Effect of Poly(sophorolipid) Functionalization on Human Mesenchymal Stem Cell Osteogenesis and Immunomodulation.

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 bioresorbable scaffolds for bone fracture healing. Toward 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 PO4-functionalized pLSL foams supported the highest degree of MSC osteogenesis as well as the highest levels of immunomodulatory factors pertinent to improve bone fracture healing. Cumulatively, these results suggest that further investigation of the long-term osteogenic commitment of MSCs in PO4-functionalized pLSL foams is warranted.

Toward zonally tailored scaffolds for osteochondral differentiation of synovial mesenchymal stem cells.

Synovium-derived mesenchymal stem cells (SMSCs) are an emerging cell source for regenerative medicine applications, including osteochondral defect (OCD) repair. However, in contrast to bone marrow MSCs, scaffold compositions which promote SMSC chondrogenesis/osteogenesis are still being identified. In the present manuscript, we examine poly(ethylene) glycol (PEG)-based scaffolds containing zonally-specific biochemical cues to guide SMSC osteochondral differentiation. Specifically, SMSCs were encapsulated in PEG-based scaffolds incorporating glycosaminoglycans (hyaluronan or chondroitin-6-sulfate [CSC]), low-dose of chondrogenic and osteogenic growth factors (TGFß1 and BMP2, respectively), or osteoinductive poly(dimethylsiloxane) (PDMS). Initial studies suggested that PEG-CSC-TGFß1 scaffolds promoted enhanced SMSC chondrogenic differentiation, as assessed by significant increases in Sox9 and aggrecan. Conversely, PEG-PDMS-BMP2 scaffolds stimulated increased levels of osteoblastic markers with significant mineral deposition. A "Transition" zone formulation was then developed containing a graded mixture of the chondrogenic and osteogenic signals present in the PEG-CSC-TGFß1 and PEG-PDMS-BMP2 constructs. SMSCs within the "Transition" formulation displayed a phenotypic profile similar to hypertrophic chondrocytes, with the highest expression of collagen X, intermediate levels of osteopontin, and mineralization levels equivalent to "bone" formulations. Overall, these results suggest that a graded transition from PEG-CSC-TGFß1 to PEG-PDMS-BMP2 scaffolds elicits a gradual SMSC phenotypic shift from chondrocyte to hypertrophic chondrocyte to osteoblast-like. As such, further development of these scaffold formulations for use in SMSC-based OCD repair is warranted. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2019-2029, 2019.

A canine in vitro model for evaluation of marrow-derived mesenchymal stromal cell-based bone scaffolds.

Tissue engineered bone grafts based on bone marrow mesenchymal stromal cells (MSCs) are being actively developed for craniomaxillofacial (CMF) applications. As for all tissue engineered implants, the bone-regenerating capacity of these MSC-based grafts must first be evaluated in animal models prior to human trials. Canine models have traditionally resulted in improved clinical translation of CMF grafts relative to other animal models. However, the utility of canine CMF models for evaluating MSC-based bone grafts rests on canine MSCs (cMSCs) responding in a similar manner to scaffold-based stimuli as human MSCs (hMSCs). Herein, cMSC and hMSC responses to polyethylene glycol (PEG)-based scaffolds were therefore compared in the presence or absence of osteoinductive polydimethylsiloxane (PDMS). Notably, the conjugation of PDMS to PEG-based constructs resulted in increases in both cMSC and hMSC osteopontin and calcium deposition. Based on these results, cMSCs were further used to assess the efficacy of tethered bone morphogenic protein 2 (BMP2) in enhancing PEG-PDMS scaffold osteoinductivity. Addition of low doses of tethered BMP2 (100 ng/mL) to PEG-PDMS systems increased cMSC expression of osterix and osteopontin compared to both PEG-PDMS and PEG-BMP2 controls. Furthermore, these increases were comparable to effects seen with up to five-times higher BMP2 doses noted in literature. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2382-2393, 2018.

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings.

