Neural progenitors: in vitro investigation and characterisation of the proliferation and differentiation into dopaminergic neurons

In der vorliegenden Arbeit wurde die in vitro Proliferation und Differenzierung neuraler mesenzephaler Progenitoren embryonaler Ratten in Abhängigkeit von den Kulturbedingungen mittels genomischer, immunhistochemischer und metabolischer Analysen untersucht. Im Mittelpunkt der Experimente stand zum einen das Entwicklungsstadium der Spenderembryonen. Die Ergebnisse dieser Versuche zeigten deutlich, dass neurale Progenitoren aus frühen embryonalen Entwicklungsstadien (E11-12) ein höheres Proliferations- und Differenzierungspotential zu Dopaminneuronen aufweisen. Da oxidativer Stress als eine Ursache für die selektive Degeneration dopaminerger Neurone bei Morbus Parkinson diskutiert wird, wurden zum anderen Untersuchungen des Einflusses des Sauerstoffpartialdrucks auf die Stoffwechselaktivität und Vitalität der Zellen durchgeführt. Dazu wurden neurale Primärzellen und Zelllinien in einem neu entwickelten bioFolie-Membransystem (BFMS) mit gasdurchlässiger Kulturoberfläche und auf herkömmlichen Multiwellplatten differenziert. Die Untersuchungen zeigten, dass der Grad der Differenzierung zu Dopaminneuronen bei hohen Sauerstoffpartialdrücken in beiden Systemen abnimmt. Eine dabei gegenüber der Kontrollkultur um 50% erhöhte Apoptoserate macht das neuartige BFMS zu einem geeigneten in vitro Modell, um den Einfluss oxidativen Stresses auf neurale Zellsysteme zu evaluieren.In the present thesis the in vitro proliferation and differentiation of neural mesencepahlic progenitors of embryonal rats were assessed by means of genomic, immunohistochemical and metabolic analysis in dependency on the culture conditions. The experiments focused on one hand on the developmental stage of the donor embryos. The results of this studies indicated, that neural precursors obtained from the early developmental stage (E11-12) show a significantly higher proliferative and differentiative potential into dopaminergic neurons. In addition, the influence of the oxygen partial pressure on the metabolic activity and vitality of the cells was examined, since oxidative stress is discussed as a cause of the selective degeneration of dopaminergic neurons in Morbus Parkinson. Therefore primary neural cells and cell lines were differentiated both in a newly developed bioFoil membrane system (BFMS) with a gas-permeable bottom and on conventional multiwell plates. The investigations showed that the degree of differentiation towards dopaminergic neurons decreased due to elevated oxygen levels in both systems. As the apoptotic rate thereby increased by 50% compared to the control culture the BFMS can be used as a suitable in vitro model for the evaluation of the influence of oxidative stress on neural cell systems

Amelogenin Peptide Extract Increases Differentiation and Angiogenic and Local Factor Production and Inhibits Apoptosis in Human Osteoblasts

Enamel matrix derivative (EMD), a decellularized porcine extracellular matrix (ECM), is used clinically in periodontal tissue regeneration. Amelogenin, EMD’s principal component, spontaneously assembles into nanospheres in vivo, forming an ECM complex that releases proteolytically cleaved peptides. However, the role of amelogenin or amelogenin peptides in mediating osteoblast response to EMD is not clear. Human MG63 osteoblast-like cells or normal human osteoblasts were treated with recombinant human amelogenin or a 5 kDa tyrosine-rich amelogenin peptide (TRAP) isolated from EMD and the effect on osteogenesis, local factor production, and apoptosis assessed. Treated MG63 cells increased alkaline phosphatase specific activity and levels of osteocalcin, osteoprotegerin, prostaglandin E2, and active/latent TGF-β1, an effect sensitive to the effector and concentration. Primary osteoblasts exhibited similar, but less robust, effects. TRAP-rich 5 kDa peptides yielded more mineralization than rhAmelogenin in osteoblasts in vitro. Both amelogenin and 5 kDa peptides protected MG63s from chelerythrine-induced apoptosis. The data suggest that the 5 kDa TRAP-rich sequence is an active amelogenin peptide that regulates osteoblast differentiation and local factor production and prevents osteoblast apoptosis

Effects of Er:YAG laser on bacteria associated with titanium surfaces and cellular response in vitro

This in vitro study examined (a) the anti-bacterial efficacy of a pulsed erbium-doped yttrium aluminum garnet (Er:YAG) laser applied to Streptococcus sanguinis or Porphyromonas gingivalis adhered to either polished or microstructured titanium implant surfaces, (b) the response of osteoblast-like cells and (c) adhesion of oral bacteria to titanium surfaces after laser irradiation. Thereto, (a) bacteria adhered to titanium disks were irradiated with a pulsed Er:YAG laser (λ = 2,940nm) at two different power settings: a lower mode (12.74J/cm2 calculated energy density) and a higher mode (63.69J/cm2). (b) After laser irradiation with both settings of sterile titanium, disks were seeded with 104 MG-63 cells/cm2. Adhesion and proliferation were determined after 1, 4, and 24h by fluorescence microscopy and scanning electron microscopy. (c) Bacterial adhesion was also studied on irradiated (test) and non-irradiated (control) surfaces. Adhered P. gingivalis were effectively killed, even at the lower laser setting, independent of the material's surface. S. sanguinis cells adhered were effectively killed only at the higher setting of 63.69J/cm2. Laser irradiation of titanium surfaces had no significant effects on (b) adhesion or proliferation of osteoblast-like MG-63 cells or (c) adhesion of both oral bacterial species in comparison to untreated surfaces. An effective decontamination of polished and rough titanium implant surfaces with a Er:YAG laser could only be achieved with a fluence of 63.69J/cm2. Even though this setting may lead to certain surface alterations, no significant adverse effect on subsequent colonization and proliferation of MG-63 cells or increased bacterial adhesion was found in comparison to untreated control surfaces

