Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The ligament rupture (LC) is lesion diagnosed in most dogs with joint problems, and the most common cause is degenerative joint disease of the knee joint. This study aimed to evaluate the action of the total fraction of mononuclear cells from autologous bone marrow (FCMO) applied intra-articular by after surgical repair of ruptured cruciate ligament. It is proposed a new technique for surgical correction of this disease, evaluating clinically the action of a polypropylene mesh as the only substitute for cranial cruciate ligament and flow of dogs. Twenty dogs underwent unilateral desmotomy cranial and caudal LC. Twenty-one days after the break, all were treated surgically by the technique of open reduction, with implantation of polypropylene replacing only the cranial cruciate ligament (CCL). Ten animals received FCMO at the time of correction. The assessments were by X-ray study, clinical examinations and biopsies at 50 and 90 days postoperatively. The surgical technique has shown rapid implementation and low soft tissue dissection, early return to ambulation and adequate strength of the implant. The group that received FCMO grew intra-articular bone at the X-ray study, however, the twenty animals showed CD34 positive in their biopsy samples, indicating a presence of stem cells in both groups. It was concluded that there was no clinical difference between the two groups until 90 days of evaluation. / A ruptura do ligamento cruzado (LC) é uma lesão diagnosticada em grande parte dos cães com problemas articulares, sendo a causa mais comum de afecção articular degenerativa da articulação do joelho. Este trabalho teve por objetivo, avaliar a ação da fração total de células mononucleares autógenas da medula óssea (FCMO) de aplicação intra-articular, após a correção cirúrgica dos ligamentos cruzados rompidos. Foi proposta uma nova técnica de correção cirúrgica desta afecção, avaliando-se clinicamente a ação da malha de polipropileno como único substituto do ligamento cruzado cranial e caudal de cães. Foram utilizados 20 cães, submetidos à desmotomia unilateral dos LC cranial e caudal. Vinte e um dias após a ruptura, todos foram tratados cirurgicamente pela técnica de redução aberta, com o implante de polipropileno, substituindo apenas o ligamento cruzado cranial (LCC). Dez animais receberam a FCMO no momento da correção. As avaliações se deram por estudo radiográfico, exames clínicos e biópsias aos 50 e 90 dias pós-operatórios. A técnica cirúrgica proposta demonstrou ser de rápida execução e pouca dissecação de tecidos moles, retorno precoce à deambulação e resistência adequada do implante. O grupo que recebeu a FCMO apresentou crescimento ósseo intra-articular ao estudo radiológico, contudo, os vinte animais apresentaram células CD34 positivas em suas amostras biopsiadas, indicando haver presença de células-tronco em ambos os grupos. Concluiu-se que não houve diferença clínica entre os dois grupos até os 90 dias de avaliação.

The issue of prosthetic infection leading to implant failure due to the formation of bacterial biofilms on biomaterial surfaces has been widely recognized as a major issue, often leading to revision surgery. The growing number of patients requiring synthetic biomaterials as implants is on the rise and so is the risk of infection arising from pre/peri-/post-operative surgical procedures. Traditional antibiotic treatment has led to the emergence of bacterial drug resistance. Therefore, the development of novel bactericidal methods to combat drug resistant microbial pathogens is the need of the hour. The first part of the thesis is an attempt to address prosthetic infection by the development of novel ultrasmall gold nanoparticles (AuNPs) which are cytocompatible and present a therapeutic dosage window for eliciting antimicrobial property. Towards this end, ultrasmall AuNPs with 0.8 nm and 1.4 nm gold core sizes, stabilized by monosulphonated triphenylphosphine ligand shells were synthesized. Such intricately designed AuNPs with ultrasmall gold cores and phosphine-based ligand chemistry were demonstrated to be highly potent bactericidal agents against staphylococci, the most common human pathogen causing biomaterial associated infection. The antibacterial efficacy of these AuNPs was significant even in mature staphylococcal biofilms. In another study, the application of high strength pulse magnetic fields (1-4 Tesla) was examined for bacterial growth inactivation in vitro. A magnetic field strength dependent decrease in