Cell-free carrier system for localized delivery of peripheral blood cell-derived engineered factor signaling: towards development of a one-step device for autologous angiogenic therapy.

Spatiotemporally-controlled delivery of hypoxia-induced angiogenic factor mixtures has been identified by this group as a promising strategy for overcoming the limited ability of chronically ischemic tissues to generate adaptive angiogenesis. We previously developed an implantable, as well as an injectable system for delivering fibroblast-produced factors in vivo. Here, we identify peripheral blood cells (PBCs) as the ideal factor-providing candidates, due to their autologous nature, ease of harvest and ample supply, and investigate wound-simulating biochemical and biophysical environmental parameters that can be controlled to optimize PBC angiogenic activity. It was found that hypoxia (3% O2) significantly affected the expression of a range of angiogenesis-related factors including VEGF, angiogenin and thrombospondin-1, relative to the normoxic baseline. While all three factors underwent down-regulation over time under hypoxia, there was significant variation in the temporal profile of their expression. VEGF expression was also found to be dependent on cell-scaffold material composition, with fibrin stimulating production the most, followed by collagen and polystyrene. Cell-scaffold matrix stiffness was an additional important factor, as shown by higher VEGF protein levels when PBCs were cultured on stiff vs. compliant collagen hydrogel scaffolds. Engineered PBC-derived factor mixtures could be harvested within cell-free gel and microsphere carriers. The angiogenic effectiveness of factor-loaded carriers could be demonstrated by the ability of their releasates to induce endothelial cell tubule formation and directional migration in in vitro Matrigel assays, and microvessel sprouting in the aortic ring assay. To aid the clinical translation of this approach, we propose a device design that integrates this system, and enables one-step harvesting and delivering of angiogenic factor protein mixtures from autologous peripheral blood. This will facilitate the controlled release of these factors both at the bed-side, as an angiogenic therapy in wounds and peripheral ischemic tissue, as well as pre-, intra- and post-operatively as angiogenic support for central ischemic tissue, grafts, flaps and tissue engineered implants.

Injectable system for spatio-temporally controlled delivery of hypoxia-induced angiogenic signalling.

While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo, namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O2). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5 °C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37 °C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol-gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3 months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.

Engineering stable topography in dense bio-mimetic 3D collagen scaffolds.

Topographic features are well known to influence cell behaviour and can provide a powerful tool for engineering complex, functional tissues. This study aimed to investigate the mechanisms of formation of a stable micro-topography on plastic compressed (PC) collagen gels. The uni-directional fluid flow that accompanies PC of collagen gels creates a fluid leaving surface (FLS) and a non-fluid leaving surface (non-FLS). Here we tested the hypothesis that the resulting anisotropy in collagen density and stiffness between FLS and non-FLS would influence the fidelity and stability of micro-grooves patterned on these surfaces. A pattern template of parallel-aligned glass fibres was introduced to the FLS or non-FLS either at the start of the compression or halfway through, when a dense FLS had already formed. Results showed that both early and late patterning of the FLS generated grooves that had depth (25 ±7 µm and 19 ±8 µm, respectively) and width (55 ±11 µm and 50 ±12 µm, respectively) which matched the glass fibre diameter (50 µm). In contrast, early and late patterning of the non-FLS gave much wider (151 ±50 µm and 89 ±14 µm, respectively) and shallower (10 ±2.7 µm and 13 ±3.5 µm, respectively) grooves than expected. The depth to width ratio of the grooves generated on the FLS remained unaltered under static culture conditions over 2 weeks, indicating that grooves were stable under long term active cell-mediated matrix remodelling. These results indicate that the FLS, characterised by a higher matrix collagen density and stiffness than the non-FLS, provides the most favourable mechanical surface for precise engineering of a stable micro-topography in 3D collagen hydrogel scaffolds.

Mechanisms of structure generation during plastic compression of nanofibrillar collagen hydrogel scaffolds: towards engineering of collagen.

Operator control of cell/matrix density of plastically compressed collagen hydrogel scaffolds critically depends on reproducibly limiting the extent of scaffold compaction, as fluid expulsion. A functional model of the compression process is presented, based on the idea that the main fluid-leaving surface (FLS) behaves as an ultrafiltration membrane, allowing fluid (water) out but retaining collagen fibrils to form a cake. We hypothesize that accumulation of collagen at the FLS produces anisotropic structuring but also increases FLS hydraulic resistance (R(FLS) ), in turn limiting the flux. Our findings show that while compressive load is the primary determinant of flux at the beginning of compression (load-dependent phase), increasing FLS collagen density (measured by X-ray attenuation) and increasing R(FLS) become the key determinants of flux as the process proceeds (flow-dependent phase). The model integrates these two phases and can closely predict fluid loss over time for a range of compressive loads. This model provides a useful tool for engineering cell and matrix density to tissue-specific levels, as well as generating localized 3D nano micro-scale structures and zonal heterogeneity within scaffolds. Such structure generation is important for complex tissue engineering and forms the basis for process automation and up-scaling.

First implantable device for hypoxia-mediated angiogenic induction.

