Photocurable extracellular matrix sealant for cessation of venous hemorrhage.

Hemorrhage is the second leading cause of death in patients under 46 years of age in the United States. Cessation of hemorrhage prevents hemorrhagic shock and tissue hypoxia. Controlling the bleed via direct pressure or tourniquet is often the first line of defense, but long-term care requires staples, hemostatic agents, or sealants that seal the vessel and restore blood flow. Here, we compare a new photocurable extracellular matrix sealant (pcECM) with low, medium, and high crosslink density formulations to a commercially available fibrin-based sealant, TISSEEL®. pcECM has potential uses in surgical and remote settings due to room temperature storage conditions and fast preparation time. Here, we determine if pcECM sealant can stop venous hemorrhage in a murine model, adhere to the wound site in vivo throughout the wound-healing process, and has the mechanical properties necessary for stopping hemorrhage. Adjusting pcECM crosslinking density significantly affected viscosity, swelling, burst strength, tensile strength, and elasticity of the sealant. 3-Dimensional ultrasound volume segmentations showed pcECM degrades to 17 ± 8% of its initial implant volume by day 28. Initially, local hemodynamic changes were observed, but returned close to baseline levels by day 28. Acute inflammation was observed near the puncture site in pcECM implanted mice, and we observed inflammatory markers at the 14-day explant for both sealants. pcECM and fibrin sealant successfully sealed the vessel in all cases, and consistently degraded over 14-28 days. pcECM is a durable sealant with tunable mechanical properties and possible uses in hemorrhage control and other surgical procedures.

Use of Polyvinyl Alcohol-Porcine Small Intestine Submucosa Stent in the Prevention of Anastomotic Leaks in the Porcine Colon.

INTRODUCTION: Gastrointestinal anastomoses are performed millions of times per year worldwide. The major complication they share is anastomotic leak. We describe the development and initial safety/efficacy of a novel luminal stent which aims to address this clinical issue. MATERIALS AND METHODS: The stent was created out of two materials, a polyvinyl alcohol core and outer layer of acellular porcine small intestine submucosa. Ten healthy pigs underwent laparotomy, a portion of the colon was transected, and the stent was placed within the colonic lumen at the site of resection. Pigs were sacrificed at the end of postoperative week 2, and postoperative week 4. A portion of the descending colon was resected, and tissue samples from the anastomosis, intentional defect scar, and normal bowel overlying the stent were sent for histopathologic examination. RESULTS: All ten animals survived the study. None developed any clinical signs of obstruction, infection, leakage, fistula, wound complications, or bleeding. No evidence of colonic leak or luminal stenosis/stricture was noted. CONCLUSIONS: The results of this study show that a polyvinyl alcohol/acellular porcine small intestine submucosa stent sewn underneath a colonic anastomosis with a 2 cm intentional defect will result in no anastomotic complications. There were also no complications from placing this stent in any pigs. Additional studies with a control group should be conducted to see if this same stent can be built in different diameters, lengths, and configurations to prevent leaks in other organs. These encouraging results will hopefully lead to decreased leaks and the need for temporary ostomies in humans.

Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis.

Cardiac fibrosis is a disease state characterized by excessive collagenous matrix accumulation within the myocardium that can lead to ventricular dilation and systolic failure. Current treatment options are severely lacking due in part to the poor understanding of the complexity of molecular pathways involved in cardiac fibrosis. To close this gap, in vitro model systems that recapitulate the defining features of the fibrotic cellular environment are in need. Type I collagen, a major cardiac extracellular matrix protein and the defining component of fibrotic depositions, is an attractive choice for a fibrosis model, but demonstrates poor mechanical strength due to solubility limits. However, plastic compression of collagen matrices is shown to significantly increase its mechanical properties. Here, confined compression of oligomeric, type I collagen matrices is utilized to resemble defining hallmarks seen in fibrotic tissue such as increased collagen content, fibril thickness, and bulk compressive modulus. Cardiomyocytes seeded on compressed matrices show a strong beating abrogation as observed in cardiac fibrosis. Gene expression analysis of selected fibrosis markers indicates fibrotic activation and cardiomyocyte maturation with regard to the existing literature. With these results, a promising first step toward a facile heart-on-chip model is presented to study cardiac fibrosis.

In Vivo Cellular Infiltration and Remodeling in a Decellularized Ovine Osteochondral Allograft.

Interest in decellularized tissues has steadily gained as potential solutions for degenerative diseases and traumatic events, replacing sites of missing tissue, and providing the relevant biochemistry and microstructure for tissue ingrowth and regeneration. Osteoarthritis, a progressive and debilitating disease, is often initiated with the formation of a focal defect in the otherwise smooth surface of articular cartilage. Decellularized cartilage tissue, which maintains the structural complexity of the native extracellular matrix, has the potential to provide a clinically relevant solution to focal defects or large tissue damage, possibly even circumventing or complementing current techniques such as microfracture and mosaicplasty. However, it is currently unclear whether implantation of decellularized cartilage in vivo may provide a mechanically and biochemically relevant platform to promote cell remodeling and repair. We examined whole decellularized osteochondral allografts implanted in the ovine trochlear groove to investigate cellular remodeling and repair tissue quality compared to empty defects and contralateral controls (healthy cartilage). At 3 months postsurgery, cells were observed in both the decellularized tissue and empty defects, although both at significantly lower levels than healthy cartilage. Qualitative and quantitative histological analysis demonstrated maintenance of cartilage features of the decellularized implant similar to healthy cartilage groups. Noninvasive analysis by quantitative magnetic resonance imaging showed no difference in T1ρ and T2* between all groups. Investigation of the mechanical properties of repair tissue showed significantly lower elasticity in decellularized implants and empty defects compared to healthy cartilage, but similar tribological quantities. Overall, this study suggests that decellularized cartilage implants are subject to cellular remodeling in an in vivo environment and may provide a potential tissue engineering solution to cartilage defect interventions.