Achieving graft endothelialization following implantation continues to be a challenge in the development of "off-the-shelf," small-caliber, arterial prostheses. Coating grafts with biomolecules to support the retention, migration, and differentiation of adherent endothelial precursor cells (EPCs) is a promising approach toward improving graft endothelialization. Designer Collagen Scl2-2 with 1 integrin binding site per strand (DC2-1X) is a Streptococcus pyogenes-derived, collagen-like protein that has previously been evaluated as a graft coating due to its ability to resist platelet aggregation and to promote attachment and migration of "late outgrowth" EPCs (EOCs). However, these prior assessments were performed in the absence of physiological shear. In addition, although DC2-1X coatings supported increased migration rates relative to native collagen coatings, EOC attachment and spreading remained inferior to collagen controls at all DC2-1X concentrations assayed. Thus, the objectives of the present work were the following: (1) to improve EOC attachment on DC2 coatings by modulating the number and spacing of DC2 integrin binding sites (IBS) and (2) to evaluate the retention, migration, and differentiation of adherent EOCs under physiological shear stress. Using single point mutations, three novel DC2 variants were generated containing either two IBS (DC2-2X) or three IBS (DC2-3X1 and DC2-3X2) per strand. After initial evaluation of the potential of each DC2 variant to support increased EOC attachment relative to DC2-1X, DC2-2X and DC2-3X1 coatings were further assessed under physiological shear for their capacity to promote EOC retention, migration, and differentiation relative to DC2-1X and collagen controls. An increase in the number of IBS from 1 to 3 significantly improved EOC retention on DC2 coatings while also supporting increased average migration rates. Moreover, EOCs on DC2-3X1 coatings showed increased gene-level expression of intermediate endothelial cell differentiation markers relative to collagen. Overall, the current results suggest that DC2-3X1 warrants further investigation as a vascular graft coating.

In vitro evaluation of a basic fibroblast growth factor-containing hydrogel toward vocal fold lamina propria scar treatment.

Scarring of the vocal fold lamina propria can lead to debilitating voice disorders that can significantly impair quality of life. The reduced pliability of the scar tissue-which diminishes proper vocal fold vibratory efficiency-results in part from abnormal extracellular matrix (ECM) deposition by vocal fold fibroblasts (VFF) that have taken on a fibrotic phenotype. To address this issue, bioactive materials containing cytokines and/or growth factors may provide a platform to transition fibrotic VFF within the scarred tissue toward an anti-fibrotic phenotype, thereby improving the quality of ECM within the scar tissue. However, for such an approach to be most effective, the acute host response resulting from biomaterial insertion/injection likely also needs to be considered. The goal of the present work was to evaluate the anti-fibrotic and anti-inflammatory capacity of an injectable hydrogel containing tethered basic fibroblast growth factor (bFGF) in the dual context of scar and biomaterial-induced acute inflammation. An in vitro co-culture system was utilized containing both activated, fibrotic VFF and activated, pro-inflammatory macrophages (MΦ) within a 3D poly(ethylene glycol) diacrylate (PEGDA) hydrogel containing tethered bFGF. Following 72 h of culture, alterations in VFF and macrophage phenotype were evaluated relative to mono-culture and co-culture controls. In our co-culture system, bFGF reduced the production of fibrotic markers collagen type I, α smooth muscle actin, and biglycan by activated VFF and promoted wound-healing/anti-inflammatory marker expression in activated MΦ. Cumulatively, these data indicate that bFGF-containing hydrogels warrant further investigation for the treatment of vocal fold lamina propria scar. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1258-1267, 2018.

Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels.

