PEG-fibrinogen Research Articles

Modelling the disease: H2S-sensitivity and drug-resistance of triple negative breast cancer cells can be modulated by embedding in isotropic micro-environment

Three-dimensional (3D) cell culture systems provide more physiologically relevant information, representing more accurately the actual microenvironment where cells reside in tissues. However, the differences between the tissue culture plate (TCP) and 3D culture systems in terms of tumour cell growth, proliferation, migration, differentiation and response to the treatment have not been fully elucidated. Tumoroid microspheres containing the MDA-MB 231 breast cancer cell line were prepared using either tunable PEG-fibrinogen (PFs) or tunable PEG-silk fibroin (PSFs) hydrogels, respectively named MDAPFs and MDAPSFs. The cancer cells in the tumoroids showed changes both in globular morphology and at the protein expression level. A decrease of both Histone H3 acetylation and cyclin D1 expression in all 3D systems, compared to the 2D cell culture, was detected in parallel to changes of the matrix stiffness. The effects of a glutathionylated garlic extract (GSGa), a slow H2S-releasing donor, were investigated on both tumoroid systems. A pro-apoptotic effect of GSGa on tumour cell growth in 2D culture was observed as opposed to a pro-proliferative effect apparent in both MDAPFs and MDAPSFs. A dedicated ad hoc 3D cell migration chip was designed and optimized for studying tumour cell invasion in a gel-in-gel configuration. An anti-cell-invasion effect of the GSGa was observed in the 2D cell culture, whereas a pro-migratory effect in both MDAPFs and MDAPSFs was observed in the 3D cell migration chip assay. An increase of cyclin D1 expression after GSGa treatment was observed in agreement with an increase of the cell invasion index. Our results suggest that the “dimensionality” and the stiffness of the 3D cell culture milieu can change the response to both the gasotransmitter H2S and doxorubicin due to differences in both H2S diffusion and changes in protein expression. Moreover, we uncovered a direct relation between the cyclin D1 expression and the stiffness of the 3D cell culture milieu, suggesting the potential causal involvement of the cyclin D1 as a bio-marker for sensitivity of the tumour cells to their matrix stiffness. Therefore, our hydrogel-based tumoroids represent a valid tunable model for studying the physically induced transdifferentiation (PiT) of cancer cells and as a more reliable and predictive in vitro screening platform to investigate the effects of anti-tumour drugs.

In vivo restoration of dystrophin expression in mdx mice using intra-muscular and intra-arterial injections of hydrogel microsphere carriers of exon skipping antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the X-linked Dytrophin gene preventing the expression of the functional protein. Exon skipping therapy using antisense oligonucleotides (AONs) is a promising therapeutic strategy for DMD. While benefits of AON therapy have been demonstrated, some challenges remain before this strategy can be applied more comprehensively to DMD patients. These include instability of AONs due to low nuclease resistance and poor tissue uptake. Delivery systems have been examined to improve the availability and stability of oligonucleotide drugs, including polymeric carriers. Previously, we showed the potential of a hydrogel-based polymeric carrier in the form of injectable PEG-fibrinogen (PF) microspheres for delivery of chemically modified 2′-O-methyl phosphorothioate (2OMePs) AONs. The PF microspheres proved to be cytocompatible and provided sustained release of the AONs for several weeks, causing increased cellular uptake in mdx dystrophic mouse cells. Here, we further investigated this delivery strategy by examining in vivo efficacy of this approach. The 2OMePS/PEI polyplexes loaded in PF microspheres were delivered by intramuscular (IM) or intra-femoral (IF) injections. We examined the carrier biodegradation profiles, AON uptake efficiency, dystrophin restoration, and muscle histopathology. Both administration routes enhanced dystrophin restoration and improved the histopathology of the mdx mice muscles. The IF administration of the microspheres improved the efficacy of the 2OMePS AONs over the IM administration. This was demonstrated by a higher exon skipping percentage and a smaller percentage of centered nucleus fibers (CNF) found in H&E-stained muscles. The restoration of dystrophin expression found for both IM and IF treatments revealed a reduced dystrophic phenotype of the treated muscles. The study concludes that injectable PF microspheres can be used as a carrier system to improve the overall therapeutic outcomes of exon skipping-based therapy for treating DMD.

Electrospun nanofiber mesh with connective tissue growth factor and mesenchymal stem cells for pelvic floor repair: Long-term study.

Pelvic organ prolapse (POP) affects many women, with an estimated lifetime risk of surgical intervention of 18.7%. There is a need for alternative approaches as the use of synthetic nondegradable mesh was stopped due to severe adverse events, and as current methods for pelvic floor repair have high POP recurrence rates. Thus, we hypothesized that electrospun degradable meshes with stem cells and growth factor were safe and durable for the long term in elderly rats. In an abdominal repair model, electrospun polycaprolactone (PCL) meshes coated with connective tissue growth factor (CTGF)/PEG-fibrinogen (PF) and rat mesenchymal stem cells were implanted in elderly female rats and removed after in average 53 weeks (53-week group). Collagen amount and production were quantified by qPCR and Western blotting. Moreover, histological appearance and biomechanical properties were evaluated. Results were compared with previous results of young rats with identical mesh implanted for 24 weeks (24-week group). The 53-week group differed from the 24-week group in terms of (1) reduced collagen III, (2) strong reduction in foreign body response, and (3) altered histological appearance. We found comparable biomechanical properties, aside from higher, not significant, mean tissue stiffness in the 53-week group. Lastly, we identified mesh components 53 weeks after implantation. This study provides new insights into future POP repair in postmenopausal women by showing how CTGF/PF-coated electrospun PCL meshes with stem cells exhibit sufficient support, biocompatibility, and no mesh-related complications long term in an abdominal repair model in elderly rats.

Abstract P1145: Hydrogel Matrix Support Of Suspension-differentiated Hipscs Impacts Resulting In Vitro Cardiac Tissue Functionality

Functional in vitro cardiac tissues are needed to provide insight into the cellular level of disease. Previously, we demonstrated hydrogel-encapsulated hiPSCs undergo cardiac differentiation and achieve cardiomyocyte (CM) maturation including T-tubule formation. To investigate the effect of encapsulation on differentiation and cardiac tissue functionality, here we compared PEG-fibrinogen (PF) microsphere (MS)-supported differentiation approach to self-aggregated embryoid body (EB) differentiation. Briefly, hiPSCs were encapsulated or formed into EBs; cardiac differentiation was initiated by Wnt activation and subsequent inhibition. CM content, contractile functionality, drug response, and electrophysiological properties were determined by flow cytometry, microscopy, and optical mapping, respectively.Initial diameter was 673±22 μm (coefficient of variance, COV=0.03) for MS and145±34 μm (COV=0.24) for EBs. The number of CMs produced (differentiation day 10) per initial hiPSC was significantly higher for MS (2.8 ± 1.8) than EBs (0.6±0.12). MS also yielded a higher percentage of CMs; MS-encapsulated hiPSC differentiation resulted in 74.7±13.8% CMs while for EBs it was 56±16.1 % (cTnT+, MF20+, n=14 and 5 batches, respectively, p<0.05). Myocardial contractility was also enhanced in cardiac MS as compared to EBs (similar CM content, day 10); maximum contraction velocity was four times higher for MS than EBs (139±17.5 μm/s versus 33.4±5.1 μm/s) and maximum relaxation velocity was fourteen times higher (97±7.2 μm/s versus 7.4±0.4 μm/s).Furthermore, cardiac MS responded more vigorously to isoproterenol (IP) and propranolol (P) (frequency +124% (IP), - 61% (P), versus +49% (IP) and -27%(P) for EB), suggesting that MS-CM β-adrenergic signaling may be more mature. Calcium transient propagation was more uniform in MS-CMs compared to EB-CMs; calcium transient duration (CTD) at 50% and 80% repolarization and calcium transient velocity were similar. In conclusion, MS supported cardiac differentiation supported higher CM content, faster contractility, and improved drug response in comparison to similarly differentiated EBs, demonstrating the potential of encapsulation to enhance cardiac tissue functionality for disease modeling.

Growth Factor Delivery for the Repair of a Critical Size Tibia Defect Using an Acellular, Biodegradable Polyethylene Glycol-Albumin Hydrogel Implant.

