This master thesis deals with the optimization of the procedure of modification of hyaluronic acid structure for the use in the artificial synovial liquids. Based on the literature research, the amino acid alanine was used for the modification of carboxylic group in the glucuronic acid. The main subject of study is the improvement of the stability and mechanical properties of synovial liquid. DLS microrheology, macrorheology, thermogravimetric analysis (TGA), multi-angle light scattering with flow-field flow fractionation (AF4-MALS) and infrared spectroscopy (FTIR) were used for characterization. The theoretical part of this theses submits review of the musculoskeletal system, role of hyaluronic acid in metabolism and summary of synovial liquid. The experimental part focuses on the measurement of the stability and mechanical properties of three artificial samples (first with no modification, second with modified hyaluronic acid and third with modified hyaluronic acid and chondroitin sulphate). These samples were compared with real horse synovial fluid and artificial viscosupplement Orthovisc®.

This diploma thesis deals with acid-base and electrolytic behavior of hyaluronan solutions at different ionic strength. Acid-base behavior of hyaluronan was investigated by acid-base titrations which were carried out with two different methods, acid and alkaline acid-base titration. Dissociation constants at different ionic strength at zero degree of dissociation and at 50% degree of dissociation were evaluated from the results of acid-base titrations. Dissociation constants obtained from acid acid-base titrations have values between 3,0 and 3,6. Dissociation constants obtained from alkaline acid-base titrations are not very informative because their values are much higher than the expected values. The study of degradation of hyaluronan during acid-base titration was performed to complete study of acid-base behavior. Electrolytic behavior of hyaluronan solution was performed by conductometric titrations in three different environments.

This diploma thesis is focused on preparation and characterization of supported lipid bilayers as simplified models of cell membranes. The bilayers were prepared from source system of lecithin liposomes in phosphate buffer using the vesicle fusion method on a cover glass sufrace hydrophilized by plasma. Three fluorescent probes – Nile red, Oregon Green DHPE and DiO – were utilized to characterize diffusion within the bilayer using fluorescence correlation spectroscopy. For this purpose Z-scan FCS, which is a method developed specially for planar samples, was used. After the process of preparation and characterization of supported lipid bilayer was optimalized, interaction between this artificial membrane and solution of hyaluronic acid in phosphate buffer was studied. It was found out, that addition of this biopolymer causes slowing the diffusion of the fluorescent probe within the bilayer.

This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.

Hyaluronic acid (HA) is widely studied as a targeting moiety to CD44 overexpressing cancer cells. Various types of nanoparticles (NPs) were modified by HA. Virus-like particles (VLPs) derived from mouse polyomavirus are an interesting class of NPs that can be modified by various targeting agents to increase their potential as gene or drug delivery vehicles for e.g. theragnostic purposes. HA has not been tested as a targeting moiety on VLPs, hence this was the focus of the current study. HA (~14 kDa) was attached to the VLPs via a bispecific Bodipy-derived fluorescent probe. To test the targeting potential of HA on comparable non-viral NPs, nanodiamonds were prepared in a similar manner. NPs functionalized with HA, together with Bodipy-labeled control variants, were tested on interactions with MDA-MB-231 cells overexpressing CD44. The NP-cell interaction via CD44 was assessed by a competitive cell-binding assay, where non-labeled HA competed for HA-binding sites at CD44 with the NPs. CD44 specific cell interactions were detected in studies with HA functionalized nanodiamonds, whereas VLP-HA* associated with cells in a less specific manner. Control VLPs with polyethylene glycol (PEG) did not interact with the cells. Results indicate that the HA targeting strategy for the VLPs requires optimization to...

