Electrical stimulation of Schwann cells on electrospun hyaluronic acid carbon nanotube fibers.

Neurofibromatosis Type 1 (NF1) is a complex genetic disorder characterized by the development of benign neurofibromas, which can cause significant morbidity in affected individuals. While the molecular mechanisms underlying NF1 pathogenesis have been extensively studied, the development of effective therapeutic strategies remains a challenge. This paper presents the development and validation of a novel biomaterial testing model to enhance our understanding of NF1 pathophysiology, disease mechanisms and evaluate potential therapeutic interventions. Our long-term goal is to develop an invitro model of NF1 to evaluate drug targets. We have developed an in vitro system to test the cellular behavior of NF1 patient derived cells on electroconductive aligned nanofibrous biomaterials with electrical stimulatory cues. We hypothesized that cells cultured on electroconductive biomaterial will undergo morphological changes and variations in cell proliferation that could be further enhanced with the combination of exogenous electrical stimulation (ES). In this study, we developed electrospun Hyaluronic Acid-Carbon Nanotube (HA-CNT) nanofiber scaffolds to mimic the axon's topographical and bioelectrical cues that influence neurofibroma growth and development. The cellular behavior was qualitatively and quantitively analyzed through immunofluorescent stains, Alamar blue assays and ELISA assays. Schwann cells from NF1 patients appear to have lost their ability to respond to electrical stimulation in the development and regeneration range, which was seen through changes in morphology, proliferation and NGF release. Without stimulation, the conductive material enhances NF1 SC behavior. Wild-type SC respond to electrical stimulation with increased cell proliferation and NGF release. Using this system, we can better understand the interaction between axons and SC that lead to tumor formation, homeostasis and regeneration.

A Fibroblast-Derived Secretome Stimulates the Growth and Invasiveness of 3D Plexiform Neurofibroma Spheroids.

Plexiform neurofibromas (PNs) occur in about a half of neurofibromatosis type 1 (NF1) patients and have garnered significant research attention due to their capacity for growth and potential for malignant transformation. NF1 plexiform neurofibroma (pNF1) is a complex tumor composed of Schwann cell-derived tumor cells (Nf1-/-) and the tumor microenvironment (TME). Although it has been widely demonstrated that the TME is involved in the formation of neurofibromas, little is known about the effects of the TME on the subsequent progression of human pNF1. Elucidating the molecular interactions between tumor cells and the TME may provide new therapeutic targets to reduce the progression of pNF1. In the present study, we focused on the contributions of fibroblasts, the most abundant cell types in the TME, to the growth of pNF1. To simulate the TME, we used a three-dimensional (3D) coculture model of immortalized pNF1 tumor cells (Nf1-/-) and primary fibroblasts (Nf1+/-) derived from pNF1 patients. We performed live-cell imaging of 3D/4D (3D in real-time) cultures through confocal microscopy followed by 3D quantitative analyses using advanced imaging software. The growth of pNF1 spheroids in 3D cocultures with fibroblasts was significantly greater than that of pNF1 spheroids in 3D monocultures. An increase in the growth of pNF1 spheroids also occurred when they were cultured with conditioned media (CM) from fibroblasts. Moreover, fibroblast-derived CM increased the invasive outgrowth and further local invasion of pNF1 spheroids. Interestingly, when small extracellular vesicles (sEVs) were depleted from the fibroblast-derived CM, the stimulation of the growth of pNF1 spheroids was lost. Our results suggest that fibroblast-derived sEVs are a therapeutic target for reducing the growth of pNF1.

Fiber alignment directs cell motility over chemotactic gradients.

