Analysis of miRNAs in Osteogenesis imperfecta Caused by Mutations in COL1A1 and COL1A2: Insights into Molecular Mechanisms and Potential Therapeutic Targets.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Impact of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum bones after sternotomy.

Median sternotomy is the surgical method of choice for many procedures where one of the main problems is the long post-operative wound healing process leading to sternal dehiscence and the development of infection. This leads to prolonged hospital stay and increased mortality due to post-operative complications. A promising solution seems to be the use of allogeneic chondrocytes for wound treatment, whose properties in the field of cartilage reconstruction are widely used in medicine, mainly in orthopedics. In the present study, we investigated the effect of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum after sternotomy. We optimized the culture conditions for the isolated chondrocytes, which were then applied to the sternal incision wound. Chondrocytes in the culture were assessed on the basis of the presence of chondrocyte-specific genes: Sox9, Aggrecan and Collagen II. In turn, the histopathological and immunohistochemical evaluation was used to assess the safety of implantation. In our work, we demonstrated the possibility of obtaining a viable culture of chondrocytes, which were successfully introduced into the sternal wound after sternotomy. Importantly, implantation of allogeneic chondrocytes showed no significant side effects. The obtained results open new possibilities for research on the use of allogeneic chondrocytes in the process of accelerating wound healing after median sternotomy.

Influence of the modifiers in polyol method on magnetically induced hyperthermia and biocompatibility of ultrafine magnetite nanoparticles.

Magnetite nanoparticles (Fe3O4 NPs) are widely tested in various biomedical applications, including magnetically induced hyperthermia. In this study, the influence of the modifiers, i.e., urotropine, polyethylene glycol, and NH4HCO3, on the size, morphology, magnetically induced hyperthermia effect, and biocompatibility were tested for Fe3O4 NPs synthesized by polyol method. The nanoparticles were characterized by a spherical shape and similar size of around 10 nm. At the same time, their surface is functionalized by triethylene glycol or polyethylene glycol, depending on the modifiers. The Fe3O4 NPs synthesized in the presence of urotropine had the highest colloidal stability related to the high positive value of zeta potential (26.03 ± 0.55 mV) but were characterized by the lowest specific absorption rate (SAR) and intrinsic loss power (ILP). The highest potential in the hyperthermia applications have NPs synthesized using NH4HCO3, for which SAR and ILP were equal to 69.6 ± 5.2 W/g and 0.613 ± 0.051 nHm2/kg, respectively. Their application possibility was confirmed for a wide range of magnetic fields and by cytotoxicity tests. The absence of differences in toxicity to dermal fibroblasts between all studied NPs was confirmed. Additionally, no significant changes in the ultrastructure of fibroblast cells were observed apart from the gradual increase in the number of autophagous structures.

Star Polymers as Non-Viral Carriers for Apoptosis Induction.

Apoptosis is a widely controlled, programmed cell death, defects in which are the source of various diseases such as neurodegenerative diseases as well as cancer. The use of apoptosis in the therapy of various human diseases is of increasing interest, and the analysis of the factors involved in its regulation is valuable in designing specific carriers capable of targeting cell death. Highly efficient and precisely controlled delivery of genetic material by low-toxic carriers is one of the most important challenges of apoptosis-based gene therapy. In this work, we investigate the effect of the star polymer with 28 poly(N,N'-dimethylaminoethyl methacrylate) arms (STAR) on human cells, according to its concentration and structure. We show that star polymer cytotoxicity increases within its concentration and time of cells treatment. Except for cytotoxic effect, we observe morphological changes such as a shrinkage, loss of shape and begin to detach. We also prove DNA condensation after star polymer treatment, one of the most characteristic feature of apoptosis. The results indicate that the use of STAR triggers apoptosis in cancer cells compared to various normal cells, what makes these nanoparticles a promising drug in therapeutic strategy, which targets apoptosis. We demonstrate highlighting potential of star polymers as an innovative tool for anti-cancer therapy.

Fibroblast and keratinocyte crosstalk: the effect of a poly(tri[ethylene glycol] ethyl ether methacrylate) thermoresponsive surface on short-term co-culture.

The treatment of difficult-to-treat wounds can be challenging. Although a number of approaches have been investigated, the healing process may be slow and unsatisfactory. An alternative approach is the use of a continuous sheet of skin cells applied over a wound which may improve cell implantation and patient recovery. To analyse the gene expression profile of fibroblast/keratinocyte co-culture on poly(tri[ethylene glycol] ethyl ether methacrylate) (P[TEGMA-EE]), a thermoresponsive biocompatible surface. Cultures were grown for 72 hours as a continuous layer on P(TEGMA-EE). Assays for genotoxicity, cell morphology, and fluorescence-assisted flow cytometry were performed to exclude adverse effects. A gene expression profile related to the extracellular matrix was investigated by microarray analysis. For fibroblast monocultures and fibroblast/keratinocyte co-cultures maintained for 72 hours on P(TEGMA-EE), no change in morphology or specific surface markers, or DNA damage (comet assay) was observed, relative to control surface. Moreover, no detrimental impact was ascertained based on microarray analysis. In response to lowered temperature, the detachment of a continuous cell layer sheet from the thermoresponsive surface was observed. When gene expression was compared between fibroblasts cultured alone and co-cultured with keratinocytes on P(TEGMA-EE), 10 genes were shown to be differentially expressed. Of these genes, six were significantly differentially expressed between cultures grown on P(TEGMA-EE) and human skin samples. Our results indicate that P(TEGMA-EE) is fully biocompatible and is therefore a suitable surface for successful preparation and recovery of two-layered fibroblast/keratinocyte co-culture as a continuous sheet of cells.