Mechanisms and Microenvironment Investigation of Cellularized High Density Gradient Collagen Matrices via Densification.

Biological tissues and biomaterials are often defined by unique spatial gradients in physical properties that impart specialized function over hierarchical scales. The structure and organization of these materials forms continuous transitional gradients and discrete local microenvironments between adjacent (or within) tissues, and across matrix-cell boundaries, which can be difficult to replicate with common scaffold systems. Here, we studied the matrix densification of collagen leading to gradients in density, mechanical properties, and fibril morphology. High-density regions formed via a fluid pore pressure and flow-driven mechanism, with increased relative fibril density (10×), mechanical properties (20×, to 94.40±18.74kPa), and maximum fibril thickness (1.9×, to >1µm) compared to low-density regions, while maintaining porosity and fluid/mass transport to support viability of encapsulated cells. Similar to the organization of the articular cartilage zonal structure, we found that high-density collagen regions induced cell and nuclear alignment of primary chondrocytes. Chondrocyte gene expression was maintained in collagen matrices, and no phenotypic changes were observed as a result of densification. Densification of collagen matrices provides a unique, tunable platform for the creation of gradient systems to study complex cell-matrix interactions. These methods are easily generalized to compression and boundary condition modalities useful to mimic a broad range of tissues.

Acellular and cellular high-density, collagen-fibril constructs with suprafibrillar organization.

Collagen is used extensively for tissue engineering due to its prevalence in connective tissues and its role in defining tissue biophysical and biological signalling properties. However, traditional collagen-based materials fashioned from atelocollagen and telocollagen have lacked collagen densities, multi-scale organization, mechanical integrity, and proteolytic resistance found within tissues in vivo. Here, highly interconnected low-density matrices of D-banded fibrils were created from collagen oligomers, which exhibit fibrillar as well as suprafibrillar assembly. Confined compression then was applied to controllably reduce the interstitial fluid while maintaining fibril integrity. More specifically, low-density (3.5 mg mL(-1)) oligomer matrices were densified to create collagen-fibril constructs with average concentrations of 12.25 mg mL(-1) and 24.5 mg mL(-1). Control and densified constructs exhibited nearly linear increases in ultimate stress, Young's modulus, and compressive modulus over the ranges of 65 to 213 kPa, 400 to 1.26 MPa, and 20 to 150 kPa, respectively. Densification also increased construct resistance to collagenase degradability. Finally, this process was amenable to creating high-density cellularized tissues; all constructs maintained high cell viability (at least 97%) immediately following compression as well as after 1 day and 7 days of culture. This method, which integrates the suprafibrillar assembly capacity of oligomers and controlled fluid reduction by confined compression, supports the rational and scalable design of a broad range of collagen-fibril materials and cell-encapsulated tissue constructs for tissue engineering applications.

Dissociated and Reconstituted Cartilage Microparticles in Densified Collagen Induce Local hMSC Differentiation.

Decellularized cartilage microparticles, and all associated native signals, are delivered to hMSC populations in a dense, type I collagen matrix. Hybrid usage of native tissue signals and the engineering control of collagen matrices show the ability to induce local infiltration and differentiation of hMSCs. Additionally, the solid cartilage microparticles inhibit bulk cell-mediated contraction of the composite.

Cell encapsulation in a magnetically aligned collagen-GAG copolymer microenvironment.

Engineered tissue microenvironments impart specialized cues that drive distinct cellular phenotypes and function. Microenvironments with defined properties, such as mechanical properties and fibril alignment, can elicit specific cellular responses that emulate those observed in vivo. Collagen- and glycosaminoglycan (GAG)-based tissue matrices have been popularized due to their biological ubiquity in a broad range of tissues and the ability to tune structure and mechanical properties through a variety of processes. Here, we investigate the combined effects of static magnetic fields, and GAG and cell encapsulation, on the structure (e.g. collagen fibril orientation) and material properties of collagen matrices. We found that magnetic fields align the collagen-GAG matrix, alter equilibrium mechanical properties and provide a method for encapsulating cells within a three-dimensional aligned matrix. Cells are encapsulated prior to polymerization, allowing for controlled cell density and eliminating the need for cell seeding. Increased relative GAG concentrations reduced the ability to magnetically align collagen fibrils, in part through a mechanism involving increased viscosity and polymerization time of the collagen-GAG solution. This work provides a functional design space for the development of pure collagen and hybrid collagen-GAG matrices in the presence of magnetic fields. Additionally, this work shows that magnetic fields are effective for the fabrication of collagen constructs with controlled fibril orientation, and can be coupled with GAG incorporation to modulate mechanical properties and the response of embedded cells.

Prestress as an optimal biomechanical parameter for needle penetration.

Drug delivery requires precise intradermal and subcutaneous injections of formulations to clinically relevant penetration depths. However, penetration depth is confounded by skin deflection, which occurs prior to and during penetration as the skin surface deforms axially with the needle, and which varies profoundly due to differing intrinsic mechanical (e.g. viscoelastic) tissue properties, disease state, aging, and ethnicity. Herein, an ex vivo model was utilized to study factors that affect skin deflection and the efficacy of injection, including prestress applied at the tissue surface, needle gauge, velocity, and actuation depth. The application of prestress minimized skin deflection during needle penetration and allowed for needle actuation to the targeted penetration depths with minimum variability. The force required to achieve target penetration depths was found to increase with prestress and decrease with needle gauge. Our findings emphasize the need for prestress applied to the skin surface to minimize variation in skin properties and administer formulations for intradermal and subcutaneous treatments with maximum precision.