High-resolution elasticity imaging for tissue engineering

An elasticity microscope provides high resolution images of tissue elasticity. With this instrument, it may be possible to monitor cell growth and tissue development in tissue engineering. To test this hypothesis, elasticity micrographs were obtained in two model systems commonly used for tissue engineering. In the first, strain images of a tissue-engineered smooth muscle sample clearly identified a several hundred micron thick cell layer from its supporting matrix. Because a one-dimensional mechanical model was appropriate for this system, strain images alone were sufficient to image the elastic properties. In contrast, a second system was investigated in which a simple one-dimensional mechanical model was inadequate. Uncultured collagen microspheres embedded in an otherwise homogeneous gel were imaged with the elasticity microscope. Strain images alone did not clearly depict the elastic properties of the hard spherical cell carriers. However, reconstructed elasticity images could differentiate the hard inclusion from the background gel. These results strongly suggest that the elasticity microscope may be a valuable tool for tissue engineering and other applications requiring the elastic properties of soft tissue at high spatial resolution (75 mum or less).     

Investigation into cell growth on collagen/chondroitin-6-sulphate gels: the effect of crosslinking agents and diamines

Artificial skin substitutes based on cultured autologous keratinocytes need to have sufficient strength and ease of handling to be utilized successfully by surgeons in the clinic. This may be achieved by crosslinking the collagen substratum on which the cells are cultured, which in this case is a collagen gel. Increased strength must be attained without detrimental effect on cell growth. The influence of potential crosslinking agents including the glycosaminoglycan, chondroitin-6-sulphate (Ch6SO4), the water soluble carbodiimide crosslinking agents 1-ethyl-3-(3-diaminopropyl) carbodiimide (EDAC), and 1,1-carbonyldiimidazole (CDI), and the polyamines putrescine, spermine and diaminohexane, on cell growth rate has been investigated. Incorporation of 20% Ch6SO4 into collagen gels caused an approximately 16% increase in keratinocyte growth, but had no significant effect on that of dermal fibroblasts. Pre-formed collagen gels (+/– Ch6SO4) were treated with the carbodiimides. This crosslinking treatment markedly inhibited fibroblast growth (EDAC 45% inhibition, CDI 70%), without affecting that of keratinocytes. Pre-formed collagen gels (+/–Ch6SO4 and carbodiimide) were treated with 0.1 M, 0.5 M or 1.0 M polyamine. Spermine inhibited the growth rate of both cell types at all concentrations tested, whereas putrescine and diaminohexane had little effect. The mechanical strength of these crosslinked gels is currently being assessed to determine the optimum composition in terms of cell growth and biocompatibility, and strength.     

Porcine collagen crosslinking, degradation and its capability for fibroblast adhesion and proliferation

Porcine dermal collagen permanently crosslinked with hexamethylene diisocyanate was investigated for its suitability as a dermal tissue engineering matrix. It was found that the chemically crosslinked collagen had far fewer free lysine groups per collagen molecule than did the uncrosslinked matrix. The ability of the matrix to support human primary fibroblast outgrowth from explants was compared for matrices that had been presoaked in various solutions, including fibroblast media, cysteine and phosphate buffered saline (PBS). It was found that superior cell outgrowth was obtained after soaking with fibroblast media and PBS. The fibroblast attachment properties of the matrix were compared against tissue culture plastic and PET. The collagen matrix showed the least amount of cell retention compared to the other to matrices, however, the general trends were similar for all three scaffolds. Longer term cultures on the collagen showed fibroblasts covering the matrix stacking up on each other and bridging natural hair follicles. However, it was also observed that the fibroblasts were not able to penetrate into the matrix structure. This was believed to result from the chemical crosslinking, as shown by the resistance of the matrix to degradation by collagenases.     

