Biomedical subjects

K M Shakesheff

Publications and source records attributed to K M Shakesheff.

At least 19 recordsLinked to original sources

PubMed2006

Supercritical carbon dioxide generated vascular endothelial growth factor encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro.

The ability to deliver, over time, biologically active vascular endothelial growth factor-165 (VEGF) through tailored designed scaffolds offers tremendous therapeutic opportunities to tissue-engineered therapies. Porous biodegradable poly(DL-lactic) acid (PLA) scaffolds encapsulating VEGF have been generated using supercritical CO2 (scCO2) and the kinetic release and angiogenic activity of these scaffolds examined in vitro and in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model. After processing through scCO2, VEGF maintained its angiogenic activity as assessed by increased tubule formation of human umbilical vein endothelial cells (HUVEC) cultured on Matrigel (VEGF = 1937 +/- 205 microm; scCO2-VEGF = 2085 +/- 234 microm; control = 1237 +/- 179 microm). VEGF release kinetics from scCO2-VEGF incorporated PLA monolith scaffolds showed a cumulative release of VEGF (2837 +/- 761 rhog/ml) over a 21 day period in culture. In addition, VEGF encapsulated PLA scaffolds increased the blood vessel network in the CAM compared to controls; control, 24.8 +/- 9.6; VEGF/PLA, 44.1 +/- 12.1 (vessels/field). These studies demonstrate that the controlled release of growth factors encapsulated into three-dimensional PLA scaffolds can actively stimulate the rapid development of therapeutic neovascularisation to regenerate or engineer tissues.

Animals↗

PubMed2006

The effect of anisotropic architecture on cell and tissue infiltration into tissue engineering scaffolds.

A common phenomenon in tissue engineering is rapid tissue formation on the outer edge of the scaffold which restricts cell penetration and nutrient exchange to the scaffold centre, resulting in a necrotic core. To address this problem, we generated scaffolds with both random and anisotropic open porous architectures to enhance cell and subsequent tissue infiltration throughout the scaffold for applications in bone and cartilage engineering. Hydroxyapatite (HA) and poly(D,L-lactic acid) (P(DL)LA) scaffolds with random open porosity were manufactured, using modified slip-casting and by supercritical fluid processing respectively, and subsequently characterised. An array of porous aligned channels (400 microm) was incorporated into both scaffold types and cell (human osteoblast sarcoma, for HA scaffolds; ovine meniscal fibrochondrocytes, for P(DL)LA scaffolds) and tissue infiltration into these modified scaffolds was assessed in vitro (cell penetration) and in vivo (tissue infiltration; HA scaffolds only). Scaffolds were shown to have an extensive random, open porous structure with an average porosity of 85%. Enhanced cell and tissue penetration was observed both in vitro and in vivo demonstrating that scaffold design alone can influence cell and tissue infiltration into the centre of tissue engineering scaffolds.

Animals↗

PubMed2006

Tissue growth in a rotating bioreactor. Part I: mechanical stability.

We develop mathematical models to provide insights into the morphology of a tissue construct formed from a single-cell suspension in culture media, within a rotating bioreactor. The bioreactor consists of a cylindrical vessel of circular cross-section rotating about its longitudinal axis with constant angular speed. Experimental studies show that at rotation rates below a critical value, the cells 'self-assemble' to form smooth 'nodules' that are approximately cylindrical with elliptical cross-section; however, at rotation rates above a critical value, an amorphous construct forms with a highly irregular boundary. The construct is denser than the surrounding culture media and histological studies indicate that the interior of the construct, which is a mix of apoptotic cells and culture media, is surrounded by an outer rim of proliferating cells and collagen. The construct is modelled as a viscous fluid drop surrounded by an extensible membrane in a (less dense) immiscible viscous fluid within a rotating bioreactor. We consider both thin-disk and slender-pipe bioreactors for which the aspect ratio, L(*)/a(*) (where L(*) and a(*) are the bioreactor length and radius, respectively), is small and large, respectively, and obtain a series of spatially 2D problems (independent of the axial coordinate). We then examine the hypothesis that the construct morphology is a result of the mechanical forces that it experiences by considering the interfacial stability of an initially circular fluid-fluid interface to small-amplitude, oscillatory perturbations. The instability is driven by the density difference between the two fluids, and we investigate the effect of the rotation rate, the (time-dependent) gravitational field, and the material and geometrical properties of the system on the stability properties.