De nos jours, la moitié des infections nosocomiales sont liées à la pose de dispositifs médicaux. Dans ce contexte, nous avons développé deux types de revêtements antimicrobiens adaptés au domaine biomédical. Le premier s'appuie sur le greffage d’un peptide comprenant une séquence d’accroche, une séquence antimicrobienne et un site de clivage spécifique d'un pathogène. La séquence antimicrobienne sera alors libérée uniquement en présence du pathogène. Malgré la réussite de l’accroche du peptide, certains paramètres restent encore à optimiser afin d’obtenir un effet antimicrobien. Le deuxième revêtement s’appuie sur la conception d’un film antimicrobien « couche-par-couche » avec l’utilisation de poly(L-arginine) (PAR) et d’acide hyaluronique (HA). L’influence de la taille des chaînes de PAR a été étudiée et seul le film construit à partir de PAR de 30 résidus possède un effet antibactérien. Avec cette PAR, nous avons démontré que HA est le seul polyanion conduisant à des propriétés antibactériennes. Ces propriétés antimicrobiennes sont maintenues lorsque d’autres homopolypeptides cationiques sont associés à HA. / Nowadays, about half of hospital-acquired infections are due to medical devices implantation. In this context, we have developed two types of antimicrobial coatings adapted to the biomedical field. The first one is based on peptide composed of an anchoring sequence, an antimicrobial sequence and a pathogen-specific cleavage site and grafted on the substrate. The antimicrobial site will be released only in the presence of the pathogen through the use of the cleavage site. Despite of the success of peptide grafting, some parameters must be optimized in order to obtain an antimicrobial effect. The second antimicrobial coating concept is based on the layer-by-layer technique by using poly(L-arginine) (PAR) and hyaluronic acid (HA). The effect of the size of PAR chains on the antimicrobial character of the coating was investigated and it is proven that only films composed with PAR of 30 residues present an antibacterial effect. Moreover HA is the only polyanion leading to such antimicrobial multilayer. It is also demonstrated that this antimicrobial properties is maintained when other cationic homopolypeptides are used in association with HA in layer-by-layer films.

Purpose: In an Orthopedic office in Northeast Tennessee clinical decision making about injection options for non-surgical candidates with knee osteoarthritis is unclear. Aims: This quality improvement project will develop a clinical guideline so that providers know criteria for choosing optimal knee alternative treatments for non-surgical knee patients. Outcome measures: An expert panel gave feedback and advice on the information presented for the injection types of Platelet Rich Plasma, Corticosteroids, Amniotic Allograft and Hyaluronic Acid. They edited the guideline then sent the edits back to be complied and edited using the Delphi method. Process and Methods: The expert feedback was then be collected in a non-identifiable fashion and the guideline was rewritten based on the panel advice. Then the guideline was then presented to the practice site and the site reviewed and rated the guideline on clarity, accuracy and ease of use. Results: The guideline was rated by the practice site as able to be adopted into practice and used at the site. Findings and Limitations: Limitations include the practice site is constantly changing and new implementations could be overlooked. The expert panel are all busy professionals and finding time to review and critique a guideline is extensive. Conclusions and Implications: This novel guideline will improve healthcare by eliciting an expert panel of orthopedics that perform injections to assist in compiling the most accurate up to date guideline through which will create enhanced decision making and overall better patient care when choosing knee injections for non surgical patients.