Functional and structural studies of tolloid-like 1 mutants associated with atrial-septal defect 6.

Inactive mammalian tolloid-like 1 (tll1) and mutations detected in tolloid-like 1 (TLL1) have been linked to the lack of the heart septa formation in mice and to a similar human inborn condition called atrial-septal defect 6 (ASD6; OMIM 613087, formerly ASD II). Previously, we reported four point mutations in TLL1 found in approximately 20% of ASD6 patients. Three mutations in the coding sequence were M182L, V238A, and I629V. In this work, we present the effects of these mutations on TLL1 function. Three recombinant cDNA constructs carrying the mutations and one wild-type construct were prepared and then expressed in HT-1080 cells. Corresponding recombinant proteins were analyzed for their metalloendopeptidase activity using a native substrate, chordin. The results of these assays demonstrated that in comparison with the native TLL1, mutants cleaved chordin and procollagen I at significantly lower rates. CD analyses revealed significant structural differences between the higher order structure of wild-type and mutant variants. Moreover, biosensor-based assays of binding interactions between TLL1 variants and chordin demonstrated a significant decrease in the binding affinities of the mutated variants. The results from this work indicate that mutations detected in TLL1 of ASD6 patients altered its metalloendopeptidase activity, structure, and substrate-binding properties, thereby suggesting a possible pathomechanism of ASD6.

Stable star polymer nanolayers and their thermoresponsiveness as a tool for controlled culture and detachment of fibroblast sheets.

In this study, we describe novel thermoresponsive star copolymer surfaces used for the first time for the culture of fibroblast sheets, followed by their detachment, controlled by a change in temperature. To date, no star polymers, or their layers, have been used for this purpose. A "grafting to" strategy was applied to obtain poly[oligo(ethylene glycol) methacrylate] star layers on functionalized solid supports. Atom transfer radical polymerization of oligo(ethylene glycol) methacrylates and glycidyl methacrylate initiated with modified poly(arylene oxindole) yielded stars with molar masses up to Mn = 380 000 g mol-1. Stars were attached to a glass substrate via the reaction between the functional epoxy groups of the stars with the amine groups of the functionalized substrate. The thickness of the layer was related to the dimensions of isolated stars in solution, which showed that multilayers were obtained. Above the phase transition temperature, polymer nanolayers were hydrophobic, thus enabling the growth of fibroblasts on their surfaces and the formation of a cell sheet. Decreasing the temperature below the phase transition temperature made the star surfaces hydrophilic. This eliminated the affinity of the surface for cells and led to detachment of the intact fibroblast sheet. These observations have shown for the first time that the star polymer architecture favors the detachment of cell sheets as compared to linear polymer analogues grafted onto supports, thus reducing the time of this process. Knowledge of the influence of the polymer topology on layer properties and cell growth and detachment can aid in the development of polymeric materials for tissue culture applications.

Nonviral Plasmid DNA Carriers Based on N,N'-Dimethylaminoethyl Methacrylate and Di(ethylene glycol) Methyl Ether Methacrylate Star Copolymers.

Star polymers with random and block copolymer arms made of cationic N,N'-dimethylaminoethyl methacrylate (DMAEMA) and nonionic di(ethylene glycol) methyl ether methacrylate (DEGMA) were synthesized via atom transfer radical polymerization (ATRP) and used for the delivery of plasmid DNA in gene therapy. All stars were able to form polyplexes with plasmid DNA. The structure and size of the polyplexes were precisely determined using light scattering and cryo-TEM microscopy. The hydrodynamic radius of a complex of DNA with star was dependent on the architecture of the star arms, the DEGMA content and the number of amino groups in the star compared to the number of phosphate groups of the nucleic acid (N/P ratio). The smallest polyplexes (Rh90°âˆ¼50 nm) with positive zeta potentials (∼15 mV) were formed of stars with N/P=6. The introduction of DEGMA into the star structure caused a decrease of polyplex cytotoxicity in comparison to DMAEMA homopolymer stars. The overall transfection efficiency using HT-1080 cells showed that the studied systems are prospective gene delivery agents. The most promising results were obtained for stars with random copolymer arms of high DEGMA content.