The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (≤180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding α5β1- and αv-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of α5β1- and αv-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.

Computational fluid dynamics for improved bioreactor design and 3D culture

The complex relationship between the hydrodynamic environment and surrounding tissues directly impacts on the design and production of clinically useful grafts and implants. Tissue engineers have generally seen bioreactors as 'black boxes' within which tissue engineering constructs (TECs) are cultured. It is accepted that a more detailed description of fluid mechanics and nutrient transport within process equipment can be achieved by using computational fluid dynamics (CFD) technology. This review discusses applications of CFD for tissue engineering-related bioreactors -- fluid flow processes have direct implications on cellular responses such as attachment, migration and proliferation. We conclude that CFD should be seen as an invaluable tool for analyzing and visualizing the impact of fluidic forces and stresses on cells and TECs.

Runx2 overexpression in bone marrow stromal cells accelerates bone formation in critically-sized femoral defects

The repair of large non-unions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause bone marrow stromal cells to lose their differentiation ability. To overcome these limitations, we have genetically engineered bone marrow stromal cells to constitutively overexpress the osteoblast specific transcription factor Runx2. In the present study, we examined Runx2-modified bone marrow stromal cells, delivered via poly(caprolactone) scaffolds loaded with type I collagen meshes, in critically-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds and empty defects. Runx2 expression in bone marrow stromal cells accelerated healing of critically-sized defects compared to unmodified bone marrow stromal cells and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects which may reduce recovery time and the need for external fixation of critically-sized defects.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.

A hydrogen tethered inhibitor of matrix metalloproteinases

During wound repair, the balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs) is crucial for the normal extra cellular matrix turnover. However, the over expression of several MMPs including MMP-1, 2, 3, 8, 9 and MMP-10, combined with abnormally high levels of activation or low expression of TIMPs, may contribute to excessive degradation of connective tissue and formation of chronic ulcers. There are many groups exploring strategies for promoting wound healing involving delivery of growth factors, cells, ECM components and small molecules. Our approach for improving the balance of MMPs is not to add anything more to the wound, but instead to neutralise the over-expressed MMPs using inhibitors tethered to a bandage-like hydrogel. Our in vitro experiments using designed synthetic pseudo peptide inhibitors have been demonstrated to inhibit MMP activity in standard solutions. These inhibitors have also been tethered to polyethylene glycol hydrogels using a facile reaction between the linker unit on the inhibitor and the gel. After tethering the inhibition of MMPs diminishes to some extent and we postulate that this arises due to poor diffusion of the MMPs into the gels. When the tethered inhibitors were tested against chronic wound fluid obtained against patients we observed over 40% inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds.

Dynamics of in vitro polymer degradation of polycaprolactone-based scaffolds : accelerated versus simulated physiological conditions

The increasing use of biodegradable devices in tissue engineering and regenerative medicine means it is essential to study and understand their degradation behaviour. Accelerated degradation systems aim to achieve similar degradation profiles within a shorter period of time, compared with standard conditions. However, these conditions only partially mimic the actual situation, and subsequent analyses and derived mechanisms must be treated with caution and should always be supported by actual long-term degradation data obtained under physiological conditions. Our studies revealed that polycaprolactone (PCL) and PCL-composite scaffolds degrade very differently under these different degradation conditions, whilst still undergoing hydrolysis. Molecular weight and mass loss results differ due to the different degradation pathways followed (surface degradation pathway for accelerated conditions and bulk degradation pathway for simulated physiological conditions). Crystallinity studies revealed similar patterns of recrystallization dynamics, and mechanical data indicated that the scaffolds retained their functional stability, in both instances, over the course of degradation. Ultimately, polymer degradation was shown to be chiefly governed by molecular weight, crystallinity susceptibility to hydrolysis and device architecture considerations whilst maintaining its thermodynamic equilibrium.

Electro-spinning of pure collagen nano-fibres – just an expensive way to make gelatin?

Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations ( p > 0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix.

In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

Application of autologous periosteal cells for the regeneration of class III furcation defects in Beagle dogs

The aim of this study was to evaluate the healing of class III furcation defects following transplantation of autogenous periosteal cells combined with b-tricalcium phosphate (b-TCP). Periosteal cells obtained from Beagle dogs’ periosteum explant cultures, were inoculated onto the surface of b-TCP. Class III furcation defects were created in the mandibular premolars. Three experimental groups were used to test the defects’ healing: group A, b-TCP seeded with periosteal cells were transplanted into the defects; group B, b-TCP alone was used for defect filling; and group C, the defect was without filling materials. Twelve weeks post surgery, the tissue samples were collected for histology, immunohistology and X-ray examination. It was found that both the length of newly formed periodontal ligament and the area of newly formed alveolar bone in group A, were significantly increased compared with both group B and C. Furthermore, both the proportion of newly formed periodontal ligament and newly formed alveolar bone in group A were much higher than those of group B and C. The quantity of cementum and its percentage in the defects (group A) were also significantly higher than those of group C. These results indicate that autogenous periosteal cells combined with b-TCP application can improve periodontal tissue regeneration in class III furcation defects.

Characterisation and culturing of adipose-derived precursor cells

This book focuses on practical applications for using adult and embryonic stem cells in the pharmaceutical development process. It emphasizes new technologies to help overcome the bottlenecks in developing stem cells as therapeutic agents. A key reference for professionals working in stem cell science, it presents the general principles and methodologies in stem cell research and covers topics such as derivitization and characterization of stem cells, stem cell culture and maintenance, stem cell engineering, applications of high-throughput screening, and stem cell genetic modification with their use for drug delivery.