Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial denaturation or contraction, weight loss, and severe calcification (13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0.2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinked sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 weeks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-50%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less than 10% in monolayers and more than 50% in floating collagen gel. A small amount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels.     

Impact of sterilization on the porous design and cell behavior in collagen sponges prepared for tissue engineering

This study investigates the impact of different sterilization processes on structural integrity and stability of collagen sponges designed for tissue engineering. Collagen sponges with uniform pore size (20 microm) were sterilized either with ethylene oxide (EO) or gamma irradiation (2.5 Mrad). Gamma-sterilized sponges showed a dramatic decrease of resistance against enzyme degradation and severe shrinkage after cell seeding. Collapsed porosity inhibited fibroblasts and barred completely the human umbilical vein endothelial cell ingrowth into the sponges. On the contrary, the porous structure and stability of EO-sterilized sponges remained almost unaltered. Fibroblasts and endothelial cells exhibited favorable proliferation and migration within sponges with normal morphology. Tubular formation by seeded endothelial cells occurred early in the first week. Therefore, we emphasize that the impact of sterilization of biomaterials is substantial and any new procedure has to be evaluated by correlating the impact of the procedure on the porous structure with cell proliferation behavior.     

Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent

The growth in refractive surgeries and corneal replacements has fueled interest in the development of a tissue-engineered cornea. This study characterizes the microstructure and biomechanical properties of film-based corneal stroma equivalents over time in culture. The increased collagen density in the films was hypothesized to result in improved mechanical properties both initially and over time. The microstructure of the film-based stromal equivalent was examined using atomic force microscopy and scanning electron microscopy; the mechanical properties, relaxed modulus, and ultimate tensile strength were quantified using uniaxial tensile testing. The dense, film-based stromal equivalent had a lamellae-like microstructure, which was notably different than the porous structure of sponges used previously. Seeded human corneal stromal fibroblasts remained on the surface of the film rather than migrating into the film and produced fibers of extracellular matrix with diameters of 35-75 nm. After an initial decrease during hydration, the relaxed modulus and ultimate tensile strength for fully hydrated collagen films were 0.4 +/- 0.2 MPa and 0.3 +/- 0.1 MPa, respectively. The mechanical properties of cell-seeded films mimicked those of control films. While further studies are needed to quantify the optical properties, the dense, lamellae-like structure of collagen films is a feasible scaffold for the development of tissue-engineered stroma.     

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.     

Comparative analysis of different collagen-based biomaterials as scaffolds for long-term culture of human fibroblasts

Biodegradable scaffolds, along with cells, are important components of most tissue-engineered consructs. In the study, there is a comparison of the behaviour of human fibroblasts cultured for up to six weeks in four diffeeent collagen-based three-dimensional matrices, in the form of sponges composed of pure native type I collagen (control), of collagen-GAG-chitosan (CGC) and of collagen cross-linked by two concentrations of diphenylphosphorylazide (DPPA-2 and DPPA-3). Variations in size and weight of the sponges, as well as fibroblast growth and migration, and total protein and collagen synthesis, are determined with time in culture. Owing to their low thermal stability, the partial denaturation and dissolution of the control sponges after incubation at 37°C lead to considerable contraction and low cell proliferation. CGC sponges, stabilised by ionic interactions between the different components, show, after six weeks, limited contraction (20%) and weight increase (10% when seeded) and high growth (threefold increase). Similar results are obtained with weakly, cross-linked (DPPA-2) collagen sponges. Highly crosslinked (DPPA-3) sponges do not contract, whereas weight gain and cell proliferation are no different from those found with CGC and DPPA-2 sponges. Similar levels of total protein and collagen synthesis shown for fibroblasts seeded in different matrices, with a slight general decrease (twofold) after three weeks, a much lower value than that observed with fibroblasts in culture within a contracted collagen gel (sixfold). Furthermore, the fraction of neo-synthesised collagen deposited in the sponges after six weeks represents more than 60% of the total, compared with only 10% obtained with fibroblasts in monolayer culture or 30% within a collagen gel. These results indicate that the matrices, particularly the CGC and DPPA-2 sponges, provide excellent supports for fibroblast growth and the formation of dermal and skin equivalents.     

