Composite tissue xenopreservation: Preliminary results of staged VCA in rat to mouse model

The time between procurement and transplantation of composite tissues, especially regarding the limited donor pool, is a challenge effecting the outcomes of the transplantation. Current preservation techniques mainly include either cold preservation with a solution or machine perfusion using blood or certain oxygen‐carrying solutions. However, none enables preservation beyond 24 h. Increasing this time to several days will provide better usage of the donor pool, safer transplantation of VCA with significant muscle content, and gives time to stabilize a patient before long surgical procedures. Herein, we described a novel strategy of xenopreservation (preservation via xenotransplantation) to preserve composite tissues for 7 days, followed by staged transplantation.

patient before long surgical procedures.Herein, we described a novel strategy of xenopreservation (preservation via xenotransplantation) to preserve composite tissues for 7 days, followed by staged transplantation.

Materials and Methods:
We used two concordant species, female Sprague Dawley rats (n = 10) and female CF-1 mice (n = 10) in this study.Four of pair of animals are used for anatomical study.The groin flap of the rat was used as a xenograft and xenotransplanted to the neck area of the carrier mouse.Cyclosporine (CsA) was administered used as immunosuppressant.After 7 days of preservation on the mouse neck, xenotransplanted groin flap (called xenopreserved flap) was re-harvested, skin and vessels samples were collected for histopathological evaluation, and the xenopreserved flap was transplanted to the donor rat's opposite groin area.Anastomoses were performed between the flap's pedicle and the femoral vessels.Clinical observation regarding inflammation and tissue perfusion of the xenopreserved flap was monitored daily.Fifteen days after the second surgical procedure, the rats were euthanized, and skin and vessel samples were collected.Histologic evaluation, including inflammatory cell numbers, was performed.Wilcoxon test was used to compare the changes in inflammation severity and p < .05 was set for statistical significance.
Results: All xenopreserved groin flaps except one survived.Mean lymphocyte count before the second operation (at the end of the xenopreservation procedure) was 20,22 ± 0.44 and reduced to 13,14 ± 0.47 at the end of 15 days, and the difference was statistically significant (p < .05).
Fatih Zor and Rezarta Kapaj have contributed equally as first author.

Conclusion:
This proof-of-concept study with preliminary results showed that xenotransplantation might be a novel strategy for preservation of VCA for a certain period of time.However, additional translational studies are needed to modulate the tissue changes following xenopreservation.

