Thermoresponsive hydrogels for mimicking three-dimensional microenvironment of mesenchymal stem cell in cartilage tissue engineering

Articular cartilage covers the bone heads of articulating joints to decrease the friction between bones. Unfortunately, articular cartilage has limited self-repair potential. Cartilage tissue engineering is a promising therapeutic approach, and its success strongly depends on our understanding and ability to mimic the complex three-dimensional microenvironment for cells and their surrounding native extracellular matrix (ECM) in articular cartilage. In particular, recreating the zonal organisation of articular cartilages makes the process more challenging. My PhD thesis aims to design and develop chitosan-based thermoresponsive matrices with tailored physical, mechanical and chemical properties to fulfill microenvironmental requirements of mesenchymal stem cells (MSCs) in order to promote functional articular cartilage regeneration. The matrices are then micro-manipulated for resembling the spatially varying architecture of articular cartilage zones. To achieve these aims, chitosan-g-poly (N-isopropylacrylamide) (CS-g-PNIPAAm) hydrogel with a random chain length of grafts was synthesized through free radical polymerization. The influence of various polymerization conditions on physical and mechanical properties was systematically investigated. Its suitability for mimicking microenvironment for MSC culture was studied using cell viability assays. The best CS-g-PNIPAAm in terms of its cell compatibility and cell culture performance was used in fabrication of microengineered constructs for regenerating the superficial zone and the middle zone of articular cartilage. Chondrogenic differentiation of embedded MSCs was evaluated through ECM components (glycosaminoglycan (GAG), total collagen and collagen type II) analysis. Cellular organisation and morphology within microchannels were determined using cell alignment and elongation quantification methods. To further control the chain length of PNIPAAm, welldefined and narrow-dispersed molecular weights of PNIPAAm were synthesized through atom transfer radical polymerization (ATRP). Influence of the polymer molecular weight on cytotoxicity and the cell death mechanisms were investigated through standard assays. Finally, chitosan/well-defined PNIPAAm (CSNI) hybrids were prepared using low/no toxic PNIPAAms. MSCs mixed with PNIPAAm solution and were seeded in the voids of the chitosan scaffolds. The phase separation of PNIPAAm at 37°C led to a hybrid matrix for MSCs. The structural characteristics of the hybrids were studied and chondrogenic differentiation of incorporated MSCs was evaluated through measuring GAG and total collagen deposition. Various copolymerization conditions of CS-g-PNIPAAm have been optimised to obtain the best hydrogel for MSC culture with desired physical and mechanical properties. After MSC proliferation, MSCs can be recovered by separating cells from the polymer solution at room temperature using the sol-gel thermo-reversible property of the CS-g-PNIPAAm copolymer. It has been demonstrated that the CS-g-PNIPAAm copolymer hydrogel can provide an appropriate microenvironment for 3D cultivation of MSCs. Biochemical analysis demonstrates that the CS-g-PNIPAAm hydrogel can support the embedded MSCs differentiation into chondrocytes in 3D. Histological and immunohistochemical stainings also confirm the increasing accumulation of GAG and collagen type II. The CS-g-PNIPAAm hydrogel with manipulated properties can be micropatterned for regenerating the superficial zone and the middle zone of articular cartilage. The cell-laden hydrogel micropatterned in 50-100 μm constructs can organize cells along the microchannel horizontal axis. The cell shape and alignment in the constructs is very similar to the superficial zone of chondrocytes of the native cartilage. Meanwhile, cells in the microchannel with the gap above 150 μm are randomly distributed which can be used to mimic the middle zone of the cartilage tissue. The cytotoxicity of PNIPAAm is molecular weight dependent, and varies with the PNIPAAm chain length. Low molecular weight PNIPAAm (degree of polymerization (DP) = 35) is inherently toxic to cells, and necrosis is the dominant cell death mechanism. Moderate-sized PNIPAAms with their DP between 100 and 200 are non-cytotoxic. For the PNIPAAm with a higher molecular weight (DP = 400, P-400), cell viability is dependent on the assay method. The P-400 hydrogel is detrimental to stem cells when the cells are covered with a thick layer of gel, and this layer may become a barrier for nutrient or oxygen delivery to cells. The CSNI hybrid matrices composed of chitosan scaffolds and well-defined PNIPAAm with a degree of polymerization of 400 (CSNI400) can provide a supporting platform for 3D stem cell culture and cartilage tissue engineering. Matrix characterization shows improved structural properties of CSNI400 in comparison with CSNI100 and the chitosan-alone scaffold. In conclusion, we are able to create and refine 3D microenvironment for stem cells through manipulation of matrices in order to enhance cell proliferation and chondrogenic differentiation. Our results reveal that graft copolymer of chitosan and PNIPAAm with tailored properties and microengineered architecture is appealing for zonal cartilage tissue engineering. The hybrid matrices from chitosan scaffolds with well-defined PNIPAAm hydrogels promote chondrogenesis, better than the graft copolymer. Graft copolymerization of chitosan and well-defined PNIPAAms (CS-g-W-PNIPAAm), microengineering of CSNI hybrids, and CS-g-W-PNIPAAm, stacking the microengineered constructs to form a macroscale 3D cartilage tissue, and in vivo implantation of engineered tissues should be addressed in future projects.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Chemical Engineering, 2015