Growth factor delivery using acellular matrices presents a promising alternative to current treatment options for bone repair in critical-size injuries. However, supra-physiological doses of the factors can introduce safety concerns that must be alleviated, mainly by sustaining delivery of smaller doses using the matrix as a depot. We developed an acellular, biodegradable hydrogel implant composed of poly(ethylene glycol) (PEG) and denatured albumin to be used for sustained delivery of bone morphogenic protein-2 (BMP2). In this study, poly(ethylene glycol)-albumin (PEG-Alb) hydrogels were produced and loaded with 7.7 μg/mL of recombinant human BMP2 (rhBMP2) to be tested for safety and performance in a critical-size long-bone defect, using a rodent model. The hydrogels were formed ex situ in a 5 mm long cylindrical mold of 3 mm diameter, implanted into defects made in the tibia of Sprague-Dawley rats and compared to non-rhBMP2 control hydrogels at 13 weeks following surgery. The hydrogels were also compared to the more established PEG-fibrinogen (PEG-Fib) hydrogels we have tested previously. Comprehensive in vitro characterization as well as in vivo assessments that include: histological analyses, including safety parameters (i.e., local tolerance and toxicity), assessment of implant degradation, bone formation, as well as repair tissue density using quantitative microCT analysis were performed. The in vitro assessments demonstrated similarities between the mechanical and release properties of the PEG-Alb hydrogels to those of the PEG-Fib hydrogels. Safety analysis presented good local tolerance in the bone defects and no signs of toxicity. A significantly larger amount of bone was detected at 13 weeks in the rhBMP2-treated defects as compared to non-rhBMP2 defects. However, no significant differences were noted in bone formation at 13 weeks when comparing the PEG-Alb-treated defects to PEG-Fib-treated defects (with or without BMP2). The study concludes that hydrogel scaffolds made from PEG-Alb containing 7.7 μg/mL of rhBMP2 are effective in accelerating the bridging of boney defects in the tibia.

The Effect of Scaffold Modulus on the Morphology and Remodeling of Fetal Mesenchymal Stem Cells.

Hydrogel materials have been successfully used as matrices to explore the role of biophysical and biochemical stimuli in directing stem cell behavior. Here, we present our findings on the role of modulus in guiding bone marrow fetal mesenchymal stem cell (BMfMSC) fate determination using semi-synthetic hydrogels made from PEG-fibrinogen (PF). The BMfMSCs were cultivated in the PF for up to 2 weeks to study the influence of matrix modulus (i.e., cross-linking density of the PF) on BMfMSC survival, morphology and integrin expression. Both two-dimensional (2D) and three-dimensional (3D) culture conditions were employed to examine the BMfMSCs as single cells or as cell spheroids. The hydrogel modulus affected the rate of BMfMSC metabolic activity, the integrin expression levels and the cell morphology, both as single cells and as spheroids. The cell seeding density was also found to be an important parameter of the system in that high densities were favorable in facilitating more cell-to-cell contacts that favored higher metabolic activity. Our findings provide important insight about design of a hydrogel scaffold that can be used to optimize the biological response of BMfMSCs for various tissue engineering applications.

A multi-cellular 3D bioprinting approach for vascularized heart tissue engineering based on HUVECs and iPSC-derived cardiomyocytes

The myocardium behaves like a sophisticated orchestra that expresses its true potential only if each member performs the correct task harmonically. Recapitulating its complexity within engineered 3D functional constructs with tailored biological and mechanical properties, is one of the current scientific priorities in the field of regenerative medicine and tissue engineering. In this study, driven by the necessity of fabricating advanced model of cardiac tissue, we present an innovative approach consisting of heterogeneous, multi-cellular constructs composed of Human Umbilical Vein Endothelial Cells (HUVECs) and induced pluripotent cell-derived cardiomyocytes (iPSC-CMs). Cells were encapsulated within hydrogel strands containing alginate and PEG-Fibrinogen (PF) and extruded through a custom microfluidic printing head (MPH) that allows to precisely tailor their 3D spatial deposition, guaranteeing a high printing fidelity and resolution. We obtained a 3D cardiac tissue compose of iPSC-derived CMs with a high orientation index imposed by the different defined geometries and blood vessel-like shapes generated by HUVECs which, as demonstrated by in vivo grafting, better support the integration of the engineered cardiac tissue with host’s vasculature.

Hydrogel Modulus Affects Proliferation Rate and Pluripotency of Human Mesenchymal Stem Cells Grown in Three-Dimensional Culture.

