Production and implantation of renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations.

BACKGROUND: It is important to identify new sources of transplantable organs because of the critical shortage of donor organs. Tissue engineering holds the potential to address this issue through the implementation of decellularization-recellularization technology. OBJECTIVE: To produce and examine acellular renal extracellular matrix (ECM) scaffolds as a platform for kidney bioengineering. METHODS: Porcine kidneys were decellularized with distilled water and sodium dodecyl sulfate-based solution. After rinsing with buffer solution to remove the sodium dodecyl sulfate, the so-obtained renal ECM scaffolds were processed for vascular imaging, histology, and cell seeding to investigate the vascular patency, degree of decellularization, and scaffold biocompatibility in vitro. Four whole renal scaffolds were implanted in pigs to assess whether these constructs would sustain normal blood pressure and to determine their biocompatibility in vivo. Pigs were sacrificed after 2 weeks and the explanted scaffolds were processed for histology. RESULTS: Renal ECM scaffolds were successfully produced from porcine kidneys. Scaffolds retained their essential ECM architecture and an intact vascular tree and allowed cell growth. On implantation, unseeded scaffolds were easily reperfused, sustained blood pressure, and were tolerated throughout the study period. No blood extravasation occurred. Pathology of explanted scaffolds showed maintenance of renal ultrastructure. Presence of inflammatory cells in the pericapsular region and complete thrombosis of the vascular tree were evident. CONCLUSIONS: Our investigations show that pig kidneys can be successfully decellularized to produce renal ECM scaffolds. These scaffolds maintain their basic components, are biocompatible, and show intact, though thrombosed, vasculature

Epigenetic regulation during fetal femur development: DNA methylation matters

Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.<br/

Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency

There is currently an unmet need for the supply of autologous, patient-specific stem cells for regenerative therapies in the clinic. Mesenchymal stem cell differentiation can be driven by the material/cell interface suggesting a unique strategy to manipulate stem cells in the absence of complex soluble chemistries or cellular reprogramming. However, so far the derivation and identification of surfaces that allow retention of multipotency of this key regenerative cell type have remained elusive. Adult stem cells spontaneously differentiate in culture, resulting in a rapid diminution of the multipotent cell population and their regenerative capacity. Here we identify a nanostructured surface that retains stem-cell phenotype and maintains stem-cell growth over eight weeks. Furthermore, the study implicates a role for small RNAs in repressing key cell signalling and metabolomic pathways, demonstrating the potential of surfaces as non-invasive tools with which to address the stem cell niche.<br/

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.<br/

TGF-<tex>\beta$</tex> 1-induced migration of bone mesenchymal stem cells couples bone resorption with formation

Bone remodeling depends on the precise coordination of bone resorption and subsequent bone formation. Disturbances of this process are associated with skeletal diseases, such as Camurati-Engelmann disease (CED). We show using in vitro and animal models that active TGF-β1 released during bone resorption coordinates bone formation by inducing migration of bone marrow stromal cells, also known as bone mesenchymal stem cells (BMSCs) to the bone resorptive sites and that this process is mediated through SMAD signaling pathway. Analysis of a mouse model carrying a CED-derived TGF-β1 mutation, which exhibits the typical progressive diaphyseal dysplasia with tibial fractures, we found high levels of active TGF-β1 in the bone marrow. Treatment with a TGF-β type I receptor inhibitor partially rescued the uncoupled bone remodeling and prevented the fractures. Thus, as TGF-β1 functions to couple bone resorption and formation, modulation of TGF-β1 activity could be an effective treatment for the bone remodeling diseases