Bioactive coatings which support the adhesion of late-outgrowth peripheral blood endothelial progenitor cells (EOCs) are actively being investigated as a means to promote rapid endothelialization of "off-the-shelf," small-caliber arterial graft prostheses following implantation. In the present work, we evaluated the behavior of EOCs on thromboresistant graft coatings based on the collagen-mimetic protein Scl2-2 and poly(ethylene glycol) (PEG) diacrylate. Specifically, the attachment, proliferation, migration, and phenotype of EOCs on PEG-Scl2-2 hydrogels were evaluated as a function of Scl2-2 concentration (4, 8, and 12 mg/mL) relative to human umbilical vein endothelial cells (HUVECs). Results demonstrate the ability of each PEG-Scl2-2 hydrogel formulation to support EOC and HUVEC adhesion, proliferation, and spreading. However, only the 8 and 12 mg/mL PEG-Scl2-2 hydrogels were able to support stable EOC and HUVEC confluence. These PEG-Scl2-2 formulations were, therefore, selected for evaluation of their impact on EOC and HUVEC phenotype relative to PEG-collagen hydrogels. Cumulatively, both gene and protein level data indicated that 8 mg/mL PEG-Scl2-2 hydrogels supported similar or improved levels of EOC maturation relative to PEG-collagen controls based on evaluation of CD34, VEGFR2, PECAM-1, and VE-Cadherin. The 8 mg/mL PEG-Scl2-2 hydrogels also appeared to support similar or improved levels of EOC homeostatic marker expression relative to PEG-collagen hydrogels based on von Willebrand factor, collagen IV, NOS3, thrombomodulin, and E-selectin assessment. Combined, the present results indicate that PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1712-1724, 2017.

Probing the response of human osteoblasts following exposure to sympathetic neuron-like PC-12 cells in a 3D coculture model.

Understanding the capacity of the sympathetic nervous system (SNS) to regulate bone homeostasis has implications for a number of metabolic diseases and may help establish connections between certain neurological conditions and bone quality. The goal of the present work was to gain a deeper understanding of the influence of the SNS on the phenotype of osteoblasts, a major cell type in bone. An in vitro coculture model with human osteoblasts and sympathetic-like, neuroendocrine pheochromocytoma-12 (PC-12) cells encapsulated within separate 3D poly(ethylene glycol) diacrylate (PEGDA) hydrogels was utilized to assess markers involved with bone ECM formation and osteoclast formation. In terms of bone ECM proteins, only osteopontin (OPN) was significantly increased in osteoblasts exposed to PC-12 cells relative to osteoblast mono-culture controls. In contrast, all bone resorption markers investigated (IL-6, TNF, IL-1ß, VEGF-A) were enhanced at the gene level and the ratio of osteoprotegerin (OPG) to RANKL was significantly decreased in osteoblasts exposed to PC-12 cells. Cumulatively, these data indicate that the SNS may substantially influence bone resorption. Because of the context-dependent nature of the SNS, future studies will characterize the secretion profile of neurotransmitters and neuropeptides from the PC-12 cells in our model. Additionally, various SNS modulating pharmacologic agents will be examined for their capacity to reduce expression of bone resorption/inflammatory markers. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 984-990, 2017.

Cell layer-electrospun mesh composites for coronary artery bypass grafts.

This work investigates the potential of cell layer-electrospun mesh constructs as coronary artery bypass grafts. These cell-mesh constructs were generated by first culturing a confluent layer of 10T½ smooth muscle progenitor cells on a high strength electrospun mesh with uniaxially aligned fibers. Cell-laden mesh sheets were then wrapped around a cylindrical mandrel such that the mesh fibers were aligned circumferentially. The resulting multi-layered constructs were then cultured for 4 wks in media supplemented with TGF-ß1 and ascorbic acid to support 10T½ differentiation toward a smooth muscle cell-like fate as well as to support elastin and collagen production. The underlying hypothesis of this work was that extracellular matrix (ECM) deposited by the cell layers would act as an adhesive agent between the individual mesh layers, providing strength to the construct as well as a source for structural elasticity at low strains. In addition, the structural anisotropy of the mesh would inherently guide desired circumferential cell and ECM alignment. Results demonstrate that the cell-mesh constructs exhibited a J-shaped circumferential stress-strain response similar to that of native coronary artery, while also displaying acceptable tensile strength. Furthermore, associated 10T½ cells and deposited collagen fibers showed a high degree of circumferential alignment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2200-2209, 2016.