The ability of tissue engineered scaffolds to direct cell behavior is paramount for scaffold design. Cell migration can be directed by various methods including chemical, adhesive, mechanical, and topographical cues. Electrospinning has emerged as a popular method to control topography and create fibrous scaffolds similar to that found in extracellular matrix. One major hurdle is limited cell infiltration and several studies have explored methods to alter electrospun materials to increase scaffold porosity; however, uniform cell distributions within scaffolds is still limited. Towards this, we investigated the motility of HUVECs on a model system of electrospun hyaluronic acid fibers under a gradient of VEGF and found that topographical cues dominate cell motility direction. Using time-lapse microscopy, cell aspect ratio, and migration angle were measured; cells were directed in a chemical gradient and/or on aligned electrospun fibers. Measurements of the persistence time demonstrated an additive effect of the chemical gradient and fiber alignment. However, when fibers were aligned perpendicular to a chemical gradient, cells were directed by fiber alignment and there was no effect of the chemical gradient. These results suggest that topographical cues may be more influential than chemical cues in directing cell motility and should be considered in material design.

Enzyme-Mediated Nerve Growth Factor Release from Nanofibers Using Gelatin Microspheres.

Spinal cord injury is a complex environment, with many conflicting growth factors present at different times throughout the injury timeline. Delivery of multiple growth factors has received mixed results, highlighting a need to consider the timing of delivery for possibly antagonistic growth factors. Cell-mediated degradation of delivery vehicles for delayed release of growth factors offers an attractive way to exploit the highly active immune response in the spinal cord injury environment. In this study, growth factor-loaded gelatin microspheres (GMS) combined with methacrylated hyaluronic acid (MeHA) were electrospun to create GMS fibers (GMSF) for delayed release of growth factors (GFs). GMS were successfully combined with MeHA while electrospinning, with an average fiber diameter of 365 ± 10 nm and 44% ± 8% fiber alignment. GMSF with nerve growth factor (NGF) was tested on dissociated chick dorsal root ganglia cells. We further tested the effect of M1 macrophage-conditioned media (M1CM) to simulate macrophage invasion after spinal cord injury for cell-mediated degradation. We hypothesized that neurons grown on GMSF with loaded NGF would exhibit longer neurites in M1CM, showing a release of functional NGF, as compared with controls. GMSF in M1CM was significantly different from MeHA in serum-free media (SFM) and M0-conditioned media (M0CM), as well as GMSF in M0CM (p < 0.05). Moreover, GMSF + NGF in all media conditions were significantly different from MeHA in SFM and M0CM (p < 0.05). The goal of this study was to develop a biomaterial system where drug delivery is triggered by immune response, allowing for more control and longer exposure to encapsulated drugs. The spinal cord injury microenvironment is known to have a robust immune response, making this immune-medicated drug release system particularly significant for directed repair.

Gradients with depth in electrospun fibrous scaffolds for directed cell behavior.

A major obstacle in creating viable tissue-engineered constructs using electrospinning is the lack of complete cellularization and vascularization due to the limited porosity in these densely packed fibrous scaffolds. One potential approach to circumvent this issue is the use of various gradients of chemical and biophysical cues to drive the infiltration of cells into these structures. Toward this goal, this study focused on creating durotactic (mechanical) and haptotactic (adhesive) gradients through the thickness of electrospun hyaluronic acid (HA) scaffolds using a unique, yet simple, modification of common electrospinning protocols. Specifically, both mechanical (via cross-linking: ranging from 27-100% modified methacrylated HA, MeHA) and adhesive (via inclusion of the adhesive peptide RGD: 0-3 mM RGD) gradients were each fabricated by mixing two solutions (one ramping up, one ramping down) prior to electrospinning and fiber collection. Gradient formation was verified by fluorescence microscopy, FTIR, atomic force microscopy, and cellular morphology assessment of scaffolds at different points of collection (i.e., with scaffold thickness). To test further the functionality of gradient scaffolds, chick aortic arch explants were cultured on adhesive gradient scaffolds for 7 days, and low RGD-high RGD gradient scaffolds showed significantly greater cell infiltration compared with high RGD-low RGD gradients and uniform high RGD or uniform low RGD control scaffolds. In addition to enhanced infiltration, this approach could be used to fabricate graded tissue structures, such as those that occur at interfaces.

Heparin-hyaluronic acid nanofibers for growth factor sequestration in spinal cord repair.

