Extremity transplantation: A review of its current state of development

The discovery of a powerful immunosuppressive, cyclosporin A, together with the development of a microsurgical free tissue transfer technique, has brought extremity transplantation closer to being a realistic possibility. In this review the authors describe the history of laboratory

research into extremity transplantation before immunosuppression, with early immunosuppres- sive agents, with cyclosporin A, and with FK-506, one of a new generation of immunosuppressives. (J HAND SURG 1993;18A:153-9.)

Liana M. Nolan, MD, and Vaughan Bowen, MD, FRCSC, MBChB, Toronto, Ontario, Canada

T he discovery of a powerful immuno-

suppressive, cyclosporin A, together with the devel- opment of a microsurgical free tissue transfer technique,

has brought the dream of extremity transplantation much closer to fruition.

Cyclosporin A has been successfully used in trans-

plantation of vital organs, but the toxic nature of its side effects still precludes its clinical application in non- life-threatening situations. The autograft, therefore, is still the workhorse of microvascular reconstruction of the upper and lower extremities, although it is clear

that allograft free tissue transplantations would have much to offer patients with mutilating limb defects.

The medical literature contains a number of articles describing laboratory research into extremity allografts in animal models. Investigators have been studying some of the surgical and scientific aspects of extremity transplantation with the use of available immuno- suppression while waiting the development of either a

less toxic immunosuppressive or some other technique

From the Divisions of Ortbopaedic and Plastic Surgery, University

of Toronto, Toronto General Hospital, Toronto, Ontario, Canada.

Supported by a grant from the Medical Research Council of Canada.

Received for publication Oct. 11, 199 1; accepted in revised form

May 15, 1992.

No benefits in any form have been received or will be received from

a commercial party related directly or indirectly to the subject of this article.

Reprint requests: Vaughan Bowen, MD, EN10-243 Toronto General

Hospital, 200 Elizabeth St., Toronto, Ontario, M5G 2C4, Canada.

3/l/41460

for avoiding the problems of rejection. At the present time the medical journals contain no reports describing clinical extremity-transplant procedures.

Important milestones in the development of extremity transplantation are listed in Table I.

Historical background

The first recorded account of an extremity transplan- tation was written in AD 1270.’ This is the story of Saint Cosmos, a physician, and Saint Damian, a surgeon. They were twins who lived in the Roman Empire in the second half of the third century. As devout and humble Christians, they cared for the sick as they trav-

eled throughout the empire. They inspired many other Christians and, as a result, were martyred by the Ro- mans in the year AD 287. Many miracles of healing

were attributed to both of them during their lifetimes and after their deaths.

The miracle of the black leg is thought to have oc- curred in AD 348 at the site of a Roman basilica ded- icated to the saints. One night in the basilica, an elderly white man with a cancerous leg was praying to the

saints. After he fell asleep, he heard Cosmos and, Dam- ian speaking. They decided that his leg was rotten and would have to be removed. After sawing off the older man’s leg, they replaced it with a leg from a recently deceased Ethiopian Moor. When the man awoke, he found that he was cured and his pain was gone. His new leg was perfectly healthy but was the leg of a black man. The man’s cancerous leg was found in the grave beside the Moor’s body. At the time that this miracle was recorded, extremity transplantation was thought to be impossible.

0363-5023/93/$1.00 + .lO THE JOURNAL OF HAND SURGERY 153

154 Nolan and Bowen

Table I. Milestones in the development of Table III. Investigations carried out before the extremity transplantation era of cyclosporin A

Year Event

3rd century BC

1962

1966

1976

1979

1982

Cosmos and Damian; miracle

of the black leg

First report of experimental

extremity transplantation

by Schwind

First experimental extremity

allograft involving immu-

nosuppression by Goldwyn

Cyclosporin A described as a

powerful immunosup-

pressive

First use of cyclosporin in

human patients for organ

grafting by Calne et al.

First report of cyclosporin A

use in extremity transplan-

tation by Black, et al.

Agent

Azothiaprine

6-Mercaptopurine

Azothiaprine hydrocor-

tisone

f Splenectomy and

thymectomy

f Exchange transfusion

or donor splenic

cells

Azothiaprine

Prednisolone

6-Mercaptopurine

Animal

Dog

Dog

Rat

Investigator

Goldwyn et al.

(1966)

Lance et al.

