Christopher A. Carruthers

A hydrogel derived from decellularized dermal extracellular matrix.

The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in vitro and in vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior.

Hydrogels derived from central nervous system extracellular matrix

Biologic scaffolds composed of extracellular matrix (ECM) are commonly used repair devices in preclinical and clinical settings; however the use of these scaffolds for peripheral and central nervous system (CNS) repair has been limited. Biologic scaffolds developed from brain and spinal cord tissue have recently been described, yet the conformation of the harvested ECM limits therapeutic utility. An injectable CNS-ECM derived hydrogel capable of in vivo polymerization and conformation to irregular lesion geometries may aid in tissue reconstruction efforts following complex neurologic trauma. The objectives of the present study were to develop hydrogel forms of brain and spinal cord ECM and compare the resulting biochemical composition, mechanical properties, and neurotrophic potential of a brain derived cell line to a non-CNS-ECM hydrogel, urinary bladder matrix. Results showed distinct differences between compositions of brain ECM, spinal cord ECM, and urinary bladder matrix. The rheologic modulus of spinal cord ECM hydrogel was greater than that of brain ECM and urinary bladder matrix. All ECMs increased the number of cells expressing neurites, but only brain ECM increased neurite length, suggesting a possible tissue-specific effect. All hydrogels promoted three-dimensional uni- or bi-polar neurite outgrowth following 7 days in culture. These results suggest that CNS-ECM hydrogels may provide supportive scaffolding to promote in vivo axonal repair.

The effect of detergents on the basement membrane complex of a biologic scaffold material.

The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin β-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold.

ECM hydrogel coating mitigates the chronic inflammatory response to polypropylene mesh.

Polypropylene has been used as a surgical mesh material for several decades. This non-degradable synthetic polymer provides mechanical strength, a predictable host response, and its use has resulted in reduced recurrence rates for ventral hernia and pelvic organ prolapse. However, polypropylene and similar synthetic materials are associated with a chronic local tissue inflammatory response and dense fibrous tissue deposition. These outcomes have prompted variations in mesh design to minimize the surface area interface and increase integration with host tissue. In contrast, biologic scaffold materials composed of extracellular matrix (ECM) are rapidly degraded in-vivo and are associated with constructive tissue remodeling and minimal fibrosis. The objective of the present study was to assess the effects of an ECM hydrogel coating on the long-term host tissue response to polypropylene mesh in a rodent model of abdominal muscle injury. At 14 days post implantation, the ECM coated polypropylene mesh devices showed a decreased inflammatory response as characterized by the number and distribution of M1 macrophages (CD86+/CD68+) around mesh fibers when compared to the uncoated mesh devices. At 180 days the ECM coated polypropylene showed decreased density of collagen and amount of mature type I collagen deposited between mesh fibers when compared to the uncoated mesh devices. This study confirms and extends previous findings that an ECM coating mitigates the chronic inflammatory response and associated scar tissue deposition characteristic of polypropylene.

Polypropylene Surgical Mesh Coated with Extracellular Matrix Mitigates the Host Foreign Body Response

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Preparation and characterization of a biologic scaffold from esophageal mucosa

Biologic scaffolds composed of extracellular matrix (ECM) are commonly used to facilitate a constructive remodeling response in several types of tissue, including the esophagus. Surgical manipulation of the esophagus is often complicated by stricture, but preclinical and clinical studies have shown that the use of an ECM scaffold can mitigate stricture and promote a constructive outcome after resection of full circumference esophageal mucosa. Recognizing the potential benefits of ECM derived from homologous tissue (i.e., site-specific ECM), the objective of the present study was to prepare, characterize, and assess the in-vivo remodeling properties of ECM from porcine esophageal mucosa. The developed protocol for esophageal ECM preparation is compliant with previously established criteria of decellularization and results in a scaffold that maintains important biologic components and an ultrastructure consistent with a basement membrane complex. Perivascular stem cells remained viable when seeded upon the esophageal ECM scaffold in-vitro, and the in-vivo host response showed a pattern of constructive remodeling when implanted in soft tissue.

From single fiber to macro-level mechanics: A structural finite-element model for elastomeric fibrous biomaterials.

In the present work, we demonstrate that the mesoscopic in-plane mechanical behavior of membrane elastomeric scaffolds can be simulated by replication of actual quantified fibrous geometries. Elastomeric electrospun polyurethane (ES-PEUU) scaffolds, with and without particulate inclusions, were utilized. Simulations were developed from experimentally-derived fiber network geometries, based on a range of scaffold isotropic and anisotropic behaviors. These were chosen to evaluate the effects on macro-mechanics based on measurable geometric parameters such as fiber intersections, connectivity, orientation, and diameter. Simulations were conducted with only the fiber material model parameters adjusted to match the macro-level mechanical test data. Fiber model validation was performed at the microscopic level by individual fiber mechanical tests using AFM. Results demonstrated very good agreement to the experimental data, and revealed the formation of extended preferential fiber orientations spanning the entire model space. We speculate that these emergent structures may be responsible for the tissue-like macroscale behaviors observed in electrospun scaffolds. To conclude, the modeling approach has implications for (1) gaining insight on the intricate relationship between fabrication variables, structure, and mechanics to manufacture more functional devices/materials, (2) elucidating the effects of cell or particulate inclusions on global construct mechanics, and (3) fabricating better performing tissue surrogates that could recapitulate native tissue mechanics.