Delayed or inadequate vascularisation is one of the major factors leading to tissue infarction and poor graft survival. Current vascularisation strategies that rely on delivering single growth factors have proved ineffective or hard to control in practise. An alternative approach has been identified by this group that relies on stimulation of physiological angiogenic factor cascades by engineering local cell-hypoxia, within a nano-fibrillar collagen material. Here we report on a novel, practical and effective implantable device for delivering engineered angiogenic signalling, on demand. Human dermal fibroblast-seeded dense-collagen depots were pre-conditioned under physiological cell-generated hypoxia to up-regulate production of key angiogenic factors, including HIF1α and VEGF(165). The level of VEGF(165) protein retained within depots (indicating general angiogenic factor production) was directly correlated to the duration of pre-conditioning. Angiogenic factor delivery from pre-conditioned, non-viable depots rapidly induced an angiogenic response within endothelial cell-seeded constructs in vitro, while implanted acellular 3D constructs incorporating such angiogenic depots in their core were infiltrated with perfused vessels by 1 week in vivo, at which stage non-angiogenic implants were minimally perfused. Depot stability, tuneability of cell/matrix composition with long clinical experience of the collagen material, together with cost effectiveness, make this angiogenic therapy a promising addition to a clinician's tool kit for improving local tissue perfusion.

Controlling physiological angiogenesis by hypoxia-induced signaling.

The full sequence of signals leading to new blood vessel formation is a physiological response to tissue hypoxia through upregulation of angiogenic factor cascades. Controlled initiation of this mechanism for therapeutic/engineered angiogenesis must rely on precisely localized hypoxia. Here we have designed a 3D in vitro model able to test the effect and predictability of spatially positioned local hypoxic stimuli using defined cell depots within a 3D collagen matrix. Cell-mediated hypoxia was engineered using human dermal fibroblasts (HDFs), to generate a local population of Hypoxia-Induced Signaling (HIS) cells. HIS cell depots released angiogenic factors which induced directional endothelial cell (EC) migration and tubule formation in a spatially defined assay system. Non-hypoxic baseline control cultures induced minimal EC migration with little tubule formation. Furthermore, depots of HIS cells, positioned in the core of 3D collagen constructs directed host vessel in-growth deep into the implant by 1 week, which was at least 7 days earlier than in non-hypoxia pre-conditioned constructs. The functionality of in vivo vascularisation was verified by real-time monitoring of O2 levels in the core of implanted constructs. These findings establish the angiogenic potential of HIS cells applicable to in vitro tissue modeling, implant vascularization and engineering predictable angiogenic therapies.

Interface integration of layered collagen scaffolds with defined matrix stiffness: implications for sheet-based tissue engineering.

Successful application of sheet-based engineering for complex tissue reconstruction requires optimal integration of construct components. An important regulator of cellular responses (such as migration and collagen deposition) mediating interface integration is matrix stiffness. In this study we developed a sheet-based 3D model of interface integration that allows control of interface matrix stiffness. Fluid was removed from acellular or fibroblast-seeded bilayer collagen hydrogel constructs, using plastic compression to increase collagen density and matrix stiffness. Cell-seeded constructs were either compressed at day 0 and cultured for 7 days (compressed culture, high stiffness) or left uncompressed during culture and compressed on day 7 (compliant-compressed culture, low stiffness). Constructs were fitted onto a mechanical testing system to measure interface adhesive strength. Analysis of stresses by finite element modelling predicted a sharp rise of stress and rapid failure at the interface. While cell-seeded constructs showed a six-fold increase in interface adhesive strength compared to acellular control constructs (p < 0.05), there was no significant difference between low- and high-stiffness cultures after 1 week. Cell migration across the interface was greater in low- compared to high-stiffness constructs at 24 h (p < 0.05); however, no significant difference was observed after 1 week. Visualization of interfaces showed fusion of the two layers in low- but not in high-stiffness constructs after 1 week of culture. The ability to regulate cellular behaviour at an interface by controlling matrix stiffness could provide an important tool for modelling the integration of sheet-based bioengineered tissues in bioreactor culture or post-implantation.

Close dependence of fibroblast proliferation on collagen scaffold matrix stiffness.

Human dermal fibroblasts (HDFs) in free-floating collagen matrices show minimal proliferation, although this may increase when the matrix is 'under tension'. We have investigated the detailed mechanics underlying one of the possible controls of this important cell behaviour, in particular the hypothesis that this is a response to substrate stiffness. Hyperhydrated collagen gels were plastic-compressed (PC) to give a predetermined collagen density and stiffness. Mechanical properties were tested using a dynamic mechanical analyser; cell number by Alamar blue assay. In the stiffest PC matrices, cell proliferation was rapid and seeding density-dependent, with a population doubling time of 2 days. In contrast, compliant attached matrices showed a 4 day lag period and a doubling time of 6 days. HDF growth was directly related to matrix stiffness, such that increasing stiffness using a range of compression levels (0-75% fluid removal) supported increasing proliferation rate, doubling times and matrix elastic modulus. HDF quiescence in compliant matrices was reversible, such that increasing stiffness in situ by compression at 1 and 5 days initiated proliferation. We conclude that collagen matrix stiffness regulates proliferation of fibroblasts (a duro-response), with important implications for understanding fibroblast-matrix feedback controls during wound healing and the design and regulation of engineered connective tissues based on collagen and other hydrogel-based scaffolds.