Growth factor (GF) delivery is a common strategy for spinal cord injury repair, however, GF degradation can impede long-term therapies. GF sequestration via heparin is known to protect bioactivity after delivery. We tested two heparin modifications, methacrylated heparin and thiolated heparin, and electrospun these with methacrylated hyaluronic acid (MeHA) to form HepMAHA and HepSHHA nanofibers. For loaded conditions, MeHA, HepMAHA, and HepSHHA fibers were incubated with soluble basic fibroblast growth factor (bFGF) or nerve growth factor (NGF) and rinsed with PBS. Control groups were hydrated in PBS. L929 fibroblast proliferation was analyzed after 24 hr of culture in either growth media or bFGF-supplemented media. Dissociated chick dorsal root ganglia neurites were measured after 3 days of cell culture in serum free media (SFM) or NGF-supplemented SFM (SFM + NGF). In growth media, fibroblast proliferation was significantly increased in loaded HepMAHA (α < .05) compared to other groups. In SFM, loaded HepMAHA had the longest average neurite length compared to all other groups. In SFM + NGF, HepMAHA and HepSHHA had increased neurite lengths compared to MeHA, regardless of loading (α < .01), suggesting active sequestration of soluble NGF. HepMAHA is a promising biomaterial for sequestering released GFs in a spinal cord injury environment and will be combined with GF filled microspheres for future studies.

Mechanical, topographical and chemical cues combined with physical therapy for peripheral nerve injuries.

Aim: The aim of this paper is to evaluate biomaterial cues combined with physical therapy (PT) on functional recovery in a rat sciatic nerve injury model. Materials & methods: Nerve growth conduits were filled with longitudinally aligned hyaluronic acid fibers and microspheres containing neurotrophic factor (growth factor [GF]). All animals received behavior and functional testing throughout the study, which concluded with measurement of compound muscle action potentials and contractile force of the gastrocnemius muscle. Results & conclusion: Including GF improved recovery of gross motor function and increased sensory pain sensation. During the 4 weeks that animals participated in PT, these groups showed higher static sciatic index scores. Including GF and PT has the potential to improve clinical outcomes following peripheral nerve injury.

Neurite growth in 3D collagen gels with gradients of mechanical properties.

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.

Biomaterial Cues to Direct a Pro-regenerative Phenotype in Macrophages and Schwann Cells.

Following peripheral nerve injury (PNI), inflammatory cues impede repair. We have previously demonstrated that spinal cord matrix (SCM) proteins and hyaluronic acid (HA) nanofibers mitigate chondroitin sulfate proteoglycan (CSPG) inhibition and promote growth in peripheral neurons. In this study, we evaluated the effects of a characteristic CSPG, chondroitin sulfate A (CSA), SCM, and HA fibers on macrophages and Schwann cells (SCs). We hypothesized that our cues would accelerate the macrophages' return to rest following classical activation (M1/pro-inflammatory) with lipopolysaccharide (LPS; 1 µg/mL) and would accelerate the transformation of SCs from an immature state following injury to a mature/pro-myelinating phenotype. LPS stimulation of the macrophages caused upregulation of inducible nitric oxide synthase (iNOS; M1 gene) and led to significantly increased cell area and decreased circularity. However, the SCM and HA nanofibers mitigated this effect, significantly reducing iNOS expression. SCs on the fibers had significantly reduced area and increased elongation. These morphological changes may have polarized the cells leading to decreased GFAP (immature gene) and increased Oct6 and Krox 20 (promyelin genes) expression. Antibody arrays were used to measure relative levels of inflammatory cytokines released by the cells. The arrays confirmed that anti-inflammatory cytokines are released from the cells when cultured with our biomaterial cues and helped identify targets for future investigation including vascular endothelial growth factor (VEGF), interleukin (IL)-10, monocyte colony stimulating factor (M-CSF) from the macrophages, Agrin, ciliary neurotrophic factor (CNTF), tissue inhibitor metalloproteinases (TIMPs)-1 from SCs, and IL-2 from both cell types. In conclusion, these results suggest that our biomaterial cues have pro-regenerative effects on both cell types and if combined may trigger cells toward regenerative programs.