Fibrous hydrogel scaffolds with cells embedded in the fibers as a potential tissue scaffold for skin repair

A novel approach was undertaken to create a potential skin wound dressing. L929 fibroblast cells and alginate solution were simultaneously dispensed into a calcium chloride solution using a three-dimensional plotting system to manufacture a fibrous alginate scaffold with interconnected pores. These cells were then embedded in the alginate hydrogel fibers of the scaffold. A conventional scaffold with cells directly seeded on the fiber surface was used as a control. The encapsulated fibroblasts made using the co-dispensing method distributed homogeneously within the scaffold and showed the delayed formation of large cell aggregates compared to the control. The cells embedded in the hydrogel fibers also deposited more type I collagen in the extracellular matrix and expressed higher levels of fgf11 and fn1 than the control, indicating increased cellular proliferation and attachment. The results indicate that the novel co-dispensing alginate scaffold may promote skin regeneration better than the conventional directly-seeded scaffold.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Biomechanical properties of single chondrocytes and chondrons determined by micromanipulation and finite-element modelling

A chondrocyte and its surrounding pericellular matrix (PCM) are defined as a chondron. Single chondrocytes and chondrons isolated from bovine articular cartilage were compressed by micromanipulation between two parallel surfaces in order to investigate their biomechanical properties and to discover the mechanical significance of the PCM. The force imposed on the cells was measured directly during compression to various deformations and then holding. When the nominal strain at the end of compression was 50 per cent, force relaxation showed that the cells were viscoelastic, but this viscoelasticity was generally insignificant when the nominal strain was 30 per cent or lower. The viscoelastic behaviour might be due to the mechanical response of the cell cytoskeleton and/or nucleus at higher deformations. A finite-element analysis was applied to simulate the experimental force-displacement/time data and to obtain mechanical property parameters of the chondrocytes and chondrons. Because of the large strains in the cells, a nonlinear elastic model was used for simulations of compression to 30 per cent nominal strain and a nonlinear viscoelastic model for 50 per cent. The elastic model yielded a Young''s modulus of 14 ± 1 kPa (mean ± s.e.) for chondrocytes and 19 ± 2 kPa for chondrons, respectively. The viscoelastic model generated an instantaneous elastic modulus of 21 ± 3 and 27 ± 4 kPa, a long-term modulus of 9.3 ± 0.8 and 12 ± 1 kPa and an apparent viscosity of 2.8 ± 0.5 and 3.4 ± 0.6 kPa s for chondrocytes and chondrons, respectively. It was concluded that chondrons were generally stiffer and showed less viscoelastic behaviour than chondrocytes, and that the PCM significantly influenced the mechanical properties of the cells.     

Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels

Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (E_c: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (E_AFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care.     

Spatio-temporal development of the endothelial glycocalyx layer and its mechanical property in vitro

The endothelial glycocalyx is a thin layer of polysaccharide matrix on the luminal surface of endothelial cells (ECs), which contains sulphated proteoglycans and glycoproteins. It is a mechanotransducer and functions as an amplifier of the shear stress on ECs. It controls the vessel permeability and mediates the blood–endothelium interaction. This study investigates the spatial distribution and temporal development of the glycocalyx on cultured ECs, and evaluates mechanical properties of the glycocalyx using atomic force microscopy (AFM) nano-indentation. The glycocalyx on human umbilical vein endothelial cells (HUVECs) is observed under a confocal microscope. Manipulation of the glycocalyx is achieved using heparanase or neuraminidase. The Young''s modulus of the cell membrane is calculated from the force–distance curve during AFM indentation. Results show that the glycocalyx appears predominantly on the edge of cells in the early days in culture, e.g. up to day 5 after seeding. On day 7, the glycocalyx is also seen in the apical area of the cell membrane. The thickness of the glycocalyx is approximately 300 nm–1 μm. AFM indentation reveals the Young''s modulus of the cell membrane decreases from day 3 (2.93 ± 1.16 kPa) to day 14 (0.35 ± 0.15 kPa) and remains unchanged to day 21 (0.33 ± 0.19 kPa). Significant difference in the Young''s modulus is also seen between the apical (1.54 ± 0.58 kPa) and the edge (0.69 ± 0.55 kPa) of cells at day 7. By contrast, neuraminidase-treated cells (i.e. without the glycocalyx) have similar values between day 3 (3.18 ± 0.88 kPa), day 14 (2.12 ± 0.78 kPa) and day 21 (2.15 ± 0.48 kPa). The endothelial glycocalyx in vitro shows temporal development in the early days in culture. It covers predominantly the edge of cells initially and appears on the apical membrane of cells as time progresses. The Young''s modulus of the glycocalyx is deduced from Young''s moduli of cell membranes with and without the glycocalyx layer. Our results show the glycocalyx on cultured HUVECs has a Young''s modulus of approximately 0.39 kPa.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

