Peter M. Crapo

An overview of tissue and whole organ decellularization processes.

Biologic scaffold materials composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. Physical methods and chemical and biologic agents are used in combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making educated decisions regarding the agents and techniques utilized during processing. An overview of decellularization methods, their effect upon resulting ECM structure and composition, and recently described perfusion techniques for whole organ decellularization techniques are presented herein.

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.

Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by 19F- and diffusion-MRI

Transplantation of human neural stem cells (hNSCs) is emerging as a viable treatment for stroke related brain injury. However, intraparenchymal grafts do not regenerate lost tissue, but rather integrate into the host parenchyma without significantly affecting the lesion cavity. Providing a structural support for the delivered cells appears important for cell based therapeutic approaches. The non-invasive monitoring of therapeutic methods would provide valuable information regarding therapeutic strategies but remains a challenge. Labeling transplanted cells with metal-based (1)H-magnetic resonance imaging (MRI) contrast agents affects the visualization of the lesion cavity. Herein, we demonstrate that a (19)F-MRI contrast agent can adequately monitor the distribution of transplanted cells, whilst allowing an evaluation of the lesion cavity and the formation of new tissue on (1)H-MRI scans. Twenty percent of cells labeled with the (19)F agent were of host origin, potentially reflecting the re-uptake of label from dead transplanted cells. Both T(2)- and diffusion-weighted MRI scans indicated that transplantation of hNSCs suspended in a gel form of a xenogeneic extracellular matrix (ECM) bioscaffold resulted in uniformly distributed cells throughout the lesion cavity. However, diffusion MRI indicated that the injected materials did not yet establish diffusion barriers (i.e. cellular network, fiber tracts) normally found within striatal tissue. The ECM bioscaffold therefore provides an important support to hNSCs for the creation of de novo tissue and multi-nuclei MRI represents an adept method for the visualization of some aspects of this process. However, significant developments of both the transplantation paradigm, as well as regenerative imaging, are required to successfully create new tissue in the lesion cavity and to monitor this process non-invasively.

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.

Physiologic Compliance in Engineered Small-diameter Arterial Constructs Based on an Elastomeric Substrate

Compliance mismatch is a significant challenge to long-term patency in small-diameter bypass grafts because it causes intimal hyperplasia and ultimately graft occlusion. Current engineered grafts are typically stiff with high burst pressure but low compliance and low elastin expression. We postulated that engineering small arteries on elastomeric scaffolds under dynamic mechanical stimulation would result in strong and compliant arterial constructs. This study compares properties of engineered arterial constructs based on biodegradable polyester scaffolds composed of either rigid poly(lactide-co-glycolide) (PLGA) or elastomeric poly(glycerol sebacate) (PGS). Adult baboon arterial smooth muscle cells (SMCs) were cultured in vitro for 10 days in tubular, porous scaffolds. Scaffolds were significantly stronger after culture regardless of material, but the elastic modulus of PLGA constructs was an order of magnitude greater than that of porcine carotid arteries and PGS constructs. Deformation was elastic in PGS constructs and carotid arteries but plastic in PLGA constructs. Compliance of arteries and PGS constructs were equivalent at pressures tested. Altering scaffold material from PLGA to PGS significantly decreased collagen content and significantly increased insoluble elastin content in constructs without affecting soluble elastin concentration in the culture medium. PLGA constructs contained no appreciable insoluble elastin. This research demonstrates that: (1) substrate stiffness directly affects in vitro tissue development and mechanical properties; (2) rigid materials likely inhibit elastin incorporation into the extracellular matrix of engineered arterial tissues; and (3) grafts with physiologic compliance and significant elastin content can be engineered in vitro after only days of cell culture.

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.