Combining growth factor releasing microspheres within aligned nanofibers enhances neurite outgrowth.

Current treatments for peripheral nerve injuries include autografts, the gold standard, and commercially available nerve growth conduits (NGCs). Autografts have several drawbacks including donor site morbidity and nerve size mismatch, which lead to incomplete recovery. However, even with these drawbacks, autografts work better then commercially available NGCs that lack sufficient cues to promote complete regeneration. This study evaluated a combination of biomaterial components that can be added to the hollow internal space of a NGC to promote and direct nerve regeneration; specifically, mechanical, chemical, and topographical cues. Methacrylated hyaluronic acid (MeHA, mechanical cue) is electrospun into aligned fibers (topographical cue), with poly-lactic-co-glycolic acid microspheres to deliver nerve growth factor (NGF, chemical cue). The properties of the scaffold were evaluated under physiological conditions using environmental scanning electron microscopy and mechanical testing. The resulting scaffolds have hydrated porosities of 35-55% and Young's modulus in the range of 0.43-2.86 MPa. Enzyme-linked immunosorbent assay showed that NGF is released from the microspheres for up to 4 weeks. Dorsal root ganglia (DRG) neurons showed that the released NGF is bioactive. DRG testing on the scaffolds also showed that the combination of NGF released from the microspheres and the aligned nanofibers significantly enhanced neurite outgrowth. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 17-25, 2018.

Positive and negative cues for modulating neurite dynamics and receptor expression.

Many current peripheral nerve repair strategies focus on delivering positive, growth promoting cues (e.g. extracellular matrix, ECM) while eliminating negative, growth inhibiting cues (e.g. chondroitin sulfate proteoglycans, CSPGs) at the injury site. We hypothesized that recapitulating the positive and negative cues of the peripheral nerve injury microenvironment would improve regeneration. First, we tested the effects of a characteristic CSPG, chondroitin sulfate A (CSA) on neurite dynamics of dissociated chick embryo dorsal root ganglion (DRG) neurons using time lapse video microscopy. DRG growth was recorded on different adhesive substrates, including a novel, porcine-derived spinal cord matrix (SCM). The SCM significantly increased frequency of neurite extension coordinated by a significant reduction in the neurites' time spent stalled. The SCM also mitigated inhibitory effects of CSA, producing longer neurites than the controls without CSA treatment. Next we aimed to elucidate receptors involved in mediating this behavior by testing the ability of CSA to upregulate cell-substrate binding receptors using flow cytometry. Our results showed a significant increase in syndecan-3 receptor expression in neurons treated with CSA. Furthermore, syndecans would most likely bind to the sulfated glycosaminoglycans measured in the SCM. Finally, we evaluated neurite growth on biomaterial scaffolds featuring CSA and SCM cues. Our results showed significantly increased neurite outgrowth on electrospun hyaluronic acid fibers with SCM and low levels of CSA. Higher incorporation of CSA maintained its inhibitory properties. Future work will evaluate coupling CSPGs with growth-permissive ECM to assess the combined effect on neurite outgrowth.

Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs.

Although there are many studies focusing on the molecular pathways underlying lung vascular morphogenesis, the extracellular matrix (ECM)-dependent regulation of mesenchymal cell differentiation in vascular smooth muscle development needs better understanding. In this study, we demonstrate that the paired related homeobox gene transcription factor Prx1 maintains the elastic ECM properties, which are essential for vascular smooth muscle precursor cell differentiation. We have found that Prx1(null) mouse lungs exhibit defective vascular smooth muscle development, downregulated elastic ECM expression, and compromised transforming growth factor (TGF)-ß localization and signaling. Further characterization of ECM properties using decellularized lung ECM scaffolds derived from Prx1 mice demonstrated that Prx1 is required to maintain lung ECM stiffness. The results of cell culture using stiffness-controlled 2-D and 3-D synthetic substrates confirmed that Prx1-dependent ECM stiffness is essential for promotion of smooth muscle precursor differentiation for effective TGF-ß stimulation. Supporting these results, both decellularized Prx1(null) lung ECM and Prx1(WT) (wild type) ECM scaffolds with blocked TGF-ß failed to support mesenchymal cell to 3-D smooth muscle cell differentiation. These results suggest a novel ECM-dependent regulatory pathway of lung vascular development wherein Prx1 regulates lung vascular smooth muscle precursor development by coordinating the ECM biophysical and biochemical properties.

Directed migration in neural tissue engineering.

Directed cell migration is particularly important in neural tissue engineering, where the goal is to direct neurons and support cells across injured nerve gaps. Investigation of the gradients present in the body during development provides an approach to guiding cells in peripheral and central nervous system tissue, but many different types of gradients and patterns can accomplish directed migration. The focus of this review is to describe current research paradigms in neural tissue gradients and review their effectiveness for directed migration. The review explores directed migration achieved through the use of chemical, adhesive, mechanical, topographical, and electrical types of gradients. Few studies investigate combined gradients, though it is known that a combination of therapies is necessary for reconnection of neuronal circuitry. To date, there has been no systematic review of gradient approaches to neural tissue engineering. By looking at effectiveness of various scaffold cue presentation and methods to combine these strategies, the potential for nerve repair is increased.

Electrospinning growth factor releasing microspheres into fibrous scaffolds.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

Jagged1 immobilization to an osteoconductive polymer activates the Notch signaling pathway and induces osteogenesis.

Treatment of nonunion fractures is a significant problem. Common therapeutics, including autologous bone grafts and bone morphogenetic proteins, show well-established limitations. Therefore, a need persists for the identification of novel clinical therapies to promote healing. The Notch signaling pathway regulates bone development. Clinically, loss-of-function mutations to the Notch ligand Jagged1 decrease bone mass and increase fracture risk. Jagged1 is also the most highly upregulated ligand during fracture repair, identifying it as a potential target to promote bone formation. Therefore, the objective of this study was to develop a clinically translatable construct comprised of Jagged1 and an osteoconductive scaffold, and characterize its activity in human mesenchymal stem cells (hMSC). We first evaluated the effects of Jagged1 directly immobilized to a novel poly(ß-amino ester) relative to indirect coupling via antibody. Direct was more effective at activating hMSC Notch target gene expression and osteogenic activity. We then found that directly immobilized Jagged1 constructs induced osteoblast differentiation. This is the first study to demonstrate that Jagged1 delivery transiently activates Notch signaling and increases osteogenesis. A positive correlation was found between Jagged1-induced Notch and osteogenic expression. Collectively, these results indicate that Jagged1 coupled to an osteogenic biomaterial could promote bone tissue formation during fracture healing.

Microfluidic generation of haptotactic gradients through 3D collagen gels for enhanced neurite growth.

We adapted a microfluidic system used previously to generate durotactic gradients of stiffness in a 3D collagen gel, to produce haptotactic gradients of adhesive ligands through the collagen gel. Oligopeptide sequences that included bioactive peptide sequences from laminin, YIGSR, or IKVAV, were grafted separately onto type I collagen using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Solutions of peptide-grafted collagen and untreated collagen were then used as source and sink input solutions, respectively, in an H-shaped microfluidic network fabricated using traditional soft lithography. One-dimensional gradients of the peptide-grafted collagen solution were generated in the channel that connected the source and sink channels, and these gradients became immobilized upon self-assembly of the collagen into a 3D fibrillar gel. The slope and average concentration of the gradients were adjusted by changing the concentration of the source solutions and by changing the length of the cross-channel. A separate, underlying channel in the microfluidic construct allowed the introduction of a chick embryo dorsal root ganglion into the network. Neurites from these explants grew significantly longer up steep gradients of YIGSR, but shallow gradients of IKVAV in comparison to untreated collagen controls. When these two gradients were presented in combination, the bias in growth acceleration was the largest and most consistent. No differences were observed in the number of neurites choosing to grow up or down the gradients in any condition. These results suggest that the incorporation of distinct gradients of multiple bioactive ligands can improve directional acceleration of regenerating axons.

