Fabrication and Characterization of Quad-Component Bioinspired Hydrogels to Model Elevated Fibrin Levels in Central Nervous Tissue Scaffolds.

Multicomponent interpenetrating polymer network (mIPN) hydrogels are promising tissue-engineering scaffolds that could closely resemble key characteristics of native tissues. The mechanical and biochemical properties of mIPNs can be finely controlled to mimic key features of target cellular microenvironments, regulating cell-matrix interactions. In this work, we fabricated hydrogels made of collagen type I (Col I), fibrin, hyaluronic acid (HA), and poly (ethylene glycol) diacrylate (PEGDA) using a network-by-network fabrication approach. With these mIPNs, we aimed to develop a biomaterial platform that supports the in vitro culture of human astrocytes and potentially serves to assess the effects of the abnormal deposition of fibrin in cortex tissue and simulate key aspects in the progression of neuroinflammation typically found in human pathologies such as Alzheimer's disease (AD), Parkinson's disease (PD), and tissue trauma. Our resulting hydrogels closely resembled the complex modulus of AD human brain cortex tissue (~7.35 kPa), promoting cell spreading while allowing for the modulation of fibrin and hyaluronic acid levels. The individual networks and their microarchitecture were evaluated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Human astrocytes were encapsulated in mIPNs, and negligible cytotoxicity was observed 24 h after the cell encapsulation.

A Bioinspired Astrocyte-Derived Coating Promotes the In Vitro Proliferation of Human Neural Stem Cells While Maintaining Their Stemness.

The repair of neuronal tissue is a challenging process due to the limited proliferative capacity of neurons. Neural stem cells (NSCs) can aid in the regeneration process of neural tissue due to their high proliferation potential and capacity to differentiate into neurons. The therapeutic potential of these cells can only be achieved if sufficient cells are obtained without losing their differentiation potential. Toward this end, an astrocyte-derived coating (HAc) was evaluated as a promising substrate to promote the proliferation of NSCs. Mass spectroscopy and scanning electron microscopy were used to characterize the HAc. The proliferation rate and the expression of stemness and differentiation markers in NSCs cultured on the HAc were evaluated and compared to the responses of these cells to commonly used coating materials including Poly-L-Ornithine (PLO), and a Human Induced Pluripotent Stem Cell (HiPSC)-based coating. The use of the HAc promotes the in vitro cell growth of NSCs. The expression of the stemness markers Sox2 and Nestin, and the differentiation marker DCX in the HAc group was akin to the expression of these markers in the controls. In summary, HAc supported the proliferation of NSCs while maintaining their stemness and neural differentiation potential.

Growth Factor Binding Peptides in Poly (Ethylene Glycol) Diacrylate (PEGDA)-Based Hydrogels for an Improved Healing Response of Human Dermal Fibroblasts.

Growth factors (GF) are critical cytokines in wound healing. However, the direct delivery of these biochemical cues into a wound site significantly increases the cost of wound dressings and can lead to a strong immunological response due to the introduction of a foreign source of GFs. To overcome this challenge, we designed a poly(ethylene glycol) diacrylate (PEGDA) hydrogel with the potential capacity to sequester autologous GFs directly from the wound site. We demonstrated that synthetic peptide sequences covalently tethered to PEGDA hydrogels physically retained human transforming growth factor beta 1 (hTGFß1) and human vascular endothelial growth factor (hVEGF) at 3.2 and 0.6 ng/mm2, respectively. In addition, we demonstrated that retained hTGFß1 and hVEGF enhanced human dermal fibroblasts (HDFa) average cell surface area and proliferation, respectively, and that exposure to both GFs resulted in up to 1.9-fold higher fraction of area covered relative to the control. After five days in culture, relative to the control surface, non-covalently bound hTGFß1 significantly increased the expression of collagen type I and hTGFß1 and downregulated vimentin and matrix metalloproteinase 1 expression. Cumulatively, the response of HDFa to hTGFß1 aligns well with the expected response of fibroblasts during the early stages of wound healing.

Modeling the effects of hyaluronic acid degradation on the regulation of human astrocyte phenotype using multicomponent interpenetrating polymer networks (mIPNs).

