Development of hybrid biomicroparticles: cellulose exposing functionalized fusion proteins.

BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected. CONCLUSIONS: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties.

Adipose-Derived Mesenchymal Stromal Cells in Basic Research and Clinical Applications.

Adipose-derived mesenchymal stromal cells (AD-MSCs) have been extensively studied in recent years. Their attractiveness is due to the ease of obtaining clinical material (fat tissue, lipoaspirate) and the relatively large number of AD-MSCs present in adipose tissue. In addition, AD-MSCs possess a high regenerative potential and immunomodulatory activities. Therefore, AD-MSCs have great potential in stem cell-based therapies in wound healing as well as in orthopedic, cardiovascular, or autoimmune diseases. There are many ongoing clinical trials on AD-MSC and in many cases their effectiveness has been proven. In this article, we present current knowledge about AD-MSCs based on our experience and other authors. We also demonstrate the application of AD-MSCs in selected pre-clinical models and clinical studies. Adipose-derived stromal cells can also be the pillar of the next generation of stem cells that will be chemically or genetically modified. Despite much research on these cells, there are still important and interesting areas to explore.

Examination of epigenetic inhibitor zebularine in treatment of skin wounds in healthy and diabetic mice.

DNA methyltransferase inhibitor zebularine was proven to induce regeneration in the ear pinna in mice. We utilized a dorsal skin wound model to further evaluate this epigenetic inhibitor in wound healing. Full-thickness excisional wounds were made on the dorsum of 2 and 10-month-old healthy BALB/c and 3 and 8-month-old diabetic (db/db) mice, followed by topical or intraperitoneal zebularine delivery. Depending on the strain, age, dose, and delivery, the zebularine treatments either had no effect or accelerated or delayed wound closure. In principle, zebularine applied topically moderately promoted wound closure in the healthy but markedly delayed in the diabetic mice, which was in line with decreased viability of cultured keratinocytes from diabetic patients exposed to zebularine. The histological analysis revealed an improvement in the architecture of restored skin in zebularine-treated mice, manifested as a distinct layered pattern resembling panniculus carnosus. The finding corresponds with the zebularine-mediated activation of the Wnt5a gene, an essential regulator of Wnt signaling, the pathway involved in hair follicle development, the process which in turn is connected with regenerative skin healing. Although zebularine did not remarkably accelerate wound healing, zebularine and other epigenetic inhibitors deserve further testing as potential drugs to improve the quality of restored skin.

Adipose-derived stromal cells for nonhealing wounds: Emerging opportunities and challenges.

Wound healing complications affect thousands of people each year, thus constituting a profound economic and medical burden. Chronic wounds are a highly complex problem that usually affects elderly patients as well as patients with comorbidities such as diabetes, cancer (surgery, radiotherapy/chemotherapy) or autoimmune diseases. Currently available methods of their treatment are not fully effective, so new solutions are constantly being sought. Cell-based therapies seem to have great potential for use in stimulating wound healing. In recent years, much effort has been focused on characterizing of adipose-derived mesenchymal stromal cells (AD-MSCs) and evaluating their clinical use in regenerative medicine and other medical fields. These cells are easily obtained in large amounts from adipose tissue and show a high proregenerative potential, mainly through paracrine activities. In this review, the process of healing acute and nonhealing (chronic) wounds is detailed, with a special attention paid to the wounds of patients with diabetes and cancer. In addition, the methods and technical aspects of AD-MSCs isolation, culture and transplantation in chronic wounds are described, and the characteristics, genetic stability and role of AD-MSCs in wound healing are also summarized. The biological properties of AD-MSCs isolated from subcutaneous and visceral adipose tissue are compared. Additionally, methods to increase their therapeutic potential as well as factors that may affect their biological functions are summarized. Finally, their therapeutic potential in the treatment of diabetic and oncological wounds is also discussed.

Alterations in peripheral blood B cells in systemic lupus erythematosus patients with renal insufficiency.

OBJECTIVE: Systemic lupus erythematosus (SLE) is one of the autoimmune diseases, believed to be closely related to hyperactivity of B cells, overproduction of autoantibodies and immune complex formation and deposition in affected tissue. The autoreactive inflammation leads to multiorgan damage with kidney dysfunction in the forefront. Studies on lupus nephritis (LN), affecting the majority of SLE patients, are mainly focused on cells causing local inflammation. The aim of our work was to detect alterations in more accessible peripheral blood B cells in the course of SLE focusing on the influence of renal insufficiency (RI) on those parameters. METHODS: We performed a comprehensive flow cytometry analysis of B cell subpopulations, analyzed gene expression patterns with qPCR, and examined serum cytokine levels with multiplex cytokine/chemokine assay. RESULTS: We discovered distribution of specific B cell subsets, especially CD38+ cells, plasmablasts, associated with the presence and severity of the disease. Changes in expression of MBD2, DNMT1 and APRIL genes were not only associated with activity of SLE but also were significantly changed in patients with RI. CONCLUSIONS: All these results shed new light on the role of circulating B cells, their subpopulations, function, and activity in the SLE with kidney manifestation.

Melanization of Bomirski hamster amelanotic melanoma cells (Ab line) depends on the type of culture medium.

INTRODUCTION: The effect of melanogenesis intensity on melanoma biology remains an open question, and the biological differences between melanotic and amelanotic melanoma cells have not yet been satisfactorily documented. As a result, the melanization of melanoma cells in in vitro cultures is not considered among experimental procedures. The aim of this study was to investigate the effect of the medium used to culture Bomirski amelanotic Ab melanoma cells on the melanogenesis process. MATERIAL AND METHODS: Amelanotic melanoma cells (Ab) were cultured in two media recommended for in vitro melanoma cell cultures, RPMI and DMEM. The melanization was evaluated by determining the melanin and tyrosinase presence in the cells using spectrophotometrical and western blot methods, respectively. Changes in Ab melanoma cells' ultrastructure were determined using electron microscopy (EM). RESULTS: The medium with higher level of tyrosine (DMEM) induced significant melanization of amelanotic melanoma cells (Ab) after only 24 h, while the RPMI medium, with a lower level of tyrosine, weakly affected melanin production. Melanization of Ab cells was paralleled by an increase in the amount of tyrosinase protein. Induced melanization was easily observed on EM-micrographs in the form of newly formed melanosomes containing melanin pigment. Melanosomes at stages from one (I) to four (IV) were observed. CONCLUSIONS: Culture medium has an important effect on the in vitro biology of amelanotic melanoma cells, since it can affect the rate of cellular melanization. The appropriate medium should be carefully selected, taking into account the known biology of the melanoma cells being used.