The enhancer landscape predetermines the skeletal regeneration capacity of stromal cells.

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

Metal-Specific Biomaterial Accumulation in Human Peri-Implant Bone and Bone Marrow.

Metallic implants are frequently used in medicine to support and replace degenerated tissues. Implant loosening due to particle exposure remains a major cause for revision arthroplasty. The exact role of metal debris in sterile peri-implant inflammation is controversial, as it remains unclear whether and how metals chemically alter and potentially accumulate behind an insulating peri-implant membrane, in the adjacent bone and bone marrow (BM). An intensively focused and bright synchrotron X-ray beam allows for spatially resolving the multi-elemental composition of peri-implant tissues from patients undergoing revision surgery. In peri-implant BM, particulate cobalt (Co) is exclusively co-localized with chromium (Cr), non-particulate Cr accumulates in the BM matrix. Particles consisting of Co and Cr contain less Co than bulk alloy, which indicates a pronounced dissolution capacity. Particulate titanium (Ti) is abundant in the BM and analyzed Ti nanoparticles predominantly consist of titanium dioxide in the anatase crystal phase. Co and Cr but not Ti integrate into peri-implant bone trabeculae. The characteristic of Cr to accumulate in the intertrabecular matrix and trabecular bone is reproducible in a human 3D in vitro model. This study illustrates the importance of updating the view on long-term consequences of biomaterial usage and reveals toxicokinetics within highly sensitive organs.

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

Induction of extracranial arteriogenesis by an arteriovenous fistula in a pig model.

BACKGROUND AND AIMS: Arteriogenesis, the positive outward remodeling and growth of pre-existent collateral vessels, holds potential as a novel treatment for ischemic vascular disease. An extracranial arteriogenesis model in a pig will allow us to study molecular changes in a complex arteriolar network in a more clinically relevant large-animal model. To increase fluid shear stress in the brain, an experimental carotid arteriovenous fistula (AVF model) in minipigs was established, providing high flow through the extracranial rete mirabile. The aim of the study was to examine whether creation of a carotid AVF can induce extracranial arteriogenesis in the pig. METHODS: Angiography was performed to demonstrate blood flow diversion. Animals were sacrificed after 0, 3 and 14 days post-surgery and both retia mirabilia were removed. Immunohistochemical analysis was performed to analyze cell proliferation and accumulation of mononuclear cells in the vessel wall. RESULTS: After 3 days of high-flow conditions, increases in vascular cell proliferation (approximately 1.5-fold; p = 0.143) and monocyte invasion (approximately 6-fold; p = 0.057) were observed when compared to animals sacrificed immediately after AVF formation. Quantitative PCR (RT-qPCR) analysis from rete mirabile tissue samples 3 days post-surgery revealed that monocyte chemoattractant protein (MCP)-1 and tissue inhibitor of metalloproteinases (TIMP)-1 were highly upregulated. Expression of the pro-arteriogenic marker, CD44, reached maximum expression level 14 days post-surgery. CONCLUSIONS: In response to high levels of shear stress produced in the pig AVF model, the onset of the arteriogenic process can be induced. This was demonstrated by enhanced cell proliferation, monocyte invasion and vascular remodeling.

The "artificial artery" as in vitro perfusion model.

Metabolic stimuli, pressure, and fluid shear stress (FSS) are major mediators of vascular plasticity. The exposure of the vessel wall to increased laminar FSS is the main trigger of arteriogenesis, the remodelling of pre-existent arterio-arteriolar anastomoses to functional conductance arteries. In this study, we have used an in vitro bioreactor to investigate cell-specific interactions, molecular mechanisms as well as time-dependent effects under laminar FSS conditions. This bioreactor termed "artificial artery" can be used for screening potential arterio-protective substances, pro-arteriogenic factors, and for investigating biomarkers of cardiovascular diseases such as cardiac diseases. The bioreactor is built up out of 14 hollow fiber membranes colonized with endothelial cells (HUVECs) on the inside and smooth muscle cells (HUASMCs) on the outside. By means of Hoechst 33342 staining as well as immunocytochemistry of ß-catenin and α-smooth-muscle-actin, a microporous polypropylene membrane was characterized as being the appropriate polymer for co-colonization. Defined arterial flow conditions (0.1 N/m2 and 3 N/m2), metabolic exchange, and cross-talk of HUVECs and HUASMCs through hollow fibers mimic physiological in vivo conditions of the vasculature. Analysing mono- and co-culture secretomes by MALDI-TOF-TOF mass spectrometry, we could show that HUVECs secreted Up4A upon 3 N/m2. A constant cellular secretion of randomly chosen peptides verified viability of the "artificial artery" for a cultivation period up to five days. qRT-PCR analyses revealed an up-regulation of KLF2 and TIMP1 as mechano-regulated genes and demonstrated arterio-protective, homeostatic FSS conditions by a down-regulation of EDN1. Expression analyses of VWF and EDN1 furthermore confirmed that RNA of both cell types could separately be isolated without cross-contamination. CCND1 mRNA expression in HUVECs did not change upon FSS indicating a quiescent endothelial phenotype. Taken together, the "artificial artery" provides a solid in vitro model to test pharmacological active compounds for their impact on arterio-damaging or arterio-protective properties on vascular response.