Implantes de titânio têm sido extensivamente utilizados na ortopedia e na odontologia principalmente como substituto de elementos dentários ausentes. O titânio é um implante metálico de escolha por ser biocompatível, resistente à corrosão e desfavorecer reações imunológicas. A influência das características superficiais do titânio no comportamento e morfologia celular tem sido amplamente investigada, mas ainda existem questões em aberto sobre a sua interação com diferentes células em um microambiente. O objetivo do presente projeto foi caracterizar o comportamento de células provenientes do cordão umbilical humano em contato com uma superfície de titânio nanotexturizada em meio osteogênico e não-osteogênico. As células da sub-íntima da veia do cordão umbilical foram isoladas e colocadas em garrafas de cultura em meio essencial mínimo. Após a subconfluência, as células foram cultivadas em placas de 24 poços (n=5) em contato com discos de titânio comercialmente puro (cpTi) polidos (grupo controle) e com discos de titânio nanotexturizados através de condicionamento ácido (grupo Ti-nano). Os parâmetros avaliados foram: proliferação e viabilidade celular, quantidade de proteína total e atividade de fosfatase alcalina, além da detecção de nódulos mineralizados. Também foi realizada a expressão gênica quantitativa de genes relacionados à adesão e diferenciação por meio de PCR em tempo real. Todos os ensaios foram realizados em triplicata e os dados analisados através do teste estatístico Sigma Plot após aplicação de testes de normalidade e homogeneidade para p<0,05. / Titanium implants have been extensively used in orthopedics and dentistry, mainly as a replacement for missing teeth. Titanium is a metal implant of choice due to its high biocompatibility and corrosion resistance, as well as absence of immune response. The influence of titanium superficial characteristics on morphology and cell behavior has been broadly investigated, but there are still questions about the interaction between titanium and different types of cells in a microenvironment. The purpose of the present investigation is to characterize the behavior of cells from human umbilical cord vein in contact with a nanotextured titanium surface cultured on osteogenic and non-osteogenic medium. Cells were obtained from umbilical cord subintimal vein layer and cultivated in minimal essential medium. After subconfluence, cells were cultured in 24-well plates (n=5) on pure commercial polished titanium discs (control group) and on nanotextured titanium discs by means of acid etching. The evaluated parameters were cell viability, alkaline phosphatase activity as well as the detection of mineralized nodules. It was also performed quantitative gene expression of genes associated to adhesion and extracellular matrix by means of Real Time PCR. All the assays were performed in triplicate and the data obtained were analyzed by means of statistical test (Sigma Plot) after normality and homogeneity tests.

The main function of mesenchymal stem cells in the body is to facilitate the restoration and regeneration of damaged tissues. They are known for the ability to differentiate into tissue originating from the mesoderm, which among others includes connective tissues. Due to this feature are MSCs being intensively examined. Different directions of differentiation can be induced by treatment of specific polypeptides, so called growth factors. In the field of tissue engineering are growth factors used to induce and accelerate the healing processes. They may be incorporated into the nanofiber carrier which is inserted into the site of injury. Cells in this area would thus be stimulated by surrounding 3D microenvironment. At the same time the scaffold provides a supply of growth factors which are able to affect metabolism, motility and differentiation of present cells. In order to induce osteogenic differentiation of human MSCs the following bioactive substances were used: TGF-β, bFGF, HGF, IGF-1, VEGF and the BMP-2 and the organic acid taurine. During 21 days lasting experiments, were these molecules added to the medium in various combinations and in the case of taurine also at two different concentrations. Cells were cultured on plastic. The best effect on cellular metabolism of MSCs, evaluated by MTS...