(1971)

Doi (1979)

In 1973 Lapchinsky et al4 induced immunologic tol- erance in dogs by performing a subtotal blood exchange

in newborn puppies. After one exchange of donor blood, the puppies were allowed to grow to the size of the mature donor over a period of 1 to 2 years. Sub-

sequently a limb al&raft was performed. Of the 12 animals in the experiment, one dog was reported to have lived for 7 years and the transplanted limb was described as having weight-bearing capabilities.

Table II. Investigations carried out

before immunosuppression

Technique

Parabiosis

Subtotal exchange transfu-

sion in newborn

Immunologic enhancement

with recipient-derived

antidonor antiserum

Animal

Rat

Dog

Rat

Investigator

Schwind (1962)

Lapchinsky et al.

(1973)

Poole et al.

(1976)

Extremity transplantation before immunosuppression

Experimental extremity transplantation began in

1961.’ Initially, the attempt to overcome immune in-

compatibility involved methods other than immuno-

suppressive drugs (Table II). Techniques involved al- teration of the immune response with methods not ap- plicable to man.

Initially, Schwind’ induced immunologic tolerance

in rats by parabiosis. Parabiosis is defined as “the ex- perimental fusing together of two individuals or em- bryos to study the effects of one partner upon the other.“3 Schwind found that rats of different genetic backgrounds could be successfully united at the point of partial amputation of a limb before the age of 2 weeks. At this stage the animals were immunologically immature and unable to mount a rejection response. Two weeks after being united, the partially amputated limb was completely severed from the donor so that the recipient animal now had an extra limb. The limbs on two recipients survived for more than 11 months.

The Journal of

HAND SURGERY

In 1976 Poole et a1.5 induced tolerance in rats by using immunologic enhancement. In one group of an- imals a recipient-derived antidonor antiserum was given

1 or more months before a limb allograft procedure. In another group the antiserum was not given until the first day after a limb allograft procedure. Rats that were given immunologic enhancement 1 month before the limb allograft procedure had longer allograft survival (21 to 207 days) than the rats who received the anti-

serum only after the operation (12 to 16 days). These early experiments were interesting, and suc-

cess was attained. Clinically, however, they were im-

practical.

Extremity transplantation with early immunosuppressive agents

Other investigators of extremity transplantation used immunosuppressive agents in techniques that were sim- ilar to those used for human vital organ transplantation (Table III).

In 1966 Goldwyn et a1.6 used azathioprine and 6-

mercaptopurine in a dog experiment involving hind limb homograft rejection with and without immunosuppres- sion. In five dogs that received homografts without im- munosuppression, rejection was evident by the sixth postoperative day. In seven of eleven dogs treated with immunosuppression, rejection was delayed and was not evident until a mean of 18 days postoperatively. The

Vol. 18A, No. 1 January 1993 Extremity transplantation 155

problem was that the dogs did not tolerate azathioprine well, and some had fatal complications.

In 1971 Lance et al.’ transplanted hind limbs beween unrelated beagles in a complicated study that combined

both pharmacologic and nonpharmacologic methods of enhancing immune tolerance. The results were en-

couraging, for long-term survival was achieved. In one group, three dogs had massive immunosuppressive ther-

apy consisting of azathioprine and hydrocortisone ac- etate, plus or minus splenectomy and thymectomy, fol- lowed by induction of immune tolerance from donor

splenic cells or exchange transfusion. Two dogs sur-

vived beyond 60 and 300 days. One dog did not reject the transplant until 200 days after the procedure.

Eight years later, in 1979, Do? described a study of rat limb homografts with and without continuous im-

munosuppression by means of a variety of combinations of azathioprine, prednisolone, and 6-mercaptopurine. Ninety-seven limb homografts were completed, making this the largest series up to that time. Nonimmunosup- pressed limbs were again rejected early, at an average of 12% days. Animals that had received immuno-

suppression with azathioprine and prednisolone had an increased survival time, although results were not as good as those reported by Lance et al.’ The longest survival times attained were only 17, 21, and 24 days.

Most animals died of side effects of the immunosup- pressive drugs.

These experiments involving the early immunosup- pressive agents not only appeared more practical, in the clinical setting, than the techniques of parabiosis, immunologic tolerance, and immunologic enhance- ment, but they also yielded better results. Unfortu- nateiy, the immunosuppressive agents used were very

toxic and the animals often died of their side effects. Investigators searched for an immunosuppressive agent with less toxic and nonfatal side effects.