Hydrogels have been used extensively with various cell types in three-dimensional (3D) culture, including with human mesenchymal stem cells (hMSCs). Here, we report on the use of poly(ethylene glycol) (PEG)-conjugated fibrinogen hydrogels to grow bone marrow-derived hMSCs in 3D culture. The initial modulus of the PEG-fibrinogen (PF) hydrogels was varied to study the influence of the stiffness on the proliferation response of the cells growing within. Shear rheology was used to quantify the changes to the initial material properties; the shear storage modulus of the PF was controlled by changing the concentration of synthetic PEG cross-linker, while keeping the fibrinogen concentration constant. Cell culture was performed during a 14-day experiment to quantify the cell proliferation response in the different modulus materials tested. The hMSCs were recovered from the hydrogels by mild enzymatic dissolution, and characterized for proliferation and cell number using cytometry. The results indicate a modulus-dependent response from the cells, and the ability to preferentially define initial hydrogel modulus that favors higher proliferation and multipotency of the hMSCs. Bioactive supplements added to the hydrogels, including exogenous fibronectin (Fn) and von willebrand factor (vWf) were used to further stimulate the proliferation response of the hMSCs in the hydrogel cultures, without altering their multipotency. These insights underscore the importance of mechanical properties in regulating cell proliferation in a 3D culture milieu. The versatility of the hydrogel culture environment and the ability to control mechanical properties for cell-fate determination further highlight the significance of a modular approach when developing materials that ultimately optimize stem cell cultures.

HiPS-MSCs differentiation towards fibroblasts on a 3D ECM mimicking scaffold.

Fibroblasts are ubiquitous cells that constitute the stroma of virtually all tissues and play vital roles in homeostasis. The poor innate healing capacity of fibroblastic tissues is attributed to the scarcity of fibroblasts as collagen-producing cells. In this study, we have developed a functional ECM mimicking scaffold that is capable to supply spatial allocation of stem cells as well as anchorage and storage of growth factors (GFs) to direct stem cells differentiate towards fibroblasts. Electrospun PCL fibers were embedded in a PEG-fibrinogen (PF) hydrogel, which was infiltrated with connective tissue growth factor (CTGF) to form the 3D nanocomposite PFP-C. The human induced pluripotent stem cells derived mesenchymal stem cells (hiPS-MSCs) with an advance in growth over adult MSCs were applied to validate the fibrogenic capacity of the 3D nanocomposite scaffold. The PFP-C scaffold was found not only biocompatible with the hiPS-MSCs, but also presented intriguingly strong fibroblastic commitments, to an extent comparable to the positive control, tissue culture plastic surfaces (TCP) timely refreshed with 100% CTGF. The novel scaffold presented not only biomimetic ECM nanostructures for homing stem cells, but also sufficient cell-approachable bio-signaling cues, which may synergistically facilitate the control of stem cell fates for regenerative therapies.

Modulation of Dental Pulp Stem Cell Odontogenesis in a Tunable PEG-Fibrinogen Hydrogel System.

Injectable hydrogels have the great potential for clinical translation of dental pulp regeneration. A recently developed PEG-fibrinogen (PF) hydrogel, which comprises a bioactive fibrinogen backbone conjugated to polyethylene glycol (PEG) side chains, can be cross-linked after injection by photopolymerization. The objective of this study was to investigate the use of this hydrogel, which allows tuning of its mechanical properties, as a scaffold for dental pulp tissue engineering. The cross-linking degree of PF hydrogels could be controlled by varying the amounts of PEG-diacrylate (PEG-DA) cross-linker. PF hydrogels are generally cytocompatible with the encapsulated dental pulp stem cells (DPSCs), yielding >85% cell viability in all hydrogels. It was found that the cell morphology of encapsulated DPSCs, odontogenic gene expression, and mineralization were strongly modulated by the hydrogel cross-linking degree and matrix stiffness. Notably, DPSCs cultured within the highest cross-linked hydrogel remained mostly rounded in aggregates and demonstrated the greatest enhancement in odontogenic gene expression. Consistently, the highest degree of mineralization was observed in the highest cross-linked hydrogel. Collectively, our results indicate that PF hydrogels can be used as a scaffold for DPSCs and offers the possibility of influencing DPSCs in ways that may be beneficial for applications in regenerative endodontics.