| INTRODUCTION
Vascularized composite allotransplantation (VCA) opened a new era and enabled reconstruction of otherwise unreconstructable defects by means of transplantation (Siemionow et al., 2009;Siemionow & Kulahci, 2007).With the gaining popularity, new applications of the VCA have been described.However, in VCA, unlike organs, matching of skin color, tone, gender, and size of the graft, in addition to blood type between donor and recipient, limits suitable donors (Cherikh et al., 2019;Datta et al., 2015;McDiarmid, 2013).In an effort to maximize donor yield for transplant, hospitals have focused on maintaining organ function until the declaration of brain death and expedient organ/tissue procurement.Despite these measures, given the national donor allocation system managed by the 59 OPOs in conjunction with the Organ Procurement and Transplantation Network (OPTN), some donor organs/VCA need to be shared across a broader geographic distance-increasing the cold ischemia time (CIT).Similar efforts per federal policy to streamline VCA allocation and improve recipient matching may indeed mandate donor VCA sharing across wider geographic distances-increasing CIT.Thus, any measures to increase the donor pool will significantly impact not only solid organ transplantation but also, more importantly, VCA (Datta et al., 2015;McDiarmid, 2013).Moreover, in some instances, general condition of the patient may not allow such lengthy general anesthesia requiring procedure (Hales & Pullen, 1982;McCutcheon & Hennessy, 2002).
Perioperative hemodynamic problems are the leading cause of early complications.As a result, transplantation or replantation of a major extremity, including large amount of muscle, following a certain time of ischemia causes serious and life-threatening complications (Gordon et al., 2011;Siemionow et al., 2010;Wang et al., 2011).
Preservation of composite tissues is a subject that is not new and may be a novel strategy for reducing the above-mentioned transplantation/replantation related perioperative complications.There are several experimental studies that report limited success of composite tissue preservation (Morgan et al., 1982;VanGiesen et al., 1983).Currently, there are tissue banks where skin, oocytes, and sperm are preserved successfully.These centers work by the cryopreservation principle.However, the ideal preservative agent and temperature are different between tissues (i.e., the ideal agent for one tissue might be fatal for another).Thus, currently, cryopreservation is not suitable for composite tissues (Bakhach, 2009a;Fahy, 1986).Although there are some encouraging cryopreservation studies, the need to preserve composite tissues has not yet been fulfilled in current plastic surgery practice (Arav et al., 2017;Rinker et al., 2007;Wang et al., 2014;Zhang et al., 1998).
Novel tissue preservation strategies that accomplish prolonged tissue oxygenation over 24 h could facilitate the logistics of VCA across geographic domains or facilitate staggering the individual transplants (hands or face) as separate procedures over a period of 1-2 days, allowing for hemodynamic recovery/stabilization and improve immunologic, neurofunctional and graft survival outcomes (Gordon et al., 2011;Siemionow & Klimczak, 2010).There is, thus, a critical unmet need for tissue preservation strategies in VCA to effectively prolong the procurement-to-transplant window beyond 24 h (Gorantla et al., 2016;Gorantla & Davis, 2017).
Xenotransplantation (XT) is born from the increasing need for organs and is a science with the potential to become one of the "bridging therapies" in the future (Starzl et al., 1993;Taniguchi & Cooper, 1997).However, XT immunology, arising from molecular mismatch and differences between species, is very different from allotransplantation immunology (Auchincloss & Sachs, 1998;Galili et al., 1999).Xenotransplantation can be between either discordant or concordant species (Calne, 1970;Chaline et al., 1994).When performed between concordant species, hyperacute rejection (HAR) does not occur (Chaline et al., 1994).Instead, acute cellular rejection occurs, which is similar to allograft rejection (Calne, 1970;Elwood et al., 1998;Najarian, 2003;Sachs et al., 2001).Although organ xenotransplantation is not a new science, there are only a few studies about vascularized composite xenotransplantation (Hebebrand et al., 1998;Tanabe et al., 2000).Thus, we hypothesized that xenopreservation (using xenotransplantation for tissue preservation) might be used to preserve vascularized composite tissues beyond 24 h, and transplantation of xeno-preserved VCA would be possible without any significant immunologic drawbacks.

| MATERIALS AND METHODS
This study was carried out in strict accordance with Institutional Animal Care and Use Committee and American Association for Laboratory Animal Care guidelines.Sample size and power calculations were conducted using SAS for windows assuming a difference between means of 7 units, a standard deviation of differences of 3 units, and a correlation of 0.5 between before and after measurements.A significance level of 0.05 was chosen for the study, and a power of 0.8 was assumed.Based on the assumptions provided, the sample size calculation indicated a total required sample size of 4.However, to account for potential losses during the study, an additional two rats were included, bringing the total sample size to 6. Additionally, four pairs of animals are used for setting up the rat-to mouse xenotransplantation model.
We used two concordant species, Sprague Dawley rats and CF-1 mice (all female).Following institutional ethical board approval, Sprague Dawley rats (n = 10) aged between 10 weeks and weighing between 250 and 300 g were used as donors.Mice (n = 10), aged around 10 weeks and weighing approximately 20 g, were used as carriers.Groin flap was used as the VCA model.All experimental subjects were followed in our Animal care center and fed in their cages with light-darkness cycles of 12 h.The feeding and water were given in habitus, and the cages were cleaned daily to avoid infections.

| Surgical procedures
All operations were performed under sterile conditions and general anesthesia, induced with intraperitoneal injection of 50 mg/kg Ketamine HCl (Ketalazar, Pfizer, Turkey) and 5 mg/kg xylazine HCl (Xylazine, Biopharm, Turkey).

| First operation
Left inguinal region of the donor rats was prepped and draped.A groin flap was planned in 1 Â 1 cm dimensions and harvested as described in the literature (Hsu et al., 2018;Zor & Siemionow, 2015).A midsagittal incision was made on the carrier mouse's neck area, and the submandibular gland was excised.The external jugular vein was found, dissected, and prepared for venous anastomosis.The sternocleidomastoid muscle was excised with electrocautery.The external carotid artery was found, dissected, and prepared for arterial anastomosis.
Later, the flap was transferred to the carrier mouse, and end-to-end arterial and venous anastomoses were performed using 11.0 Ethilon (Ethicon, Inc., Somerville, NJ) sutures.Following vessel anastomoses and confirmation of the tissue perfusion, the flap inset was performed (Figure 1a, b).The defect in the donor rat was closed primarily.To prevent hemodynamic problems, 0.25 cc of warm saline was infused via the opposite jugular vein, and 1 cc of warm saline was injected intradermally to the carrier mouse.The mice were kept in a warm environment for 1 h and then carried to their cages.Both donor rat and recipient mouse were followed up for eventual hemorrhages and kept in individual cages thereafter.In order to prevent rejection, 20 mg/kg/d Cyclosporine A was administered to the recipient mouse.
The xenografts were evaluated on a daily basis for any signs of flap failure and rejection as described in the literature (Casal et al., 2017;Kulahci et al., 2016;Ozmen et al., 2006;Zor et al., 2019).decided by for hair loss, desquamation, epidermolysis, exudation, and stiffness.During the follow-up period, any signs of acute rejection are also clinically observed using the Banff grading system (Cendales et al., 2008).

| Second operation
After 1 week, carrier mice were taken to the operation theater for the second procedure.Under general anesthesia, the transplanted groin flaps (xenopreserved flaps) were re-harvested based on the same pedicle (Figure 1c).Tissue samples were obtained from skin, subcutaneous tissue, and femoral vessels.An incision was made in the opposite groin of the respective donor rat.The femoral artery and veins were exposed and prepared for anastomosis.
The xenopreserved flap was transferred to the right inguinal region of the rat, and anastomoses were performed in an end-to-end fashion by 10.0 Ethilon sutures (Figure 1d).Following the flap inset, 2 cc of warm saline was injected intradermally.All flaps were observed daily for flap failure and rejection, as described above.Carrier mouse was sacrificed.
Fifteen days after the second operation, the study was terminated, and tissue samples were obtained from skin, subcutaneous tissue, and vascular structures.The experimental design is seen in All samples were formalin-fixed H&E stained for histopathologic evaluation by a blinded pathologist.Five-micrometer-thick sections were evaluated at 200Â magnification using Image J program, and the lymphocytes in per 100.000-pixelsquares were counted (Kondo et al., 2004).The severity of inflammation is compared between samples obtained at the end of the xeno-preservation (7 days after the first operation) and at the end of the study (15 days after the second operation).In addition to the initial power analysis that was conducted prior to the start of the study, a post-hoc power analysis was performed after the data collection was completed.Wilcoxon test is used to compare the changes in inflammation severity between the two evaluation timepoints and p < .05 was set for statistical significance.During the follow-up after the second operation, we observed no changes in the flap regarding color, edema, and temperature.All flaps were observed to be vital, with no erythema, edema, desquamation, and other signs of inflammation or rejection (Figure 4) (Cendales et al., 2008).

During
Histopathologic evaluation of the skin and vessel samples obtained from the xenopreserved flap before the second operation revealed that the skin and adnexal structures maintained their structural integrity.However, we found inflammatory infiltration in the skin.We also observed intimal and medial thickening in the arterial and venous vascular structures.
Biopsies obtained at the end of the experiment (15 days after the second operation) showed a reduction in the inflammation of the skin and adnexal structures.We also observed that intimal thickening in the vessel was reversible; however, thickening in the media layer was irreversible.Mean lymphocyte count before the second operation (at the end of the xenopreservation procedure) was 20.2 ± 0.4.However, mean lymphocyte counts 15 days after the second operation was reduced to 13.4 ± 0.4, and the difference was statistically significant ( p < .001)(Figure 5).The results of post-hoc analysis showed a power value larger than 0.99, indicating that the study had a very high likelihood of detecting significant effects or differences between groups.