Extremity transplantation with cyclosporin A

The discovery of cyclosporin A was a major step forward. Cyclosporin A was a powerful immunosup- pressive, but it was much less toxic than previously used immunosuppressives. Its discovery allowed vital

organs to be transplanted with much greater safety. The new drug gave renewed opportunity to take a look at the possibility of extremity transplantation. A new era of experimental extremity transplantation followed its introduction.

Background. Cyclosporin A is a fungal metabolite with potent immunosuppressive properties.’ It is ex- tracted from the soil fungi Trichoderma polysporum Rifai and Cylindrocarpum lucidum. Cyclosporin A con-

tains 11 amino acids, most of which are homophohic.

It is therefore lipid soluble. It was first developed as an

antifungal agent, but its antibiotic activity was limited. Bore1 et al. lo discovered its immunosuppressive activity in 1976. One year later the drug was used in animal

models and demonstrated suppression of rejection in heart and renal allografts.’ In 1979 Calne et al. I’ first described the clinical use of cyclosporin A in renal,

pancreas, and liver transplantation. Mechanism of action. The mechanism of action of

cyclosporin A is not well understood. The drug interacts

with the immune response in many ways. It affects both

humoral and cellular responses. Its main effect, how-

ever, is on cellular immunity, where it acts preferen- tially on T lymphocytes. I2 Cyclosporin A selectively

blocks early steps in T lymphocyte activation.13 It is this preferential T cell inhibition that is crucial for the

prevention of allograft rejection. I4 Rejection can be defined in many ways, and one can

use gross, microscopic, or immunologic criteria. It is necessary for each investigator to define his or her own criteria of rejection. Goldwyn et al6 noted that, micro-

scopically, rejection begins around small vascular chan- nels and predominantly involves infiltration of lym-

phoid cells. They found that, in a composite tissue allograft, skin, skeletal muscle, and connective tissue

were most affected. Doi,’ in his composite tissue al- lograft study, described the process of gross and mi-

croscopic rejection. Grossly, edema, discoloration, ul- cers, and finally necrosis were seen. Microscopically, each tissue was rejected at a different rate. Changes included edema and perivascular infiltration of lym- phocytes and polymorphonuclear cells. In muscle, thrombus formation was seen in the vessels. Fritz et al.” measured rejection in composite tissue allografts using three parameters: rejection was manifested clin-

ically by swelling, erythema, and skin changes; his- tologically by cellular infiltrate, skin atrophy, and vas- culitis; and immunologically by the presence of hem-

agglutination and cytotoxic antibodies. They described skin, muscle, and bone separately because of their dif- fering antigenicity.

Toxicity and side effects. Although much less toxic than previous immunosuppressive agents, cyclosporin A has been associated with some serious side effects.‘?

Cyclosporin A is nephrotoxic and can produce acute renal failure in the first few weeks of its use or chronic nephropathy when used over longer periods of time. Nephrotoxicity seems to be related to dose and length of treatment.

Cyclosporin A can produce hypertension. I3 It also affects the liver. Serum transaminase, alkaline phos- phate, and bilirubin levels may rise, but this does not seem to be clinically significant.

The Journal of 156 Nolan and Bowen HAND SURGERY

Other problems with the use of cyclosporin A are related to its immunosuppressive qualities. It has been

noted that there is an increased incidence of Pneumo- cystis carinii and viral infections in those receiving this

drug. Hypertrichosis and gingival hyperplasia occur in 10% to 30% of the patients and do not appear to be dose related.

Nausea, weight loss, and tremor have also been ob-

served during the initial doses of the drug. Application. Cyclosporin A is now routinely used

clinically for kidney, liver, and heart transplantation.

Results are better than with previous forms of immu- nosuppression. Success in lung and heart-lung trans-

plantation has been unprecedented, although the drug has not been so effective for pancreas and bone marrow transplants. ”

Unfortunately, the toxicity and side effects of cyclo- sporin A, although much improved over those of earlier immunosuppressives, are still too serious to allow the

drug to be used in the management of non-life-threat- ening problems. At the present time, therefore, the use

of limb allografts for extremity reconstruction is still associated with unacceptable risk, and limb transplan-

tation remains restricted to animal research.