PlGF–MMP9-engineered iPS cells supported on a PEG–fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)–fibrinogen scaffold. When assessing optimal stiffness of the PEG–fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

Influence of soluble PEG-OH incorporation in a 3D cell-laden PEG-fibrinogen (PF) hydrogel on smooth muscle cell morphology and growth

We have been able to control hydrogel compliance and cell spreading in a three-dimensional (3D) cell-laden system (hydrogel) using soluble PEG-OH. This was accomplished by encapsulating smooth muscle cells (SMCs) into poly(ethylene glycol)-fibrinogen (PEG-fibrinogen or PF) with poly(ethylene glycol)-diol (PEG-OH) as a macromolecular leachant. The cell-encapsulating hydrogels were prepared with three concentrations of soluble PEG-OH having a mass of 10 kDa (1, 5 and 10% w/v). Rheology was used to measure the elastic (storage) component of the complex shear modulus of these hydrogels, while quantitative morphometrics were used to characterize SMC morphology. PF hydrogel with a higher amount of PEG-OH displayed a lower storage modulus and a higher elongated cell morphology of SMCs. Structural changes of PF hydrogels mainly owing to gelation-induced phase separation imparted by the soluble PEG-OH in 3D cell-laden hydrogels dramatically affected both the properties of the hydrogel network including the modulus as well as cell spreading.

The effect of matrix stiffness of injectable hydrogels on the preservation of cardiac function after a heart attack

This study compares the effect of four injectable hydrogels with different mechanical properties on the post-myocardial infarction left ventricle (LV) remodeling process. The bioactive hydrogels were synthesized from Tetronic-fibrinogen (TF) and PEG–fibrinogen (PF) conjugates; each hydrogel was supplemented with two levels of additional cross-linker to increase the matrix stiffness as measured by the shear storage modulus (G′). Infarcts created by ligating the left anterior descending coronary artery in a rodent model were treated with the hydrogels, and all four treatment groups showed an increase in wall thickness, arterial density, and viable cardiac tissue in the peri-infarct areas of the LV. Echocardiography and hemodynamics data of the PF/TF treated groups showed significant improvement of heart function associated with the attenuated effects of the remodeling process. Multi-factorial regression analysis indicated that the group with the highest modulus exhibited the best rescue of heart function and highest neovascularization. The results of this study demonstrate that multiple properties of an injectable bioactive biomaterial, and notably the matrix stiffness, provide the multifaceted stimulation necessary to preserve cardiac function and prevent adverse remodeling following a heart attack.

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 α2, αv and α6 integrin subunits may play key roles in integrin-mediated osteogenesis.

Photopolymerization of cell-encapsulating hydrogels: Crosslinking efficiency versus cytotoxicity

Cell-encapsulating hydrogels used in regenerative medicine are designed to undergo a rapid liquid-to-solid phase transition in the presence of cells and tissues so as to maximize crosslinking and minimize cell toxicity. Light-activated free-radical crosslinking (photopolymerization) is of particular interest in this regard because it can provide rapid reaction rates that result in uniform hydrogel properties with excellent temporal and spatial control features. Among the many initiator systems available for photopolymerization, only a few have been identified as suitable for cell-based hydrogel formation owing to their water solubility, crosslinking properties and non-toxic reaction conditions. In this study, three long-wave ultraviolet (UV) light-activtied photoinitiators (PIs) were comparatively tested in terms of cytotoxicity, crosslinking efficiency and crosslinking kinetics of cell-encapsulating hydrogels. The hydrogels were photopolymerized from poly(ethylene glycol) (PEG) diacrylate or PEG–fibrinogen precursors using Irgacure® PIs I2959, I184 and I651, as well as with a chemical initiator/accelerator (APS/TEMED). The study specifically evaluated the PI type, PI concentration and UV light intensity, and how these affected the mechanical properties of the hydrogel (i.e. maximum storage modulus), the crosslinking reaction times and the reaction’s cytotoxicity to encapsulated cells. Only two initiators (I2959 and I184) were identified as being suitable for achieving both high cell viability and efficient crosslinking of the cell-encapsulating hydrogels during the photopolymerization reaction. Optimization of PI concentration or irradiation intensity was particularly important for achieving maximum mechanical properties; a sub-optimal choice of PI concentration or irradiation intensity resulted in a substantial reduction in hydrogel modulus.Cytocompatibility may be compromised by unnecessarily prolonging exposure to cytotoxic free radicals or inadvertently enhancing the instantaneous dose of radicals in solution, both of which are dependent on the PI type/concentration and irradiation intensity. In the absence of a radical initiator, the short exposures to long-wave UV light irradiation (up to 5min, 20mWcm–2, 365nm) did not prove to be cytotoxic to cells. Therefore, it is important to understand the relationship between PIs, light irradiation conditions and crosslinking when attempting to identify a suitable hydrogel formation process for cell encapsulating hydrogels.