Cell growth on the porous sponges prepared from poly(depsipeptide-co-lactide) having various functional groups

In tissue engineering, excellent biodegradable materials are desired as temporary scaffolds to support cell growth and disappear with the progress of tissue regeneration. We previously synthesized biodegradable poly(depsipeptide-co-lactide), poly[(Glc-Asp)-co-LA] and poly[(Glc-Lys)-co-LA], having reactive side-chain groups. Then, the effects of reactive and ionic side-chain groups on cell attachment and growth were investigated using co-polymer films with various amounts of carboxyl or amino groups. In this study, to evaluate the utility of these co-polymers as functional scaffolds for tissue regeneration, 3-dimensional porous sponges were prepared by freeze-drying method and the effects of reactive and ionic side-chain groups on cell growth and degradation behavior were investigated using co-polymer sponges with various amounts of carboxyl or amino groups. Good cell growth was observed on the co-polymer sponges. During cell culture, the co-polymer sponges exhibited various degradation rates related to the depsipeptide unit content. Three-dimensional biodegradable polymer matrices with reactive surface, controllable degradation behavior and good cell growth were successfully prepared using these co-polymers. Such kinds of co-polymer matrices are good candidate for scaffold for tissue engineering.     

Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis

Combining bovine collagen with chitosan followed by freeze-drying has been shown to produce porous scaffolds suitable for skin and connective tissue engineering applications. In this study collagen extracted from porcine and avian skin was compared with bovine collagen for the production of tissue engineered scaffolds. A similar purity of the collagen extracts was shown by electrophoresis, confirming the reliability of the extraction process. Collagen was solubilized, cross-linked by adding chitosan to the solution and freeze-dried to generate a porous structure suitable for tissue engineering applications. Scaffold porosity and pore morphology were shown to be source dependant, with bovine collagen and avian collagen resulting into the smallest and largest pores, respectively. Scaffolds were seeded with dermal fibroblasts and cultured for 35 days to evaluate the suitability of the different collagen–chitosan scaffolds for long-term tissue engineered dermal substitute maturation in vitro. Cell proliferation and scaffold biocompatibility were found to be similar for all the collagen–chitosan scaffolds, demonstrating their capability to support long-term cell adhesion and growth. The scaffolds contents was assessed by immunohistochemistry and showed increased deposition of extracellular matrix by the cells as a function of time. These results correlate with measurements of the mechanical properties of the scaffolds, since both the ultimate tensile strength and tensile modulus of the cell seeded scaffolds had increased by the end of the culture period. This experiment demonstrates that porcine and avian collagen could be used as an alternative to bovine collagen in the production of collagen–chitosan scaffolding materials.     

Nature-derived epigallocatechin gallate/duck’s feet collagen/hydroxyapatite composite sponges for enhanced bone tissue regeneration

Abstract

Scaffolds mimicking structural and chemical characteristics of the native bone tissues are critical for bone tissue engineering. Herein, we have developed and characterized epigallocatechin gallate/duck’s feet collagen/hydroxyapatite (EGCG/DC/HAp) composite sponges that enhanced the bone tissue regeneration. The three-dimensional composite sponges were synthesized by loading various amounts (i.e. 1, 5 and 10 μM) of EGCG to duck feet derived collagen followed by freeze-drying and then coating with hydroxyapatite. Several measuremental techniques were employed to examine the properties of the as-fabricated composite sponges including morphology and structure, porosity, compressive strength, etc. and as well compared with pristine duck feet derived collagen. SEM observations of EGCG/DC/HAp sponges showed the formation of a highly porous collagen matrix with EGCG embodiment. The porosity and pore size of sponges were found to increase by high EGCG content. The compressive strength was calculated as 3.54 ± 0.04, 3.63 ± 0.03, 3.89 ± 0.05, 4.047 ± 0.05 MPa for 1, 5 and 10 μM EGCG/DC/HAp sponges, respectively. Osteoblast-like cell (BMSCs isolated from rabbit) culture and in vivo experiments with EGCG/DC/HAp sponges implanted in nude mouse followed by histological staining showed enhanced cell internalization and attachment, cell proliferation, alkaline phosphatase expressions, indicating that EGCG/DC/HAp sponges have ahigh biocompatibility. Moreover, highEGCG content in the EGCG/DC/HAp sponges have led to increased cellular behavior. Collectively, the 5 μM of EGCG/DC/HAp sponges were suggested as the potential candidates for bone tissue regeneration.     