Extremity allografts differ from vital organ allografts

in that they are composite-tissue allografts. Each com- ponent of a limb allograft (skin, bone, muscle, nerves, vessels, etc.) has a different degree of antigenicity. Skin is particularly antigenic and can undergo rejection while underlying tissues remain intact.16 This requires in- creased doses of cyclosporin A with an accompanying

increase in toxicity. Extremity allografts also differ from vital organ al-

lografts in that nerve regeneration is essential to their success. Nerve repairs are not important in transplan-

tation of vital organs, but extremity transplants would be functionally useless without neurologic recovery.

Several experiments have been performed with the use of cyclosporin A in limb allografting. Hewitt and Black and their associates’6-‘8 first described such an

experiment in 1983. In inbred adult rats, they showed that marked prolongation of survival occurred with cy- closporin A as compared with conventional immuno- suppression. They subcutaneously injected recipients with cyclosporin A daily for 20 days postoperatively. Mean survival times, based on a drop in limb temper- ature below 32” as evidence of rejection, were 101 & 13 days in the cyclosporin A group compared with 18 _t 5 days in the control group.

Fumas et al. I9 wanted to determine whether indefinite survival was possible. They found that five rats treated

daily with cyclosporin for 20 days and then with main- tenance cyclosporin twice weekly after an allograft had

prolonged survival. Two of the five rats showed no evidence of rejection, and three showed evidence of skin rejection only. Allografted limbs were still alive after 404, 420, and 441 days in three of the rats. The

other two rats died of pulmonary infection on days 84

and 95. Fritz et a1.15 also transplanted adult rat limbs with

the use of cyclosporin A. Their rats received cyclospo- rin A daily for 0, 7, or 21 days or continuously until the time of death. Some rats treated for short periods of time showed signs of rejection after cyclosporin A

was discontinued. Five animals treated continuously for up to 113 days showed no signs of rejection.

In 1984 Kim et al.” described allotransplantation of adult rat limbs, comparing the effectiveness of cyclo- sporin A with azathioprine and prednisolone. Nine an-

imals received cyclosporin A continuously for 14 days (surviving 33 ? 3 days), and six animals received it for 60 days (surviving 68 days t 2 days). Cyclosporin

A was found to suppress rejection as long as treatment was continuous. They also found that there was a tem- porary posttreatment immune tolerance. Conventional agents allowed rejection while treatment was in progress and did not significantly prolong survival.

In 1983 Press et al.” reported preliminary results of limb allografts in adult rats with the use of intraperi-

toneal cyclosporin A and prednisone. Using 26 animals, they found that they were unable to prolong graft sur- vival. Loss was due to infection and sepsis. Rejection was either mild or absent at the time of loss. They concluded that their cyclosporin dose was too high. The

experiment was repeated with a decreased dose of im- munosuppressant.” Limb survival was prolonged, but skin rejection was not prevented.

In 1983 Lipson et a1.23. 24 described a modified or- thotopic limb transplantation in inbred rats. The foot and skin of the donor were removed, and recipient skin was used for coverage. No immunosuppression was used, and rejection of bone was studied. They found that, histologically, rejection was associated with ces- sation of new bone formation and eventual death of osteocytes. Bone scanning was found to be valuable for monitoring rejection.

In 1982 Silisk?. 26 reported whole-knee joint allo- grafts in rabbits in which cyclosporin A was used as the sole immunosuppressive agent. Five of 14 allografts maintained patent vessels and living bone at 12 weeks. Nonimmunosuppressed animals underwent inflamma- tory wound breakdown at 2 to 4 weeks.

Vol. 18A, No. 1 January 1993 Extremity transplantation 157

Five years later, Stark et al.” described a series of hand transplants in baboons given daily doses of cy- closporin A and prednisolone for immunosuppression. The prednisolone dose was increased if signs of rejec- tion developed. In six animals, the hands were lost between 2 and 15 days postoperatively. In the seventh

animal, the hand survived until the animal was killed at 296 days. Electromyography and nerve conduction

studies indicated that neural recovery was comparable to that seen in human replants. In all animals skin re- jection and infection were a problem.

Daniel et al.** also reported on hand transplantation in baboons. Four hand transplants and seven neurovas- cular free flap transplants were performed with the use of cyclosporin A and methylprednisolone for immu-

nosuppression. Long-term survival, ranging from 59 to 304 days, occurred in nine animals. Two animals were killed because of rejection at 26 and 71 days. Three animals had reversible rejection, and a fourth had

chronic but controllable rejection. Electrophysiologic testing suggested that sensory and motor reinnervation

will occur in the presence of high levels of cyclosporin A.

In 1987 Guzman-Stein and Shons29 described the transplantation of immature rat hind limbs with an em- phasis on functional recovery. Pinprick stimulation, muscle tone, and toe movement were used for the sub- jective assessment of results in ten allograft transplants. Muscle biopsies were used for objective evaluation. All animals demonstrated reinnervation of muscle and skin,

normal bone healing, and long bone growth. Two an- imals had episodes of rejection, which were completely reversed by a short-term increase in the immunosup- pressive dose.

In 1989 Hotokebuchi et al.3o reported limb allografts in immature rats with the use of cyclosporin A. They

studied epiphyseal plate behavior in 20 recipients and found that neither cellular architecture nor growth in length varied from those of syngrafted controls. They subsequently reported the use of cyclosporin A with whole-joint allografts in rats and described the histo-

logic changes seen in articular cartilage. In 35 major mismatched allografts, the articular cartilage of animals treated with cyclosporin A displayed normal architec- ture and viability 52 weeks after operation, despite ev- idence of skin rejection. The animals that were not treated with cyclosporin A underwent destruction of articular cartilage by 6 weeks. These results were en- couraging, although graft-versus-host disease was seen in half the cyclosporin-treated animals by the end of 1 year.

Kniha et al.32 reported orthotopic forelimb trans-

plantation in 17 immature rabbits treated with cyclo- sporin A. All limbs showed bone growth. Growth in the transplanted limb was approximately 75% to 80% of the growth in the control limb. Response to pain stimuli was seen at 2 to 3 months. Most of the animals

had a wasting syndrome, which had been previously reported in rabbits treated with cyclosporin A.33

The future of extremity transplantation

The studies involving the use of cyclosporin A in

limb allografting are encouraging. Limb rejection seems to be indefinitely prevented provided that cyclosporin A is continuously administered. Infection remains a problem with higher doses of cyclosporin A, and skin

rejection is a problem with lower doses. In immuno- suppressed allograft transplantation, bone, nerve, and cartilage appear to behave much as in replanted limbs. Unfortunately, cyclosporin A is associated with a risk

of nephrotoxicity that is still too great for limb trans- plantation to be acceptable in the human clinical situ-

ation. The future of limb transplantation rests with the discovery of immunosuppressive techniques that are

less toxic but equally effective as cyclosporin A. One possible alternative currently being studied is

FK-506. This drug, which was first described in 1987,34

is a fungal metabolite extracted from Streptomyces tsu- kubaensis and has a completely different structure from cyclosporin A. FK-506 was found through routine

screening to have immunosuppressive properties, and its effects occur at concentrations about 100 times lower than that of cyclosporin A.

Investigators have already used FK-506 for experi- mental allograft transplantation. Kuroki et al.35 de- scribed the use of this agent for limb allografts in rats with immunosuppressive results similar to those of cy-

closporin A at 1 / 50 the concentration. Arai et al. 36 have also used FK-506 in rat limb allografts. They admin- istered a 14-day course of FK-506 postoperatively and demonstrated an increase in graft survival. A subse-

quent study by the same authors demonstrated suppres- sion and reversal of skin rejection in limb allografts.37 These results are encouraging but they must be viewed with caution since Pneumocystis carinii infection oc-

curred in many of the rats receiving long-term FK-506. Other problems, such as nephrotoxicity, still have not been eliminated with this new drug; this suggests that the ultimate solution” is yet to be discovered.

Microsurgeons have the technical capability to per- form allograft extremity transplantations. Possibly FK- 506 or some other fungal metabolite will prove to be

158 Nolan and Bowen

clinically useful in human patients. Even if they do not,

immunologists will continue to investigate their prop- erties in an effort to better understand their mechanisms

of action and side effects. Once an immunosuppressive with a good side effect profile is found, the risk for this

procedure will be acceptable in human beings. Once it becomes safe to perform limb transplantation proce-

dures, it is likely that functional results will be satis- factory. The experimental work that has been carried

out indicates that function in successfully immunosup- pressed allografts will be comparable to that which fol- lows replantation.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

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