Investigators: Eric E. Sabelman, PhD and Min Hu, PhD MD

Summary: Tissue lost due to pressure sores and overt trauma needs to be replaced (e.g. skin) or reconstructed (e.g. muscle, tendon, and bone). The objective of this project is to test in rats the feasibility of using a microsurgically relocated arteriovenous loop to nourish a biomaterial matrix inoculated with the animal's own connective tissue and fat cells. Once this technique is developed, patients will no longer need to undergo secondary surgery to obtain autografts (thus reducing donor site morbidity and risk of infection and loss of function in the patient, as well as reducing surgical costs). Also, patients without suitable donor sites for autografting can be treated.

Objectives: Components of a semi-synthetic graft presently under development for repair of deep pressure sores (RR&D pilot project B92-476AP) are potentially capable of replacing free tissue grafts elsewhere than at the surface of the body. Synthetic extracellular matrices, tailored to match the properties of the lost tissue, are seeded with autologous cells and implanted into the wound cavity in muscle or connective tissue. Since thick (>2 mm) grafts with high cell populations cannot survive without a vascular supply, this is provided by a new microsurgical technique. Survival and proliferation of cells and re-integration into surrounding tissues of semi-synthetic grafts are compared to free muscle and/or fat grafts of similar dimensions.

Background: The composite artificial/cellular graft is a concept explored by others for skin grafts for burn treatment, and by us for peripheral nerve grafts (Merit Review project B588). The prior pilot project on "In Vitro Test of a Perfused Artificial/Cellular Graft For Pressure Ulcer Repair" has led to the development of a series of matrices for supporting cell inoculation, proliferation and migration based on Type I collagen (bovine and rat) with hyaluronic acid (HyA). Collagen provides a low-density microfibrous substrate for cell attachment, while HyA provides bulk and viscous damping similar to fat in the intact tissue. An alternative to the extracorporeal nutrient loop has been proposed by Drs. Lineweaver and Hui, who have experimented on mobilization and repositioning of arteriovenous pedicles using microsurgical procedures. This approach has been shown capable of providing rapid revascularization of adjacent tissues, which may be either those found naturally at the implantation site, or grafts transferred from elsewhere in the body.

Significance: Semi-synthetic grafts of all types will eliminate the need for secondary surgery to obtain autograftable tissues, reducing donor site morbidity, risk of infection and loss of function, and time and cost spent during surgery. The range of applications includes not only tissue loss due to pressure sores and overt trauma, but reconstruction following resection for cancer, repair of soft tissues surrounding joints after injury or prosthetic implantation, and, potentially, reconstruction of functional muscle, tendon and bone.

Methodology / Research Plan: The project has two phases: (1) fabrication of semi-synthetic grafts, and (2) implantation in animals with autologous grafts as controls, followed by optical and electron microscopic analysis. A preliminary implant test will be performed to establish methods of handling and positioning the fragile matrix, trimming to fit the host site, attachment to surrounding tissues (by sutures, fibrin glue, etc.), interposition of an arterio-venous pedicle between layers of the graft, and monitoring of the graft once the incision is closed. Two experimental implantation series will be performed, each involving 20 adult Sprague-Dawley rats. The first series will last 14 days, and will be an investigation of short-term effects (degree of inflammation surrounding the graft, initial sprouting of capillaries from the A-V pedicle). The second series will last 60 days, and will focus on biodegradation of the synthetic matrix and integration into the host site.

Results: Three physical forms of semi-synthetic graft have been created: (1) homogeneous 1:1 collagen:HyA dispersions of random-geometry HyA particles less than 100 µm in diameter, (2) continuous-strand HyA mats or felts, and (3) loosely-packed HyA beads 0.5-1.0 mm in diameter. These are processed into porous discs 35-60 mm in diameter and 2-5 mm thick. Methods for controlling swelling and solubility of the HyA component have been adapted from the literature. Samples of a modified Type I collagen have been provided by Nova-Gen Inc. (Fremont, CA) in addition to purchased bovine collagen and in-house prepared rat-tail collagen.

The preliminary microsurgical revascularization test was successfully completed, using a subcutaneous abdominal site. Inflammation was minimal, with rapid capillary outgrowth into the matrix within 2 weeks and preservation of the HyA component for 8 weeks. As predicted from in vitro cell ingrowth experiments, migrating cells enveloped individual HyA beads or strands, and did not form a fibrous capsule around the entire implant. A report was presented at the First Smith & Nephew International Symposium on Tissue Engineering and Biomaterials, in York, UK, July 20-23, 1997.

The abdominal site did not provide a wound margin suitable for testing integration. A small grant was obtained from the Plastic Surgery Education Foundation to explore alternate sites that would better emulate the clinical pressure sore; both latissimus and rectus abdominus muscle excision wounds are suitable, with the latter requiring less extensive mobilization of the saphenous vein to create the interposed pedicle. Funding has also been provided by Nova-Gen Inc.

A Stanford-employed Resaerch Associate, Min Hu, MD PhD, began work on the project in February, 1997. Dr. Hu, with assistance by summer intern Charlotte Tsai and medical student Ewa Komorowska-Timek, developed an alternative chemical treatment to stabilize HyA and enhance its cell adhesion. An invention disclosure was filed with Stanford Office of Technology Licensing and a small grant proposal is being submitted to the Hand Surgery Society to test this material afte4r seeding with Keratinocytes as a burn wound graft.

Funding Source: PAIRE

Funding Source: VA RR&D Merit Review - Project SAB0022

Years: 1997-2000