Positively and negatively modulating cell adhesion to type I collagen via peptide grafting.

The biophysical interactions between cells and type I collagen are controlled by the level of cell adhesion, which is dictated primarily by the density of ligands on collagen and the density of integrin receptors on cells. The native adhesivity of collagen was modulated by covalently grafting glycine-arginine-glycine-aspartic acid-serine (GRGDS), which includes the bioactive RGD sequence, or glycine-arginine-aspartic acid-glycine-serine (GRDGS), which includes the scrambled RDG sequence, to collagen with the hetero-bifunctional coupling agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The peptide-grafted collagen self-assembled into a fibrillar gel with negligible changes in gel structure and rheology. Rat dermal fibroblasts (RDFs) and human smooth muscle cells demonstrated increased levels of adhesion on gels prepared from RGD-grafted collagen, and decreased levels of adhesion on RDG-grafted collagen. Both cell types demonstrated an increased ability to compact free-floating RGD-grafted collagen gels, and an impaired ability to compact RDG-grafted gels. RDF migration on and within collagen was increased with RDG-grafted collagen and decreased with RGD-grafted collagen, and dose-response experiments indicated a biphasic response of RDF migration to adhesion. Smooth muscle cells demonstrated similar, though not statistically significant, trends. The ability to both positively and negatively modulate cell adhesion to collagen increases the versatility of this natural biomaterial for regenerative therapies.

Electrospun fibrous scaffolds with multiscale and photopatterned porosity.

The structural and mechanical properties of tissue engineered environments are crucial for successful cellular growth and tissue repair. Electrospinning is gaining wide attention for the fabrication of tissue engineered scaffolds, but the small pore sizes of these scaffolds limit cell infiltration and construct vascularization. To address this problem, we have combined electrospinning with photopatterning to create multiscale porous scaffolds. This process retains the fibrous nature of the scaffolds and permits enhanced cellular infiltration and vascularization when compared to unpatterned scaffolds. This is the first time that photopatterning has been utilized with electrospun scaffolds and is only now possible with the electrospinning of reactive macromers.

Electrospinning fibrous polymer scaffolds for tissue engineering and cell culture.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

Genipin-induced changes in collagen gels: correlation of mechanical properties to fluorescence.

Controlled crosslinking of collagen gels has important applications in cell and tissue mechanics as well as tissue engineering. Genipin is a natural plant extract that has been shown to crosslink biological tissues and to produce color and fluorescence changes upon crosslinking. We have characterized the effects of genipin concentration and incubation duration on the mechanical and fluorigenic properties of type I collagen gels. Gels were exposed to genipin (0, 1, 5, or 10 mM) for a defined duration (2, 4, 6, or 12 h). Mechanical properties were characterized using parallel plate rheometry, while fluorigenic properties were examined with a spectrofluorimetric plate reader and with a standard, inverted epifluorescent microscope. Additionally, Fourier transform infrared spectroscopy was used to characterize and track the crosslinking reaction in real-time. Genipin produced significant concentration- and incubation-dependent increases in the storage modulus, loss modulus, and fluorescence intensity. Storage modulus was strongly correlated to fluorescence exponentially. Minimal cytotoxicity was observed for exposure of L929 fibroblasts cultured within collagen gels to 1 mM genipin for 24 h, but significant cell death occurred for 5 and 10 mM genipin. We conclude that genipin can be used to stiffen collagen gels in a relatively short time frame, that low concentrations of genipin can be used to crosslink cell-populated collagen gels to affect cell behavior that is influenced by the mechanical properties of the tissue scaffold, and that the degree of crosslinking can be reliably assayed optically via simple fluorescence measurements.