Hyaluronic acid (HA) is a highly abundant component in the extracellular matrix (ECM) and a fundamental element to the architecture and the physiology of the central nervous system (CNS). Often, HA degradation occurs when an overreactive inflammatory response, derived from tissue trauma or neurodegenerative diseases such as Alzheimer's, causes the ECM in the CNS to be remodeled. Herein, we studied the effects of HA content as a key regulator of human astrocyte (HAf) reactivity using multicomponent interpenetrating polymer networks (mIPNs) comprised of Collagen I, HA and poly(ethylene glycol) diacrylate. The selected platform facilities the modulation of HA levels independently of matrix rigidity. Total astrocytic processes length, number of endpoints, the expression of the quiescent markers: Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) and Glutamate Aspartate Transporter (GLAST); the reactive markers: Glial Fibrillary Acidic Protein (GFAP) and S100 Calcium-Binding Protein ß (S100ß); and the inflammatory markers: Inducible Nitric Oxide Synthase (iNOS), Interleukin 1ß (IL-1ß) and Tumor Necrosis Factor Alpha (TNFα), were assessed. Cumulatively, our results demonstrated that the decrease in HA concentration elicited a reduction in the total length of astrocytic processes and an increase in the expression of HAf reactive and inflammatory markers.

Refined assessment of the impact of cell shape on human mesenchymal stem cell differentiation in 3D contexts.

Numerous studies have demonstrated that the differentiation potential of human mesenchymal stem cells (hMSCs) can be modulated by chemical and physical cues. In 2D contexts, inducing different cell morphologies, by varying the shape, area and/or curvature of adhesive islands on patterned surfaces, has significant effects on hMSC multipotency and the onset of differentiation. In contrast, in vitro studies in 3D contexts have suggested that hMSC differentiation does not directly correlate with cell shape. However, in 3D, the effects of cell morphology on hMSC differentiation have not yet been clearly established due to the chemical and physical properties being intertwined in 3D matrices. In this work, we studied the effects of round or elongated cell morphologies on hMSC differentiation independently of scaffold composition, modulus, crosslink density and cell-mediated matrix remodeling. The effects of cell shape on hMSC lineage progression were studied using three different cell culture media compositions and two values of scaffold rigidity. Differences in cell shape were achieved using interpenetrating polymer networks (IPNs). The mechanical and diffusional properties of the scaffolds and cell-matrix interactions were characterized. In addition, cell responses were evaluated in terms of cell spreading via gene and protein expression of differentiation markers. Cumulative results support, and extend upon previous work indicating that cell shape alone in 3D contexts does not significantly modulate hMSC differentiation, at least for the scaffold chemistry, range of modulus and culture conditions explored in this study. STATEMENT OF SIGNIFICANCE: In 2D contexts, inducing different cell shapes, by varying the curvature, area size and shape of a patterned surface, has significant effects on hMSC multipotency and the onset of cell differentiation. In contrast, in vitro studies in 3D contexts have suggested that hMSC differentiation does not directly correlate with cell shape. However, in 3D, the effects of cell morphology on hMSC differentiation have not yet been clearly established due to the chemical and physical properties being intertwined in 3D matrices. In this work, we studied the effects of round or elongated cell morphologies on the differentiation of hMSCs independently of scaffold composition, modulus, crosslink density and cell mediated matrix remodeling. Cumulative results support, and extend upon previous work indicating that cell shape alone in 3D contexts does not significantly modulate hMSCs differentiation commitment.

Collagen Based Multicomponent Interpenetrating Networks as Promising Scaffolds for 3D Culture of Human Neural Stem Cells, Human Astrocytes, and Human Microglia.

This work describes for the first time the fabrication and characterization of multicomponent interpenetrating networks composed of collagen I, hyaluronic acid, and poly(ethylene glycol) diacrylate for the 3D culture of human neural stem cells, astrocytes, and microglia. The chemical composition of the scaffolds can be modulated while maintaining values of complex moduli within the range of the mechanical performance of brain tissue (∼6.9 kPa) and having cell viability exceeding 84%. The developed scaffolds are a promising new family of biomaterials that can potentially serve as 3D in vitro models for studying the physiology and physiopathology of the central nervous system.

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.