A mathematical model of collagen lattice contraction

Two mathematical models for fibroblast–collagen interaction are proposed which reproduce qualitative features of fibroblast-populated collagen lattice contraction. Both models are force based and model the cells as individual entities with discrete attachment sites; however, the collagen lattice is modelled differently in each model. In the collagen lattice model, the lattice is more interconnected and formed by triangulating nodes to form the fibrous structure. In the collagen fibre model, the nodes are not triangulated, are less interconnected, and the collagen fibres are modelled as a string of nodes. Both models suggest that the overall increase in stress of the lattice as it contracts is not the cause of the reduced rate of contraction, but that the reduced rate of contraction is due to inactivation of the fibroblasts.     

The influence of crosslinking agents and diamines on the pore size, morphology and the biological stability of collagen sponges and their effect on cell penetration through the sponge matrix

Artificial skin substitutes based on autologous keratinocytes cultured on collagen substrata are being developed for treating patients with severe burns. The properties of the collagen substrate can be manipulated, for example, by crosslinking, to optimize desirable properties such as cell growth and penetration into the substrate, biological stability and mechanical strength. Collagen sponges crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and the diamine, diaminohexane, were used to determine the effect of crosslinking on pore size and morphology, on the stability of the crosslinked sponges both in cell culture media and during incubation with collagenase, and on the penetration of keratinocytes and fibroblasts through the sponge matrix. Crosslinking of the sponges reduced the pore size, particularly at the surface, and altered sponge morphology. After crosslinking the collagen fibers were thinner, and appeared lacy and delicate. Crosslinking also influenced sponge stability. In keratinocyte serum-free medium the pore size of plain collagen sponges increased with increasing incubation time, and crosslinking appeared to prevent this, and may have stabilized sponge structure. Incubation in serum-containing Dulbeccos minimum essential medium caused a marked reduction in pore size in both plain collagen and crosslinked collagen sponges. Crosslinking did not appear to influence this cell-free contraction of collagen sponges. Treatment of sponges with EDAC markedly increased the resistance of sponges to collagenase digestion. The penetration of both keratinocytes and fibroblasts was retarded by crosslinking the sponges. Fibroblasts penetrated through the sponges to a greater extent than keratinocytes, and their proliferation rate was faster. The total number of cells populating the crosslinked sponges after 10 days culture was approximately 50% of that on untreated collagen sponges. The mechanism responsible for this effect was different with the two crosslinkers used. Diaminohexane appeared to inhibit cell growth, whereas EDAC may have caused a decrease in cell adhesion to the sponges, without an apparent inhibition of growth rate. In terms of morphology, fibroblasts were elongated to a greater extent on crosslinked sponges, and alligned themselves along the collagen fibers. Keratinocytes grew in colonies on untreated sponges, but on crosslinked sponges they grew in isolation, with minimal cell–cell interactions. It may be necessary to reach a compromise to obtain the best combination of properties for using collagen sponges as substrata for artificial skin substitutes. © 2001 Kluwer Academic Publishers     

Inelastic behaviour of collagen networks in cell–matrix interactions and mechanosensation