Co‐expression of elastin and collagen leads to highly compliant engineered blood vessels

Elastin synthesis and physiologic compliance are significant challenges in blood vessel tissue engineering. Here, we report that a biocompatible elastomeric scaffold can support the co-expression of elastin and collagen, which likely yielded the physiologic compliance in the constructs. A biodegradable elastomer, poly(glycerol sebacate), was fabricated into highly porous tubular scaffolds. Primary baboon arterial smooth muscle cells (SMCs) were seeded in the lumen of the scaffolds followed by a 1-week culture under gentle perfusion. Circulating endothelial progenitor cells (EPCs) isolated from baboon peripheral blood was seeded directly on the smooth muscle layer in the lumen on day 8. The constructs were perfused using a pulsatile flow system for another 2 weeks before characterization. In another set of experiments, the SMCs were cultured for 7 weeks and were co-cultured for 1 week with the EPCs. Constructs obtained using either set of culture conditions contained elastin and collagen: Massons trichrome stain showed a circumferential collagen band in the constructs, and elastin was evident from its characteristic autofluorescence, Verhoffs stain, and amino acid analysis of insoluble remnants after hot alkali digestion. All constructs had a confluent cellular lumen with cells well-dispersed throughout the scaffolds. At physiologic pressures, the compliance of the 8-week construct was comparable to human arteries as observed in pressure-diameter testing. Combination of elastomeric scaffolds, co-culture of EPC and SMC, and mechanical conditioning appears to encourage the expression of a more natural extracellular matrix and lead to physiologically-relevant compliance; both are major challenges in blood vessel tissue engineering.

Small intestinal submucosa gel as a potential scaffolding material for cardiac tissue engineering

Cardiac tissue engineering typically utilizes protein-rich scaffolding materials and growth factors to improve cardiac tissue function in vitro and in vivo. The objectives of this preliminary study were (i) to investigate the potential of porcine small intestinal submucosa gel (SIS gel) in cardiac tissue engineering and (ii) to compare the function of tissues based on either SIS gel or Matrigel, a tumor-derived benchmark material. Neonatal rat cardiac cells were combined with either SIS gel or Matrigel and cultured on porous elastomeric scaffolds composed of poly(glycerol sebacate) for 13days. Tissue function was assessed by measuring contraction rates twice daily. Tissue morphology was compared qualitatively by hematoxylin and eosin staining. Normalized troponin T expression (troponin T:DNA) was compared using image analysis. SIS gel constructs contracted at significantly higher rates than Matrigel constructs on days 8-11. Normalized troponin T expression was significantly higher in SIS gel constructs compared with Matrigel constructs. In summary, this research demonstrated that: (i) SIS gel can be used to create contractile engineered cardiac tissue; (ii) SIS gel produced engineered cardiac tissues with a more physiological contraction rate and higher phenotypic protein expression based on the basic in vitro examinations performed in this study.

Hydrostatic pressure independently increases elastin and collagen co-expression in small-diameter engineered arterial constructs

Prior studies have demonstrated that smooth muscle cell (SMC) proliferation, migration, and extracellular matrix production increase with hydrostatic pressure in vitro. We have engineered highly compliant small-diameter arterial constructs by culturing primary adult baboon arterial SMCs under pulsatile perfusion on tubular, porous, elastomeric scaffolds composed of poly(glycerol sebacate) (PGS). This study investigates the effect of hydrostatic pressure on the biological and mechanical properties of PGS-based engineered arterial constructs. Pressure was raised using a downstream needle valve during perfusion while preserving flow rate and pulsatility, and constructs were evaluated by pressure-diameter testing and biochemical assays for collagen and elastin. Pressurized constructs contained half as much insoluble elastin as baboon common carotid arteries but were significantly less compliant, while constructs cultured at low hydrostatic pressure contained one-third as much insoluble elastin as baboon carotids and were similar in compliance. Hydrostatic pressure significantly increased construct burst pressure, collagen and insoluble elastin content, and soluble elastin concentration in culture medium. All arteries and constructs exhibited elastic recovery during pressure cycling. Hydrostatic pressure did not appear to affect radial distribution of SMCs, collagens I and III, and elastin. These results provide insights into the control of engineered smooth muscle tissue properties using hydrostatic pressure.