The inflammatory responses to silk films in vitro and in vivo

Silks have a long history of biomedical use as sutures. Silk can be purified, chemically modified to attach RGD sequences and processed into highly porous scaffolds for tissue engineering. We report biocompatibility studies of silk films (with or without covalently bound RGD) that were seeded with bone-marrow derived mesenchymal stem cells (MSC) and (a) cultured in vitro with human MSC or (b) seeded with autologous rat MSC and implanted in vivo. Controls for in vitro studies included tissue culture plastic (TCP; negative control), TCP with lipopolysaccharide (LPS) in the cell culture medium (positive control), and collagen films; controls for in vivo studies included collagen, PLA and TCP. After 9 h of culture, the expression of the pro-inflammatory Interleukin 1 beta (IL-1beta) and inflammatory cyclooxygenase 2 (COX-2) in human MSC were comparable for silk, collagen and TCP. After 30 and 96 h, gene expression of IL-1beta and COX-2 in MSC returned to the baseline (pre-seeding) levels. These data were corroborated by measuring IL-1beta and prostaglandin E2 levels in culture medium. The rate of cell proliferation was higher on silk films than either on collagen or TCP. In vivo, films made of silk, collagen or PLA were seeded with rat MSCs, implanted intramuscularly in rats and harvested after 6 weeks. Histological and immunohistochemical evaluation of silk explants revealed the presence of circumferentially oriented fibroblasts, few blood vessels, macrophages at the implant-host interface, and the absence of giant cells. Inflammatory tissue reaction was more conspicuous around collagen films and even more around PLA films when compared to silk. These data suggest that (a) purified degradable silk is biocompatible and (b) the in vitro cell culture model (hMSC seeded and cultured on biomaterial films) gave inflammatory responses that were comparable to those observed in vivo.     

Collagenase in keloid biopsies and fibroblasts

Trypsin-activated collagenase activity was analyzed in media from keloid biopsies as well as keloid fibroblasts using radiolabeled guinea pig skin collagen as a substrate for collagenase digestion. Collagenase activity per tissue dry weight and DNA was similar in keloid and normal dermal biopsies; however, it was significantly elevated in keloid compared to normal dermal fibroblast media. In two paired cell lines where keloid and normal dermal fibroblasts were isolated from the same patient, relative collagen synthesis and trypsin-activated collagenase were increased to a similar extent in the keloid cell lines compared to normals. The biopsy data are in agreement with other findings, and further confirm the hypothesis that increased collagen deposition in keloids does not result from decreased collagen degradation. In vitro, however, the data presented here indicate that in some cell lines increased collagen synthesis may be associated with increased trypsin-activated collagenase.     

In vivo evaluation and comparison of collagen, acetylated collagen and collagen/glycosaminoglycan composite films and sponges as candidate biomaterials

Native collagen, acetylated collagen, collagen/10% chondroitin sulphate, collagen/2.5% hyaluronic acid and collagen/20% hyaluronic acid were implanted both as film and as sponge into rat lumbar muscle for 7 and 14 d. After 7 d implantation, all materials elicited an acute inflammatory cell response characterized by numerous polymorphs and histocytes. The cell population after 14 d was principally mononuclear, i.e. leucocytes, neutrophils, macrophages, lymphocytes and fibroblasts. Both films and sponges followed a similar pattern. Native collagen elicited a subacute inflammatory response after 7 d. However, 14 d after implantation, a marked infiltration by neutrophils was apparent with subsequent degradation of existing collagen material. Acetylated collagen film evoked a much greater inflammatory cell response than native collagen. Both collagen/hyaluronic acid composites elicited a similar response. The collagen/10% chondroitin sulphate composite elicited the least inflammatory cell response at 7 d, whereas infiltration by host fibroblasts after 14 d implantation was clearly seen.     

