within pLSL foams The capacity of pLSL foams functionalized with PO4, COOH, or NH2 to promote MSC osteogenesis was assessed following 72 h of culture in osteogenic medium. Osteoactivin, osteoprotegerin, BMP-2, and COL1A1 were selected as markers for evaluation due to their previously established osteoinductive56,57 roles and due to their prior use as indicators of MSC osteoblastic differentiation58–60. MSCs cultured on pLSL-PO4 foams displayed a significant increase in secreted levels of osteoprotegerin (~1.4-fold, p = 0.035) and osteoactivin (~1.5-fold, p = 0.031) relative to unmodified pLSL-MA foams (Figure 3). Similarly, pLSL-PO4 constructs were associated with a significant increase in the potent osteoinductive factor BMP-2 (~1.4fold, p < 0.014) relative to pLSL-NH2, pLSL-COOH, and unmodified pLSL-MA foams. Although pLSL-NH2 and pLSL-COOH scaffolds appeared to support increased levels of osteoactivin relative to pLSL-MA foams, these apparent differences fell below statistical 17 ACS Paragon Plus Environment


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significance. Combined, these data indicate that the phosphate functionalization may enhance MSC osteogenic lineage progression within pLSL scaffolds.

3.3 Immunomodulatory capacity of MSCs within pLSL foams To investigate the potential of MSCs seeded in pLSL foams to modulate the inflammatory response associated with bone healing, we examined MSC release of cytokines of IL-6 and IL-8 and chemokine MCP-1 in the various foams following 72 h of culture in dexamethasone-containing osteogenic medium (Figure 4). Relative to pLSL-MA foams, there was a significant increase in IL-8 expression in MSCs seeded on pLSL-PO4 (~2.0-fold, p = 0.001) and pLSL-NH2 (~2.4-fold, p < 0.001) scaffolds. Furthermore, IL-8 expression was also elevated in MSCs seeded on pLSL-PO4 (~1.3-fold, p = 0.042) and pLSL-NH2 (~1.6-fold, p = 0.002) constructs relative to pLSL-COOH foams. In contrast, the expression of MCP-1 was slightly yet significantly reduced on pLSL-PO4 scaffolds relative pLSL-NH2 foams (~0.7-fold, p = 0.022), and no differences in MSC expression of IL-6 were observed among any of the four pLSL formulations. As previously noted, the upregulation of IL-8 is evident in the initial stages of bone healing and is associated with promoting angiogenesis through stimulation of VEGF release22,23. Thus, when coupled with the lineage progression data, the IL-8 results support the notion that functionalization of pLSLs with PO4 groups can be utilized to beneficially modulate MSC activity for bone regeneration. Due to the key role of MSCs in broader immunomodulation9,61, we also investigated the protein expression of several factors conventionally assessed in evaluating the 18 ACS Paragon Plus Environment

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immunomodulatory phenotype of MSCs: PTGES-2, IDO, COX-2 and HGF (Figure 5). MSCs cultured on pLSL-COOH foams displayed an upregulation in IDO expression relative to both pLSL-MA (~1.4 fold, p = 0.025) and pLSL-NH2 (~1.4 fold, p = 0.040) constructs. Furthermore, pLSL-PO4 foams were associated with a significant increase in IDO expression relative to pLSLMA (~1.4 fold, p = 0.031) and pLSL-NH2 (~1.4 fold, p = 0.0495) scaffolds. Similarly, COX-2 expression was elevated in MSCs cultured on pLSL-PO4 foams relative to pLSL-MA foams (~2.2 fold, p = 0.031). These combined IL-8, IL-6, MCP-1, PTGES-2, IDO, COX-2 and HGF results suggest that pLSL-based stimulation of MSC immunomodulatory factors is marker- and modification-dependent.

4. DISCUSSION pLSLs are a novel class of glycolipid based polymers that hold substantial promise for biomedical applications due to their formability, biodegradation into natural structural units and capacity to support cell adhesion40,41. In addition, we recently reported chemo-enzymatic routes to introduce clickable methacrylate moieties into LSL, which subsequently transfer to the polymer chains (pLSL-MA) following ROMP41. In the present work, we have used these clickable moieties to introduce bioactive groups into the pLSL structure toward modulating MSC differentiation and function. Towards this end, MSCs were cultured in osteogenic medium on pLSL scaffolds presenting either COOH, NH2, or PO4 functional groups, and



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protein levels of osteoinductive and immunomodulatory markers were assessed to gain initial insight into the potential of the various foams to support bone regeneration/healing. In terms of osteogenic lineage progression, MSCs cultured on PO4-functionalized pLSL foams showed a significant increase in the protein expression of osteoprotegerin and osteoactivin compared to unmodified pLSL-MA foams. Furthermore, the pLSL-PO4 foams were associated with higher BMP-2 levels compared to all other pLSL foams examined. Previous reports have demonstrated that BMP-2 and osteoactivin promote MSC osteogenesis. Specifically, Lysdahl et al. reported enhanced osteogenic differentiation of MSCs pre-treated with BMP-2 at low concentrations62, while Arosarena et al. reported similar results showing the osteoinductive effects of both osteoactivin and BMP-2 on MSC osteoblastic differentiation

in vitro56. Furthermore, Palumbo and Li demonstrated enhanced osteogenic differentiation of MSCs exposed to osteoprotegerin57. Thus, the elevated expression of these markers from MSCs cultured within pLSL-PO4 foams could imply autocrine effects which may enhance osteoblastic differentiation. The osteogenic effects of PO4 functionalization observed herein are consistent with previous studies of MSCs cultured on other materials functionalized with small functional groups42,63,64. Benoit et al. reported increased osteogenic differentiation of MSCs seeded on PO4-functionalized PEG hydrogels after 10 days in normal growth medium, whereas no increases in osteogenic markers were noted for NH2- and COOH-functionalized PEG hydrogels42. In contrast, Curran et al. reported increased osteogenic differentiation of MSCs grown on NH2-modified glass surfaces, relative to unmodified surface controls, after 14 days 20 ACS Paragon Plus Environment

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of culture in osteogenic medium63. However, they did not observe differences in osteogenic differentiation at shorter culture periods for the NH2-modified surfaces. Thus, futher studies will need to be conducted to confirm MSC osteoblastic maturation within functionalized pLSL foams. That said, previous literature indicates that increasing negative surface charge can lead to increased apatite growth rate and calcium deposition, which are relevant to bone formation43,65. Consequently, the observed osteogenic effects of the PO4 functionalized pLSLs could be mainly influenced by the greater negative charge of the phosphate group relative to the other functional groups examined. Given our long-term goal to use MSC-loaded pLSL scaffolds for bone fracture repair, we also examined the effects of foam chemistry on MSC production of factors known modulate the inflammatory response relevant to bone healing. In the PO4 and NH2-functionalized pLSL foams, we observed a significant increase in MSC IL-8 expression in comparison to unmodified pLSL-MA scaffolds. IL-8 is associated with enhanced angiogenesis22,23 and bone remodeling66 during bone healing. Conversely, MCP-1 expression by MSCs was slightly decreased in pLSLPO4 foams in comparison to pLSL-NH2 foams. Previous studies have reported impaired bone healing in macrophage-deficient67 and CCR2-deficient mice20, thus highlighting the importance of MCP-1 in in bone healing. However, the macrophage response is dependent on the inflammatory environment, as indicated by impaired fracture healing when macrophages are stimulated using amine functionalized glucans17 or lipopolysaccharide68. Therefore, the question of whether the modest decrease in MSC MCP-1 expression on pLSL-PO4 foams will negatively impact macrophage recruitment and, subsequently, bone formation would need to 21 ACS Paragon Plus Environment


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be further evaluated in vivo. Simultaneously, in the pLSL-PO4 foams, we observed significant upregulation of immunomodulatory markers IDO and COX-2. The suppression of the T-cell immune response, mediated by IDO29,30 contributes to the initiation of bone repair69. In addition, previous reports have demonstrated the vital role of COX-2 in fracture repair, mediated by PGE2 production34. Thus, the PO4 functionalized pLssssSLs may support immunomodulatory effects which contribute positively to bone fracture repair. To our knowledge, the present study is the first to investigate the effects of altering pLSL chemistry on MSC responses in a biologically-relevant 3D environment. Overall, the pLSL-PO4 scaffolds displayed the most significant induction of MSCs towards an osteoblastic lineage and release of cytokines relevant to improved bone fracture healing among the foam chemistries examined. Despite the observed differences in the more traditional immunomodulatory functions of MSCs, broader investigation of the MSC secretome will be required to more fully characterize the immunomodulatory effects of various pLSL surface chemistries61,70. It is also important to note that the presence of the anti-inflammatory glucocorticoid, dexamethasone, may have influenced the observed immunomodulatory effects associated with each foam. In addition, future studies for longer culture periods and varying copolymer compositions are needed to confirm the commitment of MSCs towards the osteoblastic lineage and to probe changes in the cell response, respectively, within functionalized pLSL foams. Future studies will also incorporate a broader panel of markers to characterize osteoblastic lineage progression and the immunomodulatory response of MSCs.


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Regardless, the present results provide initial support for the potential utility of bioresorbable, functionalized pLSLs as scaffolds for bone fracture repair.

5. CONCLUSIONS In summary, our results demonstrate that variation in the surface chemistry of pLSL foams directs the MSC response for potential use in bone fracture repair applications. Specifically, our results indicated that, overall, pLSL-PO4 foams displayed the highest osteoinductive effects on MSCs and promoted release of cytokines involved in improved bone fracture healing. Future studies are warranted to investigate the immunomodulatory response of MSCs in an inflammatory environment and to confirm the long-term commitment of MSCs towards an osteoblastic lineage within pLSL foams. Overall, bioresorbable, functionalized pLSL scaffolds could potentially be utilized in bone fracture repair applications through promoting osteoblastic lineage progression and beneficial cytokine release to improve bone healing.