An improved correlation to predict molecular weight between crosslinks based on equilibrium degree of swelling of hydrogel networks.

Accurate characterization of hydrogel diffusional properties is of substantial importance for a range of biotechnological applications. The diffusional capacity of hydrogels has commonly been estimated using the average molecular weight between crosslinks (Mc ), which is calculated based on the equilibrium degree of swelling. However, the existing correlation linking Mc and equilibrium swelling fails to accurately reflect the diffusional properties of highly crosslinked hydrogel networks. Also, as demonstrated herein, the current model fails to accurately predict the diffusional properties of hydrogels when polymer concentration and molecular weight are varied simultaneously. To address these limitations, we evaluated the diffusional properties of 48 distinct hydrogel formulations using two different photoinitiator systems, employing molecular size exclusion as an alternative methodology to calculate average hydrogel mesh size. The resulting data were then utilized to develop a revised correlation between Mc and hydrogel equilibrium swelling that substantially reduces the limitations associated with the current correlation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1339-1348, 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.

Mechanical Characterization of Hybrid Vesicles Based on Linear Poly(Dimethylsiloxane-b-Ethylene Oxide) and Poly(Butadiene-b-Ethylene Oxide) Block Copolymers.

Poly(dimethylsiloxane-ethylene oxide) (PDMS-PEO) and poly(butadiene-b-ethylene oxide) (PBd-PEO) are two block copolymers which separately form vesicles with disparate membrane permeabilities and fluidities. Thus, hybrid vesicles formed from both PDMS-PEO and PBd-PEO may ultimately allow for systematic, application-specific tuning of vesicle membrane fluidity and permeability. However, given the relatively low strength previously noted for comb-type PDMS-PEO vesicles, the mechanical robustness of the resulting hybrid vesicles must first be confirmed. Toward this end, we have characterized the mechanical behavior of vesicles formed from mixtures of linear PDMS-PEO and linear PBd-PEO using micropipette aspiration. Tension versus strain plots of pure PDMS12-PEO46 vesicles revealed a non-linear response in the high tension regime, in contrast to the approximately linear response of pure PBd33-PEO20 vesicles. Remarkably, the area expansion modulus, critical tension, and cohesive energy density of PDMS12-PEO46 vesicles were each significantly greater than for PBd33-PEO20 vesicles, although critical strain was not significantly different between these vesicle types. PDMS12-PEO46/PBd33-PEO20 hybrid vesicles generally displayed graded responses in between that of the pure component vesicles. Thus, the PDMS12-PEO46/PBd33-PEO20 hybrid vesicles retained or exceeded the strength and toughness characteristic of pure PBd-PEO vesicles, indicating that future assessment of the membrane permeability and fluidity of these hybrid vesicles may be warranted.

Osteoinductive PolyHIPE Foams as Injectable Bone Grafts.

We have recently fabricated biodegradable polyHIPEs as injectable bone grafts and characterized the mechanical properties, pore architecture, and cure rates. In this study, calcium phosphate nanoparticles and demineralized bone matrix (DBM) particles were incorporated into injectable polyHIPE foams to promote osteoblastic differentiation of mesenchymal stem cells (MSCs). Upon incorporation of each type of particle, stable monoliths were formed with compressive properties comparable to control polyHIPEs. Pore size quantification indicated a negligible effect of all particles on emulsion stability and resulting pore architecture. Alizarin red calcium staining illustrated the incorporation of calcium phosphate particles at the pore surface, while picrosirius red collagen staining illustrated collagen-rich DBM particles within the monoliths. Osteoinductive particles had a negligible effect on the compressive modulus (∼30 MPa), which remained comparable to human cancellous bone values. All polyHIPE compositions promoted human MSC viability (∼90%) through 2 weeks. Furthermore, gene expression analysis indicated the ability of all polyHIPE compositions to promote osteogenic differentiation through the upregulation of bone-specific markers compared to a time zero control. These findings illustrate the potential for these osteoinductive polyHIPEs to promote osteogenesis and validate future in vivo evaluation. Overall, this work demonstrates the ability to incorporate a range of bioactive components into propylene fumarate dimethacrylate-based injectable polyHIPEs to increase cellular interactions and direct specific behavior without compromising scaffold architecture and resulting properties for various tissue engineering applications.