Influence of gaseous ozone in peri-implantitis: bactericidal efficacy and cellular response. An in vitro study using titanium and zirconia

Dental implants are prone to bacterial colonization which may result in bone destruction and implant loss. Treatments of peri-implant disease aim to reduce bacterial adherence while leaving the implant surface intact for attachment of bone-regenerating host cells. The aims of this study were to investigate the antimicrobial efficacy of gaseous ozone on bacteria adhered to various titanium and zirconia surfaces and to evaluate adhesion of osteoblast-like MG-63 cells to ozone-treated surfaces. Saliva-coated titanium (SLA and polished) and zirconia (acid etched and polished) disks served as substrates for the adherence of Streptococcus sanguinis DSM20068 and Porphyromonas gingivalis ATCC33277. The test specimens were treated with gaseous ozone (140ppm; 33mL/s) for 6 and 24s. Bacteria were resuspended using ultrasonication, serially diluted and cultured. MG-63 cell adhesion was analyzed with reference to cell attachment, morphology, spreading, and proliferation. Surface topography as well as cell morphology of the test specimens were inspected by SEM. The highest bacterial adherence was found on titanium SLA whereas the other surfaces revealed 50-75% less adherent bacteria. P. gingivalis was eliminated by ozone from all surfaces within 24s to below the detection limit (≥99.94% reduction). S. sanguinis was more resistant and showed the highest reduction on zirconia substrates (>90% reduction). Ozone treatment did not affect the surface structures of the test specimens and did not influence osteoblastic cell adhesion and proliferation negatively. Titanium (polished) and zirconia (acid etched and polished) had a lower colonization potential and may be suitable material for implant abutments. Gaseous ozone showed selective efficacy to reduce adherent bacteria on titanium and zirconia without affecting adhesion and proliferation of osteoblastic cells. This in vitro study may provide a solid basis for clinical studies on gaseous ozone treatment of peri-implantitis and revealed an essential base for sufficient tissue regeneratio

Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo

Intervertebral disc regeneration : small and large scale systems

Objectives: The intervertebral disc (IVD) degeneration is an aberrant cell-mediated response to progressive structural failure. One of the major causes of the back pain, the IVD degeneration affects about 60% of the population, increasing the costs of the health system. Although the problem of disc degeneration has been approached from many sides, from orthopaedic surgery to molecular biology, it remains unsolved. The present study suggests a tissue engineering alternative to the current surgical therapies, focusing its attention on the regeneration of the nucleus pulposus and on the standardization of its in vitro basic methods. The first objective of this investigation is to analyze the potentiality of an injectable biomaterial combination as carrier for autologus disc cells in the IVD augmentation process. Moreover, in order to reduce the costs of tissue engineering, the second objective of this study regards the establishment of an automated system able to isolate, propagate and characterize in large scale human primary disc cells. Methods: Disc-like cells were isolated by enzymatic digestion from intervertebral disc tissue, harvested during discectomy, and expanded in monolayer cultures before seeding onto a three dimensional system. The cells were seeded onto a macroporous scaffold and subsequently encapsulated into a hydrogel. The viability of cells as well as their capability to proliferate, migrate into the biomaterial combination and synthesize the extracellular matrix was detected by cell and biochemical assays at different culture time points. Moreover, disc cells were genotypically characterized analysing by RT-PCR the expression pattern of specific genes. The disc cell isolation and the monolayer cultures procedures were furthermore performed with the automated liquid handling robot supplied from Tecan® (Tecan® freedom EVO®). Results: Disc-like cells were able to adhere and proliferate onto the three dimensional system showing a homogenous distribution into the surrounding hydrogel structure after few days of culture. Cells synthesized the required extracellular matrix and re-acquired their original genotypic features indicating their redifferentiation in vitro. The automated isolation of primary disc-like cells from human biopsies showed comparable results to the manual one. The isolated cells compared for the number, viability and morphology with the manual processing. Furthermore, cells seeded and maintained in bidimensional culture by the robot revealed the same morphology and growth rate of manually treated cells. Conclusions: This study indicates that the three dimensional developed structure can be a valid system for the treatment of early-stages IVD degeneration. Moreover, the current results obtained with the EVO® robot suggests its feasibility in the automated isolation of disc cells from human biopsy and its propagation in large scale for a future clinical application

Biomaterial : bioactive synthetic bone repair material for large oral bone

The present project aims at the development of a shapeable synthetic bone repair material, which consists of a novel and innovative combination of a synthetic ceramic scaffold and hydrogel matrix serving as signaling factor carrier system to facilitate the repair of large bone defects in oral surgery