6. ACKNOWLEDGEMENTS This work was supported by NSF Division of Materials Research (NSF-DMR) Biomaterials (BMAT) Grant 1508422 to R.A.G. and M.S.H.

7. CONFLICTS OF INTEREST The authors declare no competing financial interest. 23 ACS Paragon Plus Environment


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8. SUPPORTING INFORMATION Schematics of pLSL-MA synthesis and copolymerization, 1H NMR spectra with assignments for pLSL-MA copolymer, functionalization of pLSL-MA to form pLSL-PO4, pLSLCOOH and pLSL-NH2 via thiol-ene click reactions, representative conforcal images showing MSC attachment in different regions of the foams, and representative western blot images.

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10. FIGURE CAPTIONS Figure 1. Schematics of the functionalized pLSL polymers used to synthesize foam scaffolds: 1: pLSL-MA; 2: pLSL-NH2; 3: pLSL-COOH; 4: pLSL-PO4. Figure 2. (A) Schematic of the salt-fusion/salt-leaching method used to fabricate pLSL foams. (B) A representative SEM image of the interconnected pore structure of resulting pLSL foams, scale bar = 200 µm. (C) Representative macroscale images of the functionalized pLSL foams. 1: pLSL-MA; 2: pLSL-NH2; 3: pLSL-PO4; 4: pLSL-COOH, scale bar = 1cm. Figure 3. Relative protein levels of osteoprotegerin, osteoactivin, BMP-2, and COL1A1 associated with MSCs cultured on functionalized pLSL foams. For each marker, the results are normalized to the pLSL-MA control and represent an n = 3-4 per foam type. * Denotes a significant difference relative to pLSL-MA. # Denotes a significant difference relative to pLSLNH2. + Denotes a significant difference relative to pLSL-COOH. Error bars correspond to the standard error of the mean. Figure 4. Relative protein levels of IL-8, MCP-1, and IL-6 released by MSCs cultured on functionalized pLSL foams. For each marker, the results are normalized to the pLSL-MA control and represent an n = 3 per foam type. * Denotes a significant difference relative to the pLSL-MA control. # Denotes a significant difference relative to pLSL-NH2. + Denotes a significant difference relative to pLSL-COOH. Error bars correspond to the standard error of the mean. Figure 5. Relative protein levels for PTGES-2, IDO, COX-2 and HGF production by MSCs cultured on functionalized pLSL foams. For each marker, the results are normalized to the pLSL-MA control and represent an n = 3 per foam type. * Denotes a significant difference relative to the pLSL-MA control. # Denotes a significant difference relative to pLSL-NH2. Error bars correspond to the standard error of the mean.



Abstract Graphic 83x35mm (300 x 300 DPI)

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Figure 1. Schematics of the functionalized pLSL polymers used to synthesize foam scaffolds: 1: pLSL-MA; 2: pLSL-NH2; 3: pLSL-COOH; 4: pLSL-PO4. 165x96mm (300 x 300 DPI)



Figure 2. (A) Schematic of the salt-fusion/salt-leaching method used to fabricate pLSL foams. (B) A representative SEM image of the interconnected pore structure of resulting pLSL foams, scale bar = 200 µm. (C) Representative macroscale images of the functionalized pLSL foams. 1: pLSL-MA; 2: pLSL-NH2; 3: pLSL-PO4; 4: pLSL-COOH, scale bar = 1cm. 115x83mm (300 x 300 DPI)


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Figure 3. Relative protein levels of osteoprotegerin, osteoactivin, BMP-2, and COL1A1 associated with MSCs cultured on functionalized pLSL foams. For each marker, the results are normalized to the pLSL-MA control and represent an n = 3-4 per foam type. * Denotes a significant difference relative to pLSL-MA. # Denotes a significant difference relative to pLSL-NH2. + Denotes a significant difference relative to pLSL-COOH. Error bars correspond to the standard error of the mean. 104x57mm (300 x 300 DPI)



Figure 4. Relative protein levels of IL-8, MCP-1, and IL-6 released by MSCs cultured on functionalized pLSL foams. For each marker, the results are normalized to the pLSL-MA control and represent an n = 3 per foam type. * Denotes a significant difference relative to the pLSL-MA control. # Denotes a significant difference relative to pLSL-NH2. + Denotes a significant difference relative to pLSL-COOH. Error bars correspond to the standard error of the mean. 82x53mm (300 x 300 DPI)


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Figure 5. Relative protein levels for PTGES-2, IDO, COX-2 and HGF production by MSCs cultured on functionalized pLSL foams. For each marker, the results are normalized to the pLSL-MA control and represent an n = 3 per foam type. * Denotes a significant difference relative to the pLSL-MA control. # Denotes a significant difference relative to pLSL-NH2. Error bars correspond to the standard error of the mean. 104x57mm (300 x 300 DPI)


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