Investigators: Eric E. Sabelman, PhD and V. Rodney Hentz, MD

Project Staff: Min Hu, PhD MD, Douglas L. Eng, PhD, Greg Buncke, MD, Deborah E. Kenney, MS OTR, and Yang Cao, MD

Summary: Peripheral nerve damage impairs upper and lower limb sensorimotor function. Severe injuries of the nerves (i.e., large nerve gaps) cannot be repaired easily, if at all, and require autografting (i.e., the sacrifice of another nerve in the patient with its surgical removal). The objective of this project is to conduct limited clinical trials of an multi-component artificial nerve graft (i.e., cultured Schwann cells from the patient imbedded into commercially available collagen). This new technique promises to reduce donor site morbidity, risk of infection and loss of function and surgical costs of autograftingand and ultimately may be applicable to spinal cord injury.

Objectives: The goal of this proposal is to prepare for and conduct a limited clinical trial of a multi- component artificial nerve graft (ANG) as an alternative to an autograft when this option is not available to the participating patient, or the patient decides to forego an autograft. An ANG functionally equivalent to the autograft would enable the surgeon to repair nerve gaps now left untreated for lack of sufficient expendable nerve for autografts, and would also allow grafting of small gaps (<2 cm) now anastomosed by applying tension to the nerve. The ANG is composed of an outer tubular conduit, an internal matrix of oriented collagen, and cultured Schwann cells, emulating the nerve autograft. Since all the materials in the graft are either derived from the patient's own tissue (Schwann cells) or are FDA approved for human use, the graft can be made available to patients who have nerve gaps that cannot easily be repaired other than by an autograft or its equivalent.

Background: Peripheral nerve damage in which the matrix framework and Schwann cell population are retained (as in crush injuries) is repaired by reorganization of the matrix by the Schwann cells preparatory to regrowth of axons from neuronal cells in the spinal cord. Simple tubular ANGs without matrix or cells have been tested clinically with mixed results; it is apparent that other factors than just a guiding conduit are provided by autografts. Our approach is to re-introduce Schwann cells into the artificial graft, obviating the need to isolate and add growth factors individually. Our series of Merit Review grants (B387, B588) has tested ANGs chiefly in the rat peroneal nerve; the latest formulation of the ANG yielded functional and histological results equal to the autograft. In project B387, five monkeys had gaps of 3 cm in 6 nerves; results showed that collagen/fibrin tubes (made by the University of Utah) supported axonal regeneration as long as the ANGs remained intact.

Methods: Preparation of the ANG essentially consists of re-polymerization of solubilized collagen fibers with added cultured Schwann cells and insertion into a biodegradable conduit. Type I collagen is preferred because it is readily available, relatively inexpensive, and its properties are reasonably well understood. The conduit limits penetration of inflammatory cells into the region of axonal regrowth, as well as regeneration-promoting agents such as nerve growth factor. The conduit limits penetration of inflammatory cells into the region of axonal regrowth, as well as facilitating microsurgical reanastomosis with the proximal and distal ends of the nerve.

Research Plan: The current project (B588-3RA) has six goals, four of which have been achieved: (1) adult human Schwann cells were isolated and cultured in our laboratory as cell sources for non-clinical ANGs; (2) assays and precautions necessary to insure that clinical ANGs are free of pyrogens and immunoreactive proteins were implemented; (3) complete fabrication of a human Schwann-cell-seeded ANG was demonstrated (we cannot yet reduce the 2 to 4 weeks in culture needed for cell proliferation prior to implantation); (4) noninvasive methods were tested for evaluating motor and sensory recovery after implantation of an ANG (methods for conduction velocity and sudomotor response need further development); (5) to date, no patients have presented at VA Palo Alto HCS or Stanford suitable for ANG implantation (steps to expand the range of indications and catchment are underway, and are included in the present proposal); (6) too few past patients were available for comparison of functional outcome with a retrospective control group of patients matched for age, gender and type of injury (this lack also impacted development of the diagnostic battery). Fewer animal studies were conducted than proposed, due to staff and material shortages and to relocation of the laboratory from Stanford to VA Palo Alto HCS as required by VHA Directive 98-004.

Progress: The time schedule of the original proposal of Oct. 15, 1995, showed recruitment of clinical subjects beginning 18 months into the 3-year project. At the present time, it appears that this schedule is unrealistic. Below are listed the major factors affecting progress and the steps being taken to assure that progress is maintained:

• Availability of Clinical Subjects - It was originally thought that 6 to 12 patients per year having nerve injuries requiring autografts would present at Stanford and VA Palo Alto reconstructive microsurgery clinics. In our efforts to recruit former patients for development of diagnostic tests, we found that only 2 or 3 patients per year actually required grafts (others had simple nerve re-anastomoses). Surgeons at the Buncke Microsurgery Center at R.K. Davies Medical Center in San Francisco have agreed to participate in our test program. It will be 3 to 4 months before R.K. Davies' Institutional Review Board approves participation. We are also exploriing expanding the patient selection criteria to include brachial plexus injuries, which require larger amounts of donor nerve than distal nerve gaps.

• Availability of Biomaterials - Collagen Corporation changed its mind about allowing us to purchase research-grade collagen under a material supply agreement (the company's agreement requires that the company be given first right of refusal for any patentable technology developed using its material, which is contrary to Federal technology transfer law). It may still be possible to obtain FDA-approved bovine collagen for clinical implants by direct purchase (cost is too high for animal experimentation). We are testing other suppliers' collagen, but so far have not found an exact replacement, and will have to perform more exhaustive testing of the material prior to clinical use. The pre-clinical testing of nerve grafts made to FDA "GMP" standards will therefore take place no sooner than the fourth quarter of this fiscal year, about a one year delay.

• Availability of Human Nerve Tissue for Process Development - We had planned to begin testing human Schwann cell preparations in the third quarter after the project began. Because fewer nerve graft surgeries than expected are being performed by our immediate circle of collaborators, unused remnants of nerve could not be obtained. We then requested specimens from surgeons at R.K. Davies, and from the Human Tissue Committee at Stanford; the latter gave its approval last month. We also broadened the scope of our request for specimens to include amputated or excised tissue containing peripheral nerves. The first such specimen was obtained June 3; human peripheral nerve cells are now being cultured.

• Staffing - The original proposal specified two full-time staff (a laboratory technician-manager and a biochemist), plus a 50% research assistant. The holder of the former position, Paula Koran, left VA employment in June, 1996. After two postings of the announcement for her replacement (in Jan, 1997) with only two applicants, neither highly qualified, we decided to fill the position with a Stanford employee through an Intergovernmental Personnel Agreement, which took effect Feb, 1997. The second position was not filled until January, 1998; this person stayed in the job only 4 months before leaving to join a start-up biotechnology company. We have had several part-time student research assistants, but no one person spending 50% time on the project. We expect to have to continue short-handed, given high demand for biomedical engineers in the current economy and lag in VA pay scales behind private-sector salaries.

• Laboratory Space - Since 1987, the laboratory and microsurgical aspects of this project have been conducted in the 700 sq. ft. Plastic Surgery Research Laboratory at Stanford University. Particularly after the 1989 earthquake damaged VA research facilities, this arrangement has freed us from the need to compete with other VA researchers for scarce space. There are three reasons for reconsidering the arrangement at this time: (1) VA space in available now, (2) administrative burdens of working at Stanford have increased in recent years, (3) per VHA Directive 98-004, dated January 15, 1998, we are required to apply for a policy exemption to conduct off-site VA research. Dr. Hentz has been allocated 200 sq ft of lab space, and we are planning to share another 600 sq ft with another researcher. When the move takes place, we can expect a 1- to 2-month delay before the laboratory is fully operational again.

• Patient Follow-up - Although not yet a factor, when nerve implants are placed in patients starting in the next 6 months, we expect to need 6- to 12-month follow-up testing to ascertain success of the procedure, rather than the 6- to 7-month follow-up specified in the proposal. This change in schedule was prompted by rate of recovery of nerve function in patients we tested up to 2 years after autografting. If only patients having relatively short nerve gaps, which would yield measureable recovery in 6 months, are recruited near the end of the project, we will still need to add two months to complete follow-up compared to the original schedule.

Findings: The current project has six goals, four of which have been achieved: (1) Adult human Schwann cells were isolated and cultured as cell sources for non-clinical ANGs. (2) Assays and precautions to insure that clinical ANGs are free of pyrogens and immunoreactive proteins were implemented. (3) Complete fabrication of a human Schwann-cell-seeded ANG was demonstrated (we cannot yet reduce the 2 to 4 weeks in culture needed for cell proliferation prior to implantation). (4) Noninvasive methods were tested for evaluating motor and sensory recovery (conduction velocity and sudomotor response need further development). (5) To date, no patients have presented at VA Palo Alto HCS or Stanford suitable for ANG implantation (It was originally thought that 6 to 12 patients per year having digital nerve injuries requiring autografts could be recruited; instead, only 2 or 3 patients per year actually required grafts, the others having simple nerve re-anastomoses). Surgeons at the Buncke Microsurgery Clinic at R.K. Davies Medical Center in San Francisco have agreed to refer patients for possible ANG repairs. (6) Too few past patients were available to serve as retrospective controls for comparison of functional outcome matched for age, gender and type of injury.

Accomplishments: Poster presentations at the Society for Biomaterials conference (San Diego, CA, April, 1998) and the First International Smith & Nephew Symposium: Advances in Tissue Engineering & Biomaterials (University of York, UK, July, 1997). Attended Seventh International Symposium on Neural Regeneration (Asilomar, CA - December, 1997). Invited lecture at Dartmouth College-Thayer School of Engineering (Hanover, NH - April, 1998). Invited to present a paper on neural tissue engineering at 1998 Biomedical Engineering Society, October 10-13, 1998. Invited to propose special session at Eighth International Symposium on Neural Regeneration (December, 1999).

Funding Source: VA RR&D Merit Review - Project B588-3RA (SAB0023)

Years: 1996-1999