Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the ...levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option.
Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Müller glial cells regulate retinal repair under injury, through ...gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Müller cells under in vitro damage. BDNF, NGF, TGF-α, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Müller cells, under H2O2 and IL-1β damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-α expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H2O2 and IL-1β, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-α) and MIO-M1 (IL-6, TNF- α), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H2O2 and IL-1β. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.
The use of blood-based eye drops as therapy for various diseases of the ocular surface has become increasingly popular in ophthalmic practice during recent years. The rationale for their use is based ...on the promotion of cellular proliferation and migration thanks to the supply of metabolically active substances, in particular growth factors. Blood-derived eye drops have been used for the treatment of several ocular surface disorders, such as dry eye disease, corneal ulcer, persistent epithelial defect, neurotrophic keratitis, ocular surface burn, recurrent corneal erosion, and limbal stem-cell deficiency. Both autologous (from patients themselves) and heterologous (from adult donors or from cord blood sampled at birth)-derived products exist, and each source has specific pros and cons. Despite an extensive literature, several issues are still under debate and the aim of this manuscript is to review the indications, preparation methods and storage, characterization of content, rationale for clinical outcomes, patient stratification, length of treatment, and rationale for repeated treatments at disease relapse. A rationale based on a “5 Ws and 2 Hs” protocol is proposed as a way of thinking, with the attempt to clarify Who, Why, When, Where, What, and How to use these treatment options.
Background:The main histopathological features of abdominal aortic aneurysm (AAA) are tissue proteolysis mediated by matrix metalloproteinases (MMPs) and inflammation. This study aimed at verifying ...the presence and contribution of mesenchymal stromal cells (MSCs) to aneurysmal tissue remodeling.Methods and Results:MSCs were successfully isolated from the AAA wall of 12 male patients and were found to express mesenchymal and stemness markers. MMP-2/-9 are involved in AAA progression and their mRNA levels in AAA-MSCs resulted higher than healthy MSCs (cMSCs), especially MMP-9 (400-fold increased). Moreover, MMP-9 protein and activity were pronounced in AAA-MSCs. Immunomodulation was tested in AAA-MSCs after co-culture with activated peripheral blood mononuclear cells (PBMCs) and revealed a weak immunosuppressive action on PBMC proliferation (bromodeoxyuridine incorporation, flow cytometry assay), together with a reduced expression of anti-inflammatory molecules (HLA-G, IL-10) by AAA-MSCs compared to cMSCs. MMP-9 expression in AAA-MSCs was shown to be negatively modulated under the influence of cMSCs and exogenous IL-10.Conclusions:MSCs with stemness properties are niched in human AAA tissues and display a dysregulation of functional activities; that is, upregulation of MMP-9 and ineffective immunomodulatory capacity, which are crucial in the AAA progression; the possibility to modulate the increased MMP-9 expression by healthy MSCs and IL-10 suggests that novel therapeutic strategies are possible for slowing down AAA progression. (Circ J 2015; 79: 1460–1469)
Human mesenchymal stem cells (MSCs) possess well-known reparative abilities, but any defect of the immunomodulatory activity and/or the differentiation process may determine the development of human ...diseases, including those affecting the vascular wall. MSCs residing within the human aortic wall represent a potential cell mediator of atherosclerotic aneurysm development.
MSCs isolated from healthy and aneurysm aortas were characterized by flow cytometer and tested for differentiation properties. Healthy aorta (ha)-MSCs were then subjected to inflammatory stimuli to evaluate the microenvironmental impact on their function and involvement in vascular remodelling.
Abdominal aortic aneurysm (AAA)-MSCs were isolated from calcified and inflamed aortas of 12 patients with high serum levels of MMP-9 protein. AAA-MSCs expressed typical mesenchymal markers and, in line with the histological analysis, elevated levels of OPN, an osteogenic marker also involved in vascular remodelling. AAA-MSCs were highly osteogenic and underwent intense calcium deposition under proper stimulation; moreover, AAA-MSCs were able to differentiate into tubule-like structures in Matrigel, even if the lack of CD146 and the reduced structural stability suggested an inefficient maturation process. We further demonstrated an association between osteogenesis and inflammation; indeed, ha-MSCs cultured with either cytokines (TNF-α, IL-1β) or AAA-PBMCs showed increased expression of MMP-9 and osteogenic markers, to the detriment of the adipogenic regulator PPAR-γ. Interestingly, the culture with inflammatory cells highly stimulated ha-MSCs towards the osteogenic commitment.
AAA-MSCs displayed high osteogenic potential and pathological angiogenesis that represent crucial steps for AAA progression; we showed that the inflammatory process critically addresses human vascular MSCs towards a pathological behaviour, inducing vascular bone matrix deposition and remodelling. Inhibition of this pathway may represent a pharmacological approach against arterial calcification.
We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic ...residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.
