Effect of Cryopreservation on Lymph Node Fragment Regeneration After

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Effect of Cryopreservation on Lymph Node Fragment Regeneration After Innov Surg Sci 2018; 3(2): 139–146 Original Article Catarina Hadamitzkya, Hanes Perića,*, Sebastian J. Theobald, Klaus Friedrich Gratz, Hendrik Spohr, Reinhard Pabst and Peter M. Vogt Effect of cryopreservation on lymph node fragment regeneration after autologous transplantation in the minipig model https://doi.org/10.1515/iss-2018-0003 cryopreservation of autologous lymph nodes. Superfi- Received January 12, 2018; accepted March 10, 2018; previously cial inguinal lymph nodes were excised and conserved published online April 20, 2018 at −80 °C for 1 month. Thereafter, lymph node fragments were transplanted in the subcutaneous tissue. Abstract Results: Regeneration of the lymph nodes was assessed Introduction: Lymphoedema is a worldwide pandemic five months after transplantation. We show that lymph causing swelling of tissues due to dysfunctional transport node fragment regeneration takes place in spite of former of lymph fluid. Present management concepts are based cryopreservation. Transplanted fragments presented in conservative palliation of symptoms through manual typical histological appearance. Their draining capacity lymphatic drainage, use of compression garments, was documented by macroscopic transport of Berlin Blue manual lymph drainage, exercise, and skin care. Never- dye as well as through SPECT-CT hybrid imaging. theless, some curative options as autologous lymph node Discussion: In conclusion, our results suggest that pro- transplantation were shown to reduce lymphoedema in cesses of cryopreservation can be used in the creation of selected cases. Lately, some concern has arisen due to artificial lymph node scaffolds without major impairment reports of donor site morbidity. A possible solution could of lymph node fragments regeneration. be the development of artificial lymph node scaffolds Keywords: artificial lymph node; cryopreservation; lymph as niches of lymphatic regeneration. Engineering these node transplant; lymphoedema. scaffolds has included cryopreservation of lymph node stroma. However, the effects of cryopreservation on the regeneration capacities of these organs were unknown. Materials and methods: Here, we used the minipig animal Introduction model to assess lymphatic regeneration processes after Lymphatic fluid, products of cell catabolism, antigens and debris are transported from the tissues and organs through aCatarina Hadamitzky and Hanes Perić contributed equally to the a fine network of transparent valved vessels called lym- manuscript. *Corresponding author: Hanes Perić, myDent GmbH phatics [1, 2]. These drain into lymph node chains, where Tiergartenstrasse 114, 30559 Hannover, Germany, lymphatic fluid is progressively thickened through selec- E-mail: [email protected] tive fluid filtration into the blood capillaries of these Catarina Hadamitzky: Practice for Lympho-Vascular Diseases organs [3]. This reabsorption not only promotes homeo- Bahnhofstrasse 12, Hannover, Germany stasis of drained tissues but also allows exposure of nodal Sebastian J. Theobald: Clinic of Haematology, Haemostasis, dendritic cells and macrophages to potential pathogens, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany promoting antigen phagocytosis. Dendritic cell-driven Klaus Friedrich Gratz: Clinic of Nuclear Medicine, Hannover Medical presentation of antigen surface molecules to lymphocytes School, Hannover, Germany eventually activates tissue-specific immunity [4]. Hendrik Spohr: Institute of Functional and Applied Anatomy, Impairment of lymph node function or lymphatic trans- Hannover Medical School, Hannover, Germany port can lead to tissue swelling called lymphoedema. This Reinhard Pabst: Institute of Immunomorphology, Hannover Medical School, Hannover, Germany disease has multiple causes. Primary lymphoedema has a Peter M. Vogt: Clinic of Plastic, Hand and Reconstructive Surgery, reduced overall incidence. In developing countries, sec- Hannover Medical School, Hannover, Germany ondary lymphoedema mainly occurs due to filarial worm Open Access. © 2018 Hadamitzky C., et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 License. 140 Hadamitzky et al.: Lymph node fragment regeneration after transplantation in the minipig model infection. According to World Health Organization data, Although still in development, artificial lymph node about 40 million patients have lymphoedema due to these constructs seem to induce efficient regeneration when parasites [5, 6]. In developed countries, secondary lymphoe- biocompatible scaffolds and stromal cells are used [20]. dema is often related to oncologic causes or trauma [7]. This induces migration of immune cells into the artificial In malignant diseases such as breast cancer, mela- constructs, resulting in the presence of dendritic cells as noma or prostate cancer, draining lymph nodes participate well as B- and T-lymphocyte clusters with typical corti- in the recognition of tumour cells and have become impor- cal and paracortical distribution [21]. Some processes of tant markers for metastatic disease and overall prognosis tissue engineering associated with artificial lymph node [8]. Surgical removal of lymph nodes is often necessary. constructs apply methods of cryopreservation to allow lab- About 20%–30% of the patients develop limb lymphoe- oratory manipulation [21]. Nevertheless, it was unknown dema after axillary or inguinal lymph node removal [9, if such cryopreservation could permanently damage the 10]. Lymphoedema is a lifelong condition that reduces the regenerative capacity of nodal tissues [22]. quality of life and productivity of affected patients [10]. Its The purpose of this study was to analyse lymph continuous conservative management has high costs for node fragment regeneration after cryopreservation in the the health system [11]. minipig model. The viability of cryopreserved lymph node As the immune system and its organs show great fragments was assessed after avascular transplantation. plasticity, concepts of autologous lymph node transplan- Here, physiological drainage capacities were analysed tation were developed for the treatment of secondary lym- through SPECT-CT hybrid imaging and subsequent histo- phoedema [12–14]. The aim of these surgical procedures logic analysis provided insights on regeneration processes. is to substitute damaged lymph nodes through function- ing nodes contained in autologous tissue flaps. Clinical studies seem to show some benefit, with reported cure in Methods 32%–40% of the patients, and improvement in the great majority [12–16]. However, donor site morbidity in the form Minipig model and experimental groups of lymphoedema of the donor area was also described [17– 19]. It might therefore be important to develop alternative A total of eight wild-type Göttinger minipigs weighing 6–11 kg were donor solutions to prevent this major complication. obtained from the animal facility of the University of Göttingen Concepts of artificial lymph node engineering origi- (Relliehausen, Germany). They were housed in our animal facility, nally arose to face the challenge of patients with congenital according to German legal specifications, and fed mixed food and paucity of lymph nodes without autologous harvesting pos- water ad libitum. Animal procedures were approved by the govern- sibilities (primary lymphoedema). Nevertheless, present ment of Lower Saxony (Ref. No. 509.6-42502-07/1402). Animals were divided in two groups (Figure 1). The number of evidence of possible donor site morbidity might extend the animals allocated to each group was different, as group A was pri- potential utility of artificial lymph node constructions to oritized on being given the possibility of intra-individual limb vol- the treatment of secondary lymphoedema. ume control (negative control in the non-operated left limb). Group A Figure 1: Schematic overview of the study outline during the 6-month follow-up. Hadamitzky et al.: Lymph node fragment regeneration after transplantation in the minipig model 141 (n = 6) was therefore submitted to resection of the right lymph node microscope. Samples presenting lymph node fragments were selected under general anaesthesia, as previously described [23], with the left for further characterisation. Lymph node fragments were considered side of the animal being left intact. The single superficial groin lymph vital when they presented typical B- and T-lymphocyte distribution in node present in this species was operatively excised on the right side. the cortex and paracortex, respectively, showed uptake of Berlin blue Afferent and efferent lymphatics were carefully sharply resected in in the sinuses and had lipomatosis and fibrotic degeneration features the immediate vicinity of the organ capsule. The blood vessels of the in less than 50% of the fragment surface on at least three sections. hilum were also resected. No ligatures were applied to the organ. The Histologic analysis was performed blind by one observer. Later, his- excised lymph node was then cut transversally in two fragments of tologic probe numbers were associated with each SPECT-CT hybrid about 1-cm3 size and introduced in sterile recipients. After hermetic imaging to allow final assessment of results. closure, recipients were immediately submersed in a bath of nitric oxide. Soft plastic recipients were used to maximise the surface of thermal contact. No chemical cryoprotection was performed, as the Data analysis maximal distance for cold penetration
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