Biomedical subjects

Jan-Thorsten Schantz

Publications and source records attributed to Jan-Thorsten Schantz.

13 recordsLinked to original sources

PubMed2006

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways.

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H(2)O(2)) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint.

Apoptosis Inducing Factor↗

PubMed2006

Human circulating peripheral blood mononuclear cells for calvarial bone tissue engineering.

BACKGROUND: Large-scale production of engineered tissues requires an adequate source of expandable cells. Current strategies that involve harvesting of cells from donor tissue or bone marrow for tissue engineering are invasive and unfeasible for obtaining large quantities of cells in a clinical setting. Peripheral blood has been reported to contain circulating hematopoietic cells as well as, in significantly smaller quantities, mesenchymal cells METHODS: An adherent subset of CD14+ mononuclear cells was isolated from human peripheral venous blood and characterized in vitro by light microscopy, immunohistochemistry, flow cytometry, and quantitative differentiation assays. These cells were then evaluated for the purposes of tissue engineering in a rat calvarial defect model, in combination with biodegradable polymer matrices made from poly-e-caprolactone. Specimens were analyzed 6 weeks after implantation with histologic analysis, microcomputed tomography, and HLA immunostaining. RESULTS: CD14+ mononuclear cells were induced to differentiate into osteoblast-like cells in vitro, with areas of mineralization. In a rat calvarial defect model, tissue-engineered bone with evidence of mineralization was formed within 6 weeks. HLA immunohistochemistry demonstrated that de novo bone formation originated from the transplanted human cells. CONCLUSIONS: These findings show, for the first time, to our knowledge, the derivation of bone from human blood. They also demonstrate the utility of circulating mononuclear cells as a minimally invasive, potentially unlimited pool of cells for tissue engineering and organ regeneration.

Adult↗

PubMed2006

Cranioplasty after trephination using a novel biodegradable burr hole cover: technical case report.

OBJECTIVE AND IMPORTANCE: We have developed novel biodegradable polymer implants by using the rapid prototyping technology fused deposition modeling. Early results of a clinical pilot study for cranioplasty are presented. CLINICAL PRESENTATION: Five patients with the diagnosis of chronic subdural hematoma were included in the study. After trephination and evacuation of the subdural hematoma, burr holes (diameter, 14 mm) were closed using a biodegradable implant made of polycaprolactone. Implants were computer designed with an upper rim diameter of 16 mm and a 14 mm body diameter with a fully interconnected, honeycomb-like architecture of 400 to 600 microm in pore size. INTERVENTION: Postoperative computed tomographic scans indicated that the plugs were stably anchored in the osseous host environment with no fluid collection detectable. The postoperative course was uneventful, and patients were discharged after 5 days. Follow-up scans after 3, 6, and 12 months showed that the implants were well integrated in the surrounding calvarial bone with new bone filling the porous space. CONCLUSION: These novel polymer scaffolds made of the slow-degrading material polycaprolactone represent a suitable implant for closure of post-trephination defects.

Absorbable Implants↗

PubMed2005

Osteogenic differentiation of mesenchymal progenitor cells in computer designed fibrin-polymer-ceramic scaffolds manufactured by fused deposition modeling.

Biomimetic scaffolds offer great potentials in the development of bone analogs for tissue engineering. The studies presented in this paper focus specifically on the osteogenic potential of the novel PCL/CaP matrices and its degradation behavior. Biodegradable Polymer-ceramic Scaffolds were fabricated using the solid free form fabrication technology: Fused Deposition Modeling (FDM). The scaffold architecture was characterized by a honeycomb-like design and a complete interconnectivity of the pores. Human mesenchymal stem cells (MSCs) were seeded together with fibrin glue into PCL/CaP scaffolds and cultured in vitro for periods of up to eight weeks. Cellular adhesion, proliferation and osteogenic differentiation were assessed in these constructs using a range of histological and microscopic techniques. In additional experiments, degradation was assessed by measuring mass loss, diameter change, molecular weight change and by scanning electron micrographs. MSCs were able to adhere, migrate, and differentiate along the osteogenic lineage with in these scaffolds. The PCL/CaP scaffolds showed up to 27 fold increased degradation of compared to PCL scaffolds.

Biodegradation, Environmental↗

PubMed2005

New frontiers in calvarial reconstruction: integrating computer-assisted design and tissue engineering in cranioplasty.

Repair of large and complex calvarial defects remains a particular challenge for reconstruction. The paucity of techniques and materials emphasizes the need for alternative bone formation strategies. Recent integrative approaches suggest that successful reconstruction requires interdisciplinary teams, with surgeons interacting with imaging experts, materials scientists, and engineers. In this review, the authors present an overview of current materials used in calvarial reconstruction. Subsequently, progress in computer-designed prostheses, tissue engineering, and osteoinduction strategies is discussed. Finally, the authors discuss their experience with the integration of computer-aided fabrication of customized implants and tissue engineering for calvarial reconstruction.

Animals↗

PubMed2004

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains.

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains.

Calcium↗

PubMed2004

Degradation and cell culture studies on block copolymers prepared by ring opening polymerization of epsilon-caprolactone in the presence of poly(ethylene glycol).

Poly(epsilon-caprolactone) (PCL) and its block copolymers with poly(ethylene glycol) (PEG) were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of ethylene glycol or PEG, using zinc metal as catalyst. The resulting polymers were characterized by various analytical techniques such as (1)H NMR, SEC, DSC, IR, X-ray, ESEM, and CZE. PCL/PEG copolymers with long PCL chains presented the same crystalline structure as PCL homopolymer, whereas PEG-bearing short PCL blocks retained the crystalline structure of PEG and exhibited an amphiphilic behavior in aqueous solutions. Degradation of PCL and PCL/PEG diblock and triblock copolymers was realized in a 0.13 M, pH 7.4 phosphate buffer at 37 degrees C. The results indicated that the copolymers exhibited higher hydrophilicity and degradability compared with the PCL homopolymer. Large amounts of PEG were released from the bulk after 60 weeks' degradation. In vitro cell culture studies were conducted on scaffolds manufactured via solid free form fabrication by using primary human and rat bone marrow derived stromal cells (hMSC, rMSC). Light, scanning electron, and confocal laser microscopy, as well as immunocytochemistry, showed cell attachment, proliferation, and extracellular matrix production on the surface, as well as inside the scaffold architecture. Copolymers showed better performance in the cell culture studies than the PCL homopolymer.

Animals↗

PubMed2003

Efficacy of glow discharge gas plasma treatment as a surface modification process for three-dimensional poly (D,L-lactide) scaffolds.

Gas plasma surface modification of three-dimensional poly (D,L-lactide) scaffolds fabricated by a novel vibrating particle fabrication technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 10 days in culture using human embryonic palatal mesenchyme cells. Characterization of corresponding two-dimensional treated surfaces revealed decreased contact angle measurements of 54.2 +/- 0.6 degrees for treated surfaces compared to 72.3 +/- 0.7 degrees for control surfaces (p < 0.05). SEM of treated surfaces revealed increased surface roughness combined with marked pitting and erosion. This may contribute to increased cell adhesion. WST-1 cell proliferation assay measurements as an index of cell numbers revealed a statistically significant increase in proliferation activity on treated surfaces on days 1 and 4 compared with controls. There was a fivefold increase in WST-1 activity for both control and treated groups over 10 days. Confocal laser micrographs revealed increased cell numbers on treated specimens throughout all layers of the scaffold, indicating that the glow discharge process enhanced cell proliferation throughout the entire scaffold architecture. Scanning electron micrographs demonstrated increased cell adhesion for treated specimens at the polymer surface most evident after days 1 and 4 of culture. Alkaline phosphatase (ALP)-specific activity peaked by day 7 for control and treated surfaces, indicating cellular differentiation. There was a trend for increased protein production on the treated specimens compared with controls at the initial time points although the differences were not statistically significant. These results demonstrated that gas plasma surface modification enhances osteoblast-like cell function in a three-dimensional scaffold model.

Absorbable Implants↗

PubMed2003

Preliminary study of a polycaprolactone membrane utilized as epidermal substrate.

Solvent-cast sheets of polycaprolactone were biaxially stretched to produce 10-15 microm thick films. PCL films were found to have a tensile strength of 55 MPa which is about two and a half times stronger than native skin. One of our previous studies using non-coated PCL membranes showed that only 36% of the membrane surface was covered with keratinocytes after 9 days of culture. The present study examined the effects of coating the surface of PCL membranes with fibrin on the proliferation of keratinocytes. Qualitative analysis revealed that the cells attached and proliferated better on coated PCL films. Keratinocytes exhibited healthy cobblestone morphology and proliferated as continuous monolayers over a period of 16 days. The results indicated that fibrin coated PCL films would support the attachment and proliferation of human keratinocytes and have the potential to be applied as a matrix material for tissue engineering an epidermal equivalent.

Journal Article↗

PubMed2003

Repair of calvarial defects with customized tissue-engineered bone grafts I. Evaluation of osteogenesis in a three-dimensional culture system.

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Animals↗

PubMed2003

Repair of calvarial defects with customised tissue-engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo.

