Currently, the main method for reconstruction of extensive head and neck defects is free autograft transplantation [1–3]. However, the loss of such structures as lips, eyelids, nose makes allotransplantation of a composite facial flap the only approach allowing fully-fledged rehabilitation [4–6].
To date, 40 composite facial graft transplantation surgeries have been executed in the world. The first successful operations were performed in 2005 [7], yet this type of surgical intervention remains unique and requires involvement of highly qualified specialists in the preparation, intervention itself, further observation and rehabilitation [8]. The high immunogenicity of the skin, which increases the risks of graft rejection, is still a big problem faced by the teams performing such manipulations. Currently, there is no single approach to the intervention, with a number of solutions suggested. Humanity of experiments and preservation of life of experimental animals remain an important requirement.
To date, laboratory mice remained the animals of choice for experimental facial graft transplantations [9].
This study aimed: 1) to develop and test experimentally the composite facial graft cross-transplantation technique on minipigs; 2) to develop and test on the subjects postoperative therapy and rehabilitation courses, assess the acute rejection diagnostics approach, develop a competent immunosuppressive therapy plan; 3) to test the anesthetic aid used to reduce the risks of perioperative complications.

METHODS

The participants of the experiment carried out three series of facial graft transplantation surgeries on specially selected animals, minipigs, as biological models.
For the experiment, 26 closely related animals were selected: brothers aged from 8 to 24 months, weighing 10–20 kg [10, 11].
The surgeries involved two animals in parallel and took place in a prepared operating room. The participants used standard surgical instruments. An operating microscope was used microscopy stage. As part of the preparation for surgery, we marked the composite facial graft on one animal and, to ensure the maximum possible level of precision, used a template to repeat the same on the other animal. Collecting the grafts, we mobilized the soft tissue components of the flaps while preserving vital structures, keeping the vascular bundles intact to the level of their branching from the external carotid arteries and connecting to the jugular veins, and isolating the facial nerve for subsequent neuroraphy. The bone parts of the grafts were mobilized atraumatically with a piezosurgical tool; after transplantation, they were fastened with Conmet miniplates and miniscrews. Post-surgery, we took biopsy samples dynamically on the 7th, 14th, and 21st days. The samples were used to verify the reparative processes. In case of any signs of rejection, the biopsy samples were collected outside the adopted schedule. We took photos and recorded videos at all stages of the experiment (fig. 1, fig. 2).
We considered various combinations of flaps with the aim to include the most common flaps designs in our work (tab. 1).

Execution of the 1st stage

At the first stage, we carried out experimental facial graft cross-transplantations on five pairs of minipigs (brothers, age — 24 months, weight — 16–20 kg). In the context of these surgeries, we tested and applied the main techniques and flap designs, with the technique application involving all the key stages (fig. 11– fig. 13):
– facial musculocutaneous flap from the buccal, parotid regions;
– composite skin-musculoskeletal flap from the buccal, parotid regions and the lower jaw;
– composite skin-musculoskeletal flap form the paraorbital, buccal, parotid regions and the upper jaw.

Surgical interventions were performed under intravenous anesthesia (rometar 0.15 mg/kg + zoletil-100 2 mg/kg) without anesthetic support. The average time of surgery was 14 hours.
Post-surgery, the animals received an antibacterial drug (Baytril for 14 days) and 120 mg of prednisolone i.m. OD throughout the entire follow-up period.
On the 5th day after the operation, two animals developed edema. They were subjected to pulse therapy, and their scheduled prednisolone intake was increased to 240 mg. Five days after, we registered thrombosis of the anastomoses caused by the intensified vascular reaction to hyperergic response of the recipient's body.

Execution of the 2nd stage

At the second stage, we cross-transplanted facial grafts on four pairs of animals (two pairs — brothers, age — 24 months, weight — 20 kg; two pairs — brothers, age — 8 months, weight — 8 kg).
In this experiment, we tested cross-transplantation of the following flap designs:
– facial musculocutaneous flap from the buccal, parotid, lower paraorbital regions;
– facial musculocutaneous flap from the parotid region with auricle and buccal part.
Surgical interventions were performed with anesthetic aid, under intravenous sedation (rometar 0.15 mg/kg, zoletil-100 2 mg/kg, propofol 4 mg/kg, xyla 0.2 ml/kg) and supervision of anesthesiologists. The average time of surgery was 10 hours.
Post-surgery, the animals received 3 ml of Baytril i.m. OD (antibacterial therapy) and 16 mg of dexamethasone i.m.
OD (immunotherapy) throughout the entire follow-up period.
Same as at the 1st stage of the experiment, we registered a delayed development of rejection. Clinical manifestations were relieved by pulse therapy (360 mg of solumedrol i.m.).
On the 21st day post-surgery, we collected histological material from the place of fusion of the transplanted flap and the recipient's tissues for histological control.

