Pericardium decellularization in a one-day, two-step protocol.

Scaffolds used in tissue engineering can be obtained from synthetic or natural materials, always focusing the effort on mimicking the extracellular matrix of human native tissue. In this study, a decellularization process is used to obtain an acellular, biocompatible non-cytotoxic human pericardium graft as a bio-substitute. An enzymatic and hypertonic method was used to decellularize the pericardium. Histological analyses were performed to determine the absence of cells and ensure the integrity of the extracellular matrix (ECM). In order to measure the effect of the decellularization process on the tissue's biological and mechanical properties, residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and the tissue's tensile strength was tested. Preservation of the biomolecules, a residual genetic content below 50 ng/mg dry tissue, and maintenance of the histological structure provided evidence for the efficacy of the decellularization process, while preserving the ECM. Moreover, the acellular tissue retains its mechanical properties, as shown by the biomechanical tests. Our group has shown that the acellular pericardial matrix obtained through the super-fast decellularization protocol developed recently retains the desired biomechanical and structural properties, suggesting that it is suitable for a broad range of clinical indications.

Last twenty-years activity of cardiovascular tissue banking in Barcelona.

The Barcelona Tissue Bank was established from the merge of two previous multi-tissue banks. Potential donors are screened by Donor Center staff and multi-tissue retrieval is performed by specialized own teams. Tissue processing and preservation is performed in clean room facilities by specialised personnel. After quality control of both donor and all tissues results, the heart valves and vascular segments are stored until medical request. The aim of this report is to present the cardiovascular tissue activity and retrospectively evaluate the outcomes of the changes performed in last 20 years. Cardiovascular tissue from 4088 donors was received, specifically 3115 hearts and 2095 vascular segments were processed and evaluated. A total of 48% of the aortic valves, 68% of the pulmonary valves and 75% of the vascular segments were suitable for transplant. The main reason for discarding tissue was macroscopic morphology followed by microbiological results, for both valves and arteries. Altogether, 4360 tissues were distributed for transplantation: 2032 (47%) vascular segments, 1545 (35%) pulmonary valves and 781 (18%) aortic valves. The most common indication for aortic valve surgery was the treatment of endocarditis, while for pulmonary valves, it was congenital malformation reconstruction. Vascular segments were mainly used for reconstruction after ischemia. During this period, a number of changes were made with the goal of enhancing tissue quality, safety and efficacy. These improvements were achieved through the use of a new antibiotic cocktail, increasing of donor age criteria and changing the microbiological control strategy.

Development of a full-thickness acellular dermal graft from human skin: Case report of first patient rotator cuff patch augmentation repair.

The processing and initial testing of a new human tissue preparation is described. Full-thickness Acellular Dermal Matrix (ftADM) is the extracellular matrix (ECM) obtained by decellularization of full-thickness human skin from cadaveric donors. The safety, stability and usability of the graft are discussed with respect to the results of the residual cellular content, maintenance of ECM components, and biomechanical properties. Quantitative and qualitative analysis of the ECM demonstrated the absence of cell debris, while the native structure of human dermis was maintained. Biomechanical testing showed stiffness values comparable to other commercial products used for tendon reinforcement, suggesting that our ftADM could be successfully used not only in soft tissue regeneration surgeries, but also in tendon reinforcement. First case of ftADM in rotator cuff augmentation is described. Technical management of the patch during surgery and clinical outcomes are discussed.

Fast protocol for the processing of split-thickness skin into decellularized human dermal matrix.

BACKGROUND: Dermal scaffolds for tissue regeneration are nowadays an effective alternative in not only wound healing surgeries but also breast reconstruction, abdominal wall reconstruction and tendon reinforcement. The present study describes the development of a decellularization protocol applied to human split-thickness skin from cadaveric donors to obtain dermal matrix using an easy and quick procedure. METHODS: Complete split-thickness donor was decellularized through the combination of hypertonic and enzymatic methods. To evaluate the absence of epidermis and dermal cells, and ensure the integrity of the extracellular matrix (ECM) structure, histological analysis was performed. Residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and tensile strength was tested to measure the effect of the decellularization technique on the mechanical properties of the tissue. RESULTS: Biomolecules quantification, residual genetic content (below 50 ng/mg dry tissue) and histological structure assessment showed the efficacy of the decellularization process and the preservation of the ECM. The biomechanical tests confirmed the preservation of native properties in the acellular tissue. CONCLUSIONS: The acellular dermal matrix obtained from whole split-thickness skin donor with the newly developed decellualrization protocol, maintains the desired biomechanical and structural properties and represents a viable treatment option for patients.

Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol.

Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.

Vascularity and neuroreceptors of the pes anserinus: anatomic study.

The primary aim of this work was to evaluate the neurovascular network of the pes anserinus (PA) at its tibial insertion because the PA is often used for anterior cruciate ligament (ACL) reconstruction. Four fresh cadaver knees were injected with India ink gelatin solution and the arteries that supply blood to the PA were identified; microscopic studies of vessels and nerve fibers were also performed. Superficial and deep branches of the inferior medial genicular artery contribute to an arterial arch that courses deep to the PA insertion. A widespread array of small vessels and nerve fibers penetrate the PA insertion and course along the length of the gracilis and semitendinosus tendons. Computer analysis revealed that the mean diameter of the vessels decreased from 2201 microm at the insertion to 661 microm midway along the length of the tendon (mean tendon length = 17 cm; range = 13-21 cm); the cross-sectional area of the vessels per histologic section decreased from 336.37-137.05 microm(2). This study demonstrates that the PA insertion is well vascularized and richly innervated and that these morphological features continue along the length of the tendons.

The procurement team as a factor of bone allograft contamination.

We studied the effect of the procurement team on the risk of contamination in 270 large bone allografts retrieved from 53 non-living donors under strictly aseptic conditions.The overall contamination rate was 8.1%. When the procurement team was constituted by three or less members the contamination rate was 5.6%, while if there were four or more members the rate was 12.9%; this difference was significant in the statistical analysis.We conclude that a procurement team constituted by three or less trained members is a determinant factor in decreasing the bacterial contamination rate of bone allografts.