Jason P. Hodde

Vascular endothelial growth factor in porcine-derived extracellular matrix.

An extracellular matrix (ECM) derived from the submucosa of the porcine small intestine (SIS) has been shown to induce angiogenesis and host tissue remodeling when used as a xenogeneic bioscaffold in animal models of wound repair. In the present study, we compared the in vitro effects of SIS ECM extracts to several purified angiogenic growth factors on human dermal microvascular endothelial cell (HMEC) growth patterns. The SIS ECM was shown to induce tube formation from HMEC in a three-dimensional fibrin-based angiogenesis assay in a manner similar to that caused by the addition of vascular endothelial growth factor (VEGF). This tube formation was blocked in the presence of anti-VEGF neutralizing antibody. Western blots and ELISA procedures showed that the SIS ECM contains as much as 0.77 ng VEGF/g SIS. The closely related endothelial cell mitogen, platelet-derived growth factor (PDGF), was not detectable in the SIS extracts. We conclude that VEGF is present in the SIS extracellular matrix. The role of VEGF in SIS-induced wound repair remains unknown, but its presence in the ECM makes it a possible contributor to the angiogenic effect of SIS when this ECM is used as a tissue repair scaffold in animal models of wound repair.

Glycosaminoglycan Content of Small Intestinal Submucosa: A Bioscaffold for Tissue Replacement

Small intestinal submucosa (SIS) is a resorbable biomaterial that induces tissue remodeling when used as a xenogeneic tissue graft in animal models of vascular, urologic, dermatologic, neurologic, and orthopedic injury. Determination of the composition and structure of naturally occurring biomaterials such as SIS that promote tissue remodeling is necessary for the greater understanding of their role in wound healing. Since glycosaminoglycans (GAGs) are important components of extracellular matrix (ECM) and SIS is primarily an ECM-based material, studies were performed to identify the species of glycosaminoglycans present in SIS. Porcine SIS was chemically extracted and the extracts were analyzed for uronic acid. The extractable uronic acid content was determined to be 47.7 micromol/g (approximately 21 microg GAG/mg) of the dry weight of the SIS tissue. Using electrophoretic separation of GAGs on cellulose acetate membranes, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate A, and dermatan sulfate were identified. Digestion of specific GAGs with selective enzymes confirmed the presence of these GAG species. Two GAGs common to other tissues with large basement membrane ECM components, keratan sulfate and chondroitin sulfate C, were not detected in the SIS extracts. Identification of specific GAGs in the composition of the ECM-rich SIS provides a starting point toward a more comprehensive understanding of the structure and function of this naturally occurring biomaterial with favorable in vivo tissue remodeling properties.

Naturally occurring scaffolds for soft tissue repair and regeneration

Cell growth supports (i.e., scaffolds) that provide a conducive environment for normal cellular growth, differentiation, and angiogenesis are important components of tissue engineered grafts because rapid integration with the host is essential for long-term graft viability. While many of these scaffold materials are synthetic biodegradable polymers, others are naturally derived from mammalian tissue sources. Naturally occurring scaffold materials include small intestinal submucosa, acellular dermis, amniotic membrane tissue, cadaveric fascia, and the bladder acellular matrix graft. Upon implantation, these materials elicit a host-tissue response that initiates angiogenesis, encourages tissue deposition and culminates in restoration of structure and function specific to the grafted site. The sources, the methods of procurement and processing, and the effects of these naturally occurring materials on angiogenesis and tissue deposition are reviewed.

Small intestinal submucosa: a substrate for in vitro cell growth.

The extracellular matrix (ECM) of the small intestinal submucosa (SIS) was harvested by removing the superficial layers of the mucosa and the external muscular layers. The remaining 80 microns thick sheet was disinfected and sterilized by methods which removed all cellular components. The SIS-ECM, retaining its native 3-dimensional microarchitecture and composition, was evaluated for its ability to support in vitro cell growth. Six separate cell types were seeded either alone or in coculture with other cells upon this matrix, grown in selected media, a examined daily for time periods ranging from 48 h to 2 weeks. The six cell types tested were NIH Swiss mouse 3T3 fibroblast, NIH 3T3/j2 fibroblasts, primary human fibroblasts, primary human keratinocytes, human microvascular endothelial cells (HMECs), and an established rat osteosarcoma (ROS) cell line. All cell types showed the ability to attach a proliferate. All fibroblast cell line and the keratinocytes proliferated and/or migrated into the 3-dimensional scaffold of the SIS matrix. The ROS cells and the HMECs were confined in their growth pattern to the surface of the matrix. Coculturing of NIH 3T3/J2 fibroblasts and primary human keratinocytes resulted in a distinctive spatial orientation of the two cell types. The fibroblast populated the mid-substance of the 3-dimensional matrix and the keratinocytes formed an epidermal structure with rete ridge-like formation and stratification when the composite was lifted to an air liquid interface in culture. In summary, SIS provides a substratum with a 3-dimensional scaffold that allows for cell migration and spatial organization. The substratum is suitable for in vitro studies of the interaction between epithelial or mesenchymal cells and a naturally occurring extracellular matrix.

Endothelial cell adherence to small intestinal submucosa: an acellular bioscaffold.

Degradable biomaterials to be used as scaffolds for tissue repair will ideally be able to support new blood vessel growth. The present study evaluated the adherence of human dermal microvascular endothelial cells (HMECs) to an acellular resorbable scaffold material derived from the small intestinal submucosa (SIS). HMECs were exposed to hydrated and dehydrated forms of SIS and to plastic surfaces coated with one of four different known components of the SIS extracellular matrix: collagen Type I, collagen Type IV, fibronectin, and laminin. Results showed that adherence of HMECs to hydrated SIS was greater than to any of the other tested surfaces (P < 0.05). Exposure of HMECs to either soluble collagen Type IV or soluble fibronectin prior to exposure of these cells to hydrated SIS showed only partial inhibition of HMEC attachment. We conclude that HMECs find hydrated SIS to be a suitable substrate for adherence and that dehydration of SIS adversely affects the ability of HMECs to adhere in vitro. The cause of HMEC adherence to SIS appears to be a combination of both its composition and architecture.

Fibronectin peptides mediate HMEC adhesion to porcine-derived extracellular matrix

Extracellular matrices (ECM) derived from porcine tissues have been shown to support the successful repair and remodeling of injured tissues when evaluated in animal models. Cell-matrix interactions, including ligand-integrin associations that facilitate endothelial cell adhesion, are clearly important in the tissue remodeling process. The goal of the present study was to identify the peptide sequences within the ubiquitous protein fibronectin (FN) that may be important in the initial interactions between the host endothelial cells and the ECM scaffold. Human microvascular endothelial cells (HMEC) were seeded upon porcine ECM after having been subjected to pretreatment with peptide ligands derived from tissue FN and were allowed to attach for 20 min. Non-adherent cells were removed and the remaining, tritium-labeled cells attached to the ECM were counted. Results showed that cyclo-RGD and REDV, but not LDV or PHSRN, play a role in mediating the attachment of HMEC to porcine ECM.

Retention of Endothelial Cell Adherence to Porcine-Derived Extracellular Matrix after Disinfection and Sterilization

Extracellular matrices (ECM) derived from porcine tissue are associated with rapid and extensive repopulation with host cells when used as scaffolds for in vivo tissue repair. Cell adhesion to substrates used for tissue engineering has been studied extensively but the factors that mediate this phenomenon in ECM scaffolds following treatment with oxidants and sterilants have not been examined. Cell adhesion assays were used to examine human microvascular endothelial cell (HMEC) attachment to ECM graft materials harvested from small intestinal submucosa (SIS) and urinary bladder matrix (UBM) following decellularization and sterilization procedures designed to render the ECM safe for clinical use. HMECs were able to attach directly to these ECM scaffolds via several attachment proteins present within the ECM, including type I collagen, type IV collagen, and fibronectin. The ability of the SIS ECM and UBM ECM to support the growth and proliferation of HMEC was also examined. HMEC were able to grow to single-layer confluence on both surfaces of SIS and UBM sheets. The endothelial cells were also able to penetrate the SIS and UBM at later time points if they were seeded on the abluminal side of the ECM sheets. The ability of the processed ECM to support HMEC attachment and proliferation is similar to that reported for unprocessed ECM and may therefore play a role in the rapid remodeling response observed when these matrices are implanted in vivo as scaffolds for wound repair.