Algorithms↗

PubMed2006

Hepatic stellate cells on poly(DL-lactic acid) surfaces control the formation of 3D hepatocyte co-culture aggregates in vitro.

Evidence for the functional superiority of cells cultured as 3D aggregates or on 3D scaffolds over conventional 2D monolayer cultures has created interest in material and cell based methods that influence the formation and structure of multicellular aggregates in vitro. We have created a co-culture of primary rat hepatocytes and hepatic stellate cells on a poly(DL-lactic acid) surface, a poor substrate for rat hepatocyte adhesion, to study the dynamics of multicellular spheroid formation and the resultant cell arrangement. The poly(DL-lactic acid) surface allows dynamic and rapid interaction of hepatocytes and stellate cells to form co-culture spheroids in a complex multistage process (shown by time lapse microscopy). This spheroid morphology supports enhanced cell viability relative to a mono-culture mono-layer system (measured by lactate dehydrogenase leakage). The distribution of the aggregating cell type in the final structure is related to the mechanics of formation i.e. mainly central and peripheral. This study provides a unique and generically applicable insight into the dynamics of multicellular spheroid formation where aggregation is induced by one cell type and imposed on another. This has implications for 3D cell culture models and a wide number of currently used stromal co-culture systems.

Animals↗

PubMed2005

The influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering.

There is a clinical need for synthetic scaffolds that will promote bone regeneration. Important factors include obtaining an optimal porosity and size of interconnecting windows whilst maintaining scaffold mechanical strength, enabling complete penetration of cells and nutrients throughout the scaffold, preventing the formation of necrotic tissue in the centre of the scaffold. To address this we investigated varying slip deflocculation in order to control the resulting porosity, pore size and interconnecting window size whilst maintaining mechanical strength. Hydroxyapatite (HA) porous ceramics were prepared using a modified slip casting process. Rheological measurements of the HA slips were used to identify deflocculation conditions which resulted in changes in the cell and window sizes of the resulting ceramics. Sintered ceramics were characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Pore and window size distribution was determined by SEM. XRD analysis confirmed that the crystal structure remained HA after the sintering process. SEM showed that HA porous ceramics presented a highly interconnected porous network with average pore sizes ranging from 391+/-39 to 495+/-25 microm. The average window size varied from 73+/-5 to 135+/-7 microm. Pore diameters obtained were controllable in the range 200-500 microm. Window sizes were in the range 30-250 microm. The use of dispersant concentration allows pore and window size to be modified whilst maintaining control over porosity demonstrated by a porosity of 85% for seven different dispersant concentrations. The advantage of this approach allows the correlation between the rheological conditions of the slip and the resultant sintered ceramic properties. In particular, optimising the ceramic strength by controlling the agglomeration during the casting process.

Bone Substitutes↗

PubMed2003

Seeding cells into needled felt scaffolds for tissue engineering applications.

Tissue engineering methods are under development that will enable the repair or replacement of a variety of tissues, including articular cartilage and bone. To engineer functional tissue it is necessary that scaffolds initially be seeded with a large number of cells distributed evenly throughout the scaffold structure. It previously has been shown that, compared to static seeding conditions, seeding scaffolds under dynamic conditions facilitates high seeding densities and even distributions of cells (Li et al., Biotechnology Progress 2001;17:935-944). The efficiency of seeding HOSTE85 cells and bovine chondrocytes into needled felt scaffolds following agitation at different speeds was determined. Seeding efficiency was determined using the Hoechst 33258 assay, and cell viability was assessed using the Alamar Blue trade mark assay. The distribution of cells within the scaffolds was imaged using scanning electron microscopy. It was found that the optimum seeding conditions varied for HOSTE85 cells and bovine chondrocytes, with different agitation speeds leading to different seeding efficiencies, cell viabilities, and distributions of cells within scaffolds. The optimum agitation speeds for seeding a high number of viable cells into scaffolds so that they were arranged evenly were 300 rpm for HOSTE85 cells and 200 rpm for bovine chondrocytes.

Animals↗

PubMed2003

Surface engineering of living myoblasts via selective periodate oxidation.