Skeletal muscle injuries and muscle degenerative diseases pose significant challenges to the healthcare. Surgical interventions are restricted due to tissue availability, donor site morbidity and alterations to tissue biomechanics. Current cell-based therapies are hindered by low survival and long-term engraftment for the transplanted cells due to the lack of appropriate supportive microenvironment (cell niche) in the injured muscle. Therefore, there is a critical need for developing strategies that provide cellular and structural support in the regeneration of functional muscle. In the present work, a bioengineered cell niche mimicking the native skeletal muscle microenvironment has been developed for skeletal muscle regenerative engineering. It is hypothesized that the bioengineered scaffolds with appropriate structural and cell instructive properties will support myoblast alignment and function, as well as promote the myogenic responses in clinically relevant skeletal muscle injuries. The current work utilized a three-pronged approach to design biomaterial scaffolds to aid in skeletal muscle regeneration. In the first part, aligned poly(lactide-co-glycolide) (PLGA) fiber scaffolds mimicking the oriented muscle fiber microenvironment with fiber diameters of 335±154 nm (nanoscale), 1352±225 nm (microscale) and 3013±531 nm (microscale) were fabricated and characterized. Myoblasts were found to respond to fiber diameter as observed from the differences in cell alignment, cell elongation, cell spreading area, proliferation and differentiation. <i>In vivo</i> study demonstrated the potential of using microscale fiber scaffolds to improve myogenic potential in the <i>mdx</i> mouse model. In the second part, we designed, synthesized, and characterized an implantable glycosaminoglycan-based composite hydrogel consisting of hyaluronic acid, chondroitin sulfate and polyethylene glycol (HA-CS) with tailored structural and mechanical properties for skeletal muscle regeneration applications. We demonstrated that HA-CS hydrogels provided a suitable microenvironment for <i>in vitro</i> myoblast proliferation and differentiation. Furthermore, <i>in vivo</i> studies using a volumetric muscle loss model in the mouse quadriceps showed that HA-CS hydrogels integrated with the surrounding host tissue and facilitated <i>de novo</i> myofiber generation, angiogenesis, nerve innervation and minimized scar tissue formation. In the third part, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose dependent manner. We also demonstrated the biomimetic HA-CS hydrogel system enabled 3D encapsulation of PC12 cells secreting signaling factors and promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2088 protein/peptides from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These set of experiments not only provide critical insight on exploiting the interactions between muscle cells and their microenvironment, but they also open new avenues for developing advanced bioengineered scaffolds for regenerative engineering of skeletal muscle tissues.<br>

Spinal cord injury is a serious trauma and despite intensive research there is still no effective treatment for patients. The aim of this thesis is to study new possibilities of spinal cord injury therapy in animal models. We have focused on the use of natural materials, stem cells, gene therapy and the possibility of combining these approaches. The effect of extracellular matrix (ECM) based materials prepared by decellularization of porcine spinal cord and porcine urinary bladder on tissue regeneration after acute hemisection of the spinal cord was investigated. Another tested material was a hydrogel based on hyaluronic acid modified with RGD adhesion peptide, which was applied acutely and subacutely into the hemisection lesion. We have shown that both types of biomaterials have positive effect on regeneration of the spinal cord tissue by bridging the lesion and promotion of axonal ingrowth. In addition, ECM hydrogels promote the growth of blood vessels into the lesion site. The combination of hydrogels with mesenchymal stem cells derived from human umbilical cord (hWJ-MSCs) had synergistic effect, but since only a limited number of cells could be incorporated into hydrogels, this effect was not associated with improvement in motor skills. The limitation of ECM hydrogels is their rapid...

Use of biochemical markers (Lipoprotein phospholipase A2 and hyaluronic acid) for laboratory diagnosis of metabolic and degenerative diseases of the musculoskeletal system Abstract Musculoskeketal disorders currently belong to the most common diseases. The presented work describes the use of biochemical markers (Lipoprotein phospholipase A2 and hyaluronic acid) for laboratory diagnosis of metabolic and degenerative diseases of the musculoskeletal system. Concentrations of LP-PLA2 were significantly elevated in the patients with bone resorption compared to the control group of healthy individuals. Serum levels of Lp-PLA2 also negatively correlated with decreased levels of serum osteocalcin in patients. HA concentrations in synovial fluid did not differ from published reference values in synovial fluid. Patients who underwent arthroscopy had significantly elevated synovial HA concentration than patients who underwent total knee endoprosthesis. HA positively correlates with osmotic pressure determined by examination of osmolality in synovial fluid. Lipoprotein phospholipase A2 concentrations are elevated in patients with bone density impairment. LpPLA2 concentrations correlate with the severity of bone density impairment expressed by the T score. Hyaluronic acid concentrations in patients with knee...