Morphologic study of three collagen materials for body wall repair

BACKGROUND The search for ideal prostheses for body wall repair continues. Synthetic materials such as polypropylene mesh (PPM) are associated with healing complications. A porcine-derived collagen-based material (CBM), small intestinal submucosa (SIS), has been studied for body wall repair. Renal capsule matrix (RCM) and urinary bladder submucosa (UBS) are CBMs not previously evaluated in this application. This is the first implant study using RCM. MATERIALS AND METHODS Full-thickness muscle/fascia ventral abdominal wall defects were repaired with SIS, RCM, UBS, and PPM in rats with omentum and omentectomy. A random complete block design was used to allot implant type to each of 96 rats. Healing was evaluated at 4 and 8 weeks. Adhesion tenacity and surface area were scored. Implant site dimensions were measured at implantation and necropsy. Inflammation, vascularization, and fibrosis were histopathologically scored. Data were compared by analysis of variance (P < 0.05). RESULTS PPM produced a granulomatous foreign body response in contrast to the organized healing of CBM implants. CBM mean scores were lower than PPM scores for adhesion tenacity, surface area, and inflammation at each follow-up time for rats with omentums (P < 0.02). The CBMs had less tenacity and inflammation than PPM at each follow-up time in omentectomy groups (P < 0.008). Wound contraction was greater for PPM (P < 0.0001) for all rats. CONCLUSIONS RCM and UBS were similar to SIS invoking reduced inflammation, adhesion, and contraction compared to PPM. The fibrotic response to PPM was unique and more intense compared to CBMs. These CBM implants appear morphologically acceptable and warrant continued investigation.

Extracellular Matrix as a Strategy for Treating Chronic Wounds

The dermis normally directs all phases of skin wound healing following tissue trauma or disease. However, in chronic wounds, the dermal matrix is insufficient to stimulate healing and assistance by external factors is needed for wound closure. Although the concept of the extracellular matrix directing wound healing is not new, ideas about how best to provide the extracellular matrix components required to ‘jump-start’ the healing process are still evolving. Historically, these strategies have included use of enzyme-inhibiting dressing materials, which bind matrix metalloproteinases and remove them from the chronic wound environment, or direct application of purified growth factors to stimulate fibroblast activity and deposition of neo-matrix. More recently, the application of a structurally intact, biochemically complex extracellular matrix, designed to provide the critical extracellular components of the dermis in a single application, has allowed for the reconstruction of new, healthy tissue and restoration of tissue integrity in the previously chronic wound. This review focuses on this third mechanism as an emerging tactic in effective wound repair. Intact extracellular matrix can quickly, easily, and effectively provide key extracellular components of the dermis necessary to direct the healing response and allow for the proliferation of new, healthy tissue. Its application may promote the healing of wounds that have been refractory to other, more conventional treatment strategies, and may eventually show utility when used earlier in wound healing treatment with the goal of preventing wounds from reaching a truly chronic, nonresponsive state.

Tissue engineering a clinically useful extracellular matrix biomaterial

Implantable biomaterials are one of the most useful tools in the surgeon’s armamentarium, yet there is much room for improvement. Chronic pain, tissue erosion, and late infections are just a few of the serious complications that can occur with conventional, inert materials. In contrast, tissue-inductive materials exist today. Combinations of biologically important molecules for directing cell growth and providing structural stability can be found in naturally occuring extracellular matrices. These “soft-tissue skeletons” of Mother Nature can be harvested, processed, and provided in a medically safe and biologically active form for repairing many different tissues in the human body. The future of surgical practice may well be determined by how well these new implant materials recreate the tissues they replace.