Nanostructuring biosynthetic hydrogels for tissue engineering: A cellular and structural analysis

The nanostructuring of hydrogel scaffolds used in tissue engineering provides the ability to control cellular fate and tissue morphogenesis through cell–matrix interactions. Here we describe a method to provide nanostructure to a biosynthetic hydrogel scaffold made from crosslinked poly(ethylene glycol)–fibrinogen conjugates (PEG–fibrinogen), by modifying them with the block-copolymer Pluronic® F127. The copolymeric additive self-assembled into micelles at certain concentrations and temperatures, thereby creating nanostructures within the crosslinked hydrogel. Small-angle X-ray scattering (SAXS) and transmission electron microscopy at cryogenic temperature were used to detect Pluronic® F127 micelles embedded within the crosslinked PEG–fibrinogen hydrogels. The density and order of the micelles within the hydrogel matrix increased as the relative Pluronic® F127 concentration was raised. The transient stability of the micelles within the hydrogel network was analyzed using time-dependent swelling and Pluronic® F127 release measurements. These characterizations revealed that most of the Pluronic® F127 molecules diffuse out of the hydrogels after 4days in aqueous buffer and SAXS analysis confirmed a significant change in the structure and interactions of the micelles during this time. Cell culture experiments evaluating the three-dimensional fibroblast morphology within the matrix indicated a strong correlation between cell spreading and the hydrogel’s characteristic mesh size. The present research thereby provides a more quantitative understanding of how structural features in an encapsulating hydrogel environment can affect cell morphogenesis towards tissue regeneration.

The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes

Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24 h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical stimulation, and that the 3-D environment surrounding chondrocytes can actively participate in translating mechanical stimulation to the resident cells.

The influence of ascorbic acid, TGF-β1, and cell-mediated remodeling on the bulk mechanical properties of 3-D PEG–fibrinogen constructs

Two-dimensional cell culture studies have shown that matrix rigidity can influence cell function, but little is known about how matrix physical properties, and their changes with time, influence cell function in 3-D. Biosynthetic hydrogels based on PEGylated fibrinogen permit the initial decoupling of matrix chemical and mechanical properties, and are thus potentially attractive for addressing this question. However, the mechanical stability of these gels due to passive hydrolysis and cell-mediated remodeling has not previously been addressed. Here, we show that the bulk mechanical properties of acellular PEG–fibrinogen hydrogels significantly decrease over time in PBS regardless of matrix cross-linking density in 7 days. To compensate, smooth muscle cells (SMCs) were encapsulated and stimulated to produce their own matrix using ascorbic acid or TGF-β1. Ascorbic acid treatment improved the mechanical properties of the constructs after 14 days in less cross-linked matrices, but TGF-β1 did not. The increase in matrix modulus of the constructs was not due to an increase in type I collagen deposition, which remained low and pericellular regardless of cross-link density or the soluble factor applied. Instead, ascorbic acid, but not TGF-β1, preferentially enhanced the contractile SMC phenotype in the less cross-linked gels. Inhibition of contractility reduced cell spreading and the expression of contractile markers, and eliminated any beneficial increase in matrix modulus induced by cell-generated contraction of the gels. Together, these data show that PEG–fibrinogen hydrogels are susceptible to both hydrolysis and proteolysis, and suggest that some soluble factors may stimulate matrix remodeling by modulating SMC phenotype instead of inducing ECM synthesis in a 3-D matrix.

The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation

This study aims to explore the differential effect of scaffold composition and architecture on chondrogenic response to dynamic strain stimulation using encapsulating PEG-based hydrogels and primary bovine chondrocytes. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic materials to be used as scaffolds. Four different compositions were tested, including: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only. Primary articular chondrocytes were encapsulated in the hydrogel scaffolds and subjected to 15% dynamic compressive strain stimulation at 1-Hz frequency for 28 days. Stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the unconfined true compressive modulus by 32%, 45.4%, 33.6%, and 28.2%, compared to their static controls. The PF showed a significantly larger relative increase in the modulus in comparison to all other scaffolds tested. These results support the hypothesis that mechanical stimulation and material bioactivity have a significant effect on the reported chondrocyte response. Similar trends were observed with the swelling ratio of the constructs. These findings indicate that while stimulation causes metabolic changes in chondrocytes seeded in PEG hydrogels, the matrix bioactivity has a significant role in enhancing chondrocyte mechanotransduction in encapsulating scaffolds subjected to physical deformations.