Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation

The effects of growth factors on skeletal muscle lesions

Skeletal muscle injuries are common causes of severe long-term pain and physical disability, accounting for up to 55% of all sports injuries. The phases of the healing process after direct or indirect muscle injury are complex but clearly defined processes comprising wellcoordinated steps: degeneration, inflammation, regeneration, and fibrosis. Despite this frequent occurrence and the presence of a body of data on the pathophysiology of muscle injuries, none of the treatment strategies adopted to date have been shown to be really effective in strictly controlled trials. Most current muscle injury treatments are based on limited experimental and clinical data and/or were only empirically tested. Platelet-rich plasma (PRP) is a promising alternative approach based on the ability of autologous growth factors (GFs) to accelerate tissue healing, improve muscular regeneration, increase neovascularization and reduce fibrosis, allowing rapid recovery after muscle lesions. Thus, further experimental studies that include the quantification of specific GFs released by PRP, as well as additional data on angiogenesis, myogenesis and functional recovery are needed to ultimately validate the hypothesis of PRP efficacy in the treatment of muscle lesions and open the way for its wide clinical application

Detecting senescent fate in mesenchymal stem cells: a combined cytofluorimetric and ultrastructural approach

Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover—like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life—adds a new piece to the regenerative medicine jigsaw in an ageing society

Vitamin MK-7 enhances vitamin D3-induced osteogenesis in hMSCs: modulation of key effectors in mineralization and vascularization

The osteoblast is the bone-forming cell and is derived from mesenchymal stem cells (MSCs). Osteo-inductive substances could represent a useful therapeutic approach during the fracture repair process. The aim of this work was to evaluate the effects of vitamin MK-7, alone or in association with vitamin D3, in differentiating human MSCs (hMSCs) in vitro along the osteoblastic lineage. In particular, primary endpoints of the study include gene and protein markers of osteoblast differentiation. Considering genes involved in bone formation and mineralization, our data show that vitamin MK-7 enhances vitamin D3 gene induction of osteocalcin (OC). Among genes related to cell growth and differentiation, a specific effect of vitamin MK-7 was observed for growth differentiation factor-10 (GDF10) and insulin-like growth factor 1 (IGF1), the latter being also involved in the induction of vascular endothelial growth factors (VEGFA). Accordingly, vitamin co-supplementation greatly affected VEGFA and its receptor fms-related tyrosine kinase 1 (FLT1), a key factor in both angiogenic and osteogenic processes. These results stress the relevance of MK-7 and D3 co-supplementation in the bone-healing process as able to modulate the expression of genes involved in both mineralization and angiogenesis. Moreover, at the protein level co-association of vitamins might provide an optimal balance between induction and carboxylation of osteocalcin, essential for its functionality in the extracellular matrix (ECM). Our results may provide hints for therapeutic application of hMSCs in bone disease, clarifying mechanisms involved in stem cell-mediated bone development, and they also highlight the relevance of co-supplementation strategies, since single supplementations might result in a suboptimal effect

TOTAL KNEE PROSTHESE POLYETHYLENE WEAR REDUCTION BY A NEW METAL PART FINISHING METHOD

Purpose: The purpose of this study was to evaluate a new metal component finishing designed to improve total knee prosthesis durability. Wear of ultrahigh molecular-weight polyethylene (UHMWPE), with generation of submicrometer- and micrometer-sized particles, has been associated with osteolysis and artificial joint failure. Wear extent is influenced by several factors, some of which are related to manufacturing. Methods: UHMWPE wear was assessed in metal prosthesis components finished with the Microloy® technology and in traditionally finished components by wear simulation experiments (pin on disk and knee simulator tests) and analysis of wear debris. Results: Microloy®-finished prosthesis showed a 48,5% reduction in UHMWPE total weight loss compared with traditional components (P=0.002). A significant (p<0.05) reduction of UHMWPE debris were detected from the Microloy®-finished compared with the traditional-finished components. Conclusions: These findings suggest the Microloy® metal finishing may enhance the long-term performance of knee prostheses

Development and characterization of rhVEGF-loaded poly(HEMA-MOEP) coatings electrosynthesized on titanium to enhance bone mineralization and angiogenesis.

Osteointegration of titanium implants could be significantly improved by coatings capable of promoting both mineralization and angiogenesis. In the present study, a copolymeric hydrogel coating, poly-2-hydroxyethyl methacrylate-2-methacryloyloxyethyl phosphate (P(HEMA–MOEP)), devised to enhance calcification in body fluids and to entrap and release growth factors, was electrosynthesized for the first time on titanium substrates and compared to poly-2-hydroxyethyl methacrylate (PHEMA), used as a blank reference. Polymers exhibiting negatively charged groups, such as P(HEMA–MOEP), help to enhance implant calcification. The electrosynthesized coatings were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. MG-63 human osteoblast-like cell behaviour on the coated specimens was investigated by scanning electron microscopy, MTT viability test and osteocalcin mRNA detection. The ability of negatively charged phosphate groups to promote hydroxyapatite-like calcium phosphate deposition on the implants was explored by immersing them in simulated body fluid. Similar biological responses were observed in both coated specimens, while calcium-phosphorus globules were detected only on P(HEMA–MOEP) surfaces pretreated with alkaline solution. Testing of the ability of P(HEMA–MOEP) hydrogels to entrap and release human recombinant vascular endothelial growth factor, to tackle the problem of insufficient oxygen and nutrient delivery, suggested that P(HEMA–MOEP)-coated titanium prostheses could represent a multifunctional material suitable for bone restoration applications

Mixed Type I and Type II Collagen Scaffold for Cartilage Repair: Ultrastructural Study of Synovial Membrane Response and Healing Potential versus

The association between microfracture of the subchondral plate and a coverage scaffold has emerged as a promising strategy to treat cartilage lesions in a one-step procedure. Between different types of scaffolds (e.g. collagen, hyaluronic acid, polyglycolic acid) currently studied, type I collagen scaffold is the most used for this purpose, and is currently adopted for humans. The aim of this study was to test a novel scaffold made of mixed type I and II collagen (I-IICS) in order to define the immunological reaction of the synovial tissue and the repair capabilities induced by the collagen membrane when associated with microfracture. Eight New Zealand White rabbits, aged 180 days, were operated on bilaterally on the medial femoral condyle. A circular cartilage lesion was performed up to the calcified layer of the medial femoral condyle, and the centre of the lesion was microfractured. Randomly, one of the two lesions was covered with the I-IICS (treated), and the other was left uncovered (control). The synovial membrane reaction and the quality of the cartilage tissue repair were investigated at 2, 90, 180 and 270 days macroscopically, histomorphologically and ultrastructurally. Expression of tumor necrosis factor-alpha (TNF-α) in synovial tissue by immunocytochemistry analyses was also investigated. In the control group, at 2 days gold particles were localized mainly on synoviocyte type A, less on synoviocytes type B and on collagen bundles; in the treated group the reaction is more intense in cells in the matrix, but at 180 days controls and treated joints were very similar. The synovial membranes of the joints receiving the I-IICS did not reveal significant changes compared to the age-matched controls. Signs of inflammation were present at the 90-day time-point, and became less evident at afterwards. The degradation of the scaffolds was already evident at the 90-day time-point. The quality of the cartilage repair of the rabbits treated with the I-IICS was slightly better in 5 cases out of 6 in comparison to the controls. However, a statistically significant difference was not detected (p=0.06). Scaffolds made of mixed type I and II collagen exhibited good biocompatibility properties in vivo and favored cartilage restoration when associated with microfracture, as shown in this pilot study

Purified collagen I oriented membrane for tendon repair: an ex vivo morphological study.

Purpose: Injured tendons have limited repair ability after full-thickness lesions. Tendon regeneration properties and adverse reactions were assessed ex vivo in an experimental animal model using a new collagen I membrane. Methods: This multilamellar membrane obtained from purified equine Achilles tendon is characterized by oriented collagen I fibers and has been shown to sustain cell growth and orientation in vitro. The central third of the patellar tendon (PT) of 10 New Zealand White rabbits was sectioned and grafted with the collagen membrane; the contralateral PT was cut longitudinally (sham-operated controls). Animals were euthanized 1 or 6 months from surgery and tendons subjected to histological and Synchrotron Radiation-based Computed Microtomography (SRµCT) examination and 3D structure analysis. Results: Histological and SRuCT findings showed satisfactory graft integration with native tendon; histological examination also showed ongoing angiogenesis. Adverse side-effects (inflammation, rejection, calcification) were never observed. Conclusion: The multilamellar collagen I membrane can be considered as an effective tool for tendon defect repair and tendon augmentation