The mechanical properties of extracellular matrix proteins strongly influence cell-induced tension in the matrix, which in turn influences cell function. Despite progress on the impact of elastic behaviour of matrix proteins on cell–matrix interactions, little is known about the influence of inelastic behaviour, especially at the large and slow deformations that characterize cell-induced matrix remodelling. We found that collagen matrices exhibit deformation rate-dependent behaviour, which leads to a transition from pronounced elastic behaviour at fast deformations to substantially inelastic behaviour at slow deformations (1 μm min⁻¹, similar to cell-mediated deformation). With slow deformations, the inelastic behaviour of floating gels was sensitive to collagen concentration, whereas attached gels exhibited similar inelastic behaviour independent of collagen concentration. The presence of an underlying rigid support had a similar effect on cell–matrix interactions: cell-induced deformation and remodelling were similar on 1 or 3 mg ml⁻¹ attached collagen gels while deformations were two- to fourfold smaller in floating gels of high compared with low collagen concentration. In cross-linked collagen matrices, which did not exhibit inelastic behaviour, cells did not respond to the presence of the underlying rigid foundation. These data indicate that at the slow rates of collagen compaction generated by fibroblasts, the inelastic responses of collagen gels, which are influenced by collagen concentration and the presence of an underlying rigid foundation, are important determinants of cell–matrix interactions and mechanosensation.     

Efficient co‐cultivation of human fibroblast cells (HFCs) and adipose‐derived stem cells (ADSs) on gelatin/PLCL nanofiber

In this study, we investigated whether the nanofibers produced by natural‐synthetic polymers can probably promote the proliferation of co‐cultured adipose‐derived stem cells/human fibroblast cells (ADSs/HFCs) and synthesis of collagen. Nanofiber was fabricated by blending gelatin and poly (L‐lactide co‐ɛ‐caprolactone) (PLCL) polymer nanofiber (Gel/PLCL). Cell morphology and the interaction between cells and Gel/PLCL nanofiber were evaluated by FESEM and fluorescent microscopy. MTS assay and quantitative real‐time polymerase chain reaction were applied to assess the proliferation of co‐cultured ADSs/HFCs and the collagen type I and III synthesis, respectively. The concentrations of two cytokines including fibroblast growth factor‐basic and transforming growth factor‐β1 were also measured in culture medium of co‐cultured ADSs/HDCs using enzyme‐linked immunosorbent assay assay. Actually, nanofibers exhibited proper structural properties in terms of stability in cell proliferation and toxicity analysis processes. Gel/PLCL nanofiber promoted the growth and the adhesion of HFCs. Our results showed in contact co‐culture of ADSs/HFCs on the Gel/PLCL nanofiber increased cellular adhesion and proliferation synergistically compared to non‐coated plate. Also, synthesis of collagen and cytokines secretion of co‐cultured ADSs/HFCs on Gel/PLCL scaffolds is significantly higher than non‐coated plates. To conclude, the results suggest that Gel/PLCL nanofiber can imitate physiological characteristics in vivo and enhance the efficacy of co‐cultured ADSs/HFCs in wound healing process.Inspec keywords: biomedical materials, enzymes, adhesion, fluorescence, polymer fibres, tissue engineering, wounds, nanofibres, cellular biophysics, molecular biophysics, gelatin, biochemistry, nanomedicine, field emission scanning electron microscopy, nanofabricationOther keywords: cell morphology, cell proliferation, efficient cocultivation, HFCs, ADSs, gelatin‐PLCL nanofiber, natural‐synthetic polymers, cocultured adipose‐derived stem cells‐human fibroblast cells, FESEM, fluorescent microscopy, MTS assay, quantitative real‐time polymerase chain reaction, collagen type I synthesis, collagen type III synthesis, cytokines, transforming growth factor‐β1, fibroblast growth factor‐basic growth factor‐β1, culture medium, enzyme‐linked immunosorbent assay assay, structural properties, toxicity analysis, cellular adhesion, physiological characteristics in vivo, wound healing     

In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ∼67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.     

