Hans-Peter Wiesmann

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-β1 (TGF-β1)

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-β1 (TGF-β1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-β1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.

Synergistic effect of defined artificial extracellular matrices and pulsed electric fields on osteogenic differentiation of human MSCs

In vivo, bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an in vivo-like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone-co-lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs.

Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering

This protocol describes an effective method for the production of spherical microtissues (microspheres), which can be used for a variety of tissue-engineering purposes. The obtained microtissues are well suited for the study of osteogenesis in vitro when multipotent stem cells are used. The dimensions of the microspheres can easily be adjusted according to the cell numbers applied in an individual experiment. Thus, microspheres allow for the precise administration of defined cell numbers at well-defined sites. Here we describe a detailed workflow for the production of microspheres using unrestricted somatic stem cells from human umbilical cord blood and adapted protocols for the use of these microspheres in histological analysis. RNA extraction methods for mineralized microtissues are specifically modified for optimum yields. The duration of running the complete protocol without preparatory cell culture but including 2 weeks of microsphere incubation, histological staining and RNA isolation is about 3 weeks.

Gelatine modified monetite as a bone substitute material: An in vitro assessment of bone biocompatibility

UNLABELLED Calcium phosphate phases are increasingly used for bone tissue substitution, and the load bearing properties of these inherently brittle biomaterials are increased by inclusion of organic components. Monetite prepared using mineralization of gelatine pre-structured through phosphate leads to a significantly increased biaxial strength and indirect tensile strength compared to gelatine-free monetite. Besides the mechanical properties, degradation in physiological solutions and osteoblast and osteoclast cell response were investigated. Human bone marrow stromal cells (hBMSCs) showed considerably higher proliferation rates on the gelatine modified monetite than on polystyrene reference material in calcium-free as well as standard cell culture medium (α-MEM). Osteogenic differentiation on the material was comparable to polystyrene in both medium types. Osteoclast-like cells derived from monocytes were able to actively resorb the biomaterial. Osteoblastic differentiation and perhaps even more important the cellular resorption of the biomaterial indicate that it can be actively involved in the bone remodeling process. Thus the behavior of osteoblasts and osteoclasts as well as the adequate degradation and mechanical properties are strong indicators for bone biocompatibility, although in vivo studies are still required to prove this. STATEMENT OF SIGNIFICANCE New and unique? A low temperature precipitationprocessforcalcium anhydrous hydrogen phosphateallows for the first time to produce monolithic compact composites of monetite and gelatine. The composite is degradable and resorbable. To prove that, the question arises: what is bone biocompatibility? The reaction of both mayor cell types of bone represents this biocompatibility. Therefore, human bone marrow stromal cells were seeded revealing the materials pro-osteogenic properties. Monocyte cultivation, becoming recently focus of interest, revealed the capability of the biomaterial to be actively resorbed by derived osteoclast-like cells. Not new but necessary ismechanical characterization, which is often only investigated as uniaxial property. Here, a biaxial method is applied, to characterize the materials properties closer to its application loads.

Investigation of biomaterials by human epithelial gingiva cells: an in vitro study

IntroductionIn modern medicine and dentistry the use of biomaterials is a fast developing field of increasing interest. Especially in dentistry the interaction between biomaterials like implant materials and the soft tissue in the oral cavity is in the focus of daily research. In this context the high importance of testing materials and their surfaces concerning their biocompatibility towards corresponding cells is very likely. For this purpose this study investigates cells derived from human gingival biopsies on different materials and surfaces.MethodsCells in this study were cultivated out of human biopsies by a grow out explant technique and were sub cultivated on titanium, zirconium dioxide and collagen membrane specimens. To characterise the cells on the material surfaces used in this study immunohistochemical and histological staining techniques as well as different methods of microscopy (light microscopy and SEM) were applied.ResultsWith the aid of the explant technique and the chosen cell cultivation method it was possible to investigate the human gingiva derived cells on different materials. The data of the present study show that the human gingival cells attach and proliferate on all three tested materials by exhibiting characteristic gingival keratinocyte protein expression even after long periods of culture e.g. up to 70 days.ConclusionsIt could be shown that the three tested materials titanium, zirconium dioxide and collagen membrane (and their special surfaces) are good candidates for the application as materials in the dental gingival environment or, in the case of the collagen membrane as scaffold/cell-carrier for human gingival cells in tissue engineering.

Eggshells as natural calcium carbonate source in combination with hyaluronan as beneficial additives for bone graft materials, an in vitro study

IntroductionIn bone metabolism and the formation especially in bone substitution, calcium as basic module is of high importance. Different studies have shown that the use of eggshells as a bone substitute material is a promising and inexpensive alternative. In this in vitro study, the effects of eggshell granulate and calcium carbonate towards primary bovine osteoblasts were investigated. Hyaluronan (HA) was used as artificial extracellular matrix (ECM) for the used cells to facilitate proliferation and differentiation and to mimic the physiological requirements given by the egg in vivo.MethodsHyaluronan, eggshells, a combination of hyaluronan and eggshells and CaCO3 were applied to the cells as additive to the used standard medium (modified High Growth Enhancement Medium) in a concentration of 0,1 g/l. The effect of the additives in the culture medium was examined by proliferation tests, immunohistochemical staining (anti-collagen type I, anti-osteopontin, anti-osteonectin and anti-osteocalcin) and kinetic oxygen measurements.ResultsOur investigations revealed that all investigated additives show beneficial effect on osteoblast activity. Cell proliferation, differentiation and the metabolic activity of the differentiated cells could be influenced positively. Especially in the case cell cultures treated with eggshells the strongest effects were detected, while for the hyaluronan compared with eggshells, a weaker increase in cell activity was observed.ConclusionIn summary, it can be stated that the investigated components come into consideration as beneficial supplements for bone graft materials especially for maxillo facial surgery application.

Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro

IntroductionMaintaining or regenerating a vital pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell aggregates (spheres) were applied onto bovine and human root canal models to evaluate their potential use as pre-differentiated tissue units for dental pulp tissue regeneration.MethodsHuman dental pulp cells (DPC) were derived from wisdom teeth, cultivated into three-dimensional cell spheres and seeded onto bovine and into human root canals. Sphere formation, tissue-like and mineralization properties as well as growth behavior of cells on dentin structure were evaluated by light microscopy (LM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX).ResultsSpheres and outgrown cells showed tissue-like properties, the ability to merge with other cell spheres and extra cellular matrix formation; CLSM investigation revealed a dense network of actin and focal adhesion contacts (FAC) inside the spheres and a pronounced actin structure of cells outgrown from the spheres. A dentin-structure-orientated migration of the cells was shown by SEM investigation. Besides the direct extension of the cells into dentinal tubules, the coverage of the tubular walls with cell matrix was detected. Moreover, an emulation of dentin-like structures with tubuli-like and biomineral formation was detected by SEM- and EDX-investigation.ConclusionsThe results of the present study show tissue-like behavior, the replication of tubular structures and the mineralization of human dental pulp spheres when colonized on root dentin. The application of cells in form of pulp spheres on root dentin reveals their beneficial potential for dental tissue regeneration.

Influence of pulse ratio on codeposition of copper species with calcium phosphate coatings on titanium by means of electrochemically assisted deposition.

Aim of this study was to combine the well-known biocompatibility and ostoeconductivity of thin calcium phosphate coatings on titanium with proangiogenic signals from codeposited copper species. Copper species could be integrated in mineral layers based on hydroxyapatite by means of electrochemically assisted deposition from electrolytes containing calcium, phosphate, and copper ions. Different combinations of duration and intensity of galvanostatic pulses result in different amounts of deposited calcium phosphate and of copper species even for the same applied total charge. Absolute amounts of copper varied between 2.1 and 6.9 μg/cm², and the copper was distributed homogeneously as shown by EDX mapping. The presence of copper did not change the crystalline phase of deposited calcium phosphate (hydroxyapatite) but provoked a significant decrease in deposited amounts by factor 3 to 4. The copper was deposited mainly as Cu(I) species with a minor fraction of basic copper phosphates. Reduction of copper occurred not only at the surface of titanium but also within the hydroxyapatite coating due to the reaction with hydrogen produced by the electrolysis of water during the cathodic polarization of the substrate.

Odontoblast-like differentiation and mineral formation of pulpsphere derived cells on human root canal dentin in vitro

BackgroundThe revitalization or regeneration of the dental pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell (DPC) spheres were applied to human root canal samples to evaluate their potential adoption for physiological tissue-like regeneration of the dental root canal by odontoblastic differentiation as well as cell-induced mineral formation.MethodsDPC were cultivated into three-dimensional cell spheres and seeded on human root canal specimens. The evaluation of sphere formation, tissue-like behavior and differentiation as well as mineral formation of the cells was carried out with the aid of optical light microscopy, immunohistochemical staining and scanning electron microscopy (SEM).ResultsSpheres and cells migrated out of the spheres showed an intense cell-cell- and cell-dentin-contact with the formation of extra cellular matrix. In addition, the ingrowth of cell processes into dentinal tubules and the interaction of cell processes with the tubule walls were detected by SEM-imaging. Immunohistochemical staining of the odontoblast specific matrix proteins, dentin matrix protein-1, and dentin sialoprotein revealed an odontoblast-like cell differentiation in contact with the dentin surface. This differentiation was confirmed by SEM-imaging of cells with an odontoblast specific phenotype and cell induced mineral formation.ConclusionsThe results of the present study reveal the high potential of pulp cells organized in spheres for dental tissue engineering. The odontoblast-like differentiation and the cell induced mineral formation display the possibility of a complete or partial “dentinal filling” of the root canal and the opportunity to combine this method with other current strategies.

Electric field-assisted formation of organically modified hydroxyapatite (ormoHAP) spheres in carboxymethylated gelatin gels

UNLABELLED A biomimetic strategy was developed in order to prepare organically modified hydroxyapatite (ormoHAP) with spherical shape. The technical approach is based on electric field-assisted migration of calcium ions and phosphate ions into a hydrogel composed of carboxymethylated gelatin. The electric field as well as the carboxymethylation using glucuronic acid (GlcA) significantly accelerates the mineralization process, which makes the process feasible for lab scale production of ormoHAP spheres and probably beyond. A further process was developed for gentle separation of the ormoHAP spheres from the gelatin gel without compromising the morphology of the mineral. The term ormoHAP was chosen since morphological analyses using electron microscopy (SEM, TEM) and element analysis (EDX, FT-IR, XRD) confirmed that carboxymethylated gelatin molecules use to act as organic templates for the formation of nanocrystalline HAP. The hydroxyapatite (HAP) crystals self-organize to form hollow spheres with diameters ranging from 100 to 500nm. The combination of the biocompatible chemical composition and the unique structure of the nanocomposites is considered to be a useful basis for future applications in functionalized degradable biomaterials. STATEMENT OF SIGNIFICANCE A novel bioinspired mineralization process was developed based on electric field-assisted migration of calcium and phosphate ions into biochemically carboxymethylated gelatin acting as organic template. Advantages over conventional hydroxyapatite include particle size distribution and homogeneity as well as achievable mechanical properties of relevant composites. Moreover, specifically developed calcium ion or phosphate ion release during degradation can be useful to adjust the fate of bone cells in order to manipulate remodeling processes. The hollow structure of the spheres can be useful for embedding drugs in the core, encapsulated by the highly mineralized outer shell. In this way, controlled drug release could be achieved, which enables advanced strategies for threating bone-related diseases, e.g. osteoporosis and multiple myeloma.