On the biomechanical role of glycosaminoglycans in the aortic heart valve leaflet

While the role of collagen and elastin fibrous components in heart valve valvular biomechanics has been extensively investigated, the biomechanical role of the glycosaminoglycan (GAG) gelatinous-like material phase remains unclear. In the present study, we investigated the biomechanical role of GAGs in porcine aortic valve (AV) leaflets under tension utilizing enzymatic removal. Tissue specimens were removed from the belly region of porcine AVs and subsequently treated with either an enzyme solution for GAG removal or a control (buffer with no enzyme) solution. A dual stress level test methodology was used to determine the effects at low and high (physiological) stress levels. In addition, planar biaxial tests were conducted both on-axis (i.e. aligned to the circumferential and radial axes) and at 45° off-axis to induce maximum shear, to explore the effects of augmented fiber rotations on the fiber-fiber interactions. Changes in hysteresis were used as the primary metric of GAG functional assessment. A simulation of the low-force experimental setup was also conducted to clarify the internal stress system and provide viscoelastic model parameters for this loading range. Results indicated that under planar tension the removal of GAGs had no measureable affect extensional mechanical properties (either on- or 45° off-axis), including peak stretch, hysteresis and creep. Interestingly, in the low-force range, hysteresis was markedly reduced, from 35.96±2.65% in control group to 25.00±1.64% (p<0.001) as a result of GAG removal. Collectively, these results suggest that GAGs do not play a direct role in modulating the time-dependent tensile properties of valvular tissues. Rather, they appear to be strongly connected with fiber-fiber and fiber-matrix interactions at low force levels. Thus, we speculate that GAGs may be important in providing a damping mechanism to reduce leaflet flutter when the leaflet is not under high tensile stress.

Quantification and simulation of layer-specific mitral valve interstitial cells deformation under physiological loading.

Within each of the four layers of mitral valve (MV) leaflet tissues there resides a heterogeneous population of interstitial cells that maintain the structural integrity of the MV tissue via protein biosynthesis and enzymatic degradation. There is increasing evidence that tissue stress-induced MV interstitial cell (MVIC) deformations can have deleterious effects on their biosynthetic states that are potentially related to the reduction of tissue-level maintenance and to subsequent organ-level failure. To better understand the interrelationships between tissue-level loading and cellular responses, we developed the following integrated experimental-computational approach. Since in vivo cellular deformations are not directly measurable, we quantified the in-situ layer-specific MVIC deformations for each of the four layers under a controlled biaxial tension loading device coupled to multi-photon microscopy. Next, we explored the interrelationship between the MVIC stiffness and deformation to layer-specific tissue mechanical and structural properties using a macro-micro finite element computational model. Experimental results indicated that the MVICs in the fibrosa and ventricularis layers deformed significantly more than those in the atrialis and spongiosa layers, reaching a nucleus aspect ratio of 3.3 under an estimated maximum physiological tension of 150N/m. The simulated MVIC moduli for the four layers were found to be all within a narrow range of 4.71-5.35kPa, suggesting that MVIC deformation is primarily controlled by each tissue layers respective structure and mechanical behavior rather than the intrinsic MVIC stiffness. This novel result further suggests that while the MVICs may be phenotypically and biomechanically similar throughout the leaflet, they experience layer-specific mechanical stimulatory inputs due to distinct extracellular matrix architecture and mechanical behaviors of the four MV leaflet tissue layers. This also suggests that MVICs may behave in a layer-specific manner in response to mechanical stimuli in both normal and surgically modified MVs.

The effect of terminal sterilization on the material properties and in vivo remodeling of a porcine dermal biologic scaffold.

UNLABELLED Biologic scaffolds composed of extracellular matrix are commonly used in a variety of surgical procedures. The Food and Drug Administration typically regulates biologic scaffolds as medical devices, thus requiring terminal sterilization prior to clinical use. However, to date, no consensus exists for the most effective yet minimally destructive sterilization protocol for biologic scaffold materials. The objective of the present study was to characterize the effect of ethylene oxide, gamma irradiation and electron beam (e-beam) irradiation on the material properties and the elicited in vivo remodeling response of a porcine dermal biologic scaffold. Outcome measures included biochemical, structural, and mechanical properties as well as cytocompatibility in vitro. In vivo evaluation utilized a rodent model to examine the host response to the materials following 7, 14, and 35 days. The host response to each experimental group was determined by quantitative histologic methods and by immunolabeling for macrophage polarization (M1/M2). In vitro results show that increasing irradiation dosage resulted in a dose dependent decrease in mechanical properties compared to untreated controls. Ethylene oxide-treated porcine dermal ECM resulted in decreased DNA content, extractable total protein, and bFGF content compared to untreated controls. All ETO treated, gamma irradiated, and e-beam irradiated samples had similar cytocompatibility scores in vitro. However, in vivo results showed that increasing dosages of e-beam and gamma irradiation elicited an increased rate of degradation of the biologic scaffold material following 35 days. STATEMENT OF SIGNIFICANCE The FDA typically regulates biologic scaffolds derived from mammalian tissues as medical devices, thus requiring terminal sterilization prior to clinical use. However, there is little data and no consensus for the most effective yet minimally destructive sterilization protocol for such materials. The present study characterized the effect of common sterilization methods: ethylene oxide, gamma irradiation and electron beam irradiation on the material properties and the elicited in vivo remodeling response of a porcine dermal biologic scaffold. The results of the study will aid in the meaningful selection of sterilization methods for biologic scaffold materials.