Christopher J. Medberry

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.

Biologic scaffolds composed of central nervous system extracellular matrix

Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.

The promotion of a constructive macrophage phenotype by solubilized extracellular matrix

The regenerative healing response of injured skeletal muscle is dependent upon a heterogeneous population of responding macrophages, which show a phenotypic transition from the pro-inflammatory M1 to the alternatively activated and constructive M2 phenotype. Biologic scaffolds derived from mammalian extracellular matrix (ECM) have been used for the repair and reconstruction of a variety of tissues, including skeletal muscle, and have been associated with an M2 phenotype and a constructive and functional tissue response. The mechanism(s) behind in-vivo macrophage phenotype transition in skeletal muscle and the enhanced M2:M1 ratio associated with ECM bioscaffold use in-vivo are only partially understood. The present study shows that degradation products from ECM bioscaffolds promote alternatively activated and constructive M2 macrophage polarization in-vitro, which in turn facilitates migration and myogenesis of skeletal muscle progenitor cells.

The Effect of Source Animal Age Upon Extracellular Matrix Scaffold Properties

Biologic scaffold materials composed of mammalian extracellular matrix (ECM) are commonly used for the repair and reconstruction of injured tissues. An important, but unexplored variable of biologic scaffolds is the age of the animal from which the ECM is prepared. The objective of the present study was to compare the structural, mechanical, and compositional properties of small intestinal submucosa (SIS)-ECM harvested from pigs that differed only in age. Degradation product bioactivity of these ECM materials was also examined. Results showed that there are distinct differences in each of these variables among the various age source ECM scaffolds. The strength and growth factors content of ECM from 3-week-old animals is less than that of ECM harvested from 12, 26 or >52-week-old animals. The elastic modulus of SIS-ECM for 3 week and >52-week-old source was less than that of the 12 and 26 week source. Degradation products from all age source ECMs were chemotactic for perivascular stem cells, with the 12 week source the most potent, while the oldest source caused the greatest increase in proliferation. In summary, distinct differences exist in the mechanical, structural, and biologic properties of SIS-ECM harvested from different aged animals.

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.

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity

UNLABELLED Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8mg/mL). Only ECM concentrations >3mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution. STATEMENT OF SIGNIFICANCE Extracellular matrix (ECM) hydrogel promotes constructive tissue remodeling in many tissues. Minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form that exists in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. We here report the rheological characterization of an injectable ECM hydrogel and its concentration-dependent delivery into a lesion cavity formed after a stroke based on MRI-guidance. The concentration of ECM determined its retention within the cavity or permeation into tissue and hence influenced its interaction with the host brain. This study demonstrates the importance of understanding the structure-function relationship of biomaterials to guide particular clinical applications.

ECM hydrogel for the treatment of stroke: Characterization of the host cell infiltrate.

Brain tissue loss following stroke is irreversible with current treatment modalities. The use of an acellular extracellular matrix (ECM), formulated to produce a hydrogel in situ within the cavity formed by a stroke, was investigated as a method to replace necrotic debris and promote the infiltration of host brain cells. Based on magnetic resonance imaging measurements of lesion location and volume, different concentrations of ECM (0, 1, 2, 3, 4, 8 mg/mL) were injected at a volume equal to that of the cavity (14 days post-stroke). Retention of ECM within the cavity occurred at concentrations >3 mg/mL. A significant cell infiltration into the ECM material in the lesion cavity occurred with an average of ∼36,000 cells in the 8 mg/mL concentration within 24 h. An infiltration of cells with distances of >1500 μm into the ECM hydrogel was observed, but the majority of cells were at the tissue/hydrogel boundary. Cells were typically of a microglia, macrophage, or neural and oligodendrocyte progenitor phenotype. At the 8 mg/mL concentration, ∼60% of infiltrating cells were brain-derived phenotypes and 30% being infiltrating peripheral macrophages, polarizing toward an M2-like anti-inflammatory phenotype. These results suggest that an 8 mg/mL ECM concentration promotes a significant acute endogenous repair response that could potentially be exploited to treat stroke.

In vivo degradation of 14C-labeled porcine dermis biologic scaffold.

Biologic scaffold materials are used for repair and reconstruction of injured or missing tissues. Such materials are often composed of allogeneic or xenogeneic extracellular matrix (ECM) manufactured by decellularization of source tissue, such as dermis. Dermal ECM (D-ECM) has been observed to degrade and remodel in vivo more slowly than other biologic scaffold materials, such as small intestinal submucosa (SIS-ECM). Histologic examination is a common method for evaluating material degradation, but it lacks sensitivity and is subject to observer bias. Utilization of (14)C-proline labeled ECM is a quantitative alternative for measuring degradation of ECM scaffolds. Using both methods, the amount of degradation of D-ECM and SIS-ECM was determined at 2, 4, and 24 weeks post-implantation in a rodent model. Results utilizing (14)C liquid scintillation counting (LSC) analysis showed distinct differences in degradation at the three time points. D-ECM material in situ stayed the same at 76% remaining from 2 to 4 weeks post-implantation, and then decreased to 44% remaining at 24 weeks. In the same time period, implanted SIS-ECM material decreased from 72% to 13% to 0%. Visual examination of device degradation by histology overestimated degradation at 2 weeks and underestimated device degradation at 24 weeks, compared to the (14)C method.

Injectable Extracellular Matrix Hydrogels as Scaffolds for Spinal Cord Injury Repair.

Restoration of lost neuronal function after spinal cord injury (SCI) still remains a big challenge for current medicine. One important repair strategy is bridging the SCI lesion with a supportive and stimulatory milieu that would enable axonal rewiring. Injectable extracellular matrix (ECM)-derived hydrogels have been recently reported to have neurotrophic potential in vitro. In this study, we evaluated the presumed neuroregenerative properties of ECM hydrogels in vivo in the acute model of SCI. ECM hydrogels were prepared by decellularization of porcine spinal cord (SC) or porcine urinary bladder (UB), and injected into a spinal cord hemisection cavity. Histological analysis and real-time qPCR were performed at 2, 4, and 8 weeks postinjection. Both types of hydrogels integrated into the lesion and stimulated neovascularization and axonal ingrowth into the lesion. On the other hand, massive infiltration of macrophages into the lesion and rapid hydrogel degradation did not prevent cyst formation, which progressively developed over 8 weeks. No significant differences were found between SC-ECM and UB-ECM. Gene expression analysis revealed significant downregulation of genes related to immune response and inflammation in both hydrogel types at 2 weeks post SCI. A combination of human mesenchymal stem cells with SC-ECM did not further promote ingrowth of axons and blood vessels into the lesion, when compared with the SC-ECM hydrogel alone. In conclusion, both ECM hydrogels bridged the lesion cavity, modulated the innate immune response, and provided the benefit of a stimulatory substrate for in vivo neural tissue regeneration. However, fast hydrogel degradation might be a limiting factor for the use of native ECM hydrogels in the treatment of acute SCI.