David Christian Kraft

Fibronectin adsorption, cell adhesion, and proliferation on nanostructured tantalum surfaces

The interaction between dental pulp derived mesenchymal stem cells (DP-MSCs) and three different tantalum nanotopographies with and without a fibronectin coating is examined: sputter-coated tantalum surfaces with low surface roughness <0.2 nm, hut-nanostructured surfaces with a height of 2.9 +/- 0.6 nm and a width of 35 +/- 8 nm, and dome structures with a height of 13 +/- 2 nm and a width of 52 +/- 14 nm. Using ellipsometry, the adsorption and the availability of fibronectin cell-binding domains on the tantalum surfaces were examined, as well as cellular attachment, proliferation, and vinculin focal adhesion spot assembly on the respective surfaces. The results showed the highest fibronectin mass uptake on the hut structures, with a slightly higher availability of cell-binding domains and the most pronounced formation of vinculin focal adhesion spots as compared to the other surfaces. The proliferation of DP-MSCs was found to be significantly higher on dome and hut surfaces coated with fibronectin compared to the uncoated flat tantalum surfaces. Consequently, the results presented in this study indicate that fibronectin-coated nanotopographies with a vertical dimension of less than 5 nm influence cell adhesion. This rather interesting behavior is argued to originate from the more available fibronectin cell-binding domains observed on the hut structures.

Accelerated bone ingrowth by local delivery of strontium from surface functionalized titanium implants

Studies have indicated systemic treatment with strontium (Sr) as a potential route to increase bone quality and formation around osseointegrating implants. However, adverse effects are linked to such treatment. In this study we present a surface modification method designed for sustained local release of Sr from implants. The four groups used were prepared by a magnetron co-sputtering process and selected on the basis of Sr release data. The composition, morphology and mechanical stability of the coatings were analyzed and the Sr release profiles were investigated in vitro by washout experiments. Mesenchymal stem cells were cultured on the different coatings to evaluate potential cytotoxic effects and the effect on cellular proliferation. No indication of toxicity was found. A rodent study demonstrated a significant increase in direct bone-to-implant contact and peri-implant bone volume, for several of the groups, four weeks after implantation when compared to a Grade 4 titanium reference group. Median values of bone-to-implant contact and new bone formation was found to be 19% and 53%, respectively, for the best group compared to 0% for both parameters with respect to the Grade 4 titanium reference. The results indicate that this method may have applications within the orthopedic and dental industry.

Preincubation with the proteasome inhibitor MG-132 enhances proteasome activity via the Nrf2 transcription factor in aging human skin fibroblasts

Abstract:  Strategies that lead to the upregulation of the proteasome are known to elicit beneficial consequences to the organism by countering oxidative stress–associated disorders, such as protein conformational diseases, cancer, and aging. Mild treatment with proteasome inhibitors has been previously demonstrated to stimulate proteasome activity and cellular resistance against oxidative injury. However, the mechanism for this action has not been clearly defined. We examined the role of the nuclear factor‐E2‐related factor 2 (Nrf2) in fibroblasts, a key transactivator of the antioxidant response pathway, in the regulation of the proteasome by its inhibitor MG‐132. Here, we demonstrate that the stimulation of the proteasome by low levels of MG‐132 can be abrogated by small interfering RNAs (siRNAs) targeted against Nrf2. Consistently, cells that constitutively express Nrf2 exhibit elevated levels of proteasome activities. We further investigate how its beneficial effects, that is, proteasome stimulation, are manifested in young and replicative‐senescent cells. Our data underscore that manipulation of Nrf2 by the administration of pharmacologically low levels of proteasome inhibitors may prove to be an alternatively potent strategy for inducing long‐term protective effects against oxidative stress.

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cells capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.

Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity

For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC-mature exhibited higher osteocalcin and alkaline phosphatase gene expression and activity than PDSC-immature. Pulsating fluid flow (PFF) stimulated nitric oxide production within 5 min by PDSC-mature but not by PDSC-immature. In PDSC-mature, PFF induced prostaglandin E(2) production, and cyclooxygenase 2 gene expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs and produce more bone in vivo. These data suggest that PDSCs with a mature osteogenic phenotype might be preferable for bone tissue engineering to restore, for example, maxillofacial defects, because they might be able to perform mature bone cell-specific functions during bone adaptation to mechanical loading in vivo.

