Search

Crosslinking of tryptophan (Trp) containing copolypeptides with varying ratios of benzyl-l-glutamate (BLG) and Nα-(carbobenzyloxy)-l-lysine (Z-Lys) is achieved by the selective reaction with hexamethylene-bis-TAD (bisTAD). Conversion of the resulting organogels into biocompatible hydrogels by full BLG or Z-Lys deprotection is demonstrated. Moreover, diffusion controlled deprotection allows the design of macroscopic hybrid organohydrogels comprising hydrophilic as well as hydrophobic regions at a desired ratio and position. FTIR and SEM analysis confirm the coexistence of both hydrophilic and hydrophobic segments in one copolypeptide piece. Selective loading of hydrogel and organogel segments with hydrophilic and hydrophobic dyes, respectively, is observed on macroscopic amphiphilic gels and films. These materials offer significant potential as dual-loaded drug release gels as well as tissue engineering platforms.

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The role of substrate mechanics in guiding mesenchymal stem cell (MSC) fate has been the focus of much research over the last decade. More recently, the complex interplay between substrate mechanics and other material properties such as ligand chemistry and substrate degradability to regulate MSC differentiation has begun to be elucidated. Additionally, there are several changes in the presentation of these material properties as the dimensionality is altered from two- to three-dimensional substrates, which may fundamentally alter our understanding of substrate-induced mechanotransduction processes. In this review, an overview of recent findings that highlight the material properties that are important in guiding MSC fate decisions is presented, with a focus on underlining gaps in our existing knowledge and proposing potential directions for future research.

Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function.

Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.

Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure., (Copyright © 2013 John Wiley & Sons, Ltd.)

Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Alterations in bone tissue composition during osteoporosis likely disrupt the mechanical environment of bone cells and may thereby initiate a mechanobiological response. It has proved challenging to characterize the mechanical environment of bone cells in vivo, and the mechanical environment of osteoporotic bone cells is not known. The objective of this research is to characterize the local mechanical environment of osteocytes and osteoblasts from healthy and osteoporotic bone in a rat model of osteoporosis. Using a custom-designed micromechanical loading device, we apply strains representative of a range of physical activity (up to 3000 με) to fluorescently stained femur samples from normal and ovariectomized rats. Confocal imaging was simultaneously performed, and digital image correlation techniques were applied to characterize cellular strains. In healthy bone tissue, osteocytes experience higher maximum strains (31,028 ± 4213 με) than osteoblasts (24,921 ± 3,832 με), whereas a larger proportion of the osteoblast experiences strains >10,000 με. Most interestingly, we show that osteoporotic bone cells experience similar or higher maximum strains than healthy bone cells after short durations of estrogen deficiency (5 weeks), and exceeded the osteogenic strain threshold (10,000 με) in a similar or significantly larger proportion of the cell (osteoblast, 12.68% vs. 13.68%; osteocyte, 15.74% vs. 5.37%). However, in long-term estrogen deficiency (34 weeks), there was no significant difference between bone cells in healthy and osteoporotic bone. These results suggest that the mechanical environment of bone cells is altered during early-stage osteoporosis, and that mechanobiological responses act to restore the mechanical environment of the bone tissue after it has been perturbed by ovariectomy., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chondrogenic priming (CP) of mesenchymal stem cells (MSCs) and coculture of MSCs with human umbilical vein endothelial stem cells (HUVECs) both have been shown to significantly increase the potential for MSCs to undergo osteogenic differentiation and mineralization in vitro and in vivo. Such strategies mimic cartilage template formation or vascularization that occur during endochondral ossification during early fetal development. However, although both chondrogenesis and vascularization are crucial precursors for bone formation by endochondral ossification, no in vitro bone tissue regeneration strategy has sought to incorporate both events simultaneously. The objective of this study is to develop an in vitro bone regeneration strategy that mimics critical aspects of the endochondral ossification process, specifically (1) the formation of a cartilage template and (2) subsequent vascularization of this template. We initially prime the MSCs with chondrogenic growth factors, to ensure the production of a cartilage template, and subsequently implement a coculture strategy involving MSC and HUVECs. Three experimental groups were compared; (1) CP for 21 days with no addition of cells; (2) CP for 21 days followed by coculture of HUVECs (250,000 cells); (3) CP for 21 days followed by coculture of HUVECs and MSCs (250,000 cells) at a ratio of 1:1. Each group was cultured for a further 21 days in osteogenic media after the initial CP period. Biochemical (DNA, Alkaline Phosphatase Activity, Calcium, and Vessel Endothelial Growth Factor) and histological analyses (Alcian blue, alizarin red, CD31(+), and collagen type X) were performed 1, 2, and 3 weeks after the media switch. The results of this study show that CP provides a cartilage-like template that provides a suitable platform for HUVEC and MSC cells to attach, proliferate, and infiltrate for up to 3 weeks. More importantly we show that the use of the coculture methodology, rudimentary vessels are formed within this cartilage template and enhanced the mineralization potential of MSCs. Taken together these results indicate for the first time that the application of both chondrogenic and vascular priming of MSCs enhances the mineralization potential of MSCs in vitro while also allowing the formation of immature vessels.

Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47°C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47°C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis followed by a cascade of remodelling responses.

Recent in vivo studies have proposed that integrin αvβ3 attachments between osteocyte cell processes and the extracellular matrix may facilitate mechanosensation in bone. However the role of these attachments in osteocyte biochemical response to mechanical stimulus has yet to be investigated. With this in mind, the objective of this study was to determine the effect of blocking integrin αvβ3 function on the biochemical response of osteocytes to mechanical stimulus. Antagonists specific to integrin subunit β3 were used to block integrin αvβ3 on MLO-Y4 mouse osteocytes. After treatment, cells were subjected to laminar oscillatory fluid flow stimulus (1 Pa, 1 Hz) for one hour. Fluorescent staining was performed to visualise cell morphology. Prostaglandin E2 (PGE2) release was assayed using an enzyme immunoassay and qRT-PCR was used to analyse the relative expression of cyclooxygenase-2 (COX-2), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). Our results show that blocking integrin αvβ3 disrupts osteocyte morphology, causing a reduction in spread area and process retraction. Integrin αvβ3 blocking also disrupted COX-2 expression and PGE2 release in response to fluid shear stress. Taken together, the results of this study indicate that integrin αvβ3 is essential for the maintenance of osteocyte cell processes and also for mechanosensation and mechanotransduction by osteocytes. A better understanding of this process may lead to the development of novel treatments for bone pathologies where mechanosensitivity is thought to be compromised., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17β-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis., (© Georg Thieme Verlag KG Stuttgart · New York.)

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast-osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro., (© 2013 Elsevier Ltd. All rights reserved.)

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment and strain-induced flow of interstitial fluid is believed to be the primary mediator of mechanical stimuli to bone cells in vivo. In vitro investigations have shown that bone cells produce important biochemical signals in response to fluid flow applied using parallel-plate flow chamber (PPFC) systems. However, the exact mechanical stimulus experienced by the cells within these systems remains unclear. To fully understand this behaviour represents a most challenging multi-physics problem involving the interaction between deformable cellular structures and adjacent fluid flows. In this study, we use a fluid-structure interaction computational approach to investigate the nature of the mechanical stimulus being applied to a single osteoblast cell under fluid flow within a PPFC system. The analysis decouples the contribution of pressure and shear stress on cellular deformation and for the first time highlights that cell strain under flow is dominated by the pressure in the PPFC system rather than the applied shear stress. Furthermore, it was found that strains imparted on the cell membrane were relatively low whereas significant strain amplification occurred at the cell-substrate interface. These results suggest that strain transfer through focal attachments at the base of the cell are the primary mediators of mechanical signals to the cell under flow in a PPFC system. Such information is vital in order to correctly interpret biological responses of bone cells under in vitro stimulation and elucidate the mechanisms associated with mechanotransduction in vivo.

Severe heat-shock to bone cells caused during orthopaedic procedures can result in thermal damage, leading to cell death and initiating bone resorption. By contrast, mild heat-shock has been proposed to induce bone regeneration. In this study, bone cells are exposed to heat-shock for short durations occurring during surgical cutting. Cellular viability, necrosis and apoptosis are investigated immediately after heat-shock and following recovery of 12, 24 h and 4 days, in osteocyte-like MLO-Y4 and osteoblast-like MC3T3-E1 cells, using flow cytometry. The regeneration capacity of heat-shocked Balb/c mesenchymal stem cells (MSCs) and MC3T3-E1s has been investigated following 7 and 14 day's recovery, by quantifying proliferation, differentiation and mineralization. An immediate necrotic response to heat-shock was shown in cells exposed to elevated temperatures (45°C, 47°C and most severe at 60°C). A longer-term apoptotic response is induced in MLO-Y4s and, to a lesser extent, in MC3T3-E1s. Heat-shock-induced differentiation and mineralization by MSCs. These findings indicate that heat-shock is more likely to induce apoptosis in osteocytes than osteoblasts, which might reflect their role as sensors detecting and communicating damage within bone. Furthermore, it is shown for the first time that mild heat-shock (less than equal to 47°C) for durations occurring during surgical cutting can positively enhance osseointegration by osteoprogenitors.

