Thomas W. Gilbert

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.

Reprint of: Extracellular matrix as a biological scaffold material: Structure and function.

Biological scaffold materials derived from the extracellular matrix (ECM) of intact mammalian tissues have been successfully used in a variety of tissue engineering/regenerative medicine applications both in preclinical studies and in clinical applications. Although it is recognized that the materials have constructive remodeling properties, the mechanisms by which functional tissue restoration is achieved are not well understood. There is evidence to support essential roles for both the structural and functional characteristics of the biological scaffold materials. This paper provides an overview of the composition and structure of selected ECM scaffold materials, the effects of manufacturing methods upon the structural properties and resulting mechanical behavior of the scaffold materials, and the in vivo degradation and remodeling of ECM scaffolds with an emphasis on tissue function.

Quantification of DNA in Biologic Scaffold Materials

Biological scaffold materials composed of extracellular matrix (ECM) are routinely used for a variety of clinical applications ranging from the treatment of chronic skin ulcers to hernia repair and orthopaedic soft tissue reconstruction. The tissues and species from which the ECM is harvested vary widely as do the methods used to remove the cellular component of the source tissues. The efficacy of decellularization procedures can be quantified by examination of the DNA that remains in the ECM. The objective of the present study was to determine the DNA content and fragment length in both laboratory produced and commercially available ECM scaffold materials. Results showed that the majority of DNA is removed from ECM devices but that small amounts remained in most tested materials.

Macrophage participation in the degradation and remodeling of extracellular matrix scaffolds.

Biologic scaffolds composed of extracellular matrix (ECM) are widely used to facilitate remodeling and reconstruction of a variety of tissues in both preclinical animal studies and human clinical applications. The mechanisms by which such scaffolds influence the host tissue response are only partially understood, but it is logical that the mononuclear macrophage cell population plays a central role. The present study evaluated the role of macrophages that derive from peripheral blood in the degradation of ECM scaffolds. An established rat body wall reconstruction model was used to evaluate the degradation of carbodiimide (CDI)-crosslinked scaffolds composed of porcine small intestinal submucosa (SIS), noncrosslinked SIS, and autologous body wall. To assess the role of circulating macrophages in the degradation process, the degradation of each scaffold was assessed with and without macrophage depletion caused by administration of clodronate-containing liposomes. Results showed that peripheral blood monocytes are required for the early and rapid degradation of both SIS scaffolds and autologous body wall, and that CDI crosslinked SIS is resistant to macrophage-mediated degradation.

Degradation and Remodeling of Small Intestinal Submucosa in Canine Achilles Tendon Repair

BACKGROUND Extracellular matrix derived from porcine small intestinal submucosa is used for the repair of musculotendinous tissues. Preclinical evaluation and clinical use have suggested that small intestinal submucosa extracellular matrix degrades rapidly after implantation and can be replaced by host tissue that is functionally and histologically similar to the normal tissue. METHODS The present study analyzed the temporal degradation of a ten-layer multilaminate device of small intestinal submucosa extracellular matrix used for the repair of canine Achilles tendon and examined the corresponding histological appearance of the remodeled tissue during the course of scaffold degradation. Devices were fabricated from small intestinal submucosa extracellular matrix labeled with 14C. The amount of 14C remaining in the remodeled graft was measured by liquid scintillation counting at three, seven, fourteen, twenty-eight, sixty, and ninety days after surgery. Blood, urine, feces, and other parenchymal tissues were also harvested to determine the fate of scaffold degradation products. Tissue specimens were prepared for routine histological analysis to examine the morphology of the remodeled graft at each time-point. RESULTS The small intestinal submucosa extracellular matrix graft degraded rapidly, with approximately 60% of the mass lost by one month after surgery, and the graft was completely resorbed by three months after surgery. The graft supported rapid cellular infiltration and host tissue ingrowth. By ninety days after surgery, the remodeled small intestinal submucosa extracellular matrix consisted of a dense collagenous tissue with organization, cellularity, and vascularity similar to that of normal tendon. CONCLUSIONS Small intestinal submucosa extracellular matrix is rapidly degraded after implantation for the repair of a musculotendinous tissue in this canine Achilles tendon repair model and is replaced by the deposition and organization of host tissue that is histologically similar to that of normal tissue.

The Effects of Processing Methods upon Mechanical and Biologic Properties of Porcine Dermal Extracellular Matrix Scaffolds

Biologic materials from various species and tissues are commonly used as surgical meshes or scaffolds for tissue reconstruction. Extracellular matrix (ECM) represents the secreted product of the cells comprising each tissue and organ, and therefore provides a unique biologic material for selected regenerative medicine applications. Minimal disruption of ECM ultrastructure and content during tissue processing is typically desirable. The objective of this study was to systematically evaluate effects of commonly used tissue processing steps upon porcine dermal ECM scaffold composition, mechanical properties, and cytocompatibility. Processing steps evaluated included liming and hot water sanitation, trypsin/SDS/TritonX-100 decellularization, and trypsin/TritonX-100 decellularization. Liming decreased the growth factor and glycosaminoglycan content, the mechanical strength, and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for all). Hot water sanitation treatment decreased only the growth factor content of the ECM (p ≤ 0.05). Trypsin/SDS/TritonX-100 decellularization decreased the growth factor content and the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). Trypsin/Triton X-100 decellularization also decreased the growth factor content of the ECM but increased the ability of the ECM to support in vitro cell growth (p ≤ 0.05 for both). We conclude that processing steps evaluated in the present study affect content, mechanical strength, and/or cytocompatibility of the resultant porcine dermal ECM, and therefore care must be taken in choosing appropriate processing steps to maintain the beneficial effects of ECM in biologic scaffolds.

Cell orientation determines the alignment of cell-produced collagenous matrix

In healing ligaments and tendons, the cells are not aligned and collagen matrix is not organized as in normal tissues. In addition, the mechanical properties of the tissues are abnormal. We hypothesized that the lack of alignment of the collagen matrix results from random orientation of the cells seen in the healing area. To test this hypothesis, a novel in vitro model was used in which the orientation of cells could be controlled via microgrooves, and alignment of the collagen matrix formed by these cells could be easily observed. It is known that cells align uniformly along the direction of microgrooves; therefore MC3T3-E1 cells, which produce large amounts of collagen, were grown on silicone membranes with parallel microgrooves (10 microm wide x 3 microm deep) in the surface. As a control, the same cells were also grown on smooth silicone membranes. Cells on both the microgrooved and smooth silicone surfaces produced a layer of readily visible collagen matrix. Immunohistochemical staining showed that the matrix consisted of abundant type I collagen. Polarized light microscopy of the collagen matrix revealed the collagen fibers to be parallel to the direction of the microgrooves, whereas the collagen matrix produced by the randomly oriented cells on the smooth membranes was disorganized. Thus, the results of this study suggest that the orientation of cells affects the organization of the collagenous matrix produced by the cells. The results also suggest that orienting cells along the longitudinal direction of healing ligaments and tendons may lead to the production of aligned collagenous matrix that more closely represents the uninjured state. This may enhance the mechanical properties of healing ligaments and tendons.

Functional Skeletal Muscle Formation with a Biologic Scaffold

Biologic scaffolds composed of extracellular matrix (ECM) have been used to reinforce or replace damaged or missing musculotendinous tissues in both preclinical studies and in human clinical applications. However, most studies have focused upon morphologic endpoints and few studies have assessed the in-situ functionality of newly formed tissue; especially new skeletal muscle tissue. The objective of the present study was to determine both the in-situ tetanic contractile response and histomorphologic characteristics of skeletal muscle tissue reconstructed using one of four test articles in a rodent abdominal wall model: 1) porcine small intestinal submucosa (SIS)-ECM; 2) carbodiimide-crosslinked porcine SIS-ECM; 3) autologous tissue; or 4) polypropylene mesh. Six months after surgery, the remodeled SIS-ECM showed almost complete replacement by islands and sheets of skeletal muscle, which generated a similar maximal contractile force to native tissue but with greater resistance to fatigue. The autologous tissue graft was replaced by a mixture of collagenous connective tissue, adipose tissue with fewer islands of skeletal muscle compared to SIS-ECM and a similar fatigue resistance to native muscle. Carbodiimide-crosslinked SIS-ECM and polypropylene mesh were characterized by a chronic inflammatory response and produced little or no measurable tetanic force. The findings of this study show that non-crosslinked xenogeneic SIS scaffolds and autologous tissue are associated with the restoration of functional skeletal muscle with histomorphologic characteristics that resemble native muscle.

Chemoattraction of Progenitor Cells by Remodeling Extracellular Matrix Scaffolds

The chemotactic properties of a biologic scaffold composed of extracellular matrix (ECM) and subjected to in vivo degradation and remodeling were evaluated in a mouse model of Achilles tendon reconstruction. Following a segmental resection of the Achilles tendon in both C57BL/6 and MRL/MpJ mice, the defect was repaired with either an ECM scaffold composed of urinary bladder matrix (UBM) or resected autologous tendon. The surgically repaired and the contralateral tendons were harvested at 3, 7, and 14 days following surgery from each animal. Chemotaxis of multipotential progenitor cells toward the harvested tissue was quantified using a fluorescent-based cell migration assay. Results showed greater migration of progenitor cells toward tendons repaired with UBM-ECM scaffold compared to both the tendons repaired with autologous tissue and the normal contralateral tendon in both the MRL/MpJ and C57BL/6 mice. The magnitude and temporal pattern of the chemotactic response differed between the two mouse strains.

Surface Characterization of Extracellular Matrix Scaffolds

Extracellular matrix (ECM) scaffolds prepared from different tissue sources or using different methods have been demonstrated to have distinctive effects upon cell adhesion patterns and the ability to support and maintain differentiated phenotypes. It is unknown whether the molecular composition or the ultrastructure of the ECM plays a greater role in determining the phenotype of the cells with which it comes into contact. However, when implanted, the topology and ligand landscape of the material will determine the host molecules that bind and the type and behavior of cells that mediate the host response. Therefore, a comprehensive understanding of surface characteristics is essential in the design of scaffolds for specific clinical applications. The surface characteristics of ECM scaffolds derived from porcine urinary bladder, small intestine, and liver as well as the effects of two commonly used methods of chemical cross-linking upon UBM were investigated. Electron microscopy and time of flight secondary ion mass spectroscopy were used to examine the surface characteristics of the scaffolds. The results show that ECM scaffolds have unique morphologic and structural properties which are dependant on the organ or tissue from which the scaffold is harvested. Furthermore, the results show that the surface characteristics of an ECM scaffold are changed through chemical cross-linking.