The use of allograft bone is effective in the treatment of large bone loss following tumour removal or surgery. However, it is not osteogenic due to a lack of viable osteogenic cells and the remaining marrow material is potentially harmful to the recipient. Sterilisation techniques, such as gamma irradiation, are routinely used to improve the safety of these grafts; however this fails to remove the immunogenic material and may diminish the bones innate properties. Thus, wash techniques are being developed to remove the deleterious marrow, whilst retaining the native properties of the bone so that through tissue engineering, pre-osteogenic cells may be added to aid osseointegration. To this end, this study utilised a novel wash process (developed by the National Health Service Blood and Transplant Tissue services (NHSBT)) on whole human femoral heads, to assess the resulting material’s suitability as a biological scaffold for bone tissue engineering (BTE). Following the wash process, marrow removal efficiency was analysed by biochemical testing and histological assessment, and biocompatibility of fresh-frozen and washed human bone was assessed using extract cytotoxicity assays with BM-MSCs. The results showed a marrow removal efficiency of 99.5%, leaving a material with only 16.7 ng DNA/100mg of dry material, and which histologically displayed minimal cellular content demonstrating that this was an efficient wash process producing an acellular biological scaffold material (<50ng DNA/100mg bone). Extract cytotoxicity testing indicated the material was biocompatible. Uniaxial compression to failure was performed on 1cm3 cubes using bone samples from mirrored location of bilaterally halved femoral heads, with one half washed, whilst the other was fresh-frozen. A random orientated “clinical” model was also utilised, with samples processed as fresh-frozen, washed and irradiated for comparative assessment. There was no significant change in the mechanical strength of the washed material compared to fresh-frozen samples or between sterilisation types, suggesting the washed bone was mechanically comparable to existing bone allograft stock. BM-MSCs from both young (≤50 years) and old donors (≥70 years) were seeded on washed bone cubes from young and old donors, and cultured in standard or osteogenic media. Samples were analysed at 0, 14 and 28 day timepoints for cell viability, osteogenic gene expression, alkaline phosphatase activity and histological analysis. Results indicated significant fold increases in cell metabolism at day 14 and 28, in both medium types compared to day 0 (p≤0.001). QRT-PCR data showed increased expression of osteogenic markers RUNX2 (p≤0.001), osteopontin (p≤0.001) and osteocalcin (p≤0.001) in both standard and osteogenic media with significantly higher RUNX2 and osteocalcin in osteogenic medium samples at day 28. Expression of osteogenic genes was significantly higher in young donor cells seeded on the washed bone compared to old donor cells, as was expression in BM-MSCs cultured on old donor bone compared to young bone. This implies that the washed bone was able to induce osteogenic differentiation in BM-MSCs, that young donor cells were better able to differentiate than old, and that old donor bone was better able to induce osteogenic activity. Additionally, patient-matched BM-MSCs and ASCs, and BM-MSCs and BM-MNCs were seeded onto washed bone cubes and cultured for 28 days in standard or osteogenic media, with gene expression and metabolic activity assessed. The washed bone was able to induce osteogenic differentiation of ASCs. Moreover, BM-MNCs when cultured on washed bone also expressed osteogenic genes, indicative of osteogenic differentiation. These results indicate the efficacy of a novel wash process in producing a biological acellular scaffold suitable for bone tissue engineering. Interestingly, data also suggests that the age of the cell donor and bone donor may effect osteogenic differentiation of seeded cells which has significant implications clinically.

Células-tronco humanas derivadas da polpa dental (hDPSCs) e células-tronco humanas derivadas de tecido adiposo (AhSCs) são células multipotentes capazes de diferenciação osteogênica in vitro e in vivo, e promissoras fontes de células para a engenharia de tecido ósseo, dada a sua facilidade de expansão, isolamento e diferenciação. É de grande interesse compreender qual é o melhor tipo celular para diferenciação osteogênica, assim, o objetivo deste estudo foi comparar o potencial de diferenciação osteogênica in vitro e in vivo entre hDPSCs e hASCs. Foram isoladas e estabelecidas seis populações de células-tronco de hDPSCs (entre 7-12 anos) e seis da hASC (de indivíduos com idade entre 30-49 anos). Após a indução in vitro, a diferenciação osteogênica foi comprovado através das colorações de fosfatase alcalina (9 dias) e vermelho de alizarina (14 e 21 dias). A quantificação da mineralização da matriz após 21 dias de diferenciação osteogênica revelou 2,24 mais ossificação das hDPSCs em relação às hASCs. Para realizar o experimento in vivo, foram triados seis biomateriais para verificar qual melhor biomaterial para o nosso modelo, defeito crítico em calvária de Ratos Wistar não imunossuprimidos, com três amostras de hDPSCs. Após 45 dias, CellCeram(TM) exibiu a melhor neoformação óssea in vivo, e foi selecionado para comparar os potenciais osteogênicos in vivo entre hDPSCs e hASCs. Células (10e6) foram associadas a discos de 4,5 mm CellCeram(TM), grupo controle foi realizado através do transplante do biomaterial livre de células. Neoformação óssea foi mensurada 45 dias após a cirurgia através da coloração histológica de hematoxilina / eosina. A formação óssea total foi quantificada através da análise de imagens de todas as ilhas de ossificação. A associação entre hDPSCs e CellCeram(TM) promoveu 7,24 vezes mais neoformação óssea quando comparado com a associação entre esse mesmo material e hASCs (p <0,0001). A utilização de células-tronco adultas para regeneração óssea é uma ótima abordagem para uso terapêutico, e calcular ou predizer o potencial osteogênico das células utilizadas é extremamente importante e necessário para futura aplicação em novas estratégias de bioengenharia de tecido ósseo / Human dental pulp stem cells (hDPSCs) and human adipose-derived stem cells (AhSCs) are multipotent cells capable of undergoing osteogenesis in vitro and in vivo, and promising cell-source populations for bone tissue engineering given their easiness of isolation, expansion and differentiation. It is of great interest to understand which is the best cell type for osteogenic differentiation, thus the aim of this study is to compare the in vitro and the in vivo osteogenic differentiation potentials between DPSCs and ASCs. We isolated six stem cell populations from DPSCs (aged 7-12 years) and six from ASCs (from subjects aged 30-49 years) and cell culture was established. After in vitro induction the populations were able to undergo osteogenic differentiation, as evidenced by alkaline phosphatase (9 days) and alizarin red S (14 and 21 days) stainings. Quantification of matrix mineralization after 21 days of osteogenic differentiation revealed an enhancement of 2.24-fold increase between hDPSCs and hASCs differentiation. To perform the in vivo experiment, we promoted a screening of six scaffolds to find out which would be best scaffold to our model, a calvarial critical-sized defect in Wistar non-immunosuppressed rats, with three different culture samples of hDPSCs. After 45 days, CellCeram(TM) displayed the best in vivo bone neoformation, and was used to compare the in vivo osteogenic potentials between hDPSCs and hASCs. Cells (10e6) were associated to 4.5 mm CellCeram(TM) discs, and control groups were performed transplanting the biomaterial free of cells. Bone healing was measured through histological hematoxylin/eosin staining 45 days after surgery. Newly formed bone was also evaluated by total bone island surface quantification through image analysis. The association between hDPSCs and CellCeram(TM) induced a mean of 7.24 times more bone formation when compared to the association between this same material and hASCs (p<0.0001). The use of adult stem cells for bone regeneration is a robust therapeutic option, and calculate or predicts the osteogenic potential of the cell used are extremely important and necessary to future application, and translation to new strategies in bone tissue engineering

Cationic steroid antimicrobials (CSAs or ceragenins) are a novel class of synthetic, cholic acid-based mimics of endogenous antimicrobial peptides. These small molecule compounds display broad bactericidal activity against gram-negative and gram-positive bacteria, potent ability against fungal pathogens, and cidal effects against drug resistant and multidrug resistant microbes. Implantable medical devices provide an abiotic surface upon which bacteria and fungi can accumulate--thereby leading to localized or systemic infection. We proposed that CSA antibiotics can be incorporated into medical device surface coatings which can be optimized for the active release or elution of the CSA compounds over time to prevent device-associated infections. This report will discuss the progress of developing and testing coating systems for 3 such devices: cardiac implantable electronic devices (CIED), silicone nasal splints, and breast tissue expanders. In the case of CIEDs, an envelope material containing CSA was created using bioresorbable polymers. We found that this envelope elutes CSA antibiotics and kills all surrounding bacteria or fungi in both planktonic and biofilm forms within 1 hour of exposure. We also developed a nasal splint coating which is directly adhered to the surface of the silicone splint. This coating system demonstrated more than 8 days of protective ability (full microbicidal activity to the detection limit) against Candida albicans, and reduced microbial growth of P. aeruginosa, Candida auris, and MRSA for approximately 6 days. Lastly, in the case of tissue expanders, we developed a layered coating which displays fully-reductive antimicrobial activity against MRSA for 8 days with reintroduction of bacteria every 24 hours. Additionally, this work will discuss our investigations into the secondary properties of ceragenin compounds. On the basis of studies which have demonstrated the pro-osteogenic properties of CSA, we probed the mechanism of this effect. We studied the potential effects of ceragenins on the proliferation, differentiation, and migration of bone-derived mesenchymal stem cells (MSCs). We have determined the absence of any positive proliferative effects of ceragenins on these cells; however, we have demonstrated the significant migration-promoting chemoattractant properties of CSA. In the case of CSA-13, we have observed up to a 400% increase in migration compared to the control. Also, we demonstrated that the P2X7 receptor is strongly implicated in the cellular mechanism of this effect. Our studies of the differentiation-promoting properties of CSA on MSCs have been largely inconclusive, but further investigations are proposed in this report. Lastly, this work includes a report on our investigations into the potential synergistic interactions between CSA-131/CSA-44 with amphotericin B or caspofungin, two commonly used antifungal agents.