The inherent biocompatibility of fibrin hydrogels makes them an attractive material for use in a wide range of tissue engineering applications. Despite this, their relatively low stiffness and high compliance limits their potential for certain orthopaedic applications. Enhanced mechanical properties are desirable so as to withstand surgical handling and in vivo loading after implantation and additionally, can provide important cues to cells seeded within the hydrogel. Standard methods used to enhance the mechanical properties of biological scaffolds such as chemical or thermal crosslinking cannot be used with fibrin hydrogels as cell seeding and gel formation occurs simultaneously. The objective of this study was to investigate the use of plastic compression as a means to improve the mechanical properties of chondrocyte-seeded fibrin hydrogels and to determine the influence of such compression on cell viability within these constructs. It was found that the application of 80% strain to fibrin hydrogels for 30 min (which resulted in a permanent strain of 47.4%) produced a 2.1-fold increase in the subsequent compressive modulus. Additionally, chondrocyte viability was maintained in the plastically compressed gels with significant cellular proliferation and extracellular matrix accumulation observed over 28 days of culture. In conclusion, plastic compression can be used to modulate the density and mechanical properties of cell-seeded fibrin hydrogels and represents a useful tool for both in theatre and in vitro tissue engineering applications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In stem cell biology, focus has recently turned to the influence of the intrinsic properties of the extracellular matrix (ECM), such as structural, composition and elasticity, on stem cell differentiation. Utilising collagen-glycosaminoglycan (CG) scaffolds as an analogue of the ECM, this study set out to determine the effect of scaffold stiffness and composition on naive mesenchymal stem cell (MSC) differentiation in the absence of differentiation supplements. Dehydrothermal (DHT) and 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC) crosslinking treatments were used to produce three homogeneous CG scaffolds with the same composition but different stiffness values: 0.5, 1 and 1.5 kPa. In addition, the effect of scaffold composition on MSC differentiation was investigated by utilising two glycosaminoglycan (GAG) types: chondroitin sulphate (CS) and hyaluronic acid (HyA). Results demonstrated that scaffolds with the lowest stiffness (0.5 kPa) facilitated a significant up-regulation in SOX9 expression indicating that MSCs are directed towards a chondrogenic lineage in more compliant scaffolds. In contrast, the greatest level of RUNX2 expression was found in the stiffest scaffolds (1.5 kPa) indicating that MSCs are directed towards an osteogenic lineage in stiffer scaffolds. Furthermore, results demonstrated that the level of up-regulation of SOX9 was higher within the CHyA scaffolds in comparison to the CCS scaffolds indicating that hyaluronic acid further influences chondrogenic differentiation. In contrast, enhanced RUNX2 expression was observed in the CCS scaffolds in comparison to the CHyA scaffolds suggesting an osteogenic influence of chondroitin sulphate on MSC differentiation. In summary, this study demonstrates that, even in the absence of differentiation supplements, scaffold stiffness can direct the fate of MSCs, an effect that is further enhanced by the GAG type used within the CG scaffolds. These results have significant implications for the therapeutic uses of stem cells and enhance our understanding of the physical effects of the in vivo microenvironment on stem cell behaviour., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Various forms of mechanical stimulation have been shown to enhance chondrogenesis of mesenchymal stem cells (MSCs). However, the response of MSCs undergoing chondrogenesis to such signals has been shown to depend on the temporal application of loading. The objective of this study was to determine the effect of dynamic compression on cartilage-matrix-specific gene expression and to relate this response to the local biochemical environment and cell phenotype at the time of loading. At 0, 7, 14, and 21 days extracellular matrix (ECM) deposition within MSC-seeded agarose hydrogels due to transforming growth factor-β3 stimulation was determined biochemically and histologically, and then reverse transcription-polymerase chain reaction was used to examine the effects of dynamic compression on cartilage-matrix-specific gene expression. The results of these experiments show that the local environment in the core of the constructs is more favorable for chondrogenesis in comparison to the annulus, as evident from both ECM synthesis and gene expression. Additionally, we found that the response of the cells to mechanical stimulus varied with both the spatial region within the constructs and the temporal application of loading. Dynamic compression applied at day 21 was found to enhance levels of cartilage matrix gene expression following a peak in expression levels at day 14 in free swelling constructs, suggesting that mechanical signals play a key role in the maintenance of a chondrogenic phenotype. The application of mechanical stimulus to enhance cartilage ECM synthesis may be an important tool in regenerative medicine-based cartilage repair. The results of this study suggest that a chondrogenic phenotype and/or a well-developed pericellular matrix must first be established before dynamic compression can have a positive effect on cartilage-matrix-specific gene expression.

