An optimized non-destructive protocol for testing mechanical properties in decellularized rabbit trachea.

Successful tissue-engineered tracheal transplantation relies on the use of non-immunogenic constructs, which can vascularize rapidly, support epithelial growth, and retain mechanical properties to that of native trachea. Current strategies to assess mechanical properties fail to evaluate the trachea to its physiological limits, and lead to irreversible destruction of the construct. Our aim was to develop and evaluate a novel non-destructive method for biomechanical testing of tracheae in a rabbit decellularization model. To validate the performance of this method, we simultaneously analyzed quantitative and qualitative graft changes in response to decellularization, as well as in vivo biocompatibility of implanted scaffolds. Rabbit tracheae underwent two, four and eight cycles of detergent-enzymatic decellularization. Biomechanical properties were analyzed by calculating luminal volume of progressively inflated and deflated tracheae with microCT. DNA, glycosaminoglycan and collagen contents were compared to native trachea. Scaffolds were prelaminated in vivo. Native, two- and four-cycle tracheae showed equal mechanical properties. Collapsibility of eight-cycle tracheae was significantly increased from -40cm H2O (-3.9kPa). Implantation of two- and four-cycle decellularized scaffolds resulted in favorable flap-ingrowth; eight-cycle tracheae showed inadequate integration. We showed a more limited detergent-enzymatic decellularization successfully removing non-cartilaginous immunogenic matter without compromising extracellular matrix content or mechanical stability. With progressive cycles of decellularization, important loss of functional integrity was detected upon mechanical testing and in vivo implantation. This instability was not revealed by conventional quantitative nor qualitative architectural analyses. These experiments suggest that non-destructive, functional evaluation, e.g. by microCT, may serve as an important tool for mechanical screening of scaffolds before clinical implementation. STATEMENT OF SIGNIFICANCE: Decellularization is a front-running strategy to generate scaffolds for tracheal tissue-engineering. Preservation of biomechanical properties of the trachea during this process is paramount to successful clinical transplantation. In this paper, we evaluated a novel method for biomechanical testing of decellularized trachea. We detected important loss of functional integrity with progressive cycles of decellularization. This instability was not revealed by our quantitative nor qualitative analyses. These experiments suggest that the technique might serve as a performant, non-destructive tool for mechanical screening of scaffolds before clinical implementation.

Dynamic reconstruction of complex abdominal wall defects with the pedicled innervated vastus lateralis and anterolateral thigh PIVA flap.

BACKGROUND AND AIM: Reconstruction of large and chronically infected recurrent abdominal wall defects with exposed bowel in a scarred wound environment, when component release has been previously performed but failed, is a veritable challenge. We use a pedicled innervated vastus lateralis muscle with a fasciocutaneous anterolateral thigh flap (PIVA flap) to restore the continuity of the abdominal wall with vascularised tissues and create a dynamic component that improves the functional outcome. MATERIALS AND METHODS: A one-stage PIVA flap was used in 15 patients with grade 4 transmural chronically infected defects. They had a mean of 4.53 previous laparotomies and important co-morbidities. We determined post-operative reconstructive abdominal wall strength using a validated quality-of-life (QoL) hernia-related questionnaire and modified it to quantify donor-site morbidity at the thigh. We measured the maximal force generated at 60°/s and the force velocity at 120°/s by isokinetic dynamometric analysis at 3 and 12 months. Electromyography (EMG) was performed 12 months after the reconstruction to analyse the contractile integrity of the vastus lateralis segment. A two-sided sign test was used to analyse data. RESULTS: All transmural chronic wounds healed without recurrence. Dynamometric strength increased significantly in the abdominal wall musculature (p < 0.016) and in the donor thigh (p < 0.023) between 3 months and 12 months after the intervention, which reflected in the EMG outcome and the high scores in the QoL measurements after 12 months. CONCLUSIONS: The PIVA flap revascularises the scarred milieu, adds a dynamic component to improve function and may reach up to the xiphoid process. Donor-site morbidity is limited.

Prefabrication and prelamination strategies for the reconstruction of complex defects of trachea and larynx.

Complex tracheal and laryngeal defects can be reconstructed using prelamination and prefabrication techniques. Three clinical situations are described in detail in the article. In short segment restenosis defects within scarred surroundings, we restore the fibrocartilaginous defect with a radial forearm fascia flap prelaminated with buccal mucosa or cartilage. This provides a newly vascularized inner lining to the tracheal defect and restores the tubular convexity. For long segment defects we need a technique that can withstand respiratory forces. We use a heterotopic prefabrication strategy to vascularize a tracheal allograft wrapped in forearm fascia. Chimerism is created by replacing donor respiratory epithelium with buccal mucosa of the recipient. After orthotopic transfer, this chimerism allows immunosuppression to be tapered and stopped when bronchoscopy shows mucosal integrity of the new trachea, since the recipient epithelium replaces the allogeneic inner tracheal lining by means of a chronic rejection process. A distinct situation occurs after resection of a unilateral larynx tumor, which usually results in a total laryngectomy with loss of both vocal cords, since reconstruction of the hemilarynx is considered too complex. First, we prefabricate a nearby four-ring autologous tracheal segment using radial forearm fascia. In a second stage, this orthotopically vascularized trachea restores the laryngeal structure with the aim to conserve one vocal cord and thus speech. Orthotopic and heterotopic prelamination and prefabrication strategies offer efficient and reproducible solutions for the restoration of challenging short and long segment tracheal defects, as well as unilateral laryngeal defects. The series in this review article are based on previous studies and case reports. The level of evidence is III-"Study of nonconsecutive patients, without a universally applied gold standard: case-control study".

Multilevel cervical reconstruction with no remaining hardware: the potential of a vascularised fibular strut graft.

We present a case of a cervical chordoma extending from C3 to C6, treated by surgical resection followed by reconstruction in two stages with a vascularised fibular strut graft anteriorly and fibular allografts posteriorly. The goal was to achieve a stable reconstruction without any metal hardware to allow for increased accuracy of the adjuvant radiotherapy. The advantages of a vascularised fibula flap to realise this are discussed.

Learning curve in tracheal allotransplantation.

The first vascularized tracheal allotransplantation was performed in 2008. Immunosuppression was stopped after forearm implantation and grafting of the recipient mucosa to the internal site of the transplant. Nine months after forearm implantation, the allograft was transplanted to the tracheal defect on the radial blood vessels. Since then, four additional patients have undergone tracheal allotransplantation, three (patients 2-4) for long-segment stenosis and one (patient 5) for a low-grade chondrosarcoma. Our goal was to reduce the time between forearm implantation and orthotopic transplantation and to determine a protocol for safe withdrawal of immunosuppressive therapy. Following forearm implantation, all transplants became fully revascularized over 2 months. Withdrawal of immunosuppression began 4 months after graft implantation and was completed within 6 weeks in cases 2-4. Repopulation of the mucosal lining by recipient cells, to compensate for the necrosis of the donor mucosa, was not complete. This resulted in partial loss of the allotransplant in patients 2-4. In patient 5, additional measures promoting recipient cell repopulation were made. The trachea may be used as a composite tissue allotransplant after heterotopic revascularization in the forearm. Measures to maximize recipient cell repopulation may be important in maintaining the viability of the transplant after cessation of immunosuppression.

Prospective trial of adipose-derived regenerative cell (ADRC)-enriched fat grafting for partial mastectomy defects: the RESTORE-2 trial.

AIMS: Women undergoing breast conservation therapy (BCT) for breast cancer are often left with contour defects and few acceptable reconstructive options. RESTORE-2 is the first prospective clinical trial using autologous adipose-derived regenerative cell (ADRC)-enriched fat grafting for reconstruction of such defects. This single-arm, prospective, multi-center clinical trial enrolled 71 patients post-BCT with defects ≤150 mL. METHODS: Adipose tissue was collected via syringe lipoharvest and then processed during the same surgical procedure using a closed automated system that isolates ADRCs and prepares an ADRC-enriched fat graft for immediate re-implantation. ADRC-enriched fat graft injections were performed in a fan-shaped pattern to prevent pooling of the injected fat. Overall procedure times were less than 4 h. The RESTORE-2 protocol allowed for up to two treatment sessions and 24 patients elected to undergo a second procedure following the six month follow-up visit. RESULTS: Of the 67 patients treated, 50 reported satisfaction with treatment results through 12 months. Using the same metric, investigators reported satisfaction with 57 out of 67 patients. Independent radiographic core laboratory assessment reported improvement in the breast contour of 54 out of 65 patients based on blinded assessment of MRI sequence. There were no serious adverse events associated with the ADRC-enriched fat graft injection procedure. There were no reported local cancer recurrences. Injection site cysts were reported as adverse events in ten patients. CONCLUSION: This prospective trial demonstrates the safety and efficacy of the treatment of BCT defects utilizing ADRC-enriched fat grafts.

Abdominal aplasia cutis congenita: case report and review of the literature.

A case of bilateral abdominal aplasia cutis congenita without skull defect is reported and was treated successfully by a combination of allografts and growth factors delivered by allogenic cultured keratinocytes.

Arterialized venous free flaps for soft-tissue reconstruction of digits: a 40-case series.

When there is not sufficient local tissue available for reconstruction of one or multiple digital defects, distant flap reconstructions are required. Available thin flaps with defined arterial inflow and venous outflow are limited and require the sacrifice of an arterial pedicle. In addition, a functional limitation can occur at the donor site. During the last decades, experimental and clinical attempts have been made in using venous flaps, based just on the venous network for in- and outflow. A 40-case series of arterialized venous free flaps in soft-tissue reconstruction of the digits and hand is presented. Neovessel formation induced by an intact venous plexus within the flap proved clinically to be the survival mechanism. Postoperative congestion was present in all flaps and subsided within 14 days; 92 percent of all flaps eventually survived. In 57.5 percent of cases, total flap survival was observed; in 17.5 percent of flaps, a superficial epidermolysis occurred, not requiring further surgery; in 17.5 percent of flaps, a full-thickness skin necrosis developed, requiring grafting (minor complication); in 7.5 percent of reconstructions, total flap necrosis was observed. Thus, in the authors' experience, when conventional local flaps are not available, arterialized venous free flaps proved to be a successful solution for soft-tissue reconstruction in digits and hands. The authors present the main indications and advantages of arterialized venous free flaps, emphasizing the essential technical steps and the most frequent pitfalls in treatment.

Edge deepithelialization: a method to prevent leakage when tubed free skin flap is used for pharyngoesophageal reconstruction.

BACKGROUND: Anastomosis leakage remains a major source of morbidity and mortality in patients who receive pharyngoesophageal reconstruction, especially when a free skin flap is used as the substitute. This makes free skin flaps less favored than other substitutes in pharyngoesophageal reconstruction. However, a free skin flap provides many advantages for pharyngoesophageal reconstruction and, in some circumstances, may be the only choice. To reduce the incidence of postoperative leakage, we developed a technical modification called edge-deepithelialization for skin flap to ensure a more secure anastomosis. METHODS: Twenty-four patients who received tubed free skin flaps for cervical esophagus or hypopharynx reconstruction during a 7-year period were included in this study. Group 1 (n = 12) received flap edge-deepithelialization before undergoing the anastomosis, and Group 2 (n = 12) received the conventional anastomosis method. Preoperative nutritional status was evaluated by serum albumin level. All the patients were followed up for at least 1 year (1 approximately 14 years; mean, 7.3 years). Postoperative leakage was detected by clinical symptoms and signs or by routine esophagography. RESULTS: Postoperative anastomosis leakage developed in 1 patient in Group 1 (8.3%) and 5 patients in Group 2 (41.67%). The difference in the leakage rate between these groups was of statistical significance (P =.0001). There were no significant differences in the preoperative serum albumin level noted between these 2 groups (t test, P >.05). Older age was not associated with a higher leakage rate. Among the 6 leakage cases, 1 was treated conservatively, and the other 5 needed an additional 1 to 5 procedures for the management of leakage. All the leakages were cured during the follow-up period. CONCLUSIONS: With the flap edge deepithelialized, a more secure and reliable anastomosis can be obtained. This makes the free skin flap an easier and safer alternative in pharyngoesophageal reconstruction, especially when laparotomy and enteric flaps are contraindicated.