In vivo performance of decellularized tracheal grafts in the reconstruction of long length tracheal defects: Experimental study.

BACKGROUND: The repair of long-segment tracheal lesions remains an important challenge. Nowdays no predictable and dependable substitute has been found. Decellularized tracheal scaffolds have shown to be a promising graft for tracheal transplantation, since it is non-immunogenic. OBJECTIVE: Evaluate in vivo decellularized tracheal allografts performance to replace long tracheal segment. METHODS: Forty-five swines underwent surgery as follows: Fifteen trachea donors and 30 receptors of decellularized trachea allografts. The receptors were randomly divided in five groups (n = 6). In groups I and II, donor trachea segment was decellularized by 15 cycles with sodium deoxycholate and deoxyribonuclease, in group II, the allograft was reinforced with external surgical steel wire. Groups, III, IV, and V decellularization was reduced to seven cycles, supplemented with cryopreservation in group IV and with glutaraldehyde in group V. A 10 rings segment was excised from the receptor swine and the decellularized trachea graft was implanted to re-establish trachea continuity. RESULTS: Both decellularization cycles caused decreased stiffness. All trachea receptors underwent euthanasia before the third post-implant week due to severe dyspnea and trachea graft stenosis, necrosis, edema, inflammation, hemorrhage, and granulation tissue formation in anastomotic sites. Histologically all showed total loss of epithelium, separation of collagen fibers, and alterations in staining. CONCLUSIONS: Both decellularization techniques severely damaged the structure of the trachea and the extracellular matrix of the cartilage, resulting in a no functional graft, in spite of the use of surgical wire, cryopreservation or glutaraldehyde treatment. An important drawback was the formation of fibrotic stenosis in both anastomosis.

Cytokines secretion from human mesenchymal stem cells induced by bovine bone matrix.

BACKGROUND: Bovine bone matrix is a natural material that has been used in the treatment of bone lesions. In this study, bovine bone matrix Nukbone® (NKB) was investigated due its osteoconductive and osteoinductive properties. This biomaterial induces CBFA-1 activation and osteogenic differentiation, although the cytokines involved in these processes is still unknown. OBJECTIVE: The aim of this work was to determine the influence of NKB on the pro-osteoblastic and anti-osteoblastic cytokines secretion from human mesenchymal stem cells (hMSCs). METHODS: The hMSCs were cultured onto NKB and cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ and TNF-α were analized at 0-14 days by immunoassay. In addition, hemocompatibility of NKB and characterization of hMSCs were evaluated. RESULTS: NKB induces an increase on pro-osteoblastic cytokine secretion IL-4 and a decrease on anti-osteoblastic cytokine IL-6 secretion, at days 7 and 14 of cell culture. Interestingly, there was no statistical difference between secretion profiles of others cytokines analized. CONCLUSIONS: The up-regulation of IL-4 and down-regulation of IL-6, and the secretion profiles of other cytokines examined in this work, are findings that will contribute to the understanding of the role of NKB, and similar biomaterials, in bone homeostasis and in the osteoblastic differentiation of hMSCs.

Tenascin-C and Integrin α9 Mediate Interactions of Prostate Cancer with the Bone Microenvironment.

Deposition of the extracellular matrix protein tenascin-C is part of the reactive stroma response, which has a critical role in prostate cancer progression. Here, we report that tenascin C is expressed in the bone endosteum and is associated with formation of prostate bone metastases. Metastatic cells cultured on osteo-mimetic surfaces coated with tenascin C exhibited enhanced adhesion and colony formation as mediated by integrin α9ß1. In addition, metastatic cells preferentially migrated and colonized tenascin-C-coated trabecular bone xenografts in a novel system that employed chorioallantoic membranes of fertilized chicken eggs as host. Overall, our studies deepen knowledge about reactive stroma responses in the bone endosteum that accompany prostate cancer metastasis to trabecular bone, with potential implications to therapeutically target this process in patients. Cancer Res; 77(21); 5977-88. ©2017 AACR.

Absorbable bioprosthesis for the treatment of bile duct injury in an experimental model.

INTRODUCTION: Cholecystectomy is a common surgical procedure in which complications may occur, such as injury to the biliary tract, which are associated with high morbidity. The aim of this study was to demonstrate the efficacy of a polymer-based absorbable bioprosthesis with bone scaffold for the treatment of bile duct injury in an animal model. MATERIALS AND METHODS: An absorbable bioprosthesis was used to replace the common bile duct in 15 pigs which were divided into 3 groups with different follow-ups at 1, 3 and 6 months. The animals were anesthetized at these time points and laboratory tests, Magnetic Resonance Cholangiopancreatogram [MRCP], Choledochoscopy using Spyglass and Endoscopic retrograde Cholangiopancreatogram [ERCP] were performed. After radiological evaluation was complete, the animals were euthanized and histological and immunohistochemical analyses were performed. RESULTS: Liver function tests at different time points demonstrated no significant changes. No mortality or postoperative complications were found in any of the experimental models. Imaging studies ([MRCP], [ERCP] and Choledochoscopy with SpyGlass(™)) showed absence of stenosis or obstruction in all the experimental models. DISCUSSION: Histological and immunohistochemical staining (CK19 and MUC5+) revealed the presence of biliary epithelium with intramural biliary glands in all the experimental models. There was no stenosis or obstruction in the bile duct. CONCLUSIONS: The bioprosthesis served as scaffolding for tissue regeneration. There was no postoperative complication at 6 months follow-up. This bioprosthesis could be used to replace the bile duct in cancer or bile duct injury. The bioprosthesis may allow different modeling depending on the type of bile duct injury.

Isolation of human mesenchymal stem cells and their cultivation on the porous bone matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Selection of appropriate end-points (pCR vs 2yDFS) for tailoring treatments with prediction models in locally advanced rectal cancer.

PURPOSE: Personalized treatments based on predictions for patient outcome require early characterization of a rectal cancer patient's sensitivity to treatment. This study has two aims: (1) identify the main patterns of recurrence and response to the treatments (2) evaluate pathologic complete response (pCR) and two-year disease-free survival (2yDFS) for overall survival (OS) and their potential to be relevant intermediate endpoints to predict. METHODS: Pooled and treatment subgroup analyses were performed on five large European rectal cancer trials (2795 patients), who all received long-course radiotherapy with or without concomitant and/or adjuvant chemotherapy. The ratio of distant metastasis (DM) and local recurrence (LR) rates was used to identify patient characteristics that increase the risk of recurrences. FINDINGS: The DM/LR ratio decreased to a plateau in the first 2 years, revealing it to be a critical follow-up period. According to the patterns of recurrences, three patient groups were identified: 5-15% had pCR and were disease free after 2 years (excellent prognosis), 65-75% had no pCR but were disease free (good prognosis) and 15-30% had neither pCR nor 2yDFS (poor prognosis). INTERPRETATION: Compared with pCR, 2yDFS is a stronger predictor of OS. To adapt treatment most efficiently, accurate prediction models should be developed for pCR to select patients for organ preservation and for 2yDFS to select patients for more intensified treatment strategies.

Postoperative 5-FU based radiochemotherapy in rectal cancer: retrospective long term results and prognostic factors of a pooled analysis on 1,338 patients.

AIM: To evaluate survival outcomes of patients in pStage II-III rectal cancer treated with adjuvant 5-fluorouracil-based radiochemotherapy and to retrospectively analyze the impact of prognostic variables on local control, metastasis-free survival and cause-specific survival. PATIENTS AND METHODS: A total of 1,338 patients, treated between 1985-2005 for locally advanced rectal cancer, who underwent surgery and postoperative 5-fluorouracil-based chemoradiation, were selected. RESULTS: The actuarial 5- and 10-year outcomes were: local control 87.0%-84.1%, disease-free survival 61.6%-52.1%, metastasis-free survival 72.0%-67.2%, cause-specific survival 70.4%-57.5%, and overall survival 63.8%-53.4%. Better outcomes were observed in patients with IIA, IIIA stage. Multivariate analyses showed that variables significantly affecting metastasis-free survival were pT4 and pN2, while for cancer-specific survival those variables were age >65 years, pT4, pN1, pN2, distal tumors and number of lymph nodes removed ≤ 12. CONCLUSION: This study confirmed that among stage II-III rectal cancer patients there are subgroups of patients with different clinical outcomes.

Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane.

Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

Outcomes of clinical T4M0 extra-peritoneal rectal cancer treated with preoperative radiochemotherapy and surgery: a prospective evaluation of a single institutional experience.

BACKGROUND: Our objective was evaluate the outcome of primary clinical T4M0 extraperitoneal rectal cancer treated by neoadjuvant radiochemotherapy. Prognosis of clinical T4 rectal cancer is poor. Preoperative chemoradiation therapy may be beneficial. The results obtained are unclear due to lack of objective and strictly applied staging methods. METHODS: Patients with primary, clinical, T4MO, extraperitoneal rectal cancer, defined by transrectal ultrasonography, computed tomography or magnetic resonance imaging, were considered. Intraoperative radiotherapy and adjuvant chemotherapy were employed in some patients after curative resection (R0). Variables influencing the possibility to perform an R0 resection and a sphincter-saving procedure were investigated as predictors of outcome. RESULTS: 100 patients were included. R0 resection was performed in 78 patients. R0 resection rate was greater in females (93% vs 67%) and in responders to neoadjuvant chemoradiation (94% vs 60%). The ability to perform a sphincter-saving procedure was 57%, greater in middle rectal location (85% vs 51%) and in responders to the chemoradiation (70% vs 47%). Median follow-up was 31 months (range, 4-136). Local recurrences were found in 7 patients (10%). Five-year local control in R0 patients was 90% and better in the IORT group (100%). Distant relapse occurred in 24 patients (30%). Five-year overall survival was 59%, and was better after an R0 versus an R1 or R2 resection (68% vs 22%). Overall and disease free survival in R0 patients improved after overall downstaging. Adjuvant chemotherapy given in addition to the neoadjuvant therapy did not appear to offer benefit in improving survival. CONCLUSION: A multimodal approach enabled us to obtain a 5-year overall survival of about 60%. IORT increased local control. The role of adjuvant chemotherapy needs to be further investigated.