Crosslinking and the resultant changes in mechanical properties have been shown to influence cellular activity within collagen biomaterials. With this in mind, we sought to determine the effects of crosslinking on both the compressive modulus of collagen-glycosaminoglycan scaffolds and the activity of osteoblasts seeded within them. Dehydrothermal, 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde crosslinking treatments were first investigated for their effect on the compressive modulus of the scaffolds. After this, the most promising treatments were used to study the effects of crosslinking on cellular attachment, proliferation, and infiltration. Our experiments have demonstrated that a wide range of scaffold compressive moduli can be attained by varying the parameters of the crosslinking treatments. 1-Ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde treatments produced the stiffest scaffolds (fourfold increase when compared to dehydrothermal crosslinking). When cells were seeded onto the scaffolds, the stiffest scaffolds also showed increased cell number and enhanced cellular distribution when compared to the other groups. Taken together, these results indicate that crosslinking can be used to produce collagen-glycosaminoglycan scaffolds with a range of compressive moduli, and that increased stiffness enhances cellular activity within the scaffolds.

The pore structure of three-dimensional scaffolds used in tissue engineering has been shown to significantly influence cellular activity. As the optimal pore size is dependant on the specifics of the tissue engineering application, the ability to alter the pore size over a wide range is essential for a particular scaffold to be suitable for multiple applications. With this in mind, the aim of this study was to develop methodologies to produce a range of collagen-glycosaminoglycan (CG) scaffolds with tailored mean pore sizes. The pore size of CG scaffolds is established during the freeze-drying fabrication process. In this study, freezing temperature was varied (−10 degrees C to −70 degrees C) and an annealing step was introduced to the process to determine their effects on pore size. Annealing is an additional step in the freeze-drying cycle that involves raising the temperature of the frozen suspension to increase the rate of ice crystal growth. The results show that the pore size of the scaffolds decreased as the freezing temperature was reduced. Additionally, the introduction of an annealing step during freeze-drying was found to result in a significant increase (40%) in pore size. Taken together, these results demonstrate that the methodologies developed in this study can be used to produce a range of CG scaffolds with mean pore sizes from 85 to 325 microm. This is a substantial improvement on the range of pore sizes that were possible to produce previously (96-150 microm). The methods developed in this study provide a basis for the investigation of the effects of pore size on both in vitro and in vivo performance and for the determination of the optimal pore structure for specific tissue engineering applications.

Artificial tissue growth requires cells to proliferate and differentiate within the host scaffold. As cell function is governed by mechano-sensitive selection, tissue type is influenced by the microscopic forces exposed to the cells, which is a product of macroscopically straining the scaffold. Accordingly, the microscopic strain environment within a CG scaffold is offered here. Using muCT to characterise CG scaffold architecture, two high-resolution 3D FE models were used to predict the deformation mechanics. While also providing an analysis of region-specific features, such as relative density, pore diameters and microstructural elastic stability, the deformation patterns afforded strains to be inferred for seeded cells. The results indicate a regional dependence, in terms of architectural and mechanical properties. Specifically, the peripheral regions demonstrated the lowest volume fraction, the highest stress concentrations and the greatest potential for elastic instability. Conversely, the mid-region exhibited the most homogeneous environment. Based on the proviso of mechano-sensitive proliferation and differentiation, the findings suggest cell function will vary between CG scaffold regions. Further work should investigate the possibility of improving the fabrication process in order to deliver a construct in line with the mid-region, or alternatively, isolation of the mid-region may prove beneficial for cell culturing., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Articular cartilage has a poor intrinsic capacity for self-repair. The advent of autologous chondrocyte implantation has provided a feasible method to treat cartilage defects. However, the associated drawbacks with the isolation and expansion of chondrocytes from autologous tissue has prompted research into alternative cell sources such as mesenchymal stem cells (MSCs) which have been found to exist in the bone marrow as well as other joint tissues such as the infrapatellar fat pad (IFP), synovium and within the synovial fluid itself. In this work we assessed the chondrogenic potential of IFP-derived porcine cells over a 6 week period in agarose hydrogel culture in terms of mechanical properties, biochemical content and histology. It was found that IFP cells underwent robust chondrogenesis as assessed by glycosaminoglycan (1.47+/-0.22% w/w) and collagen (1.44+/-0.22% w/w) accumulation after 42 days of culture. The 1Hz dynamic modulus of the engineered tissue at this time point was 272.8 kPa (+/-46.8). The removal of TGF-beta3 from culture after 21 days was shown to have a significant effect on both the mechanical properties and biochemical content of IFP constructs after 42 days, with minimal increases occurring from day 21 to day 42 without continued supplementation of TGF-beta3. These findings further strengthen the case that the IFP may be a promising cell source for putative cartilage repair strategies., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