Growth of fibroblast and vascular smooth muscle cells in fibroin/collagen scaffold

In our recent study, a novel fibroin/collagen scaffold with improved mechanical properties and controllable porous structure was prepared through freeze–drying method. In this research, the cyto-compatibility was further studied, using fibroblast and vascular smooth muscle cells (VSMC) as the model cells. MTT results indicated that the growth of fibroblast and VSMC both further improved in the fibroin/collagen scaffold than in pure fibroin scaffolds. The confocal and SEM results showed that fibroblast cells and VSMCs had better adhesion and spreading properties in the fibroin/collagen scaffolds. Although further studies, such as the extracellular matrix production and the functional gene expression, are necessary to clarify the biocompatibility of the fibroin/collagen scaffolds, the present results indicate that the fibroin/collagen scaffold is a new scaffold material suitable for tissue engineering. On the other hand, the mild and all-aqueous preparation processes also make it possible to embed different growth factors inside the scaffolds to maximize cell functions and the formation of specific tissues.     

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging.     

The effect of gold nanorods on cell-mediated collagen remodeling

Cardiac fibroblasts, the noncontractile cells of the heart, contribute to myocardial maintenance through the deposition, degradation, and organization of collagen. Adding polyelectrolyte-coated gold nanorods to three-dimensional constructs composed of collagen and cardiac fibroblasts reduced contraction and altered the expression of mRNAs encoding beta-actin, alpha-smooth muscle actin, and collagen type I. These data show that nanomaterials can modulate cell-mediated matrix remodeling and suggest that the targeted delivery of nanomaterials can be applied for antifibrotic therapies.     

Quantification of the passive and active biaxial mechanical behaviour and microstructural organization of rat thoracic ducts

Mechanical loading conditions are likely to play a key role in passive and active (contractile) behaviour of lymphatic vessels. The development of a microstructurally motivated model of lymphatic tissue is necessary for quantification of mechanically mediated maladaptive remodelling in the lymphatic vasculature. Towards this end, we performed cylindrical biaxial testing of Sprague–Dawley rat thoracic ducts (n = 6) and constitutive modelling to characterize their mechanical behaviour. Spontaneous contraction was quantified at transmural pressures of 3, 6 and 9 cmH₂O. Cyclic inflation in calcium-free saline was performed at fixed axial stretches between 1.30 and 1.60, while recording pressure, outer diameter and axial force. A microstructurally motivated four-fibre family constitutive model originally proposed by Holzapfel et al. (Holzapfel et al. 2000 J. Elast. 61, 1–48. (doi:10.1023/A:1010835316564)) was used to quantify the passive mechanical response, and the model of Rachev and Hayashi was used to quantify the active (contractile) mechanical response. The average error between data and theory was 8.9 ± 0.8% for passive data and 6.6 ± 2.6% and 6.8 ± 3.4% for the systolic and basal conditions, respectively, for active data. Multi-photon microscopy was performed to quantify vessel wall thickness (32.2 ± 1.60 µm) and elastin and collagen organization for three loading conditions. Elastin exhibited structural ‘fibre families’ oriented nearly circumferentially and axially. Sample-to-sample variation was observed in collagen fibre distributions, which were often non-axisymmetric, suggesting material asymmetry. In closure, this paper presents a microstructurally motivated model that accurately captures the biaxial active and passive mechanical behaviour in lymphatics and offers potential for future research to identify parameters contributing to mechanically mediated disease development.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Fibroblast growth and polymorphonuclear granulocyte activation in the presence of a new biologically active sol–gel glass

The search for chemical devices to be used in clinical orthopaedics must find substances that are biocompatible and do not elicit inflammatory responses in vivo. To this end, a new form of glass has been prepared, composed of 8.1% CaO, 2.9% P2O5, 6.7% N2O5 and 82.3% SiO2, using sol–gel procedures. In order to evaluate the in vitro biocompatibility of this glass, the proliferation of cultured murine fibroblasts and the activation of human polymorphonuclear leukocytes has been studied. The performance of the sol–gel glass has been compared with that of a biocompatible non-resorbable soda–lime glass. Unlike the soda–lime glass, the sol–gel glass neither caused the inhibition of fibroblast growth nor elicited a marked inflammatory response by polymorphonuclear leukocytes, as demonstrated by chemiluminescence assay for reactive oxygen metabolites.