Children with autism spectrum disorder (ASD) show worse oral health than their peers. Their access to health services is, at present, inadequate: few high-quality interventions have been designed and ...implemented to improve their care procedures so far. The purpose of this study is to describe an experience of dental care supported by Information and Communication Technologies (ICT), for children with ASD in a public health service. In our study, 59 children (mean age 9.9 years; SD = 5.43) participated in the MyDentist project. It integrates classic dental care techniques with new practices for desensitization and fear control, delivered through an enhanced customized ICT-based intervention aiming at familiarizing the child with ASD with the medical setting and procedures. Two questionnaires were filled out by parents to describe the acceptability of the MyDentist experience for their children. Significant results were shown from T0 (before initiating MyDentist) to T1 (after 6 months of the MyDentist experience) regarding improved oral hygiene and cooperation during dental treatments. Families positively assessed the use of ICT support. In conclusion, the project demonstrated acceptability by parents, suggesting that public health dental care and prevention can be successfully implemented without resorting to costly pharmacological interventions (with potential side effects), taking better care of children’s health.
Umbilical cord platelet-rich plasma (C-PRP) has more growth factors and anti-inflammatory molecules compared with autologous PRP (A-PRP) derived from peripheral blood. The aim of this study was to ...compare intra-articular C-PRP or A-PRP injections in terms of safety and clinical efficacy for the treatment of patients with hip osteoarthritis (OA). This study investigated the results of 100 patients with hip OA treated with three weekly ultrasound-guided injections of either C-PRP or A-PRP. Clinical evaluations were performed before the treatment and after two, six, and twelve months with the HHS, WOMAC, and VAS scores. No major adverse events were recorded. Overall, the improvement was limited with both treatments. Significant improvements in VAS (p = 0.031) and HHS (p = 0.011) were documented at two months for C-PRP. Patients with a low OA grade (Tonnis 1-2) showed a significantly higher HHS improvement with C-PRP than A-PRP at twelve months (p = 0.049). C-PRP injections are safe but offered only a short-term clinical improvement. The comparative analysis did not demonstrate benefits compared with A-PRP in the overall population, but the results are influenced by OA severity, with C-PRP showing more benefits when advanced OA cases were excluded. Further studies are needed to confirm the most suitable indications and potential of this biological injective approach.
The clinical use of endothelial progenitor cells is hampered by difficulties in obtaining an adequate number of functional progenitors. This study aimed to establish whether human thoracic aortas ...harvested from healthy multiorgan donors can be a valuable source of angiogenic progenitors. Immunohistochemical tissue studies showed that two distinct cell populations with putative stem cell capabilities, one composed of CD34+ cells and the other of c-kit+ cells, are present in between the media and adventitia of human thoracic aortas. Ki-67+ cells with high growth potential were located in an area corresponding to the site of CD34+ and c-kit+ cell residence. We thus isolated cells (0.5 approximately 2.0 x 10(4) aortic progenitors per 25 cm2) which, upon culturing, coexpressed molecules of mesenchymal stromal cells (i.e., CD44+, CD90+, CD105+) and showed a transcript expression of stem cell markers (e.g., OCT4, c-kit, BCRP-1, Interleukin-6) and BMI-1. Cell expansion was adequate for use in a clinical setting. A subset of cultured cells acquired the phenotype of endothelial cells in the presence of vascular endothelial growth factor (e.g., increased expression of KDR and von Willebrand factor positivity), as documented by flow cytometry, immunofluorescence, electron microscopy, and reverse transcription-polymerase chain reaction assays. An in vitro angiogenesis test kit revealed that cells were able to form capillary-like structures within 6 hours of seeding. This study demonstrates that thoracic aortas from multiorgan donors yield mesenchymal stromal cells with the ability to differentiate in vitro into endothelial cells. These cells can be used for the creation of an allogenic bank of angiogenic progenitors, thus providing new options for restoring vascularization at ischemic sites. Disclosure of potential conflicts of interest is found at the end of this article.
Age-related macular degeneration (AMD) is one of the leading causes of visual loss in western countries, it has no cure, and its incidence will grow in the future, for the overall population aging. ...Albino rats with retinal degeneration induced by exposure to high-intensity light (light-damage, LD) have been extensively used as a model of AMD to test neuroprotective agents. Among them, trophic factors (NGF and BDNF) have been shown to play a significant role in photoreceptors' survival. Interestingly, cord blood serum (CBS) is an extract full of chemokines and trophic factors; we, therefore, hypothesized that CBS could be an excellent candidate for neuroprotection. Here, we investigate whether CBS-based eye drops might mitigate the effects of light-induced retinal degeneration in albino rats. CBS treatment significantly preserved flash-electroretinogram (f-ERG) response after LD and reduced the "hot-spot" extension. Besides, CBS-treated animals better preserved the morphology of the outer nuclear layer, together with a reduction in microglia migration and activation. Interestingly, the treatment did not modulate reactive gliosis and activation of the self-protective mechanism (FGF2). In conclusion, our results suggest that CBS-based eye drops might be successfully used to mitigate retinal neurodegenerative processes such as AMD.
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