bacterial viability with a concomitant increase in the production of reactive oxygen species (ROS) and longer doubling times were recorded. The mechanism of action was explained through an analytical model which involves ion-transport interference of essential ions like Ca2+ and Mg2+ and disruption of FeS clusters leading to inactivation of bacterial redox enzymes. On the contrary, such high magnetic fields did not pose any detrimental effects to eukaryotic cells under similar exposure. Additionally, the potency of low intensity direct current electric field (DC EF: 1V/cm) against biofilm formation by methicillin resistant Staphylococcus aureus (MRSA) was explored on antimicrobial surfaces of hydroxyapatite and Zinc oxide (HA-xZnO; x = 0, 5, 7.5 and 10 wt%). An EF exposure time dependent decline in the viability and stability of MRSA biofilms were noted. Further, EF treatment resulted in bacterial membrane depolarization and reduced biofilm formation on HA-ZnO composites, independent of the substrate composition. In summary, the above three studies were cases of the developmental methods to address prothetic infection. The second part of the thesis is focused on the development of magneto-responsive biomaterials as implants for orthopaedic applications. Under this category, the sintering/ hot pressing of hydroxyapatite-magnetite (HA-xFe3O4; x = 0, 5, 10, 20 and 40 wt%) powders in oxidizing and inert atmospheres was carried out and the resulting phases and microstructure were characterized. A detailed analysis of the phase assemblage by Rietveld refinement of the X-ray diffraction (XRD) data and Mössbauer spectroscopy revealed the major retention of Fe3O4 along with wustite (FeO) formation under reducing conditions while hematite (α-Fe2O3) was the oxidized product of conventional sintering in ambient atmosphere. A good correlation between the unit cell volume increases in HA observed from Rietveld refinements and Fe incorporation into the apatite lattice from Mössbauer spectral parameters was evident. Further, the Mössbauer data analysis indicated a preferential occupancy of Fe at the Ca(1) site under oxidizing conditions and Ca(2) site in inert atmosphere. The above phase analyses were further confirmed by X-ray photoelectron spectroscopy (XPS), Infrared spectroscopy (FT-IR) and CHN analysis. The microstructure of the hot-pressed samples observed under transmission electron microscope (TEM) divulged similar phases as deduced from XRD as well as the formation of translational Moire fringe patterns due to inference of overlapping crystal planes of HA and Fe3O4 in the HA-40 wt% composite. Such translational Moire fringes suggest a preferred arrangement and orientation of the crystallites resulting from hot-pressing, which correlated well with the room temperature magnetic measurements made with the help of a vibrating sample magnetometer (VSM). The compositional similarity of Fe doping in HA to that of the tooth enamel and bone presents these HA-Fe3O4 composites as potent dental/ orthopaedic implant materials. In the conclusive study, the hot-pressed HA-xFe3O4 composites were tested for their efficacy in supporting the osteogenesis of human mesenchymal stem cells (hMSCs) assisted by intermittent static magnetic field exposure. The magneto-responsive substrates were applied as platforms for the culture of hMSCs and the effect of static magnetic field (SMF) exposure on the viability, proliferation and differentiation of hMSCs were elucidated. With a mild compromise in viability, SMF triggered the osteogenic differentiation of hMSCs mediated by proliferative arrest in the G0/G1 phase and elevated intracellular calcium levels. The early bone marker genes - Runx2, Col IA and ALP were significantly up regulated upon SMF exposure on pure HA and HA-Fe3O4 composites. Further, the late osteogenic markers – OCN and OPN were detected exclusively in the HA-xFe3O4 (x = 10 and 40 wt%) composites. Matrix mineralization was enhanced and CaP nodules were detected on similar SMF treated HA-Fe3O4 composites. A substrate magnetization and time dependent modulation of gene expression was recorded which corroborated well with the temporal trending of osteogenic genes during bone development. In conclusion, substrate magnetization can be applied as a tool to modulate the behavior of stem cells and direct them towards osteogenic lineage. Such a pertinent combination of substrate magnetization and external magnetic field stimulation can be applied synergistically for stem cell based bone tissue engineering applications.