Collagen-mimetic hydrogels promote human endothelial cell adhesion, migration and phenotypic maturation.

This work evaluates the response of human aortic endothelial cells (HAECs) to thromboresistant collagen-mimetic hydrogel coatings toward improving the biocompatibility of existing "off-the-shelf" small-caliber vascular grafts. Specifically, bioactive hydrogels - previously shown to support α1/α2 integrin-mediated cell adhesion but to resist platelet activation - were fabricated by combining poly(ethylene glycol) (PEG) with a 120 kDa, triple-helical collagen-mimetic protein(Scl2-2) containing the GFPGER adhesion sequence. Analysis of HAECs seeded onto the resulting PEG-Scl2-2 hydrogels demonstrated that HAEC adhesion increased with increasing Scl2-2 concentration, while HAEC migration rate decreased over this same concentration range. In addition, evaluation of HAEC phenotype at confluence indicated significant differences in the gene expression of NOS3, thrombomodulin, and E-selectin on the PEG-Scl2-2 hydrogels relative to PEG-collagen controls. At the protein level, however, only NOS3 was significantly different between the PEG-Scl2-2 and PEG-collagen surfaces. Furthermore, PECAM-1 and VE-cadherin expression on PEG-Scl2-2 hydrogels versus PEG-collagen controls could not be distinguished at either the gene or protein level. Cumulatively, these data indicate the PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. In future studies, the Scl2-2 protein can potentially be modified to include additional extracellular matrix or cytokine binding sites to further improve endothelial cell responses.

In depth examination of impact of secondary reactive species on the apparent decoupling of poly(ethylene glycol) diacrylate hydrogel average mesh size and modulus.

Poly(ethylene glycol) diacrylate (PEGDA) hydrogels are widely used in biotechnology due to their in situ crosslinking capacity and tunable physical properties. However, as with all single component hydrogels, the modulus of PEGDA networks cannot be tailored independently of mesh size. This interdependence places significant limitations on their use for defined, 3D cell-microenvironment studies and for certain controlled release applications. The incorporation of secondary reactive species (SRS) into PEGDA hydrogels has previously been shown to allow the identification of up to 6 PEGDA hydrogel formulations for which distinct moduli can be obtained at consistent average mesh size (or vice versa). However, the modulus and mesh size ranges which can be probed by these formulations are quite restricted. This work presents an in-depth study of SRS incorporation into PEGDA hydrogels, with the goal of expanding the space for which "decoupled" examination of modulus and mesh size effects is achievable. Towards this end, over 100 PEGDA hydrogels containing either N-vinyl pyrrolidone or star PEG-tetraacrylate as SRS were characterized. To our knowledge, this is the first study to demonstrate that SRS incorporation allows for the identification of a number of modulus ranges that can be probed at consistent average mesh size (or vice versa).

Evaluation of the Osteoinductive Capacity of Polydopamine-Coated Poly(ε-caprolactone) Diacrylate Shape Memory Foams.

Recently, a novel shape memory polymer foam based on the photopolymerization of poly(ε-caprolactone) diacrylate (PCLDA) has been developed. These PCLDA foams enter a temporary softened state when briefly treated with warm saline (T saline > T m of PCLDA), allowing them to conform to irregular bone defect "boundaries" prior to shape setting. When coated with a mechanically stable polydopamine (PD) layer, these PCLDA foams have previously been demonstrated to induce hydroxyapatite deposition. In the present study, the osteoinductivity of these "self-fitting" PD-coated PCLDA (PD-PCLDA) materials was evaluated relative to uncoated PCLDA (U-PCLDA) controls using bone marrow-derived human mesenchymal stem cells (h-MSCs). When cultured in the absence of osteogenic media supplements, PD-PCLDA scaffolds expressed similar levels of Runx2, alkaline phosphatase, and osteopontin protein as U-PCLDA scaffolds cultured in the presence of osteogenic media supplements. In addition, PD-PCLDA scaffolds cultured without osteogenic supplements did not significantly promote undesired lineage progression (e.g., adipogenesis or chondrogenesis) of h-MSCs. Cumulatively, these data indicate that PD-PCLDA materials display increased osteoinductivity relative to U-PCLDA substrates. Future studies will examine tethered osteogenic factors or peptides toward augmenting the osteoinductive properties of the PD-PCLDA foams.

Characterization of sequential collagen-poly(ethylene glycol) diacrylate interpenetrating networks and initial assessment of their potential for vascular tissue engineering.

Collagen hydrogels have been widely investigated as scaffolds for vascular tissue engineering due in part to the capacity of collagen to promote robust cell adhesion and elongation. However, collagen hydrogels display relatively low stiffness and strength, are thrombogenic, and are highly susceptible to cell-mediated contraction. In the current work, we develop and characterize a sequentially-formed interpenetrating network (IPN) that retains the benefits of collagen, but which displays enhanced mechanical stiffness and strength, improved thromboresistance, high physical stability and resistance to contraction. In this strategy, we first form a collagen hydrogel, infuse this hydrogel with poly(ethylene glycol) diacrylate (PEGDA), and subsequently crosslink the PEGDA by exposure to longwave UV light. These collagen-PEGDA IPNs allow for cell encapsulation during the fabrication process with greater than 90% cell viability via inclusion of cells within the collagen hydrogel precursor solution. Furthermore, the degree of cell spreading within the IPNs can be tuned from rounded to fully elongated by varying the time delay between the formation of the cell-laden collagen hydrogel and the formation of the PEGDA network. We also demonstrate that these collagen-PEGDA IPNs are able to support the initial stages of smooth muscle cell lineage progression by elongated human mesenchymal stems cells.

Poly(sophorolipid) structural variation: effects on biomaterial physical and biological properties.

Diacetylated lactonic sophorolipids (polyLSL[6'Ac,6″Ac]), a biosurfactant, can be efficiently polymerized by ring-opening metathesis polymerization (ROMP). In this paper, enzyme-mediated chemical transformations are developed to regioselectively modify LSL[6'Ac,6″Ac] at sophorose primary hydroxyl positions (6' and 6″). The resulting modified LSLs were polymerized to expand polyLSL structural diversity, that is, polyLSL[6'OH,6″Ac], polyLSL[6'OH,6″OH], polyLSL[6'Bu,6″Ac], polyLSL[6'N3,6″Ac], and polyLSL[6'MA,6″Ac]. Controlled placement of azide and methacrylate at sophorolipid moieties enables the use of "click" reactions to introduce bioactive groups. Thermal analyses of polyLSLs showed that the acylation pattern at sugar moieties has a remarkable effect on chain stiffness and crystallinity. Films of polyLSL[6'Ac,6″Ac], polyLSL[6'OH,6″Ac], and polyLSL[6'Bu,6″Ac] exhibited nonbrittle behaviors with compressive elastic moduli ranging from ∼1.5 to ∼4.9 MPa. PolyLSLs were cytocompatible with human mesenchymal stem cells (h-MSCs), and examination of material-induced osteogenic cell lineage progression uncovered a dependence on polyLSL substitution at sophorose 6'-sites. This research reveals opportunities to regulate polyLSL physical properties and cell response behaviors by variation in substituents at polyLSL sophorolipid moieties.

Influence of pressurized cyclic stretch and endothelial cell presence on multipotent stem cell osteogenic commitment.

Applied mechanical stretch and blood vessel invasion are key stimuli to which progenitor cells are exposed in post-natal endochondral bone formation. Understanding the combined effects of cyclic stretch and endothelial cell (EC) presence on multipotent stem cell (MSC) osteogenesis therefore has the potential to lead to improved MSC-based bone regeneration strategies. Toward this goal, 10T1/2 mouse MSCs were encapsulated in tubular poly(ethylene glycol) diacrylate [PEGDA] hydrogels with moduli within the "osteogenic" range in order to induce osteogenesis. Half of the constructs were fabricated with a luminal EC layer. All of the EC(+) (EC(+)/dyn(+)) and half of the EC(-) constructs (EC(-)/dyn(+)) were subjected to pressurized cyclic stretch in the absence of osteogenic media supplements, with remaining EC(-) constructs (EC(-)/dyn(-)) serving as static controls. At day 10 of culture, expression of the bone extracellular matrix protein osteopontin was over 3.3- and 1.9-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. At day 22 of culture, osteopontin levels could not be statistically distinguished from day 0 in the EC(+)/dyn(+) constructs and were one-third less than day 0 in the EC(-)/dyn(+) constructs. In contrast, at day 22 levels of an osteogenic marker alkaline phosphatase (AP) were over 2.4- and 1.4-fold higher in the EC(+)/dyn(+) and EC(-)/dyn(+) constructs, respectively, relative to day 0. Furthermore, at day 22 matrix mineralization in both dynamic groups was increased over 2.5-fold and over 9-fold relative to the EC(-)/dyn(-) and day 0 groups, respectively. Cumulatively, these results suggest that pressurized cyclic stretch alone significantly increases the rate/degree of osteogenesis relative to static culture. However, EC presence combined with pressured cyclic stretch appears to further enhance the rate/degree of MSC osteogenesis and/or to support a distinct osteogenic "fingerprint" compared to that promoted by cyclic stretch alone.

