Extracellular matrix composition analysis of human articular cartilage for the development of organ-on-a-chip.

INTRODUCTION: Articular cartilage has a complex extracellular matrix (ECM) that provides it a defined architecture for its load-bearing properties. The complete understanding of ECM components is imperative for developing biomimetic organ-on-a-chip tissue construct. OBJECTIVE: This study aimed to decellularize and characterize the ECM for its protein profiling to generate a niche for enhanced chondrocyte proliferation. METHODS: Articular cartilage scrapings were subjected to mechanical and collagenase digestion, followed by sodium dodecyl sulfate (SDS) treatment for 8 h and 16 h. The de-cellularization efficiency was confirmed by hematoxylin & eosin, alcian blue, masson's trichrome staining, and scanning electron microscopy (SEM). The ECM protein profile was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a bottom-up approach. RESULTS: Histological characterization revealed void lacunae that lacked staining for cellular components. The ECM, sulfated glycosaminoglycan content, and collagen fibers were preserved after 8 h and 16 h of de-cellularization. The SEM ultrastructure images showed that few chondrocytes adhered to the ECM after 8 h and cell-free ECM after 16 h of de-cellularization. LC-MS/MS analysis identified 66 proteins with heterotypic collagen types COL1A1-COL6A1, COL14A1, COL22A1 and COL25A1 showed moderate fold change and expression levels, while COL18A1, COL26A1, chondroitin sulfate, matrix metalloproteinase-9 (MMP9), fibronectin, platelet glycoprotein 1 beta alpha (GP1BA), vimentin, bone morphogenetic protein 6 (BMP6), fibroblast growth factor 4 (FGF4) and growth hormone receptor (GHR) showed maximum fold change and expression levels. CONCLUSIONS: The standardized de-cellularization process could preserve majority of ECM components, providing structural integrity and architecture to the ECM. The Identified proteins quantified for their expression levels provided insight into engineering the ECM composition for developing cartilage-on-a-chip.

Preparation, characterization, and xenotransplantation of the caprine acellular dermal matrix.

BACKGROUND: Caprine skin is a promising biomaterial for tissue-engineering applications. However, tissue processing is required before its xenogenic use. AIMS: Therefore, the purpose of this study was to evaluate the structural integrity and biocompatibility of the caprine skin after de-epithelialization, using sodium chloride (NaCl) and trypsin solutions, followed by de-cellularization using sodium dodecyl sulfate (SDS) solution. MATERIALS & METHODS: The caprine skin was de-epithelialized using NaCl (2-4 mol/L) and trypsin (0.25%-0.5%) followed by the treatment of SDS (1%-4%) solution over a period of time. Acellularity of the prepared matrix was confirmed histologically and characterized by appropriate staining, scanning electron microscopy (SEM), DNA quantification, and Fourier-transform infrared (FTIR) spectroscopy. The caprine acellular dermal matrix (CADM) was used for the repair of spontaneously occurring abdominal hernia in ten buffaloes. The biocompatibility of the CADM was evaluated using clinical, hematological, biochemical, and anti-oxidant parameters. RESULTS: Histologically, the skin treated with 0.25% trypsin in 4 mol/L NaCl for 8 hours resulted in complete de-epithelialization. Further treatment with 2% SDS for 48 hours demonstrated complete acellularity and orderly arranged collagen fibers. The SEM confirmed a preservation of collagen arrangement within CADM. The DNA content was significantly (P < .05) lower in CADM (46.20 ± 7.94 ng/mg) as compared to fresh skin (662.56 ± 156.11 ng/mg) indicating effective acellularity. The FTIR spectra showed characteristic collagen peaks of amide A, amide B, amide I, amide II, and amide III in CADM. All the 10 animals recovered uneventfully and remained sound. Hematological, biochemical, and anti-oxidants findings were unremarkable. CONCLUSION: Results indicated the acceptance and biocompatibility of the xenogenic caprine acellular dermal matrix for abdominal hernia repair in buffaloes without complications.

Recent advances in re-engineered liver: de-cellularization and re-cellularization techniques.

Allogeneic transplantation is the definitive treatment for patients with end-stage liver disease but is limited by donor shortage and very high cost. Through de-cellularization and re-cellularization methods, re-engineered liver may provide a promising alternative for treating patients with end-stage liver disease. To achieve this, the prevention of the native extracellular matrix ultrastructure plays a central role in de-cellularization protocol; the re-seeding cell types, as well as re-seeding strategies, need more explorations in re-cellularization protocol. Some success of this approach has been published in a rat model; however, the re-engineered liver remains functional in vivo for only several hours, which suggests that the recent protocol may be far from the ideal target. This Review highlights the challenges still to be overcome and presents an overview and summary of methods of de-cellularization and re-cellularization strategies, together with a view on future directions that may lead to the regeneration of a functional liver.

Automated procedure for biomimetic de-cellularized lung scaffold supporting alveolar epithelial transdifferentiation.

The optimal method for creating a de-cellularized lung scaffold that is devoid of cells and cell debris, immunologically inert, and retains necessary extracellular matrix (ECM) has yet to be identified. Herein, we compare automated detergent-based de-cellularization approaches utilizing either constant pressure (CP) or constant flow (CF), to previously published protocols utilizing manual pressure (MP) to instill and rinse out the de-cellularization agents. De-cellularized lungs resulting from each method were evaluated for presence of remaining ECM proteins and immunostimulatory material such as nucleic acids and intracellular material. Our results demonstrate that the CP and MP approaches more effectively remove cellular materials but differentially retain ECM proteins. The CP method has the added benefit of being a faster, reproducible de-cellularization process. To assess the functional ability of the de-cellularized scaffolds to maintain epithelial cells, intra-tracheal inoculation with GFP expressing C10 alveolar epithelial cells (AEC) was performed. Notably, the CP de-cellularized lungs were able to support growth and spontaneous differentiation of C10-GFP cells from a type II-like phenotype to a type I-like phenotype.