Imaging the Cardiac Extracellular Matrix.

Cardiovascular disease is the global leading cause of death. One route to address this problem is using biomedical imaging to measure the molecules and structures that surround cardiac cells. This cellular microenvironment, known as the cardiac extracellular matrix, changes in composition and organization during most cardiac diseases and in response to many cardiac treatments. Measuring these changes with biomedical imaging can aid in understanding, diagnosing, and treating heart disease. This chapter supports those efforts by reviewing representative methods for imaging the cardiac extracellular matrix. It first describes the major biological targets of ECM imaging, including the primary imaging target of fibrillar collagen. Then it discusses the imaging methods, describing their current capabilities and limitations. It categorizes the imaging methods into two main categories: organ-scale noninvasive methods and cellular-scale invasive methods. Noninvasive methods can be used on patients, but only a few are clinically available, and others require further development to be used in the clinic. Invasive methods are the most established and can measure a variety of properties, but they cannot be used on live patients. Finally, the chapter concludes with a perspective on future directions and applications of biomedical imaging technologies.

A Mineralized Collagen-Polycaprolactone Composite Promotes Healing of a Porcine Mandibular Defect.

A tissue engineering approach to address craniofacial defects requires a biomaterial that balances macro-scale mechanical stiffness and strength with the micron-scale features that promote cell expansion and tissue biosynthesis. Such criteria are often in opposition, leading to suboptimal mechanical competence or bioactivity. We report the use of a multiscale composite biomaterial that integrates a polycaprolactone (PCL) reinforcement structure with a mineralized collagen-glycosaminoglycan scaffold to circumvent conventional tradeoffs between mechanics and bioactivity. The composite promotes activation of the canonical bone morphogenetic protein 2 (BMP-2) pathway and subsequent mineralization of adipose-derived stem cells in the absence of supplemental BMP-2 or osteogenic media. We subsequently examined new bone infill in the acellular composite, scaffold alone, or PCL support in 10 mm dia. ramus mandibular defects in Yorkshire pigs. We report an analytical approach to quantify radial, angular, and depth bone infill from micro-computed tomography data. The collagen-PCL composite showed improved overall infill, and significantly increased radial and angular bone infill versus the PCL cage alone. Bone infill was further enhanced in the composite for defects that penetrated the medullary cavity, suggesting recruitment of marrow-derived cells. These results indicate a multiscale mineralized collagen-PCL composite offers strategic advantages for regenerative repair of craniofacial bone defects.

Incorporation of the Amniotic Membrane as an Immunomodulatory Design Element in Collagen Scaffolds for Tendon Repair.

Tendon injuries often require surgical intervention and even then result in poor outcomes due to scar formation and repeated failure. Biomaterial implants offer the potential to address multiple underlying concerns preventing improved tendon repair. Here, we describe modifications to the composition of an anisotropic collagen-glycosaminoglycan (CG) scaffold biomaterial, incorporating amniotic membrane (AM)-derived matrix to alter the inflammatory response and establish conditions for improved regenerative repair. We explored two methods of AM matrix incorporation to address multiple concerns associated with tendon repair. Amniotic membrane-derived matrix was incorporated directly into the scaffold microstructure during fabrication to form a C/AM composite. Alternatively, decellularized amniotic matrix was wrapped around the traditional collagen-chondroitin sulfate (C/CS) scaffold to form a core-shell composite (C/CS plus AM wrap) in a manner similar to current collagen membrane wraps used in rotator cuff and Achilles tendon surgeries to improve the mechanical strength of the repair. Human mesenchymal stem cells (MSCs) cultured within these materials were evaluated for metabolic health and immunomodulatory gene expression in response to inflammatory media challenge of interleukin 1 ß and tumor necrosis factor α. The scaffolds were able to maintain MSC metabolic activity in all media conditions over the course of a 7 day culture. Expression of genes encoding for pro-inflammatory cytokines were down-regulated in AM containing scaffolds, suggesting the potential to employ AM-modified CG scaffolds for tendon-repair applications.

Naturally derived biomaterials for addressing inflammation in tissue regeneration.

Tissue regeneration strategies have traditionally relied on designing biomaterials that closely mimic features of the native extracellular matrix (ECM) as a means to potentially promote site-specific cellular behaviors. However, inflammation, while a necessary component of wound healing, can alter processes associated with successful tissue regeneration following an initial injury. These processes can be further magnified by the implantation of a biomaterial within the wound site. In addition to designing biomaterials to satisfy biocompatibility concerns as well as to replicate elements of the composition, structure, and mechanics of native tissue, we propose that ECM analogs should also include features that modulate the inflammatory response. Indeed, strategies that enhance, reduce, or even change the temporal phenotype of inflammatory processes have unique potential as future pro-regenerative analogs. Here, we review derivatives of three natural materials with intrinsic anti-inflammatory properties and discuss their potential to address the challenges of inflammation in tissue engineering and chronic wounds.

Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds.

Adult tendon wound repair is characterized by the formation of disorganized collagen matrix which leads to decreases in mechanical properties and scar formation. Studies have linked this scar formation to the inflammatory phase of wound healing. Instructive biomaterials designed for tendon regeneration are often designed to provide both structural and cellular support. In order to facilitate regeneration, success may be found by tempering the body's inflammatory response. This work combines collagen-glycosaminoglycan scaffolds, previously developed for tissue regeneration, with matrix materials (hyaluronic acid and amniotic membrane) that have been shown to promote healing and decreased scar formation in skin studies. The results presented show that scaffolds containing amniotic membrane matrix have significantly increased mechanical properties and that tendon cells within these scaffolds have increased metabolic activity even when the media is supplemented with the pro-inflammatory cytokine interleukin-1 beta. Collagen scaffolds containing hyaluronic acid or amniotic membrane also temper the expression of genes associated with the inflammatory response in normal tendon healing (TNF-α, COLI, MMP-3). These results suggest that alterations to scaffold composition, to include matrix known to decrease scar formation in vivo, can modify the inflammatory response in tenocytes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1332-1342, 2016.

The effect of glycosaminoglycan content on polyethylenimine-based gene delivery within three-dimensional collagen-GAG scaffolds.

The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.

The use of bioinspired alterations in the glycosaminoglycan content of collagen-GAG scaffolds to regulate cell activity.

The design of biomaterials for regenerative medicine can require biomolecular cues such as growth factors to induce a desired cell activity. Signal molecules are often incorporated into the biomaterial in either freely-diffusible or covalently-bound forms. However, biomolecular environments in vivo are often complex and dynamic. Notably, glycosaminoglycans (GAGs), linear polysaccharides found in the extracellular matrix, are involved in transient sequestration of growth factors via charge interactions. Biomaterials mimicking this phenomenon may offer the potential to amplify local biomolecular signals, both endogenously produced and exogenously added. GAGs of increasing sulfation (hyaluronic acid, chondroitin sulfate, heparin) were incorporated into a collagen-GAG (CG) scaffold under development for tendon tissue engineering. Manipulating the degree of GAG sulfation significantly impacts sequestration of growth factors from the media. Increasing GAG sulfation improved equine tenocyte metabolic activity in normal serum (10% FBS), low serum (1% FBS), and IGF-1 supplemented media conditions. Notably, previously reported dose-dependent changes in tenocyte bioactivity to soluble IGF-1 within the CG scaffold were replicated by using a single dose of soluble IGF-1 in scaffolds containing increasingly sulfated GAGs. Collectively, these results suggest that CG scaffold GAG content can be systematically manipulated to regulate the sequestration and resultant enhanced bioactivity of growth factor signals on cell behavior within the matrix.