Karin Berr

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.

Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

BackgroundFacial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation.MethodsMurine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and β-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR.ResultsESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17.ConclusionSupplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.

Scaffold-free microtissues: differences from monolayer cultures and their potential in bone tissue engineering.

ObjectivesThe paper’s aim is to review dentin hypersensitivity (DHS), discussing pain mechanisms and aetiology.Materials and methodsLiterature was reviewed using search engines with MESH terms, DH pain mechanisms and aetiology (including abrasion, erosion and periodontal disease).ResultsThe many hypotheses proposed for DHS attest to our lack of knowledge in understanding neurophysiologic mechanisms, the most widely accepted being the hydrodynamic theory. Dentin tubules must be patent from the oral environment to the pulp. Dentin exposure, usually at the cervical margin, is due to a variety of processes involving gingival recession or loss of enamel, predisposing factors being periodontal disease and treatment, limited alveolar bone, thin biotype, erosion and abrasion.ConclusionsThe current pain mechanism of DHS is thought to be the hydrodynamic theory. The initiation and progression of DHS are influenced by characteristics of the teeth and periodontium as well as the oral environment and external influences. Risk factors are numerous often acting synergistically and always influenced by individual susceptibility.Clinical relevanceWhilst the pain mechanism of DHS is not well understood, clinicians need to be mindful of the aetiology and risk factors in order to manage patients’ pain and expectations and prevent further dentin exposure with subsequent sensitivity.ObjectivesCell-based therapies for bone augmentation after tooth loss and for the treatment of periodontal defects improve healing defects. Usually, osteogenic cells or stem cells are cultivated in 2D primary cultures, before they are combined with scaffold materials, even though this means a loss of the endogenous 3D microenvironment for the cells. Moreover, the use of single-cell suspensions for the inoculation of scaffolds or for the direct application into an area of interest has the disadvantages of low initial cell numbers and susceptibility to unwanted cellular distribution, respectively.Materials and methodsWe addressed the question whether an alternative to monolayer cultures, namely 3D microtissues, has the potential to improve osteogenic tissue engineering and its clinical outcome.ResultsBy contrast, to monolayer cultures, osteogenic differentiation of 3D microtissues is enhanced by mimicking in vivo conditions. It seems that the osteogenic differentiation in microtissues is enhanced by strong integrin–extracellular matrix interaction and by stronger autocrine BMP2 signaling. Moreover, microtissues are less prone to wash out by body fluids and allow the precise administration of large cell numbers.ConclusionMicrotissue cultures have closer characteristics with cells in vivo and their enhanced osteogenic differentiation makes scaffold-free microtissues a promising concept in osteogenic tissue engineering.Clinical relevanceMicrotissues are particularly suitable for tissue engineering because they improve seeding efficiency of biomaterials by increasing the cell load of a scaffold. This results in accelerated osteogenic tissue formation and could contribute to earlier implant stability in mandibular bone augmentation.

Biocompatibility of Osteogenic Predifferentiated Human Cord Blood Stem Cells with Biomaterials and the Influence of the Biomaterial on the Process of Differentiation

Modern cell-based bone reconstruction therapies offer new therapeutic opportunities and tissue engineering represents a more biological-oriented approach to heal bone defects of the skeleton. Human unrestricted somatic stem cells (USSCs) derived form umbilical cord blood offer new promising aspects e.g., can differentiate into osteogenetic cells. Furthermore these cells have fewer ethical and legal restrictions compared to embryonic stem cells (ESCs). The purpose of this study was to evaluate the compatibility of osteogenic pre-differentiated USSCs with various biomaterials and to address the question, whether biomaterials influence the process of differentiation of the USSCs. After osteogenic differentiation with DAG USSCs were cultivated with various biomaterials. To asses the biocompatibility of USSCs the attachment and the proliferation of the cells on the biomaterial were measured by a CyQUANT® assay, the morphology was analyzed by scanning electron microscopy and the influence of the gene expression was analyzed by real time PCR. Our results provide evidence that insoluble collagenous bone matrix followed by β-tricalciumphosphate is highly suitable for bone tissue engineering regarding cell attachment and proliferation. The gene expression analysis indicates that biomaterials influence the gene expression of USSCs. These results are in concordance with our previous study with ESCs.

Compatibility of Embryonic Stem Cells with Biomaterials

Periodontal bone defects and atrophy of the jaws in an aging population are of special concern. Tissue engineering using embryonic stem cells (ESCs) and biomaterials may offer new therapeutic options. The purpose of this study is to evaluate the compatibility of ESCs with biomaterials and the influence of biomaterials on the osteogenic gene expression profile. Therefore, ESCs are cultured with various biomaterials. The cytocompatibility of murine ESCs is measured regarding the proliferation of the cells on the materials by CyQUANT ® assay, the morphology by scanning electron microscopy, and the influence on the gene expression by real time PCR. The results show that insoluble collagenous bone matrix, followed by β-tricalciumphosphate, is most suitable for bone tissue engineering regarding cell proliferation, and phenotype. The gene expression analysis indicates that biomaterials do influence the gene expression of ESCs. Our results provide new insight into the cytocompatibility of ESCs on different scaffolds.

Embryonic stem cells induce ectopic bone formation in rats.

BACKGROUND Surgery often leads to massive destruction of the skeleton. Cell-based bone reconstruction therapies promise new therapeutic opportunities for the repair of bone. Embryonic stem cells (ESCs) can be differentiated into osteogenic cells and are a potential cell source for bone tissue engineering. The purpose of this in vivo study was to investigate the bone formation in various constructs containing ESCs (with and without micromass technology) and insoluble collagenous bone matrix (ICBM). METHODS Murine ESCs were cultured as monolayer cultures as well as micromasses and seeded on ICBM. These constructs were implanted in immunodeficient rats. After one week, one, two and three months CT-scans were performed to detect any calcifications and the rats were sacrificed. RESULTS The radiological examination shows a steep increase of the mineralized tissue in group 1 (ICBM+seeded ESC). This increase can be considered as statistical significant. In contrast, the volume of the mineralization in group 2 (ICBM+ESC-spheres) and group 3 (ESC-spheres) does not increase significantly during the study. CONCLUSION ESCs in combination with ICBM do promote ectopic bone formation in vivo. Thus, this cell population as well as the biomaterial ICBM might be promising components for bone tissue engineering.

Impact of DAG stimulation on mineral synthesis, mineral structure and osteogenic differentiation of human cord blood stem cells ☆ ☆☆

It remains unexplored in what way osteogenic stimulation with dexamethasone, ascorbic acid and β-glycerol phosphate (DAG) influences the process of mineralization, the composition and structure of the assembled mineral. Therefore, we analyzed and characterized biomineralization in DAG-stimulated and unstimulated 3D human unrestricted somatic stem cell (USSC) cultures. Microspheres were analyzed by histological staining, scanning electron microscopy (SEM), semi-quantitative energy-dispersive X-ray spectroscopy (EDX), quantitative wavelength-dispersive X-ray spectroscopy (WDX), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Raman spectroscopy. Mineral material was detected by SEM and histological staining in both groups, and showed structural differences. DAG influenced the differentiation of USSCs and the formation, structure and composition of the assembled mineral. SEM showed that cells of the +DAG spheres exhibited morphological signs of osteoblast-like cells. EDX and WDX confirmed a Ca-P mineral in both groups. Overall, the mineral material found showed structural similarities to the mineral substance of bony material.

Improvement of the Cell-loading Efficiency of Biomaterials by Inoculation with Stem Cell-based Microspheres, in Osteogenesis

In critical-size bone defects, autologous or allogenic cells are required in addition to compatible biomaterials for the successful defect healing. State of the art inoculation methods of biomaterials are based on the application of cell suspensions to the biomaterial. However, only less amounts of cells can be applied and sufficient adhesion to the material is required. Therefore, it was investigated whether the advantages of stem cell-based microspheres and insoluble collagenous bone matrix (ICBM) scaffolds can be combined which can lead to an advancement in cell seeding on biomaterials. Microspheres were produced from unrestricted somatic stem cells from human umbilical cord blood and were mounted on ICBM scaffolds. Following the incubation with osteogenic or control medium, the constructs were analyzed histologically after 3, 7, 14, and 28 days. Alizarin Red S and von Kossa staining revealed microsphere mineralization after 3 days in osteogenic and after 14 days in control medium. Meanwhile, a time-dependent increase in tissue, growing out of the microspheres, was detected. Our results provide evidence that microsphere–ICBM constructs are promising candidates for approaches of bone regeneration. They allow the transfer of substantially high numbers of cells in partially mineralized constructs.

Laboratory Procedures – Culture of Cells and Tissues

Tissue engineering and cell cultivation are common methods of basic research that help to provide an understanding of the biology of cells and tissues. Even today there remain many challenges to face. These methods may support the control of processes that are necessary for the construction and design of artificial living tissues. The potential to improve, maintain, and restore tissue function may enhance quality of life and health care, for instance by overcoming the shortages of donor tissues and organs. Tissue engineering and cell cultivation include cell sourcing, manipulation of cell function, and the construction of living tissues.