In the literature there are conflicting reports on the optimal scaffold mean pore size required for successful bone tissue engineering. This study set out to investigate the effect of mean pore size, in a series of collagen-glycosaminoglycan (CG) scaffolds with mean pore sizes ranging from 85 microm to 325 microm, on osteoblast adhesion and early stage proliferation up to 7 days post-seeding. The results show that cell number was highest in scaffolds with the largest pore size of 325 microm. However, an early additional peak in cell number was also seen in scaffolds with a mean pore size of 120 microm at time points up to 48 h post-seeding. This is consistent with previous studies from our laboratory which suggest that scaffold specific surface area plays an important role on initial cell adhesion. This early peak disappears following cell proliferation indicating that while specific surface area may be important for initial cell adhesion, improved cell migration provided by scaffolds with pores above 300 microm overcomes this effect. An added advantage of the larger pores is a reduction in cell aggregations that develop along the edges of the scaffolds. Ultimately scaffolds with a mean pore size of 325 microm were deemed optimal for bone tissue engineering.

The mechanical properties of tissue engineering scaffolds are critical for preserving the structural integrity and functionality during both in vivo implantation and long-term performance. In addition, the mechanical and structural properties of the scaffold can direct cellular activity within a tissue-engineered construct. In this context, the aim of this study was to investigate the effects of dehydrothermal (DHT) treatment on the mechanical and structural properties of collagen-glycosaminoglycan (CG) scaffolds. Temperature (105-180 degrees C) and exposure period (24-120 h) of DHT treatment were varied to determine their effect on the mechanical properties, crosslinking density, and denaturation of CG scaffolds. As expected, increasing the temperature and duration of DHT treatment resulted in an increase in the mechanical properties. Compressive properties increased up to twofold, while tensile properties increased up to 3.8-fold. Crosslink density was found to increase with DHT temperature but not exposure period. Denaturation also increased with DHT temperature and exposure period, ranging from 25% to 60% denaturation. Crosslink density was found to be correlated with compressive modulus, whilst denaturation was found to correlate with tensile modulus. Taken together, these results indicate that DHT treatment is a viable technique for altering the mechanical properties of CG scaffolds. The enhanced mechanical properties of DHT-treated CG scaffolds improve their suitability for use both in vitro and in vivo. In addition, this work facilitates the investigation of the effects of mechanical properties and denaturation on cell activity in a 3D environment.

In this study, we examined the effects of varying collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. Three different collagen contents (0.25%, 0.5% and 1% collagen) and two different dehydrothermal (DHT) crosslinking processes [1] 105 degrees C for 24 h and [2] 150 degrees C for 48 h were investigated. These scaffolds were assessed for (1) pore size, (2) permeability (3) compressive strength and (4) cell viability. The largest pore size, permeability rate, compressive modulus, cell number and cell metabolic activity was all found to occur on the 1% collagen scaffold due to its increased collagen composition and the DHT treatment at 150 degrees C was found to significantly improve the mechanical properties and not to affect cellular number or metabolic activity. These results indicate that doubling the collagen content to 1% and dehydrothermally crosslinking the scaffold at 150 degrees C for 48 h has enhanced mechanical and biological properties of the scaffold making it highly attractive for use in bone tissue engineering.

Marrow stromal cell (MSC) populations, which are a potential source of undifferentiated mesenchymal cells, and culture scaffolds that mimic natural extracellular matrix are attractive options for orthopaedic tissue engineering. A type I collagen-glycosaminoglycan (CG) scaffold that has previously been used clinically for skin regeneration was recently shown to support expression of bone-associated proteins and mineralisation by MSCs cultured in the presence of osteogenic supplements. Here we follow RNA markers of osteogenic differentiation in this scaffold. We demonstrate that transcripts of the late stage markers bone sialoprotein and osteocalcin are present at higher levels in scaffold constructs than in two-dimensional culture, and that considerable gene induction can occur in this scaffold even in the absence of soluble osteogenic supplements. We also find that bone-related gene expression is affected by pore size, mechanical constraint, and uniaxial cyclic strain of the CG scaffold. The data presented here further establish the CG scaffold as a potentially valuable substrate for orthopaedic tissue engineering and for research on the mechanical interactions between cells and their environment, and suggest that a more freely-contracting scaffold with larger pore size may provide an environment more conducive to osteogenesis than constrained scaffolds with smaller pore sizes.