Reconstruction of Epidural Fat to Prevent Epidural Fibrosis after Laminectomy in Rabbits

Background: Laminectomy can effectively decompress the spinal cord and expand the vertebral canal. However, the fibrosis that appears may cause adherence and recompression of the spinal cord or/ and nerve root, which may cause Failed Back Syndrome (FBS) and make the re-exposure process more difficult. Reconstruction of the epidural fat may be an ideal method to achieve satisfactory results. Methods: Thirty-six New Zealand rabbits were randomly divided into three groups: control, ECM, and ECM+aMSCs groups. Saline, ECM gel, and ECM+aMSC complex were placed respectively at the fifth lumbar vertebrate of the rabbits. Epidural fat and fibrosis formation were detected by MRI and histologically at the 4th, 8th, and 12th weeks. Quantitative RT-PCR was used to detect the expression of IL-6 and TGF-β. Results: MRI and Oil Red O staining revealed epidural fat formation at the 12th week in the ECM+aMSCs group. H&E staining showed that the numbers of fibroblasts in the ECM gel and ECM+aMSCs groups were less than the control group at the 4th and 8th weeks (P<0.05). Masson’s trichrome staining showed that the proportion of collagen fibers in the ECM gel and ECM+aMSCs group was lower than the control group (P<0.05). Quantitative RT-PCR showed the expressions of TGF-β and IL-6 were lower in the ECM gel and ECM+aMSCs group than the control group (P<0.05) at the 4th week, but higher at the 8th week. Conclusion: We successfully reconstructed the epidural fat with ECM gel and aMSC complex; additionally, IL-6 and TGF-β cytokines were lower at early stage after laminectomy.

Phenotypic and Cellular Characteristics of a Stromal Vascular Fraction/Extracellular Matrix Gel Prepared Using Mechanical Shear Force on Human Fat

The retention of fat-derived grafts remains a challenge for regenerative medicine. Fat aspirates from patients undergoing liposuction were prepared into standard Coleman fat grafts or further isolated using mechanical shear force to prepare a stromal vascular fraction (SVF)/extracellular matrix (ECM) gel. The retention rate of the SVF/ECM gel was significantly higher than that of the Coleman fat at 3, 14, 28, and 60 days following transplantation on the backs of nude mice. The viscosity of the fat was directly proportional to the shearing force. Although the mechanical isolation did not affect the total number of cells, it significantly decreased the number of living cells. Flow cytometry showed a greater number of mesenchymal stem cells, supra-adventitial (SA)-adipose stromal cells (ASCs), and adipose-derived stem cells but a lower number of endothelial progenitor cells in the SVF/ECM gel than in the Coleman fat. Thus, mechanical isolation of fat can increase the pluripotency of adipocytes, which can improve graft retention in cell therapy.

New Insight into Natural Extracellular Matrix: Genipin Cross-Linked Adipose-Derived Stem Cell Extracellular Matrix Gel for Tissue Engineering

The cell-derived extracellular matrix (ECM) is associated with a lower risk of pathogen transfer, and it possesses an ideal niche with growth factors and complex fibrillar proteins for cell attachment and growth. However, the cell-derived ECM is found to have poor biomechanical properties, and processing of cell-derived ECM into gels is scarcely studied. The gel provides platforms for three-dimensional cell culture, as well as injectable biomaterials, which could be delivered via a minimally invasive procedure. Thus, in this study, an adipose-derived stem cell (ADSC)-derived ECM gel was developed and cross-linked by genipin to address the aforementioned issue. The genipin cross-linked ADSC ECM gel was fabricated via several steps, including rabbit ADSC culture, cell sheets, decellularization, freeze–thawing, enzymatic digestion, neutralization of pH, and cross-linking. The physicochemical characteristics and cytocompatibility of the gel were evaluated. The results demonstrated that the genipin cross-linking could significantly enhance the mechanical properties of the ADSC ECM gel. Furthermore, the ADSC ECM was found to contain collagen, fibronectin, biglycan, and transforming growth factor (TGF)-β1, which could substantially maintain ADSC, skin, and ligament fibroblast cell proliferation. This cell-derived natural material could be suitable for future regenerative medicine and tissue engineering application.

Superparamagnetic nanoparticles targeting cancer cells invading the extracellular matrix.

3083 Background: Metastatic cancer cells secrete matrix metalloproteinases (MMP), which allow cancer cells to burrow their way to the nearby vasculature. Therefore, MMPs are potential targets for anticancer treatment. Nanoparticles (NPs) can be crafted to adhere to the ECM and subsequently be released in response to advance of invasive cells. The objective of the study is to elucidate the interaction of cancer cells in a three-dimensional culture, with functionalized SPIONS targeted to the extracellular matrix around them. Methods: SPIONS, 15-20 nm in size were functionalized using different coatings: ficoll 400, sucrose, lysine-arginine, dextran50,000. Cellular uptake studies using cervical adenocarcinoma HeLa cell lines were performed to determine the optimum formulation taken up by the cells, using electron microscopy and Prussian blue staining for optical microscopy. Two types of extracellular matrices were used: Rat-tail Collagen type I and ECM gel from Engelbreth-Holm-Swarm murine sarcoma with NPs embedded in them. To assess the capability of cells to invade the matrix, cells were grown on the surface of the matrices for 1 week To evaluate the ability to grow, expand and migrate inside the matrix, cells were embedded within the matrix and left for 14 days. Comparison was made in the presence or absence of SPIONS. Results: Sucrose-coated SPIONS were taken up the best by HeLa cell lines as evaluated by MRI. MMP-1 Secretion allowed HeLa cell invasion of collagen type-1 matrix unidirectionally. Cells could adhere, proliferate, differentiate and migrate in the absence of SPIONS. Cells positive for MMP-9 invaded ECM gel from Engelbreth-Hol-Swarm murine sarcoma matrix also only in the absence of SPIONS. Cells that were found engulfing SPIONS showed morphological features of apoptosis as nuclear pyknosis and karryorhexis. Conclusions: Targeting NPs to the ECM surrounding cancer cells that have developed a metastatic potential represents an attractive platform for cancer therapeutics. The findings show a great promise for development of new theranostic agents, that can be directed to the tumor environment using external magnetic fields, with subsequent suppression of invasion and even destroying malignant cells.

Reconstruction of Kidney-Like Tissue Using ECM Gel and Rat Embryonic Kidney Fragments

Organ engineering remains a challenge to researchers. We tried to reconstruct kidney-like tissue using tissue engineering technique. Kidney fragments were isolated from rat E16 embryonic kidneys and seeded on either ECM gel or on polyglycolic acid (PGA) fibers, then implanted in vivo into the subcutaneous tissue of nude mice for 2 and 3 weeks, respectively. As a negative control, ECM alone was implanted. Results showed that kidney like tissue could be formed in ECMfragment constructs after 2 or 3 weeks of implantation, including the formation of glomeruli, tubules and collecting ducts. In addition, these structures seemed more mature in 3 weeks specimens than that of 2 weeks. Further development of this model may lay a solid foundation for kidney tissue engineering.

ECM is required for skeletal muscle differentiation independently of muscle regulatory factor expression

Transcription of specific skeletal muscle genes requires the expression of the muscle regulatory factor myogenin. To assess the role of the extracellular matrix (ECM) in skeletal muscle differentiation, the specific inhibitors of proteoglycan synthesis, sodium chlorate and β-d-xyloside, were used. Treatment of cultured skeletal muscle cells with each inhibitor substantially abolished the expression of creatine kinase and α-dystroglycan. This inhibition was totally reversed by the addition of exogenous ECM. Myoblast treatment with each inhibitor affected the deposition and assembly of the ECM constituents glypican, fibronectin, and laminin. These treatments did not affect MyoD, MEF2A, and myogenin expression and nuclear localization. Differentiated myoblast treatment with RGDS peptides completely inhibited myogenesis without affecting the expression or nuclear localization of myogenin. Integrin-mediated signaling of focal adhesion kinase was partially inhibited by chlorate and β-d-xyloside, an effect reversed by the addition of exogenous ECM gel. These results suggested that the expression of myogenin is not sufficient to successfully drive skeletal muscle formation and that ECM is required to complete the skeletal muscle differentiation process.