| DISCUSSION
The time between procurement and transplantation of composite tissues, especially regarding the limited donor pool, is a challenge effecting the outcomes of the transplantation, and there is an unmet need for preservation of VCA beyond 24 h.Here, we described the usage of xenotransplantation for preservation of composite tissues, which is named "composite tissue xenopreservation," and successful F I G U R E 3 Xenopreserved flap is seen at 7 days on the carrier mouse.Note that the flap looks quite healthy.
F I G U R E 4 Xenopreserved flap is seen at 15 days after the second operation.Inset shows the groin flap immediately after transplantation to the rat.
The number of inflammatory cells before and 15 days after the second operation.Note that, the inflammation, secondary to xenotransplantation significantly resolved following the return of the flap to the corresponding donor rat (**p < .001).
preservation of VCA allograft for 7 days in a carrier animal.This time is enough to perform any detailed tissue typing, allocation of the allograft to the best matching recipient over a larger geographical area, perform any kind of preconditioning to the recipient and relocate the recipient if needed.It is also enough period to observe any acute rejection during the xeno-preservation.The follow-up time after the second surgery was decided to be 15 days, which is before the remodeling phase of the wound healing and after the resolution of the surgical inflammation.However, additional studies with longer follow-up periods are needed to understand the long-term outcomes.
Concordant xenografting or xenotransplantation is transplantation of organs between closely related species, such as between primates or rodents.This definition was based on the difference in speed between hyperacute and acute rejection in allogeneic transplantation (Kemp, 1998).Johnsson et al. described more than 100 days of survival of CsA treated concordant heart xenotransplants (Johnsson et al., 1997).On the other hand, Wang et al. showed that when no immunosuppressive is given, concordant heart xenotransplants survived around 4 days, while the major immune reaction was acute vascular rejection with lymphocyte infiltration (Wang et al., 2005).In our study, we used concordant rat-to-mouse model and avoided HAR.We also suppressed acute rejection using cyclosporine as recommended in the literature (Roslin et al., 1992).Our findings were in accordance with the literature regarding lymphocytedominant xenoresponse and vascular endothelium as the primary target, which explains acute rejection of one groin at POD 5.However, additional studies are needed to determine the best immunosuppression protocol for xenotransplantation between concordant species.
After the xenopreserved groin flaps were retransferred to the original donors, no immune suppression was administered to the subjects, and there was no sign of rejection.This finding supports the thought that the changes in the tissue caused by xenotransplantation would be reversible to a great extent.In addition, the histopathological changes in the flaps were also reversible.The total time for preservation of groin flap was 7 days, which is another factor for the reversibility of the changes related to the xenotransplantation.It is obvious that the inflammatory changes during the xenopreservation process may be irreversible if preservation is too long.Thus, advanced studies are needed to clarify the exact mechanism and timing of this process.By better understanding the xenopreservation, reducing the tissue changes and reversal of these changes could be possible.
Cryopreservation is one of the few techniques that enable longterm preservation of cells and tissues.Composite tissue cryopreservation has been advocated based on the same principles, but finding the proper protocol for the diverse structure of the composite tissue has been the real obstacle.The ideal agent and freezing and thawing temperatures might be good for one of the tissues but fatal for another (He et al., 2021).Cryopreserved tissues are sensitive to apoptotic damage, especially during the freezing and thawing process (Komorowska-Timek et al., 2002;Saito et al., 1993).Bakhach et al. have cryopreserved human digits and transferred them to rabbits.Three cryopreserved digital segments have been xenotransplanted to the rabbit neck area after a cryopreservation period of 4-12 weeks.
One of the segments was thrombosed after the rabbit tore the fibrin lytic way.The second and third rabbits were lost due to cardiopulmonary arrest on the first and fifth postoperative days (Bakhach, 2009b).
Despite the limited number, this study is significant as it has shown that composite tissue xenotransplantation between species is possible.Xenotransplantion has been notorious for very high rates of failures when it was first suggested.The discovery of the Gal epitope and the finding of the concordant and discordant species was a serious advancement in the field (Lei et al., 2022;Sykes & Sachs, 2022).
Recently, Griffith et al. reported genetically modified porcineto-human cardiac xenotransplantation.The transplanted porcine heart had normal function for nearly 50 days, without typical xenograft rejection findings (Griffith et al., 2022).This clinical application showed that xenotransplantation is a valid option; however, the authors concluded that further studies are needed for better understanding of the mechanisms and future success.
In this study, composite tissue preservation has been achieved through xenotransplantation for the first time.As a preliminary study, we obtained encouraging results.However, studies including big animal models are needed to translate this strategy into possible clinical use.Regarding a possible human application, concordant species are needed, or genetically modified species, which are concordant to human, may be created (Sachs et al., 2001).A recent study about heart xenotransplantation reported survival of porcine hearts in baboons for up to 57 days, which is enough for preservation (Langin et al., 2018).
The most crucial progress in preventing HAR has been the development of the Gal knockout pigs.This technology is yet in its prime steps, but there is serious hope for the future.Theoretically, when human tissue is carried to Gal knockout pigs, HAR will not develop.
Still, more studies are needed to establish the exact molecular mismatches and pathways.Another critical issue that must be addressed is the zoonoses, which are prominent in such close species.This proof-of-concept study with preliminary results has some limitations.We think that additional studies with larger sample size are needed with new time points and longer preservation and followup periods.Also, translation of this murine model first to large animals and later to human applications will be needed.Moreover, mechanistic studies with more detailed immunological analysis for deeply explaining the changes related to xenopreservation period will also be valuable.
As a conclusion, composite tissue xenopreservation offers a novel strategy and encouraging alternative for preservation of composite tissue blocks.The changes related to xenotransplantation seem to be reversible to some extent.Additional studies with larger animals and longer periods of xenopreservation are needed.
In the early postoperative period, skin flap was monitored clinically by color, warmth, and refill.Color changes, such as erythema and pallor, were noted as the first signs of rejection.Advanced rejection was F I G U R E 1 (a) rat groin flap is transferred to mouse neck area.Vessel anastomoses are performed between femoral vessels and carotid artery and jugular vein.(b) Following vessel anastomosis, the flap inset is performed.Note the nipple of the rat.(c) At the end of 7 days of xenopreservation, the rat groin flap is re-harvested, tissue samples are obtained.* indicates the xenopreseved flap.(d) Xenopreserved groin (*) flap is transferred to corresponding donor rat, vessel anastomoses are performed.

