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.

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.

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 freefloating 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.

The purpose of this study was to assess the biological revitalization and mechanical integrity of Strattice™ Reconstructive Tissue Matrix, a porcine-derived acellular dermal matrix, in vivo over time. We expanded the traditional subcutaneous model to incorporate biologic matrix scaffolds large enough to allow evaluation of mechanical properties in addition to the assessment of histological changes. Hematoxylin and eosin histology staining was used to evaluate cellular and tissue changes, and a mechanical testing frame was used to measure the ultimate tensile stress and Young’s modulus of the implanted material over time. Cell infiltration and blood vessel formation into the porcine-derived acellular dermal matrix were evident at 2 weeks and increased with implantation time. Mechanical remodeling resulted in an initial decrease in ultimate tensile stress, not associated with cell infiltration, followed by a significant increase in material strength, concurrent with histological evidence of new collagen synthesis. Young’s modulus followed a similar trend.

We compared fascial wounds repaired with non-cross-linked intact porcine-derived acellular dermal matrix versus primary closure in a large-animal hernia model. Incisional hernias were created in Yucatan pigs and repaired after 3 weeks via open technique with suture-only primary closure or intraperitoneally placed porcine-derived acellular dermal matrix. Progressive changes in mechanical and biological properties of porcine-derived acellular dermal matrix and repair sites were assessed. Porcine-derived acellular dermal matrix–repaired hernias of additional animals were evaluated 2 and 4 weeks post incision to assess porcine-derived acellular dermal matrix regenerative potential and biomechanical changes. Hernias repaired with primary closure showed substantially more scarring and bone hyperplasia along the incision line. Mechanical remodeling of porcine-derived acellular dermal matrix was noted over time. Porcine-derived acellular dermal matrix elastic modulus and ultimate tensile stress were similar to fascia at 6 weeks. The biology of porcine-derived acellular dermal matrix–reinforced animals was more similar to native abdominal wall versus that with primary closure. In this study, porcine-derived acellular dermal matrix–reinforced repairs provided more complete wound healing response compared with primary closure.

Recent studies have shown that the anterior ectosylvian sulcal cortex (AESc) and the rostral suprasylvian sulcal cortex (RSSSc) of the cat play integral roles in behavioral and collicular responses to multisensory stimuli. However, substantially more multisensory superior colliculus (SC) neurons are affected by blockade of the AESc than the RSSSc. Although both cortical regions project directly to the SC, a possible explanation for this differential effect is that the AESc may also relay an indirect corticotectal signal via the RSSSc that is reduced when the AESc is deactivated. This possibility was examined by placing orthograde tracer in the auditory field AES (FAES), visual AEV, or between these two regions of the AESc. FAES injections produced labeled boutons in the posterior-lateral bank of the RSSSc, while those placed in AEV failed to label the RSSSc. However, injections between the FAES and AEV regions revealed terminal label in both the posterior lateral bank and fundus. These observations and other studies showing connections between somatosensory portions of the AESc and RSSSc are consistent with the hypothesis that signals from the AESc can take both direct and indirect (through the RSSSc) corticotectal routes to influence processing in the SC.

The use of human tissue-derived autografts and allografts continues to be the gold standard in anterior cruciate ligament (ACL) repair. However, autografts and allografts have their own set of associated risks. Many alternative options, including synthetic replacements, have failed to demonstrate long-term success. In this study, sterile acellular porcine bone-tendon-bone (BTB) xenografts were created using a proprietary process and tested against BTB autografts in goats for 13 and 52 weeks. At 13 weeks, all xenograft-implanted animals ( n = 9) had subjective hind leg motor function (HLMF) that was categorized as either normal (score = 0) or a slight limp (score = 1) compared with two of nine autograft-implanted animals having a moderate limp (score = 2). At 39 weeks, there was HLMF improvement with each autograft-implanted and xenograft-implanted animal having normal HLMF or only a slight limp. At 13 weeks, six of nine animals in each group achieved normal anterior drawer scores, which increased to nine of nine animals in each group by 39 weeks. Both autografts and xenografts exhibited minimal inflammation with excellent integration of the fibrous tendon portion of the graft to host bone, as evidenced histologically by Sharpey’s fiber formation. At 52 weeks, maximum mechanical load at failure for xenografts was 1092.0 ± 586.4 N compared with 1037.0 ± 422.6 N for autografts. These results demonstrate that a sterile acellular porcine BTB xenograft can perform equivalently to BTB autograft in a caprine model of ACL repair.

During development, neurites are directed by gradients of attractive and repulsive soluble (chemotactic) cues and substrate-bound adhesive (haptotactic) cues. Many of these cues have been extensively researched in vitro, and incorporated into strategies for nerve and spinal cord regeneration, primarily to improve the regenerative environment. To enhance and direct growth, we have developed a system to create 1D gradients of adhesion through a 3D collagen gel using microfluidics. We test our system using collagen grafted with bioactive peptide sequences, IKVAV and YIGSR, from laminin — an extra-cellular matrix (ECM) protein known to strongly influence neurite outgrowth. Gradients are established from ∼0.37mg peptide/mg collagen – 0, and ∼0.18 mg peptide/mg collagen – 0 of each peptide and tested using chick dorsal root ganglia (DRG). Neurite growth is evaluated 5 days after gradient formation. Neurites show increased growth in the gradient system when compared to control and biased growth up the gradient of peptides. Growth in YIGSR-grafted collagen increased with steeper gradients, whereas growth in IKVAV-grafted collagen decreased with steeper gradients. These results demonstrate that neurite growth can be enhanced and directed by controlled, immobilized, haptotactic gradients through 3D scaffolds, and suggest that including these gradients in regenerative therapies may accelerate nerve and spinal cord regeneration.