Evaluation of functions and tissue compatibility of poly (D,L-lactic-co-glycolic acid) seeded with human dermal fibroblasts

In tissue engineering and wound-healing applications, dermal substitutes are used to provide fibroblasts with the mechanical support for their growth and then to facilitate the skin formation. In this study, three-dimensional porous poly(lactic-co-glycolic acid) (PLGA) 65/35 scaffolds were prepared and then the composites of the scaffolds and human fetal dermal fibroblasts were fabricated as a tissue-engineered dermal substitute. The function and tissue compatibility of the artificial dermal substitute were evaluated at the levels of gene expression (by RT-PCR) and protein expression (total collagen quantities), as well as by histological and immunohistochemical analysis. The PCR products indicated that the mRNA of type-I collagen, mainly secreted by the fibroblasts onto the PLGA scaffolds, was clearly expressed after 4 weeks. The amount of total collagen synthesized from the cells was shown to increase gradually during the initial culture period and slightly decreased afterwards. After 8 weeks of culture, the fibroblasts were well attached and migrated entirely throughout the pores of the PLGA scaffold with normal function. Furthermore, the positively stained type-I collagen was intensively detected throughout the pores. These results suggest that the function and tissue compatibility may be important criteria in evaluating an artificial tissue-engineered skin.     

Evaluation of functions and tissue compatibility of poly (D,L-lactic-co-glycolic acid) seeded with human dermal fibroblasts

In tissue engineering and wound-healing applications, dermal substitutes are used to provide fibroblasts with the mechanical support for their growth and then to facilitate the skin formation. In this study, three-dimensional porous poly(lactic-co-glycolic acid) (PLGA) 65/35 scaffolds were prepared and then the composites of the scaffolds and human fetal dermal fibroblasts were fabricated as a tissue-engineered dermal substitute. The function and tissue compatibility of the artificial dermal substitute were evaluated at the levels of gene expression (by RT-PCR) and protein expression (total collagen quantities), as well as by histological and immunohistochemical analysis. The PCR products indicated that the mRNA of type-I collagen, mainly secreted by the fibroblasts onto the PLGA scaffolds, was clearly expressed after 4 weeks. The amount of total collagen synthesized from the cells was shown to increase gradually during the initial culture period and slightly decreased afterwards. After 8 weeks of culture, the fibroblasts were well attached and migrated entirely throughout the pores of the PLGA scaffold with normal function. Furthermore, the positively stained type-I collagen was intensively detected throughout the pores. These results suggest that the function and tissue compatibility may be important criteria in evaluating an artificial tissue-engineered skin.     

The impact of vacuum freeze-drying on collagen sponges after gas plasma sterilization

The sterilization of porous collagen sponges remains a challenging procedure. Gamma irradiation denatures collagen, resulting in dramatic changes to its structure. Ethylene oxide leaves toxic residues requiring weeks to evaporate. This study investigated the impact on cell behavior of gas plasma treatment when combined with vacuum freeze-drying. The goal of this procedure is to eliminate the molecules of hydrogen peroxide remaining after the sterilization process, together with their decomposition products, from the scaffolds. These molecules hinder the immediate use of the porous designs. Collagen and EDC/NHS-heparinized collagen scaffolds were sterilized with gas plasma. H2O2 released by the collagen specimens was measured by peroxidase test both immediately and also 1 week after the plasma treatment. Further measurements were done 24, 36, 48 and 72 h after vacuum freeze-drying. The activity of these scaffolds was further evaluated in relation to the proliferation, migration and differentiation of human umbilical vein endothelial cells (HUVECs). Both immediately after exposure to gas plasma and also 1 week later, the collagen designs contained significantly higher concentrations of H2O2 than scaffolds having also undergone vacuum freeze-drying. This procedure achieved faster decontamination of the remaining H2O2. Following vacuum freeze-drying, sponges already allowed HUVEC proliferation after 48 h, but in non-lyophilized specimens after gas plasma treatment alone, cell death occurred as early as only 1 week later. These data highlight the advantages of carrying out vacuum freeze-drying following gas plasma sterilization. The results show the substantial impact of sterilization of porous materials made for tissue engineering.     

Dynamics of the healing of skin wounds in the horse as compared with the rat.

The dynamics of skin wound healing were studied in three horses with either full thickness skin excision or with subcutaneously implanted polyvinyl alcohol sponges. Granulation tissue and reactive granuloma were harvested from three anatomical sites and were analyzed by morphological and biochemical methods at three time intervals. PVA sponges were also implanted in rats and studied by similar methods. No effect of wound or implant location on morphology or density of the sample tissue was found. In the horse, neutrophil infiltration was found in the tissue of wounds less than one day old. This tissue actively synthesized collagen and showed high activity of collagenase. The activity of prolyl hydroxylase (PH), however, was low. At later time sampling period (3, 7, 14 days), both granulation and granuloma tissue showed increasing PH activity, high activity of collagenase, and decreasing rate of collagen synthesis. Collagen content also increased with time. The reactive granuloma tissue found in rats showed less connective tissue reactivity than in the horse as seen by the dynamics of the morphological and biochemical changes of the tissue. We conclude that the healing in the horse is rather prompt and excessive and may tend toward abnormal repair reactions.     

Behavior of fibroblasts on a porous hyaluronic acid incorporated collagen matrix

A hyaluronic acid (HA) incorporated porous collagen matrix was fabricated at -70 degrees C by lyophilization. The HA incorporated collagen matrix showed increased pore size in comparison with collagen matrix. Biodegradability and mechanical properties of matrices were controllable by varying the ultraviolet (UV) irradiation time for cross-linking collagen molecules. Addition of HA to collagen matrix did not effect ultimate tensile stress after UV irradiation. HA incorporated collagen matrices demonstrated a higher resistance against the collagenase degradation than collagen matrix. In an in vitro investigation of cellular behavior using dermal fibroblasts on the porous matrix, HA incorporated collagen matrix induced increased dermal fibroblast migration and proliferation in comparison with collagen matrix. These results suggest that the HA incorporated collagen porous matrix assumes to enhance dermal fibroblast adaptation and regenerative potential.     

Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.     

Tendon cell contraction of collagen-GAG matrices in vitro: effect of cross-linking

The contraction of connective tissue cells can play important roles in wound healing and pathological contractures. The effects of this contractile behavior on cell-seeded constructs for tissue engineering have not yet been investigated. The goal of this work was to investigate in vitro tendon cell-mediated contraction of collagen-glycosaminoglycan (GAG) matrices cross-linked using selected methods. Highly porous collagen-GAG sponges were seeded with calf tendon cells and the projected area and DNA content of the sponges measured at 3, 7, 14, and 21 days post-seeding. Immunohistochemistry was performed to determine if alpha-smooth muscle actin (SMA) was associated with the cell contraction of the matrices. Dehydrothermal (DHT) treatment alone was not sufficient to resist contraction by the seeded tendon fibroblasts. Cross-linking of the collagen-GAG sponges to the extent that the modulus was three times that of sponges treated by DHT alone was necessary to resist contraction. SMA was seen in the cytoplasm of most cells in all sponges at all time periods. The results provide a rational basis for the determination of the mechanical properties of collagen matrices required for engineering certain connective tissues.     

Control of pore structure and size in freeze-dried collagen sponges.

Because of many suitable properties, collagen sponges are used as an acellular implant or a biomaterial in the field of tissue engineering. Generally, the inner three-dimensional structure of the sponges influences the behavior of cells. To investigate this influence, it is necessary to develop a process to produce sponges with a defined, adjustable, and homogeneous pore structure. Collagen sponges can be produced by freeze-drying of collagen suspensions. The pore structure of the freeze-dried sponges mirrors the ice-crystal morphology after freezing. In industrial production, the collagen suspensions are solidified under time- and space-dependent freezing conditions, resulting in an inhomogeneous pore structure. In this investigation, unidirectional solidification was applied during the freezing process to produce collagen sponges with a homogeneous pore structure. Using this technique the entire sample can be solidified under thermally constant freezing conditions. The ice-crystal morphology and size can be adjusted by varying the solute concentration in the collagen suspension. Collagen sponges with a very uniform and defined pore structure can be produced. Furthermore, the pore size can be adjusted between 20-40 microm. The thickness of the sponges prepared during this research was 10 mm.