Figure 2 (
Figure 2 (Video 1).The primary outcome measure was survival of the groin flap in both carrier mouse and rat.Secondary outcome measure was the severity of the inflammation at the end of the xeno-preservation and then 15 days after the VCA transplantation.As the immune reaction between concordant species is same as acute cellular rejection in the allogeneic transplantation setting, only the lymphocyte counts were analyzed in the tissues.
the set-up of the model (n = 4) transplanted mice died during the early postoperative period of the xenotransplantation due to hemodynamic complications.The flap size of these animals was reduced to 1 Â 1 cm to prevent perioperative hemodynamic instability during the set-up.A total of six xenopreservation was performed and no early postoperative complications were seen during the first and second operations.Of the six xenopreserved groin flaps, five flaps survived, and no problems related to vascular insufficiency, thrombosis, or rejections were observed in these five flaps.One xenopreserved flap was lost due to rejection at the 5th day of the first operation.Clinical observation revealed that at the end of the xenopreservation period (7 days after the xenotransplantation), all xenotransplanted flaps except one survived without any signs of rejection and morphologic changes (clinical Banff Grade 0) in the F I G U R E 2 Experimental design is shown.skin (Figure 3).One xenotransplanted flap showed skin necrosis with vital subcutaneous tissues and was rejected at the 5th day of the transplantation.Thus, this flap is not included in the lymphocyte count analysis.The remaining five xenopreserved flaps were re-harvested at the second operation.Both artery and vein of the flaps were observed to be patent in all flaps.All xenopreserved flaps were transferred successfully to the corresponding donor rat, and no vascular complications were observed following the second transplantation (Video 2).