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.

Aldehydes↗

PubMed2003

Maggots and wound healing: an investigation of the effects of secretions from Lucilia sericata larvae upon interactions between human dermal fibroblasts and extracellular matrix components.

BACKGROUND: Through clinical observation, Lucilia sericata (greenbottle fly) larvae are credited with exerting the following beneficial effects upon a chronic nonhealing wound: removal of necrotic tissue ('debridement'), disinfection of the wound and active promotion of granulation tissue formation. As a major cellular component of granulation tissue, fibroblasts play an extensive role in healing. The composition of extracellular matrix (ECM) located in the wound is another important factor, partaking in a dynamic feedback loop with the fibroblasts that produce it. Fibroblast-ECM interactions therefore exert considerable influence upon new tissue formation. OBJECTIVES: To investigate the effects of L. sericata larval excretory/secretory products (ES) upon the behaviour of fibroblasts, seeded upon ECM component surfaces. METHODS: ES were collected by washing freshly hatched larvae in phosphate-buffered saline. Human dermal neonatal fibroblast cells were seeded upon fibronectin- or collagen-coated surfaces, together with untreated (or 'native') ES, heat-treated ES, or no ES (ES blank). Following incubation, fibroblast adhesion was determined using an adenosine triphosphate assay and, for confirmation, a total nucleic acid content assay. Cell spreading was observed using microscopy. The effect of ES upon fibronectin structure was observed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Peptide sequencing of suspected fibronectin fragments was performed using an 'electrospray' type time of flight mass spectrometer and 'Peptident' database search. RESULTS: ES significantly reduced fibroblast adhesion to both fibronectin and, to a lesser extent, collagen. Cell spreading was also reduced, yet cells remained viable. For both spreading and adhesion, heat-treated ES exerted significantly less activity than native, untreated ES. However, they still exhibited significant activity when compared with the ES blank. ES appeared to modify fibroblast adhesion indirectly via proteolytic fragmentation of the fibronectin protein surface. CONCLUSIONS: L. sericata larval secretions modify fibroblast adhesion and spreading across ECM protein surfaces, while keeping cells viable. Proteolytic activity of the ES played a significant role. If transferred to the wound situation, such alteration of fibroblast-ECM interactions may enhance new tissue formation.

Amino Acid Sequence↗

PubMed2003

Novel osteoinductive biomimetic scaffolds stimulate human osteoprogenitor activity--implications for skeletal repair.

The development of new bone formation strategies offers tremendous therapeutic implications in a variety of musculoskeletal diseases. One approach involves harnessing the regenerative capacity of osteoprogenitor bone cells in combination with biomimetic scaffolds generated from appropriate scaffold matrices and osteoinductive factors. The aims of our study were to test the efficacy of two innovative osteoinductive agents: the osteoblast stimulating factor-1 (osf-1), an extracellular matrix-associated protein, and osteoinductive extracts of Saos-2 cells on human osteoprogenitor cells. Saos-2 extracted osteoinductive factors significantly stimulated alkaline phosphatase specific activity in basal and osteogenic conditions. Osf-1 significantly stimulated chemotaxis, total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase specific activity at concentrations as low as 10 pg/ml compared with control cultures. Osteoinductive factors present in Saos-2 cell extracts and osf-1 promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on 3-D scaffolds. The successful generation of 3-D biomimetic structures incorporating osf-1 or osteoinductive factors from Saos-2 cells indicates their potential for de novo bone formation that exploits cell-matrix interactions.

Aged↗

PubMed2002

Interactions of 3T3 fibroblasts and endothelial cells with defined pore features.

The colonization of biodegradable polymer scaffolds with cell populations has been established as the foundation for the engineering of a number of tissues, including cartilage, liver, and bone. Within these scaffolds, the cells encounter a porous environment in which they must migrate across the convoluted polymer surface to generate a homogenous cell distribution. Predicting the interactions between cells and pores is important if scaffold characteristics are to be optimized. Therefore, we investigated the behavior of two model cell types over a range of defined pore features. These pore features range from 5 to 90 microm in diameter and have been fabricated by photolithographic techniques. Quantitatively, the behavior of the cells is dependent on three factors: 1) percentage cell coverage of the surface; 2) pore size; and 3) cell type. Fibroblast cells displayed a co-operative pattern of cell spreading in which pores with diameters greater than the cell dimensions were bridged by groups of cells using their neighbors as supports. Endothelial cells were unable to use neighbors as support structures and failed to bridge pores greater than the cell diameter.

3T3 Cells↗

PubMed2002

Recent advances in tissue engineering: an invited review.

Tissue formation within the body, as part of a development or repair process, is a complex event in which cell populations self-assemble into functional units. There is intense academic, medical, and commercial interest in finding methods of replicating these events outside the body. This interest has accelerated with the demonstration of the engineering of skin and cartilage tissue in the laboratory and there is now worldwide activity in the in vitro regeneration of tissues including nerve, liver, bone, heart valves, blood vessels, bladder, and kidney. Approaches to tissue engineering center on the need to provide signals to cell populations to promote cell proliferation and differentiation. This review considers recent advances in methods of providing these signals to cells using examples of progress in the engineering of complex tissues.

Absorbable Implants↗

PubMed2001

A comparison of the adhesion of mammalian cells and Staphylococcus epidermidis on fibronectin-modified polymer surfaces.

The modification of polymer surfaces to mimic the extracellular matrix, and therefore, stimulate cell growth via receptor-mediated interactions, is one approach used to promote the integration of tissue-engineering scaffolds and biomaterials into the body. However, by optimizing surfaces for tissue integration it is likely that bacterial adhesion may also be affected, resulting in a greater risk of biomaterial-related infection. This could be detrimental to both the implant and the patient because biomaterial related infections are particularly resistant to host defenses and antibiotics. In this study, we analyzed the adhesion of a Staphylococcus epidermidis clinical isolate and 3T3 rat fibroblasts to tissue culture plastic coated with varying concentrations of fibronectin (Fn). Bacterial adhesion was always lower than tissue culture plastic and appeared to decrease with increasing Fn concentrations. Mammalian cell adhesion to Fn exceeded adhesion to tissue culture plastic but did not differ significantly over the range of protein concentrations or between 1 and 4 h of incubation. In most cases, the total surface coverage did not vary with time or Fn concentration, indicating that maximal cell adhesion and spreading occurred rapidly and at low protein concentrations. This study suggests that, by controlling the density of proteins or ligands on a surface, we can potentially optimize mammalian cell adhesion without stimulating bacterial adhesion, hence reducing the likelihood of infection.

3T3 Cells↗

PubMed2001

Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid).

The immobilization of adhesion peptide sequences (such as RGD) at the surfaces of poly(alpha-hydroxyacid)s, including poly(lactic acid) (PLA), is complicated by an absence of functional groups to support covalent attachment. We demonstrate a method to overcome this problem, by attaching the peptide to poly(L-lysine) (PLL), which immobilizes the sequence through adsorption at the poly(alpha-hydroxyacid) surface. When coated using a 0.01% w/v solution of PLL-GRGDS, bovine aortic endothelial cells seeded upon the modified PLA showed a marked increase in spreading over unmodified PLA. However, inhibition of the cell-spreading effect occurred when using higher concentrations of PLL-GRGDS, which we attribute to the PLL component. This inhibitory effect can be challenged by increasing the amount of GRGDS attached to each PLL molecule. Potentially, this is a flexible method of surface modification that can engineer many different types of tissue engineering scaffolds with a variety of biomolecules, thus allowing initial cell adhesion to be controlled.

Animals↗

PubMed2001

Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification.

