Although the link between altered stem cell properties and tissue aging has been recognized, the molecular and cellular processes of tendon aging have not been elucidated. As tendons contain stem/progenitor cells (TSPC), we investigated whether the molecular and cellular attributes of TSPC alter during tendon aging and degeneration. Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained. Senescence analysis showed a premature entry into senescence of the A-TSPC, a finding accompanied by an upregulation of p16INK4A. To identify age-related molecular factors, we performed microarray and gene ontology analyses. These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton. Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments. Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell–matrix interactions and the ROCK kinase pathway might be key players in TSPC aging. Taken together, we propose that during tendon aging and degeneration, the TSPC pool is becoming exhausted in terms of size and functional fitness. Thus, our study provides the first fundamental basis for further exploration into the molecular mechanisms behind tendon aging and degeneration as well as for the selection of novel tendon-specific therapeutical targets.

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors – α1β1, α2β1 and α11β1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2β1- or α11β1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.

Acute ankle injuries are among the most common injuries in emergency departments. However, there are still no standardized examination procedures or evidence-based treatment. Therefore, the aim of this study was to systematically search the current literature, classify the evidence, and develop an algorithm for the diagnosis and treatment of acute ankle injuries. We systematically searched PubMed and the Cochrane Database for randomized controlled trials, meta-analyses, systematic reviews or, if applicable, observational studies and classified them according to their level of evidence. According to the currently available literature, the following recommendations have been formulated: i) the Ottawa Ankle/Foot Rule should be applied in order to rule out fractures; ii) physical examination is sufficient for diagnosing injuries to the lateral ligament complex; iii) classification into stable and unstable injuries is applicable and of clinical importance; iv) the squeeze-, crossed leg- and external rotation test are indicative for injuries of the syndesmosis; v) magnetic resonance imaging is recommended to verify injuries of the syndesmosis; vi) stable ankle sprains have a good prognosis while for unstable ankle sprains, conservative treatment is at least as effective as operative treatment without the related possible complications; vii) early functional treatment leads to the fastest recovery and the least rate of reinjury; viii) supervised rehabilitation reduces residual symptoms and re-injuries. Taken these recommendations into account, we present an applicable and evidence-based, step by step, decision pathway for the diagnosis and treatment of acute ankle injuries, which can be implemented in any emergency department or doctor's practice. It provides quality assurance for the patient and promotes confidence in the attending physician.

Human mesenchymal stem cells (hMSC) are a heterogeneous cell population, which is reflected in varying morphological and biological properties. Three subpopulations with intrinsic characteristics can be distinguished: small rapidly self-renewing cells, spindle-shaped cells and large, flattened cells. Unfortunately, it has neither been possible to morphologically define these distinct cells consistently, nor to relate them to specific surface marker features. Here, the primary hMSC subpopulations of three donors are clearly defined by maximum cell diameter and area. Furthermore, these cells were stained for the putative hMSC surface markers CD105, CD90 as well as CD73, and evaluated by three-colour flow cytometry and simultaneous multicolour immunocytochemistry. Interestingly, cell cultures with a high rate of triple-positive hMSC featured a higher content of rapidly self-renewing cells. On the other hand, a higher fraction of flattened cells correlated with a loss of one or more hMSC surface markers. The expression of CD73 showed the highest heterogeneity. Immunocytochemistry further confirmed that flattened cells mainly lack CD73 expression, whereas rapidly self-renewing cells were steadily positive for all three hMSC markers. In the literature, hMSC properties are especially conceded to rapidly self-renewing cells, whereas flattened cells have been suggested to represent early stages of lineage-specific progenitors. We reveal that among the recently suggested surface markers, CD73 is the most sensitive, as it seems to be down-regulated in the early stages of differentiation. Our morphological and immunocytochemical characterization of hMSC subpopulations indicates the yield of early multipotent hMSC and thereby provides a quality control approach for hMSC culturing.

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-kappaB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-alpha (TNF-alpha), which is known to be a key regulator of the NF-kappaB pathway. Therefore, we hypothesized that TNF-alpha may lead to MSC invasion and proliferation by activation of the NF-kappaB pathway. TNF-receptor 1 and 2, NF-kappaB (p65), and IkappaB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-alpha caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-alpha strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-kappaB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-alpha stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-kappaB pathway caused a significant reduction in the basal proliferation rate. TNF-alpha stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-alpha led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-kappaB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-alpha-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-kappaB signal transduction pathway.