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Composite Cell/Tissue Replacement for Nerve and Pressure Sore Repair
Eric E Sabelman, PhD; Robert Keeley, MD; William C Linaweaver, ML; Tanya Atagi, MS; Paula Kadlcik, BS Joseph M Rosen, MD; VA Medical Center; White River Junction, VT
Abstract - Grafts made from artificial and natural biomaterials combined with a patient's own cells hold promise for replacing tissues lost to injury and disease. This concept evolved from a technique explored by others for cell-seeded and vascular grafts skin grafts used for burn treatment and by us for peripheral nerve grafts. The concept is based on two premises: 1) that the human body's capacity for regeneration is limited because in maturity it lacks the positional and migratory direction information present in the embryo, which may be provided by the artificial extracellular matrix, and 2) that a completely synthetic matrix lacks the capacity to integrate with intact tissue and respond to functional demands, which is provided by the cellular component. Incorporation of living cells is a decision based on the ability of cells to secrete attachment and growth factors in response to the demands of their environment, obviating the need to isolate and replace such factors individually.
Peripheral nerve graft - As an example, the function of the artificial peripheral nerve graft is to guide regenerating axons across gaps in nerves too long to be bridged surgically. The recovering damaged nerve normally has a high population of Schwann cells which proliferate after injury. These cells originally made up the myelin sheaths of axons. Schwann cells secrete diffusible nerve growth factor and repair the extracellular matrix by depositing basement membrane, in preparation for extending regenerating axons into the damaged region. We have experimented with neonatal rat Schwann cells in otherwise acellular collagen-matrix grafts, implanted into 10P14 mm gaps in adult rat peroneal nerves, with autografts as controls. Ten of 14 (71%) live Schwann cell-containing repairs regenerated across the gap, while only 57% of collagen-only repairs bridged the gap.
Preparation of the graft essentially consists of repolymerization of solubilized collagen, mixing with cultured Schwann cells before polymerization is complete, and insertion into a conduit. The matrix maintains the donor Schwann cells inside the graft, and the conduit facilitates surgery and reduces scar-forming inflammation within its lumen. The latest iteration of the matrix structural design is based on multi- stranded oriented collagen, which provides channels parallel to the nerve axis to guide regenerating axons (Figure 1). A 1 to 2 mm diameter graft replaces a single nerve fascicle; for large nerves, fascicles in proximal and distal stumps would be reconnected with individual parallel grafts.
Figure 1. Multi-stranded collagen provides channels parallel to the nerve axis to guide regenerating axons.
Pressure sore repair - Healing of subcutaneous tissue could be accelerated by creating a synthetic tissue having the same mechanical and geometric properties as the lost tissue, inoculating it with cells, and externally supplying nutrients, oxygen and protection against infection. In the pressure sore, the vascular supply to the injury is impaired and the interior of an artificial graft would only be vascularized slowly. The proposed solution is to provide an artificial capillary bed - a branching network of permeable tubes similar to the straight tubes used for artificial nerve grafts - which can be connected either to vessels at a distance from the injury, or to an extracorporeal supply of cell culture medium. The latter perfusion system takes the place of the blood supply until replaced by it; it also provides a means for infusing high-dose antibiotics to combat infection, and for raising hydrostatic pressure to resist compression.
As presently envisioned, the composite graft would consist of 1) a number of layers of artificial capillaries made of biodegradable biocompatible materials; 2) cell-supporting gel (e.g.,collagen) filling the spaces between capillary layers; 3) culture medium flowing through the synthetic capillary bed, which may include hemoglobin substitutes and antibiotics at concentrations that could not be tolerated systemically; 4) pumps and reservoirs for supplying the liquid medium; 5) sensors and controls for regulating dissolved oxygen, pH and other parameters; and 6) an artificial skin sealed to the patient's skin outside the wound.
Conclusion - While neonatal rat cells are suitable for populating experimental grafts, primary cultures of human fetal cells are less suitable for seeding grafts for clinical use (for economic, if not ethical, reasons). Encapsulated xenogeneic or allogeneic cells would not work, since it is desired that donor cells have direct contact with host cells and extracellular matrix. Perhaps eventually genetically modified "universal" donor cells derived from serially propagated fetal or adult cell lines will become available. In the meantime, we propose that autogeneic donor cells be obtained from minute amounts of the patient's tissue; in the case of peripheral nerves, this can be fragments of the nerve distal to the injury.
Republished from the 1994 Rehabilitation R&D Center Progress Report. For current information about this project, contact Eric E Sabelman.