Proteasomal Oscillation during Mild Heat Shock in Aging Human Skin Fibroblasts

Abstract:  Augmentation of proteasome machinery is emerging as a significant gerontomodulatory consequence of hormetic stimulation, such as mild heat stress. This study describes the phenomenon we term hormetic proteasomal oscillation, wherein mildly heat‐stressed human fibroblasts (41°C, 1 h) display an adaptation response pattern in proteasome activity. Remarkably, such response appears to be diverse in severely heat‐stressed or senescent fibroblasts. This proteasomal oscillation, as an innate cellular reaction to heat and aging, however, is independent of 20S proteasome protein levels and nuclear factor‐E2‐related factor 2 (Nrf2) transactivation.

Geroprotection by glycerol : Insights to its mechanisms and clinical potentials

Abstract:  Chaperones, particularly the heat‐shock proteins, are considered as key players in the maintenance of protein homeostasis and are associated with longevity and cellular immortalization. In this study, we investigated the geroprotective activity of the chemical chaperone glycerol. Glycerol showed significant chaperoning activity in refolding heat‐denatured luciferase in vivo and in protecting cells from heat stress‐induced cytotoxicity. This was accompanied by decrease in p53, an upregulation of a stress chaperone mortalin/mtHsp70, and an increase in proteasome activity in the presence of oxidative stress.

Glycerol stimulates innate chaperoning, proteasomal and stress-resistance functions: implications for geronto-manipulation

Aging is associated with accumulation of toxic intracellular and extracellular protein aggregates. Cells manage “aged” proteins by mobilizing their molecular chaperones or heat shock proteins that are also considered as determinants of lifespan in diverse species. In this study, we tested whether an exogenous addition of the non-toxic chemical chaperone ‘glycerol’ could elicit stress and geronto-protective activities. We found that glycerol enhanced chaperoning of heat-denatured proteins. In addition to stimulating proteasome activity, glycerol led to an increased expression of the stress chaperone ‘mortalin’ and decreased p53 function in human cells. Glycerol-fed worms exhibited thermo-tolerance and lower level of age-associated auto-fluorescence. Through the combined stimulation of the proteasome and chaperoning activities of mortalin, in particular, glycerol treatment resulted in increased survival and fitness against oxidative- and heat-stress. These results may have significant implications in the use of glycerol as a candidate geronto-modulator in development of practical interventions for “healthy aging”.

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D) polycaprolactone (PCL) – hyaluronic acid – tricalcium phosphate (HT-PCL) scaffold. Population doubling (PD), alkaline phosphatase (ALP) activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1) empty defects vs. HT-PCL scaffolds; (2) PCL scaffolds vs. HT-PCL scaffolds; and (3) autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV) were assessed with micro-computed tomography (μCT) and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

Human dental pulp cells exhibit bone cell-like responsiveness to fluid shear stress

BACKGROUND AIMS For engineering bone tissue to restore, for example, maxillofacial defects, mechanosensitive cells are needed that are able to conduct bone cell-specific functions, such as bone remodelling. Mechanical loading affects local bone mass and architecture in vivo by initiating a cellular response via loading-induced flow of interstitial fluid. After surgical removal of ectopically impacted third molars, human dental pulp tissue is an easily accessible and interesting source of cells for mineralized tissue engineering. The aim of this study was to determine whether human dental pulp-derived cells (DPC) are responsive to mechanical loading by pulsating fluid flow (PFF) upon stimulation of mineralization in vitro. METHODS Human DPC were incubated with or without mineralization medium containing differentiation factors for 3 weeks. Cells were subjected to 1-h PFF (0.7 ± 0.3 Pa, 5 Hz) and the response was quantified by measuring nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, and gene expression of cyclooxygenase (COX)-1 and COX-2. RESULTS We found that DPC are intrinsically mechanosensitive and, like osteogenic cells, respond to PFF-induced fluid shear stress. PFF stimulated NO and PGE₂ production, and up-regulated COX-2 but not COX-1 gene expression. In DPC cultured under mineralizing conditions, the PFF-induced NO, but not PGE₂, production was significantly enhanced. CONCLUSIONS These data suggest that human DPC, like osteogenic cells, acquire responsiveness to pulsating fluid shear stress in mineralizing conditions. Thus DPC might be able to perform bone-like functions during mineralized tissue remodeling in vivo, and therefore provide a promising new tool for mineralized tissue engineering to restore, for example, maxillofacial defects.