The ability to generate new bone for skeletal use is a major clinical need. Biomimetic scaffolds that interact and promote osteoblast differentiation and osteogenesis offer a promising approach to the generation of skeletal tissue to resolve this major health-care issue. In this study we examine the ability of surface-modified poly(lactic acid) (PLA) films and poly(lactic-co-/glycolic acid) (PLGA) (75:25) porous structures to promote human osteoprogenitor adhesion, spreading, growth, and differentiation. Cell spreading and adhesion were examined using Cell Tracker green fluorescence and confocal microscopy. Osteogenic differentiation was confirmed with alkaline phosphatase activity as well as immunocytochemistry for type I collagen, core binding factor-1 (Cbfa-1), and osteocalcin. Poor cell growth was observed on nonmodified PLA films and PLGA scaffolds. The polymers were then coupled with RGD peptides [using poly(L-lysine), or PLL] and physical adsorption as well as PLA films presenting adsorbed fibronectin (FN). Both modifications enhanced cell attachment and spreading. On PLA-FN and PLA-PLL-GRGDS films, the osteoblast response was dose dependent (20 pmol/L to 0.2 micromol/L FN and 30 nmol/L to 30 micromol/L PLL-GRGDS) and significant at concentrations as low as 2 nmol/L FN and 30 nmol/L PLL-GRGDS. With optimal concentrations of FN or RGD, adhesion and cell spreading were comparable to tissue culture plastic serum controls. In PLGA (75:25) biodegradable porous scaffolds, coated with FN, PLL-GRGDS, or fetal calf serum for 24 h in alpha MEM alone, prior to growth in dexamethasone and ascorbate-2-phosphate for 4-6 weeks, extensive osteoblast impregnation was observed by confocal and fluorescence microscopy. Cell viability in extended culture was maintained as analyzed by expression of Cell Tracker green and negligible ethidium homodimer-1 (a marker of cell necrosis) staining. Alkaline phosphatase activity, type I collagen, Cbfa-1, and osteocalcin expression were observed by immunocytochemistry. Mineralization of collagenous matrix took place after 4 weeks, which confirmed the expression of the mature osteogenic phenotype. These observations demonstrate successful adhesion and growth of human osteoprogenitors on protein- and peptide-coupled polymer films as well as migration, expansion, and differentiation on three-dimensional biodegradable PLGA scaffolds. The use of peptides/proteins and three-dimensional structures that provide positional and environmental information indicate the potential for biomimetic structures coupled with appropriate factors in the development of protocols for de novo bone formation.

Aged↗

PubMed2001

Synthesis and characterisation of a degradable poly(lactic acid)-poly(ethylene glycol) copolymer with biotinylated end groups.

Poly(lactic acid)-poly(ethylene glycol)-biotin (PLA-PEG-biotin) is a degradable polymer with protein resistant properties that can undergo rapid surface engineering in aqueous media to create biomimetic surfaces. Surface engineering of this polymer is dependent on biomolecular interactions between the biotin end group and the protein avidin. Given the vigorous conditions of synthesis, it is essential that the manufacture of the polymer does not alter the biotin structure or its molecular recognition. Equally, it is important that the incorporation of biotin does not adversely affect the physicochemical properties of the polymer. (1)H NMR provides evidence of biotin attachment and structural integrity. (1)H NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analysis shows there is no significant effect on bulk properties induced by the biotin end group. Surface plasmon resonance (SPR) and fluorescent spectroscopy studies using the 2-(4'-hydroxyazobenzene) benzoic acid (HABA)/avidin complex show that the biotin moieties binding capabilities are not impaired by the synthesis.

Avidin↗

PubMed2001

Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability.

A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

3T3 Cells↗

PubMed2001

Growth factor release from tissue engineering scaffolds.

Synthetic scaffold materials are used in tissue engineering for a variety of applications, including physical supports for the creation of functional tissues, protective gels to aid in wound healing and to encapsulate cells for localized hormone-delivery therapies. In order to encourage successful tissue growth, these scaffold materials must incorporate vital growth factors that are released to control their development. A major challenge lies in the requirement for these growth factor delivery mechanisms to mimic the in-vivo release profiles of factors produced during natural tissue morphogenesis or repair. This review highlights some of the major strategies for creating scaffold constructs reported thus far, along with the approaches taken to incorporate growth factors within the materials and the benefits of combining tissue engineering and drug delivery expertise.

Bioartificial Organs↗

PubMed2000

Surface plasmon resonance analysis of dynamic biological interactions with biomaterials.

Surface plasmon resonance (SPR) is an optical technique that is widely gaining recognition as a valuable tool to investigate biological interactions. SPR offers real time in situ analysis of dynamic surface events and, thus, is capable of defining rates of adsorption and desorption for a range of surface interactions. In this review we highlight the diversity of SPR analysis. Examples of a wide range of applications of SPR are presented, concentrating on work relevant to the analysis of biomaterials. Particular emphasis is given to the use of SPR as a complimentary tool, showing the broad range of techniques that are routinely used alongside SPR analysis.

Animals↗