The Relationship Between Blood Lead Level and Preeclampsia

Introduction: Lead is a heavy metal to which people are commonly exposed. One of the possible mechanisms of tissue damages caused by this toxic metal is oxidative stress, which in turn may cause numerous pregnancy complications such as preeclampsia. The present study was conducted to determine the relationship between maternal Blood Lead Level (BLL) and preeclampsia. Methods: The present case-control study was conducted on 158 pregnant women admitted to a hospital in Zanjan, Iran, from August 2015 to March 2016. To measure their BLL, 1.5 cc of blood was drawn from each participant. The demographic and obstetric details of the patients were recorded in a form. The potentiometric method was used to test the samples. The data obtained were analyzed by SPSS version 22, using Mann-Whitney U test, the Chi square, independent-samples t-test, Pearson product-moment correlation, and simple linear regression analysis. Results: The mean BLL was 6.24±1.74 µg/dl in the control group and 8.04±3.4 µg/dl in the preeclampsia group. The two groups were matching in terms of the mother's age and education and the household income. A significant relationship was observed between BLL and preeclampsia (p=0.028), as per every unit of increase (1 µg/dl) in BLL, systolic blood pressure increased by 0.014 mm Hg and diastolic blood pressure by 0.013 mm Hg (p=0.004). Conclusion: The results obtained suggest a relationship between BLL and preeclampsia. Global health measures should be taken to remove the exposure to lead so as to reduce its absorption by pregnant wome

Effect of collagen hydrogel containing Lavandula officinalis essential oil nanoemulsion in wound healing of infectious burn

Background and Objectives: The main cause of mortality in burn patients is infection from burns. Drug-resistant bacteria are the main causes of wound infection, so alternative antibiotic therapies hold significant importance. The objective of this study was to examine the impact of a collagen hydrogel that contains a nanoemulsion of Lavandula essential oil on the healing process of infected burn wounds. Materials and Methods: In this experimental study, 20 rats were randomly divided after applying burns with a 10 mm diameter hot plate and infecting the wounds with multidrug-resistant Pseudomonas aeruginosa into four groups, including a positive control, a negative control, the first experiment (collagen hydrogel), and the second experiment (collagen hydrogel containing Lavandula essential oil nanoemulsion). On the 4th, 11th, and 18th days, tissue samples were taken for pathology studies. The important parameters in burn wound healing with hematoxylin and eosin and Masson's trichrome staining methods were investigated and scored according to Abramov’s method. Results: Based on the pathology findings, experimental groups 1 and 2 compared to the negative and positive control groups were effective in rat infection wound healing. The hydrogel scaffold in the experimental groups increased fibroblasts and angiogenesis compared to the control groups. Epithelization was noticed only in the hydrogel group containing nanoemulsion. Conclusion: The study findings suggest that the use of collagen hydrogel with Lavandula essential oil nanoemulsion has potential as a wound dressing. This is because it has the potential to effectively promote healing and act as an antibacterial agent to prevent infections

Preparation of bilayer tissue-engineered polyurethane/poly-L-lactic acid nerve conduits and their in vitro characterization for use in peripheral nerve regeneration

Abstract Background Due to loss of peripheral nerve structure and/or function resulting from trauma, accidents, and other causes, peripheral nerve injuries continue to be a major clinical problem. These injuries can cause partial or total loss of sensory, motor, and autonomic capabilities as well as neuropathic pain. PNI affects between 13 and 23 out of every 100,000 people annually in developed countries. Regeneration of damaged nerves and restoration of function after peripheral nerve injury remain significant therapeutic challenges. Although autologous nerve graft transplantation is a viable therapy option in several clinical conditions, donor site morbidity and a lack of donor tissue often hinder full functional recovery. Biomimetic conduits used in tissue engineering to encourage and direct peripheral nerve regeneration by providing a suitable microenvironment for nerve ingrowth are only one example of the cutting-edge methods made possible by this field. Many innate extracellular matrix (ECM) structures of different tissues can be successfully mimicked by nanofibrous scaffolds. Nanofibrous scaffolds can closely mimic the surface structure and morphology of native ECMs of many tissues. Methods In this study, we have produced bilayer nanofibrous nerve conduit based on poly-lactic acid/polyurethane/multiwall carbon nanotube (PLA/PU/MWCNT), for application as composite scaffolds for static nerve tissue engineering. The contact angle was indicated to show the hydrophilicity properties of electrospun nanofibers. The SEM images were analyzed to determine the fiber’s diameters, scaffold morphology, and endometrial stem cell adhesion. Moreover, MTT assay and DAPI staining were used to show the viability and proliferation of endometrial stem cells. Results The constructed bilayer PLA/PU/MWCNT scaffolds demonstrated the capacity to support cell attachment, and the vitality of samples was assessed using SEM, MTT assay, and DAPI staining technique. Conclusions According to an in vitro study, electrospun bilayer PLA/PU/MWCNT scaffolds can encourage the adhesion and proliferation of human endometrial stem cells (hEnSCs) and create the ideal environment for increasing cell survival

Brain Tumors in Elderly

Brain tumors in elderly are increasing as the number of people, who comprise the older population, does.About half of the patients with brain tumors appear to be over 60 years of age.In this review article, Glioblastoma multiform, as the most common malignant tumor of the central nervous system in elderly is discussed in details of definition, prognosis, diagnosis, treatment and differential diagnosis. Other tumors such as meningioma, pituitary adenoma, CNS lymphoma and metastasis are also included to be reviewed.  Treatment plans, either conservative or aggressive, classic or novel, approved or under investigation, are presented. Furthermore different attitudes of treatment in the past and recently are also argued. Conventional therapy, Surgery, Radiotherapy, chemotherapy radioimmunotherapy, hormonal therapy and some other novel methods of treatments are discussed in details for the glioma.Determining factors which may be associated to the patient's response to each treatment planare also discussed. Finally, some age related issues are provided to be paid attention to consider an old patient with brain tumor, and planning an optimal treatment in order to make the best management decisions.Until recently,  peoplewith brain tumors in elderly, were used to be treated in conservative plans and often were excluded of the clinical trials but now the number of patients who desire and receive more aggressive therapy for brain tumors is increasing

Injectable hydrogels in central nervous system: Unique and novel platforms for promoting extracellular matrix remodeling and tissue engineering

Repairing central nervous system (CNS) is difficult due to the inability of neurons to recover after damage. A clinically acceptable treatment to promote CNS functional recovery and regeneration is currently unavailable. According to recent studies, injectable hydrogels as biodegradable scaffolds for CNS tissue engineering and regeneration have exceptionally desirable attributes. Hydrogel has a biomimetic structure similar to extracellular matrix, hence has been considered a 3D scaffold for CNS regeneration. An interesting new type of hydrogel, injectable hydrogels, can be injected into target areas with little invasiveness and imitate several aspects of CNS. Injectable hydrogels are being researched as therapeutic agents because they may imitate numerous properties of CNS tissues and hence reduce subsequent injury and regenerate neural tissue. Because of their less adverse effects and cost, easier use and implantation with less pain, and faster regeneration capacity, injectable hydrogels, are more desirable than non-injectable hydrogels. This article discusses the pathophysiology of CNS and the use of several kinds of injectable hydrogels for brain and spinal cord tissue engineering, paying particular emphasis to recent experimental studies

Defining the role of 17β-estradiol in human endometrial stem cells differentiation into neuron-like cells

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17β-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17β-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17β-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17β-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering. © 2020 International Federation for Cell Biolog

Defining the role of 17 beta-estradiol in human endometrial stem cells differentiation into neuron-like cells

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17 beta-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17 beta-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17 beta-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17 beta-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering