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.

Fabrication using a rapid prototyping system and in vitro characterization of PEG-PCL-PLA scaffolds for tissue engineering

n the field of tissue engineering new polymers are needed to fabricate scaffolds with specific properties depending on the targeted tissue. This work aimed at designing and developing a 3D scaffold with variable mechanical strength, fully interconnected porous network, controllable hydrophilicity and degradability. For this, a desktop-robot-based melt-extrusion rapid prototyping technique was applied to a novel tri-block co-polymer, namely poly(ethylene glycol)-block-poly(epsi-caprolactone)-block-poly(DL-lactide), PEG-PCL-P(DL)LA. This co-polymer was melted by electrical heating and directly extruded out using computer-controlled rapid prototyping by means of compressed purified air to build porous scaffolds. Various lay-down patterns (0/30/60/90/120/150°, 0/45/90/135°, 0/60/120° and 0/90°) were produced by using appropriate positioning of the robotic control system. Scanning electron microscopy and micro-computed tomography were used to show that 3D scaffold architectures were honeycomb-like with completely interconnected and controlled channel characteristics. Compression tests were performed and the data obtained agreed well with the typical behavior of a porous material undergoing deformation. Preliminary cell response to the as-fabricated scaffolds has been studied with primary human fibroblasts. The results demonstrated the suitability of the process and the cell biocompatibility of the polymer, two important properties among the many required for effective clinical use and efficient tissue-engineering scaffolding.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

Direct writing of chitosan scaffolds using a robotic system

Purpose – This paper aims to present a novel rapid prototyping (RP) fabrication methods and preliminary characterization for chitosan scaffolds. Design – A desktop rapid prototyping robot dispensing (RPBOD) system has been developed to fabricate scaffolds for tissue engineering (TE) applications. The system is a computer-controlled four-axis machine with a multiple-dispenser head. Neutralization of the acetic acid by the sodium hydroxide results in a precipitate to form a gel-like chitosan strand. The scaffold properties were characterized by scanning electron microscopy, porosity calculation and compression test. An example of fabrication of a freeform hydrogel scaffold is demonstrated. The required geometric data for the freeform scaffold were obtained from CT-scan images and the dispensing path control data were converted form its volume model. The applications of the scaffolds are discussed based on its potential for TE. Findings – It is shown that the RPBOD system can be interfaced with imaging techniques and computational modeling to produce scaffolds which can be customized in overall size and shape allowing tissue-engineered grafts to be tailored to specific applications or even for individual patients. Research limitations/implications – Important challenges for further research are the incorporation of growth factors, as well as cell seeding into the 3D dispensing plotting materials. Improvements regarding the mechanical properties of the scaffolds are also necessary. Originality/value – One of the important aspects of TE is the design scaffolds. For customized TE, it is essential to be able to fabricate 3D scaffolds of various geometric shapes, in order to repair tissue defects. RP or solid free-form fabrication techniques hold great promise for designing 3D customized scaffolds; yet traditional cell-seeding techniques may not provide enough cell mass for larger constructs. This paper presents a novel attempt to fabricate 3D scaffolds, using hydrogels which in the future can be combined with cells.

Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the pupillary light reflex and circadian rhythm

Recently discovered intrinsically photosensitive melanopsin retinal ganglion cells contribute to the maintenance of pupil diameter, recovery and post-illumination components of the pupillary light reflex and provide the primary environmental light input to the suprachiasmatic nucleus for photoentrainment of the circadian rhythm. This review summarises recent progress in understanding intrinsically photosensitive ganglion cell histology and physiological properties in the context of their contribution to the pupillary and circadian functions and introduces a clinical framework for using the pupillary light reflex to evaluate inner retinal (intrinsically photosensitive melanopsin ganglion cell) and outer retinal (rod and cone photoreceptor) function in the detection of retinal eye disease.

Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work we assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer‐designed gyroid architecture fabricated by stereolithography to a random‐pore architecture resulting from salt‐leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than tenfold higher permeability due to the absence of size‐limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds, and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture, gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt‐leached scaffolds were covered with a cell‐sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolongs the time of static culture before overgrowth of cells at the scaffold periphery occurs. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds, and to create tissue engineering grafts with designed, pre‐fabricated vasculature.