We have demonstrated in Part I of this study [see Schantz, J.-T., et al., Tissue Eng. 2003;9(Suppl. 1): S-113-S-126; this issue] that bone marrow-derived progenitor cells and calvarial osteoblasts could be successfully directed into the osteogenic lineage and cultured in three-dimensional (3-D) polycaprolactone (PCL) scaffolds. The objective of the second part of the study was to evaluate and to compare tissue engineered cell-polymer constructs using calvarial osteoblasts (group I) and mesenchymal progenitor cells (MPCs; group II) for the reconstruction of critical-size and three-dimensionally complex cranial defects. In 30 New Zealand White rabbits, bilateral parietal critical-size defects were created. On the basis of computed tomography scans, customized PCL scaffolds with precisely controlled microarchitecture were fabricated, using a rapid prototyping technology. Bone marrow-derived progenitor cells and osteoblasts were isolated and expanded in culture. Osteoblasts (group I) and mesenchymal progenitor cells (group II) were seeded in combination with a fibrin glue suspension into 40 PCL scaffolds. After incubating for 3 days in static culture, the PCL scaffold-cell constructs as well as nonseeded PCL scaffolds (control group) were implanted into 15-mm-diameter calvarial defects. Reconstruction of the cranium and bone formation were evaluated after 3 months. In vivo results indicated osseous tissue integration within the implant and functionally stable restoration of the calvarium. Islands of early bone formation could be observed in X-ray radiographs and in histological sections. Implants showed a high cell:ECM ratio and a dense vascular network. Mechanical testing of the reconstructed area revealed partial integration with the surrounding corticocancellous calvarial bone. The amount (area) of calcification, measured by clinical computed tomography, indicated that cell-seeded constructs measured about 60% more than unrepaired or unseeded scaffolds. Mechanical investigations revealed that stiffness reached 52 +/- 29 and 44 +/- 16 MPa for MPC- and osteoblast-seeded scaffolds, respectively. The yield strength for the push-out tests was 180 +/- 36 N for normal calvarial bone, 90 +/- 1 N for unrepaired site, and 106 +/- 10 N for unseeded constructs, which is about 60% of normal bone strength. MPC- and osteoblast-seeded scaffolds indicated a yield strength of 149 +/- 15 and 164 +/- 42 N, respectively, which is about 85-90% of normal bone. This study demonstrated that customized biodegradable polymeric implants may be used to deliver osteogenic cells and enhance bone formation within critically-sized defects in vivo. The use of rapid prototyping technology to produce scaffolds with controlled external geometry and microarchitecture offers new possibilities in the functional and aesthetic reconstruction of complex craniofacial defects.

Animals↗

PubMed2002

Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology.

Due to their osteogenic germination potential, periosteum-derived osteoprogenitor cells are a potential source for tissue engineering a bone graft that could be used to regenerate skeletal defects. In this study we evaluated if ectopic bone formation could be induced by a construct made of human periosteal cells and a novel scaffold architecture whose mechanical properties are in the range of cancellous bone. Biopsies from human calvarial periosteum were harvested and cells were isolated from the inner cambial layer. Fifty thousand periosteal cells were seeded into the scaffolds measuring 6 x 6 x 2 mm. The cell-scaffold constructs were cultured for a period of 3 weeks prior to implantation into balb C nude mice. Mice were sacrificed and implants were analyzed 6 and 17 weeks postoperatively. Immunohistochemical analysis confirmed the osteoblastic phenotype of the seeded cells. Formation of focal adhesions and stress fibers could be observed in both scaffold architectures. Three-dimensional cell proliferation was observed after 2 weeks of culturing with centripetal growth pattern inside the pore network. The deposition of calcified extracellular matrix was observed after 3 weeks of culturing. In vivo, endochondral bone formation with osteoid production was detectable via von Kossa and Osteocalcin staining after 6 and 17 weeks. Histology and SEM revealed that the entire scaffold/bone grafts were penetrated by a vascular network. This study showed the potential of bone tissue engineering by using human periosteal cells in combination with a novel scaffold technology.

Animals↗

PubMedDate not supplied

Evaluation of a tissue-engineered membrane-cell construct for guided bone regeneration.

PURPOSE: Currently, a number of bioresorbable and biodegradable membranes used for guided bone regeneration lead to incomplete tissue regeneration. Poor mechanical properties, short degradation time, and the lack of integrated biologic components result in the inability to create and maintain an appropriate environment and to actively support tissue remodeling. In the present study, the osteogenic potential of human calvarial periosteal cells in combination with ultrathin polycaprolactone (pc2) membranes of a slow biodegradation rate was investigated. MATERIALS AND METHODS: In vitro and in vivo analyses of the tissue-engineered constructs were conducted using imaging techniques, immunohistochemistry, and histology. Two types of membranes were investigated. Group 1 consisted of a plain membrane, and in group 2 membranes were treated with sodium hydroxide. RESULTS: In vitro results showed that osteoblast-like cells attached and proliferated on the membranes with the formation of extracellular matrix. Sodium hydroxide-treated membranes showed enhanced cell attachment and proliferation kinetics, resulting in a dense cellular layer after 2 weeks in culture. In vivo mineralized tissue formation in association with vascularization was observed. Extracellular matrix calcification with nodule formation was detected via histology as well as scanning electron microscopy. DISCUSSION: PCL membranes support the attachment, growth, and osteogenic differentiation of human primary osteoblast-like cells. Sodium hydroxide-treated membranes demonstrated increased cell attachment resulting from increased hydrophilicity. CONCLUSION: These findings have potential application in the development of a new generation of osteoconductive membranes.

Absorbable Implants↗