Tissue engineering is a promising approach that combines the interactions of biomaterials, cells, and growth factors to stimulate tissue growth and regeneration. As such, selecting a suitable biomaterial is vital to the success of the procedure. Ideally, the material should show similarity to the extracellular matrix in the structure and relative stiffness, and biofunctionality beside others to provide a comfortable environment for the cells. Additionally, the biomaterial properties should allow for the effective diffusion of relevant growth factors and nutrients throughout the material to enable cell growth. Because peptides are composed of amino acids found naturally within the human body, they are considered non-toxic and biocompatible. Ultrashort peptides are peptides with three to seven amino acids that can be self-assembled into helical fibers forming scaffolds of supramolecular structures. These peptide hydrogels formed a highly porous network of nanofibers which can quickly solidify into nanofibrous hydrogels that resemble the extracellular matrix (ECM) and provide a 3D environment for cells with suitable mechanical properties. Furthermore, we can easily tune the stiffness of these peptide hydrogels by just increasing peptide concentration, thus providing a wide range of peptide hydrogels with different stiffness for 3D cell culture applications. Herein we describe the use of ultrashort peptide hydrogels for the maintenance and the differentiation of human mesenchymal stem cells into the osteogenic lineage. Furthermore, we develop a three dimensional (3D) biomimicry acute myeloid leukemia (AML) disease model using biomaterial from a tetramer ultrashort self-assembling peptide. In addition, we evaluate the potential application of peptide hydrogels as a hemostatic agent. The results presented in this study suggest that our biomimetic ultrashort tetrapeptide hydrogels are an excellent candidate for tissue engineering and biomedical applications.

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ABSTRAKT:Isolering och karaktärisering av mesenkymala stamceller från periodontalligamentet hos friskatänderSYFTE: Att isolera och odla celler från periodontalligamentet samt karaktärisera dem sommesenkymala stamceller.MATERIAL OCH METOD: Friska premolarer gjordes tillgängliga vid ortodontiskaextraktioner. Den mellersta 1/3 av periodontalligamentet skrapades varpå en enzymatiskmetod användes för isolering av individuella celler. Resulterande celler odlades understandardiserade metoder. Karaktärisering av celler skedde genom flödescymetri med 2 olikapaneler av cellyta markörer; en för etablerat positiva uttryck och en för kända negativauttryck hos mesenkymala stamceller. Möjlighet av celler att differentieras in vitro tilladipocyter och osteocyter testades genom tillförsel av specifika substanser till odlingsmediet.RESULTAT: Celler från 11 av 13 tänder isolerades och odlades framgångsrikt adherenta tillodlingsytan i upp till 8 generationer. Celluttryck av de positiva markörerna CD73, CD90 samtCD44 bekräftades genom flödescymetri. Inget uttryck observerades för den negativa panelenCD45, CD34, CD11b, CD19 eller HLA class II. Uttrycket av CD105 kunde inte fastställas pgaofullständigt data. Försök till differentiering av celler till adipocyter och osteocyter visade påfenotypiska förändringar efter 21 dagar.SLUTSATS: Den här studien har bidragit till framgångsrik isolering och delvis karaktäriseringav mesenkymala stamceller från periodontalligamentet hos friska tänder. En icke-invasivmetod av detta slag, resulterande i tillgång till denna cellpopulation utgör ett lovande verktygför framtida studier med goda möjligheter till ytterligare kunskap applicerbart till kliniskasituationer inom tandvården. / ABSTRACT:Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament ofHealthy TeethAIM: To isolate and culture viable cells from the periodontal ligament and confirming theiridentity as mesenchymal stem cells.METHODS AND MATERIALS: Healthy premolars were collected at the time oforthodontic extractions. The middle 1/3 of the periodontal ligament was scraped andsubsequent cell isolation was performed using an enzymatic method; yielding single cellisolates. Cells were cultured and maintained under standard culture conditions. Cellcharacterization was performed by flow cytometry using two sets of cell surface markers; oneknown to be present and one known to be absent in mesenchymal stem cells. Ability of thecells for in vitro differentiation into adipogenic and osteogenic lineages was tested usingspecifically formulated media supplements.RESULTS: Cells were successfully isolated from 11 of 13 teeth and were maintained asadherent cultures for up to 8 generations. Cellular expression of positive markers; CD73, CD90and CD44 were confirmed by flow cytometry. For the negative marker panel, expression ofCD45, CD34, CD11b, CD19 and HLA class II were not detectable. The expression of CD105was inconclusive. As determined by phenotypic changes, cells appeared to have undergoneadipogenic and osteocytic differentiation at 21 days.CONCLUSION: This study has resulted in successful isolation and partial characterization ofmesenchymal stem cells from the periodontal ligament of healthy teeth. Non-invasive accessto these cells, provides an excellent tool for future studies, potentially leading to beneficialknowledge transferable to the dental clinical situation.