Execution of the 3rd stage

At the 3rd stage, we cross-transplanted facial grafts on four pairs of animals (four pairs — brothers, age — 8 months, weight — 10 kg). Analysis of the results of the previous stages allowed us to adjust perioperative therapy and the anesthesia protocol. Intra- and post-surgery, we subjected the animals to immunosuppressive therapy [12].
To prevent immediate loss of grafts for immunological reasons, we determined blood group compatibility and performed the microlimphocytotoxic test on the eve of the operation. The fact that each animal was both a donor and a recipient simultaneously was factored in. Individual blood compatibility was checked with the help of room temperature crossmatching.
Based on the results of a series of immunological tests, we made four pairs of animals that underwent a total of eight transplantation surgeries. In each case, the individual compatibility and the microlymphiphocytotoxic tests returned negative.
In this experiment, we continued testing composite flap designs, namely:
– facial musculocutaneous flap from the buccal and parotid regions, with neuroanastomoses made in the region of facial nerve branches;
– facial musculocutaneous flap from the parotid region with external part of the auricle, buccal region, with neuroanastomoses made in the region of facial nerve branches (fig. 14).
Surgical intervention was performed with anesthetic aid under intravenous sedation (zoletil-100 2 mg/kg, propofol — 4 mg/kg, xyla — 0.2 ml/kg). The average time of surgery was 8 hours.
Based on the additional advice received through consultations with transplantologists and anesthesiologists, we adjusted the drug therapy as follows.
Pre-surgery: 8 hours before intervention — low molecular weight heparins (clexane), s.c.; antibiotic therapy — 1 ml of interspectin i.v. 30 minutes before the incision.
Intraoperatively, two pairs of subjects received: 0.15 mg/kg of Prograf i.v.; heparin before the blood flow was resumed.
Post-surgery, experimental models received: antibiotics (1 ml of interspectin per 10 kg of weight i.m. OD) for 14 days with the aim to prevent secondary bacterial complications; immunosuppressive drug (Solumedrol 160 mg/m) throughout the follow-up period.
We did not register pronounced manifestations of flap rejection post-surgery. The persisting edema were attributed to the volume of intervention and hypersecretion of the salivary gland.

RESULTS

We had the subjects surviving long-term at all stages of the experiment, which indicates humane use of animals. Post-surgery, their vital functions remained unchanged (tab. 2). We succeeded in improving the survival rate of models after surgical interventions (tab. 3).
Histological examination (fig. 15) of the recipient–donor boundaries revealed the ongoing primary adhesion process, which prevents acute rejection as it is described in the Banff classification [13, 14].
Figure 15 shows the skin and the subcutaneous tissue, consisting of two fragments, separated by the wound.
The first fragment (recipient) is a skin flap with platysma. The skin is a set of ordinary layers with signs of keratinization and accompanying elements (hair follicles, sebaceous glands). Fatty tissue includes vessels of various sizes. Platysma is of the usual structure, it consists of longitudinal and transverse muscle fibers. In the deep layer, there are glandular structures.
The second fragment is the skin flap with platysma. The skin is a set of ordinary layers with signs of keratinization and accompanying elements (hair follicles, sebaceous glands). Fatty tissue includes vessels of various sizes. The typical platysma of longitudinal and transverse muscle fibers has narrow strands of granulation tissue penetrating it. The vessels contain form elements.
The wound is a narrow slit filled with granulation tissue of low cellularity. The granulation tissue mainly consists of small capillaries and interlayers of connective tissue with thin fibrils. It is practically not infiltrated with polymorphonuclear leukocytes (neutrophils), lymphocytes. They are found only in the surface layer under a patch of necrotic epidermis. Along the wound slit, infiltration with multinucleated cells can only be seen from the side of the first fragment.
tab. 4 shows the results of graft retention depending on the therapy regimens in the peri- and postoperative periods. It should be noted that the response is more effective in the cases where acute rejection reactions were purposefully relieved.

DISCUSSION

Even with the histological analysis confirming graft healing, it is necessary to closely observe the dynamics of the processes post-surgery and adjust the immunosuppressive therapy regimen with minimum possible delay following registration of signs of the acute tissue rejection reaction.
Having analyzed the results of our experiment and considered the cases of development of acute graft rejection, we concluded that it is necessary to continue development and testing of the immunosuppression regimen, which is consistent with the results other researchers have arrived at [15]. Another group of researchers has discovered that the features of the composite graft play a role in the development of rejection in one of its components [16], which leads to loss of the skin part of the flap while its muscle components remains.
Thus, the question is raised about the need to select objective methods for diagnosing the state of all components of the flap. Also, compared to single organ transplantation, surgeries involving composite grafts require greater attention to the specific features of such grafts.

CONCLUSIONS

The experimentally tested composite facial graft cross-transplantation technique allows all members of the team (surgeons, anesthesiologists, transplantologists, immunologists) to practice and improve their skills involved in the preparation, conduct of the surgery and postoperative rehabilitation of face transplant patients. Extended anesthetic aid was registered to decrease the operating time and improve survival rate of the subjects post-surgery.
The immunosuppressive therapy applied at this stage of the experiment requires further adjustment and testing to reduce the risk of development of acute or chronic rejection.
The emphasis on the unique features of composite grafts may allow additional, more specific treatment, which can multiply the life expectancy of patients with such grafts. Given the above, it is worth considering the possibility of using alemtuzumab perioperatively in addition to the plan typically followed in the context of transplantation surgeries.