Influence of select extracellular matrix proteins on mesenchymal stem cell osteogenic commitment in three-dimensional contexts.

Growth factors have been shown to be powerful mediators of mesenchymal stem cell (MSC) osteogenic differentiation. However, their use in tissue engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically promote MSC osteogenic differentiation in the absence of exogenous growth factors via the manipulation of scaffold material properties would be beneficial. The current work examines the influence of select extracellular matrix (ECM) proteins on MSC osteogenesis toward the goal of developing scaffolds with intrinsically osteoinductive properties. Fibrinogen (FG), fibronectin (FN) and laminin-1 (LN) were chosen for evaluation due to their known roles in bone morphogenesis or bone fracture healing. These proteins were conjugated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels and their effects on encapsulated 10T½ MSCs were evaluated. Specifically, following 1week of culture, mid-term markers of various MSC lineages were examined in order to assess the strength and specificity of the observed osteogenic responses. PEG-LN gels demonstrated increased levels of the osteogenic transcription factor osterix relative to day 0 levels. In addition, PEG-FG and PEG-LN gels were associated with increased deposition of bone ECM protein osteocalcin relative to PEG-FN gels and day 0. Importantly, the osteogenic response associated with FG and LN appeared to be specific in that markers for chondrocytic, smooth muscle cell and adipocytic lineages were not similarly elevated relative to day 0 in these gels. To gain insight into the integrin dynamics underlying the observed differentiation results, initial integrin adhesion and temporal alterations in cell integrin profiles were evaluated. The associated results suggest that α(2), α(v) and α(6) integrin subunits may play key roles in integrin-mediated osteogenesis.

An approach for assessing hydrogel hydrophobicity.

Hydrogel hydrophobicity is an important modulator of mammalian cell behavior, drug payload release, and medical device fouling. Contact angle and protein adsorption measures are two common methods for evaluating hydrogel hydrophobicity. However, protein adsorption is a complex phenomenon which is challenging to interpret in terms of gel hydrophobicity alone. In addition, the permeability of hydrogels can be problematic for contact angle assessment, as this method can only be strictly applied to smooth, solid, and nonpermeable surfaces. Therefore, the development of a technique for measuring hydrogel hydrophobicity which is simple, sensitive, and independent of variations in gel permeability would significantly advance the ability to finely tune this variable. The present technical note develops a method for quantifying the hydrophobicity of hydrogels by exploiting their capacity to swell differentially in solvents of distinct polarities. To validate this technique, hydrogels of varying hydrophobicities were prepared by combining hydrophilic poly(ethylene glycol) diacrylate (PEGDA) with either hydrophobic 3-(trimethoxysilyl) propyl methacrylate (TMSPM) or hydrophilic 2-hydroxyethyl methacrylate (HEMA). The ratio of hydrogel swelling in 70% isopropanol to that in water was termed the hydrophobicity index (H-index) and was determined for each gel type. The measured H-indices reflected known differences in the hydrophobicities of HEMA, TMSPM, and PEGDA and, in contrast to contact angle assessments, appeared to be independent of variations in hydrogel permeability. In addition, the trend in H-indices agreed well with the trend in protein adsorption across hydrogel formulations, although the H-indices appeared to be able to resolve more subtle differences in gel hydrophobicity than protein adsorption measures.