Jung Park

TiO2 Nanotube Surfaces: 15 nm—An Optimal Length Scale of Surface Topography for Cell Adhesion and Differentiation†

Studies of biomimetic surfaces in medicine and biomaterial fields have explored extensively how the micrometer-scale topography of a surface controls cell behavior, but only recently has the nanoscale environment received attention as a critical factor for cell behavior. Several investigations of cell interactions have been performed using surface protrusion topographies at the nanoscale; such topographies are typically based on polymer demixing, ordered gold cluster arrays, or islands of adhesive ligands at distinct length scales. Recent work has indicated that the fabrication of ordered TiO2 nanotube layers with controlled diameters can be achieved by anodization of titanium in adequate electrolytes. Such surfaces can almost ideally be used as nanoscale spacing models for size-dependent cellular response. This is particularly important as these studies are carried out on titanium surfaces—a material used for clinical titanium implantations for the purpose of bone, joint, or tooth replacements. Therefore, principles elucidated from this work can guide implant surface modifications toward an optimized surface geometry and profile to best fit and cell interactions for adequate bone growth.

Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix

The ultimate goal in the design of biomimetic materials for use in tissue engineering as permanent or resorbable tissue implants is to generate biocompatible scaffolds with appropriate biomechanical and chemical properties to allow the adhesion, ingrowth, and survival of cells. Recent efforts have therefore focused on the construction and modification of biomimetic surfaces targeted to support tissue-specific cell functions including adhesion, growth, differentiation, motility, and the expression of tissue-specific genes. Four decades of extensive research on the structure and biological influence of the extracellular matrix (ECM) on cell behavior and cell fate have shown that three types of information from the ECM are relevant for the design of biomimetic surfaces: (1) physical properties (elasticity, stiffness, resilience of the cellular environment), (2) specific chemical signals from peptide epitopes contained in a wide variety of extracelluar matrix molecules, and (3) the nanoscale topography of microenvironmental adhesive sites. Initial physical and chemical approaches aimed at improving the adhesiveness of biomaterial surfaces by sandblasting, particle coating, or etching have been supplemented by attempts to increase the bioactivity of biomaterials by coating them with ECM macromolecules, such as fibronectin, elastin, laminin, and collagens, or their integrin-binding epitopes including RGD, YIGSR, and GFOGER. Recently, the development of new nanotechnologies such as photo- or electron-beam nanolithography, polymer demixing, nano-imprinting, compression molding, or the generation of TiO2 nanotubes of defined diameters (15–200 nm), has opened up the possibility of constructing biomimetic surfaces with a defined nanopattern, eliciting tissue-specific cellular responses by stimulating integrin clustering. This development has provided new input into the design of novel biomaterials. The new technologies allowing the construction of a geometrically defined microenvironment for cells at the nanoscale should facilitate the investigation of nanotopography-dependent mechanisms of integrin-mediated cell signaling.

Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes.

Large bone defects resulting from nonunion fractures or tumour resections are common clinical problems. Recent studies have shown bone morphogenetic protein-2 (BMP-2) gene transfer using adenoviral vectors to be a promising new therapeutic approach. However, comparative studies of different vectors are required to identify the optimal system for possible clinical trials. This study compares the use of liposome-mediated and adenoviral gene transfer for the generation of autologous BMP-2-producing bone marrow stromal cells (BMSC). Primary BMSC isolated from the rat femur were treated ex vivo with either an adenovirus or a liposome carrying human BMP-2 cDNA. The genetically modified cells were evaluated in vitro and transplanted into critical size defects in the rat mandible in vivo. BMSC treated with a reporter gene vector or untreated BMSC served as controls. The newly formed tissue was analysed by in situ hybridization, radiography and immunohistochemistry. Both groups of genetically modified cells produced BMP-2 for at least 2 weeks, and markers of new bone matrix such as osteopontin and osteocalcin were observed within 2 weeks following gene transfer. In the liposome group, the critical size defects were found completely healed at 6 weeks after the gene transfer, whereas the more efficient adenoviral gene transfer allowed for complete bone healing within 4 weeks. None of the three control groups showed bone healing, not even after 8 weeks. Thus, both liposome-mediated and adenoviral BMP-2 gene transfer to primary BMSC are suitable methods to achieve the healing of critical size bone defects in rats. As liposomes have proven sufficient for this purpose and offer several advantages over any other vector, such as ease of preparation, theoretically no limitation of the size of the DNA, and less immunological and safety problems, they may represent the best vector system for future clinical trials of bone regeneration by BMP-2 gene therapy.

Narrow window in nanoscale dependent activation of endothelial cell growth and differentiation on TiO2 nanotube surfaces.

Critical features of biomimetic materials used for vascular grafts and stents are surface structure and chemical features of the implant material supporting adhesion, proliferation, and differentiation of endothelial cells and smooth muscle cells, the major cell types of blood vessels. Recently, experimental evidence from several laboratories have indicated a strong stimulation of cellular activities on vertically aligned TiO(2) nanotube surfaces in comparison to amorphous TiO(2) surfaces. Conflicting reports exist, however, concerning the nanoscale dimension, and the role of the chemistry and crystallinity of the nanotubes in eliciting cell responses. Here we demonstrate that 15 nm nanotubes provide a substantially stronger stimulation of differentiation of mesenchymal cells to endothelial cells and smooth muscle cells than 70-100 nm nanotubes, while high rates of apoptosis were seen on 100 nm nanotubes. Also endothelial cell adhesion, proliferation, and motility were several-fold higher on 15 nm than on 100 nm nanotubes. Furthermore, our data indicate a clear dominance of the nanoscale geometry on endothelial cell behavior over surface chemistry and crystallinity of the TiO(2) nanotube surface. These findings indicate that fine-tuning of TiO(2) surfaces at nanoscale will be an essential parameter in optimizing endothelial cell and smooth muscle cell responses to vascular implants.

Improved attachment of mesenchymal stem cells on super-hydrophobic TiO2 nanotubes

Self-organized layers of vertically orientated TiO(2) nanotubes providing defined diameters ranging from 15 up to 100nm were grown on titanium by anodic oxidation. These TiO(2) nanotube layers show super-hydrophilic behavior. After coating TiO(2) nanotube layers with a self-assembled monolayer (octadecylphosphonic acid) they showed a diameter-dependent wetting behavior ranging from hydrophobic (108+/-2 degrees ) up to super-hydrophobic (167+/-2 degrees ). Cell adhesion, spreading and growth of mesenchymal stem cells on the unmodified and modified nanotube layers were investigated and compared. We show that cell adhesion and proliferation are strongly affected in the super-hydrophobic range. Adsorption of extracellular matrix proteins as fibronectin, type I collagen and laminin, as well as bovine serum albumin, on the coated and uncoated surfaces showed a strong influence on wetting behavior and dependence on tube diameter.

Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells

Adult primary mesenchymal cells of different origin which can be obtained with minor donor site morbidity are considered for articular cartilage repair. This study aims at a comparison of their chondrogenic potential.

Synergistic control of mesenchymal stem cell differentiation by nanoscale surface geometry and immobilized growth factors on TiO2 nanotubes.

The aim of this study is to elucidate whether combined environmental signals provided by nanoscale topography and by growth factors control cell behavior of mesenchymal stem cells (MSCs) in a synergistic or simply additive manner. Chondrogenic and osteogenic differentiation of MSCs is studied on vertically aligned TiO(2) nanotubes of size 15 and 100 nm with and without immobilized bone morphogenetic protein-2 (BMP-2). Although BMP-2 coating stimulates both chondrogenic and osteogenic differentiation of MSCs, the response strongly depends on the surface nanoscale geometry of the BMP-2-coated nanotubes. Chondrogenic differentiation is strongly supported on 100 nm BMP-2-coated nanotubes, but not on 15 nm nanotubes, which induce spreading and de-differentiation of chondrocytes. A similar response is observed with primary chondrocytes, which maintain their chondrogenic phenotype on BMP-2-coated 100 nm nanotubes, but de-differentiate on 15 nm nanotubes. In contrast, osteogenic differentiation is greatly enhanced on 15 nm but not on 100 nm BMP-2-coated nanotubes as shown previously. Furthermore, covalent immobilization of BMP-2 rescues MSCs from apoptosis occurring on uncoated 100 nm TiO(2) nanotube surfaces. Thus, combined signals provided by BMP-2 immobilized to a defined lateral nanoscale spacing geometry seem to contain environmental cues that are able to modulate a lineage-specific decision of MSC differentiation and cell survival in a synergistic manner.

Deficiency in the LIM-only protein Fhl2 impairs skin wound healing

After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359–369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736–748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of α-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

Cell‐based resurfacing of large cartilage defects: Long‐term evaluation of grafts from autologous transgene‐activated periosteal cells in a porcine model of osteoarthritis

OBJECTIVE To investigate the potential of transgene-activated periosteal cells for permanently resurfacing large partial-thickness cartilage defects. METHODS In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno-associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls. RESULTS All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad-stimulated periosteal cells had adopted a chondrocyte-like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad-stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix. CONCLUSION Large partial-thickness cartilage defects can be resurfaced efficiently with hyaline-like cartilage formed by transgene-activated periosteal cells. The long-term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.

Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage

According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond “terminal” differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates – depending on the developmental stage – substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP+Osx+ cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate.