Cecilia Aulin

A Poly(Lactic Acid-Co-Caprolactone)–Collagen Hybrid for Tissue Engineering Applications

A biodegradable hybrid scaffold consisting of a synthetic polymer, poly(lactic acid-co-caprolactone) (PLACL), and a naturally derived polymer, collagen, was constructed by plastic compressing hyperhydrated collagen gels onto a flat warp-knitted PLACL mesh. The collagen compaction process was characterized, and it was found that the duration, rather than the applied load under the test conditions in the plastic compression, was the determining factor of the collagen and cell density in the cell-carrying component. Cells were spatially distributed in three different setups and statically cultured for a period of 7 days. Short-term biocompatibility of the hybrid construct was quantitatively assessed with AlamarBlue and qualitatively with fluorescence staining and confocal microscopy. No significant cell death was observed after the plastic compression of the interstitial equivalents, confirming previous reports of good cell viability retention. The interstitial, epithelial, and composite tissue equivalents showed no macroscopic signs of contraction and good cell proliferation with a two- to threefold increase in cell number over 7 days. Quantitative analysis showed a homogenous cell distribution and good biocompatibility. The results indicate that viable and proliferating multilayered tissue equivalents can be engineered using the PLACL-collagen hybrid construct in the space of several hours.

In situ cross-linkable hyaluronan hydrogel enhances chondrogenesis

The present work describes the feasibility of a cross‐linkable injectable hyaluronan hydrogel for cartilage repair. The hydrogel used is a two‐component system based on aldehyde‐modified hyaluronan and hydrazide‐modified polyvinyl alcohol, which are rapidly cross‐linked in situ upon mixing. The in vitro study showed that chondrocytes and mesenchymal cells cultured in the gel form cartilage‐like tissue, rich in glycosaminoglycans, collagen type II and aggrecan. In a rabbit animal model the injection of the hydrogel improved the healing of a full‐thickness cartilage defect created in the knee as compared to non‐treated controls. This rabbit study showed that the regenerated cartilage defects stained more intensely for type II collagen upon treatment with the hydrogel. The hyaluronan‐based hydrogel may be used as a delivery vehicle for both growth factors and/or cells for cartilage repair. The in vivo study also indicated that the hydrogel alone has a beneficial effect on cartilage regeneration. Copyright

Extracellular matrix–polymer hybrid materials produced in a pulsed-flow bioreactor system

Cell adhesion, interaction with material, cell proliferation and the production of an extracellular matrix (ECM) are all important factors determining the successful performance of an engineered scaffold. Scaffold design should aim at creating structures which can guide cells into forming new, functional tissue. In this study, the concept of in situ deposition of ECM by human dermal fibroblasts onto a compliant, knitted poly (ethyleneterephtalate) support is demonstrated, creating in vitro produced ECM polymer hybrid materials for tissue engineering. Comparison of cells cultured under static and dynamic conditions were examined, and the structure and morphology of the materials so formed were evaluated, along with the amount collagen deposited by the seeded cells. In vitro produced ECM polymer hybrid scaffolds could be created in this way, with the dynamic culture conditions increasing ECM deposition. Histological analysis indicated a homogenous distribution of cells in the 1 mm thick scaffold, surrounded by a matrix‐like structure. ECM deposition was observed throughout the materials wigh 81.6 µg/cm2 of collagen deposited after 6 weeks. Cell produced bundles of ECM fibres bridged the polymer filaments and anchored cells to the support. These findings open hereto unknown possibilities of producing materials with structure designed by engineering together with biochemical composition given by cells. Copyright

Bulk collagen incorporation rates into knitted stiff fibre polymer in tissue-engineered scaffolds: the rate-limiting step

Fabrication of tissue‐engineered constructs in vitro relies on sufficient synthesis of extracellular matrix (ECM) by cells to form a material suitable for normal function in vivo. Collagen synthesis by human dermal fibroblasts grown in vitro on two polymers, polyethylene terephthalate (PET) and polyglycolic acid (PGA), was measured by high‐performance liquid chromatography (HPLC). Cells were either cultured in a dynamic environment, where meshes were loaded onto a pulsing tube in a bioreactor, or in a static environment without pulsing. Collagen synthesis by cells cultured on a static mesh increased by six‐fold compared to monolayer culture, and increased by up to a further 5.4‐fold in a pulsed bioreactor. However, little of the collagen synthesized was deposited onto the meshes, almost all being lost to the medium. The amount of collagen deposited onto meshes was highest when cells were cultured dynamically on PET meshes (17.6 µg), but deposition still represented only 1.4% of the total synthesized. Although total collagen synthesis was increased by the use of 3D culture and the introduction of pulsing, the results suggest that the limiting factor for fabrication of a tissue‐engineered construct within practical timescales is not the amount of collagen synthesized but the quantity retained (i.e. deposited) within the construct during culture. This may be enhanced by systems which promote or assemble true 3D multi‐layers of cells. Copyright

USING cells as micro factories for ECM polymer hybrid material production

For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. In this study, we prepared crosslinked porous tubular structures from poly(trimethylene carbonate) (PTMC), in which smooth muscle cells (SMCs) were seeded and cultured in a pulsatile bioreactor mimicking the physiological conditions. PTMC was synthesized and porous tubular structures were prepared by dipping coating, cross-linking by g-irradiation, and leaching. SMCs were seeded into the porous structures by perfusion and then the constructs were cultured in a pulsatile bioreactor system. The morphologies, mechnical properties were analyzed and SMCs attachment and proliferation were evaluated by histology studies and CyQuant. Flexible tubular structures were obtained by dip coating with 3mm inner diameter and 1mm wall thickness. The porosity of the structures in wet state reached 85 vol% and the pore sizes were 60-150 mm. PTMC tubular structures showed comparable tensile strength and higher elongation compared with natural blood vessels. A pulsatile bioreactor system mimicking the conditions in vivo (dynamic pressure 70 mmHg, 75 beats/min) was successfully built. Experiements showed 7-day dilation was <10% and variation of diameter at each pulse was <1%. SMCs were homogeneously seeded in the porous scaffolds by perfusion. SMCs proliferate well to form confluent cell layer during a time period of up to 14 days, leading to constructs with even better mechanical performance. PTMC Porous tubular structures were prepared with good microstructures, elasticity and biocompatibility. SMCs were seeded and proliferated well in pulsatile bioreactor system and significant improvement of mechnical strength was observed.

Synthesis and surface activation of synthetic biodegradable polymers as support for cell produced ECM

Introduction: For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. The applicability of poly(trimethylene carbonate) (PTMC), random copolymers of TMC and e-caprolactone poly (TMC-CL), and networks based on these polymers as scaffolding materials was investigated. Methods: TMC-based (co)polymers were synthesized by ringopening polymerization. Tubular structures were prepared by dipping glass mandrels in polymer solutions containing dispersed, sieved sugar particles, followed by g-irradiation and cross-linking, and leaching. For mechanical- and biocompatibility tests, films of different thicknesses were prepared by compression molding, solvent casting, and spin-coating. Results and Discussion: PTMC and poly(TMC-CL) are flexible materials, with E-modulus values below 10 MPa and elongations at break higher than 500%. After g-irradiation in vacuo at 25– 100 kGy, networks with gel contents up to 73 wt% were obtained. The networks showed excellent creep resistance under static and dynamic loading conditions. Good cell attachment and proliferation behavior of mesenchymal stem cells, endothelial cells, and smooth muscle cells on polymer films and networks was observed. In lipase solutions, the films degraded substantially within one month by surface erosion. Porous tubular structures, with pore sizes in the range of 80 – 130 mm and a porosity of approximately 85%, could readily be prepared. A pulsatile bioreactor that allows mechanical stimulation of smooth muscle cells and endothelial cells seeded in the porous structures is being constructed. Conclusions: TMC-based (co)polymers and networks are flexible, elastic, biocompatible, and biodegradable. Porous tubular scaffolds based on these materials have much potential in tissue engineering of small diameter blood vessels.Our bodies are constantly exposed to different sorts of mechanical forces, from muscle tension to wound healing. Connective tissue adapts its extracellular matrix (ECM) to changes in mechanical load and the influence of mechanical stimulation on fibroblasts has been studied for a long time [1, 2]. When exposed to forces, fibroblasts are known to respond with expression and remodeling of ECM proteins, in particular collagen type I [3]. In this study the effect of dynamic culture conditions on human dermal fibroblasts was evaluated in terms of deposition and remodeling of ECM, with the aim of producing an ECM based scaffold. The fibroblasts were grown on compliant polymer supports either in a bioreactor with a pulsating flow or under static conditions. By applying dynamic culture conditions, the collagen deposition on the polymer supports increased fivefold. Scanning electron microscopy showed that polymer fibers were well integrated with cells and ECM and alignment along the polymer fibers was observed. Scaffold design should aim at creating structures that can help guiding the cells to form new, functional tissue. The presented system may present a new way of producing designed extracellular matrix based scaffolds for tissue engineering.Synthesis and surface activation of synthetic biodegradable polymers as support for cell produced ECMWe have previously demonstrated that porous poly-(epsiloncalprolactone) films with regularly spaced, controlled pore sizes provide adhesion and support for cultured dermal fibroblasts. We have determined the effects of applying various sized porous films (n¼3 for each treatment) on 4mm punch biopsy wounded mice to assess wounding response. Films with pores ranging in size from 3–20 microns, elicited a mild lymphocytic and foreign body perifollicular immune response, regardless of pore size but this treatment failed to significantly shorten wound healing time or increase the rate of wound closure. By 21 days after wounding the grafted porous films had become fully incorporated into or completely biodegraded in the wounded tissue. Finally, we assessed the proof of principle that live cultured fibroblasts can be delivered using porous films and sustained in model SCID mouse wounds. Human fibroblasts (30,000 cells) were subconfluently cultured on 5 micron porous films. These cell/film combinations were then transplanted onto wounded mice but failed to significantly affect wound healing. However, these transplanted fibroblast cells were readily detected using anti-human HLA antibodies in wounded SCID mice skin 21 days after treatment, when the wounds had completely healed. Taken together, these data demonstrate for the first time the feasibility of using porous films to deliver living human cells into skin wounds as part of our aim to use cell therapy to improve the wound healing response.The aim of this work is to develop an artificial artery for use in bypass surgery. The hybrid artery consists of a porous tubular scaffold made from a polyurethane elastomer. The surface of the polymer is then modified with recombinant proteins in order to encourage the growth of organised layers of vascular cells.

Designing Extracellular Matrix Scaffolds by Dynamic culture of fibroblasts

Introduction: For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. The applicability of poly(trimethylene carbonate) (PTMC), random copolymers of TMC and e-caprolactone poly (TMC-CL), and networks based on these polymers as scaffolding materials was investigated. Methods: TMC-based (co)polymers were synthesized by ringopening polymerization. Tubular structures were prepared by dipping glass mandrels in polymer solutions containing dispersed, sieved sugar particles, followed by g-irradiation and cross-linking, and leaching. For mechanical- and biocompatibility tests, films of different thicknesses were prepared by compression molding, solvent casting, and spin-coating. Results and Discussion: PTMC and poly(TMC-CL) are flexible materials, with E-modulus values below 10 MPa and elongations at break higher than 500%. After g-irradiation in vacuo at 25– 100 kGy, networks with gel contents up to 73 wt% were obtained. The networks showed excellent creep resistance under static and dynamic loading conditions. Good cell attachment and proliferation behavior of mesenchymal stem cells, endothelial cells, and smooth muscle cells on polymer films and networks was observed. In lipase solutions, the films degraded substantially within one month by surface erosion. Porous tubular structures, with pore sizes in the range of 80 – 130 mm and a porosity of approximately 85%, could readily be prepared. A pulsatile bioreactor that allows mechanical stimulation of smooth muscle cells and endothelial cells seeded in the porous structures is being constructed. Conclusions: TMC-based (co)polymers and networks are flexible, elastic, biocompatible, and biodegradable. Porous tubular scaffolds based on these materials have much potential in tissue engineering of small diameter blood vessels.Our bodies are constantly exposed to different sorts of mechanical forces, from muscle tension to wound healing. Connective tissue adapts its extracellular matrix (ECM) to changes in mechanical load and the influence of mechanical stimulation on fibroblasts has been studied for a long time [1, 2]. When exposed to forces, fibroblasts are known to respond with expression and remodeling of ECM proteins, in particular collagen type I [3]. In this study the effect of dynamic culture conditions on human dermal fibroblasts was evaluated in terms of deposition and remodeling of ECM, with the aim of producing an ECM based scaffold. The fibroblasts were grown on compliant polymer supports either in a bioreactor with a pulsating flow or under static conditions. By applying dynamic culture conditions, the collagen deposition on the polymer supports increased fivefold. Scanning electron microscopy showed that polymer fibers were well integrated with cells and ECM and alignment along the polymer fibers was observed. Scaffold design should aim at creating structures that can help guiding the cells to form new, functional tissue. The presented system may present a new way of producing designed extracellular matrix based scaffolds for tissue engineering.Synthesis and surface activation of synthetic biodegradable polymers as support for cell produced ECMWe have previously demonstrated that porous poly-(epsiloncalprolactone) films with regularly spaced, controlled pore sizes provide adhesion and support for cultured dermal fibroblasts. We have determined the effects of applying various sized porous films (n¼3 for each treatment) on 4mm punch biopsy wounded mice to assess wounding response. Films with pores ranging in size from 3–20 microns, elicited a mild lymphocytic and foreign body perifollicular immune response, regardless of pore size but this treatment failed to significantly shorten wound healing time or increase the rate of wound closure. By 21 days after wounding the grafted porous films had become fully incorporated into or completely biodegraded in the wounded tissue. Finally, we assessed the proof of principle that live cultured fibroblasts can be delivered using porous films and sustained in model SCID mouse wounds. Human fibroblasts (30,000 cells) were subconfluently cultured on 5 micron porous films. These cell/film combinations were then transplanted onto wounded mice but failed to significantly affect wound healing. However, these transplanted fibroblast cells were readily detected using anti-human HLA antibodies in wounded SCID mice skin 21 days after treatment, when the wounds had completely healed. Taken together, these data demonstrate for the first time the feasibility of using porous films to deliver living human cells into skin wounds as part of our aim to use cell therapy to improve the wound healing response.The aim of this work is to develop an artificial artery for use in bypass surgery. The hybrid artery consists of a porous tubular scaffold made from a polyurethane elastomer. The surface of the polymer is then modified with recombinant proteins in order to encourage the growth of organised layers of vascular cells.