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1.
Sci Rep ; 10(1): 5330, 2020 03 24.
Article in English | MEDLINE | ID: mdl-32210309

ABSTRACT

Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.


Subject(s)
Bioprinting/methods , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Bone Matrix/metabolism , Bone and Bones/metabolism , Cell Differentiation , Coculture Techniques , Durapatite/chemistry , Endothelial Cells/cytology , Gelatin/chemistry , Humans , Hydrogels/chemistry , Ink , Mesenchymal Stem Cells/metabolism , Neovascularization, Physiologic/physiology , Osteogenesis/physiology , Printing, Three-Dimensional
2.
Acta Biomater ; 52: 159-170, 2017 04 01.
Article in English | MEDLINE | ID: mdl-27965173

ABSTRACT

In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM. STATEMENT OF SIGNIFICANCE: The clickECM was produced by the incorporation of azide-functionalized sugar analogues into the extracellular glycans of fibroblast cell cultures by metabolic oligosaccharide engineering. By introducing these azide groups into the glycan structures, we enabled this cell-derived ECM for bioorthogonal click reactions. Click chemistry provides extremely specific reactions with high efficiency, high selectivity, and high reaction yields. We could show that the azide functionalities within the clickECM are chemically accessible. Based on our here described clickECM technique it will be possible to create and investigate new clickECM materials with tunable bioactive properties and additional functionalities, which offers a promising approach for basic and applied research in the field of biomaterial science, biomedical applications, and tissue engineering.


Subject(s)
Azides/chemistry , Biocompatible Materials/chemical synthesis , Click Chemistry/methods , Extracellular Matrix Proteins/chemistry , Extracellular Matrix/chemistry , Fibroblasts/chemistry , Cell-Free System/chemistry , Cells, Cultured , Humans , Materials Testing
3.
J Biotechnol ; 205: 101-10, 2015 Jul 10.
Article in English | MEDLINE | ID: mdl-25562421

ABSTRACT

Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVß3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.


Subject(s)
Monocytes/cytology , Osteoblasts/cytology , Osteoclasts/metabolism , Tissue Culture Techniques/methods , Animals , Biomarkers/metabolism , Bone Matrix/metabolism , Bone Resorption/pathology , Cattle , Cell Differentiation , Cell Size , Humans , Osteoclasts/cytology , Polystyrenes/pharmacology
4.
Cytotherapy ; 11(2): 245-55, 2009.
Article in English | MEDLINE | ID: mdl-19152153

ABSTRACT

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.


Subject(s)
Adult Stem Cells/cytology , Biomedical Research , Embryonic Stem Cells/cytology , Immunotherapy, Adoptive , Neoplasms/therapy , Adult Stem Cells/physiology , Biomedical Research/ethics , Biomedical Research/methods , Biomedical Research/trends , Cell Culture Techniques , Cell Differentiation , Cell Movement , Cell Transdifferentiation , Diagnostic Imaging , Embryonic Stem Cells/physiology , Gene Expression Profiling , Germany , Hematopoietic Stem Cell Mobilization , Humans , Regenerative Medicine/trends , Stem Cell Niche
5.
Spinal Cord ; 41(4): 211-9, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12669085

ABSTRACT

We describe the case of a 47-year-old female who sustained a C5/6 fracture with C6 complete spinal cord injury 26 years ago. She presented with increased spasticity of the lower extremities, the abdominal wall and episodes of autonomic dysreflexia. Imaging of the spine revealed post-traumatic kyphosis at the level of the injury and degenerative changes of the lumbar spine with marked facet joint hypertrophy at the level of L4/5 causing severe spinal canal stenosis. Discussants of this case comment on the possible pathophysiological mechanisms causing autonomic dysreflexia, especially the development of degenerative changes, Charcot arthropathy and the role of tethering mechanisms. The diagnostic options and management approaches are also discussed.


Subject(s)
Spinal Cord Injuries/complications , Spinal Stenosis/etiology , Diagnosis, Differential , Female , Humans , Lumbosacral Region/pathology , Magnetic Resonance Imaging , Middle Aged , Prognosis , Spinal Cord Injuries/classification , Spinal Cord Injuries/pathology , Spinal Cord Injuries/surgery , Spinal Fractures/etiology , Spinal Fractures/pathology , Spinal Fractures/surgery , Spinal Stenosis/pathology , Spinal Stenosis/surgery
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