Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

Engineered Smooth Muscle Tissues: Regulating Cell Phenotype with the Scaffold

Culturing cells on three-dimensional, biodegradable scaffolds may create tissues suitable either for reconstructive surgery applications or as novel in vitro model systems. In this study, we have tested the hypothesis that the phenotype of smooth muscle cells (SMCs) in three-dimensional, engineered tissues is regulated by the chemistry of the scaffold material. Specifically, we have directly compared cell growth and patterns of extracellular matrix (ECM) (e.g. , elastin and collagen) gene expression on two types of synthetic polymer scaffolds and type I collagen scaffolds. The growth rates of SMCs on the synthetic polymer scaffolds were significantly higher than on type I collagen sponges. The rate of elastin production by SMCs on polyglycolic acid (PGA) scaffolds was 3.5 +/- 1.1-fold higher than that on type I collagen sponges on Day 11 of culture. In contrast, the collagen production rate on type I collagen sponges was 3.3 +/- 1.1-fold higher than that on PGA scaffolds. This scaffold-dependent switching between elastin and collagen gene expression was confirmed by Northern blot analysis. The finding that the scaffold chemistry regulates the phenotype of SMCs independent of the scaffold physical form was confirmed by culturing SMCs on two-dimensional films of the scaffold materials. It is likely that cells adhere to these scaffolds via different ligands, as the major protein adsorbed from the serum onto synthetic polymers was vitronectin, whereas fibronectin and vitronectin were present at high density on type I collagen sponges. In summary, this study demonstrates that three-dimensional smooth muscle-like tissues can be created by culturing SMCs on three-dimensional scaffolds, and that the phenotype of the SMCs is strongly regulated by the scaffold chemistry. These engineered tissues provide novel, three-dimensional models to study cellular interaction with ECM in vitro.     

支架材料对棕色脂肪源性干细胞向起搏细胞诱导分化的影响

目的观察不同三维支架材料对棕色脂肪来源干细胞（BADSCs）诱导分化成起搏细胞的效果,为构建生物起搏器提供实验依据。 方法将培养7 d的原代BADSCs分别种植到胶原海绵、明胶海绵和透明质酸水凝胶3种不同的材料中,在不同时间用光镜和扫描电镜观察细胞-支架复合体中细胞形态学的变化,免疫荧光染色检测心肌细胞、起搏细胞相关蛋白的表达。采用单因素方差分析。 结果细胞在3种支架上均能存活、增殖,LIVE/DEAD检测显示,培养3 d的胶原海绵、明胶海绵和透明质酸水凝胶3种细胞-支架复合物死细胞率分别为（46.35±1.50）%、（47.00±1.60）%和（1.76±1.08）%,其中细胞在透明质酸水凝胶中死亡率最低,并且细胞-透明质酸水凝胶复合物可自发性地搏动,三组比较差异具有统计学意义（F = 37.56,P < 0.05）。培养至2周时,胶原海绵、明胶海绵和透明质酸水凝胶中Connexin45细胞阳性率分别为（10.67±1.25）%、（13.67±1.25）%和（21.00±1.60）%,差异有统计学意义（F = 9.435,P < 0.01）,HCN2细胞阳性率分别为（11.00±1.60）%、（14.00±2.16）%和（34.33±3.68）%,差异有统计学意义（F = 17.52,P < 0.01）,HCN4细胞阳性率分别为（18.67±2.05）%、（13.00±1.60）%和（66.00±2.94）%,差异有统计学意义（F = 27.96,P < 0.01）,Sr细胞阳性率分别为（13.00±1.63）%、（14.33±1.24）%和（75.33±3.30）%,差异有统计学意义（F = 36.40,P < 0.01）,水凝胶中Connexin45、HCN2、HCN4和Sr的细胞阳性率均高于胶原海绵和明胶海绵,差异均具有统计学意义（P < 0.05）。 结论BADSCs在胶原海绵、明胶海绵和透明质酸水凝胶中均能很好地生长和分化,但透明质酸水凝胶更适用于组织工程化起搏器的构建。     

Transplantation of autologous rabbit BM-derived mesenchymal stromal cells embedded in hyaluronic acid gel sponge into osteochondral defects of the knee

BACKGROUND: Mesenchymal stromal cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, suggesting that these cells are an attractive cell source for cartilage tissue engineering approaches. Various methods, such as using hyaluronan-based materials, have been employed to improve transplantation for repair. Our objective was to study the effects of autologous transplantation of rabbit MSC with hyaluronic acid gel sponges into full-thickness osteochondral defects of the knee. METHODS: Rabbit BM-derived MSC were cultured and expanded with fibroblast growth factor (FGF). Specimens were harvested at 4 and 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type II collagen and CD44. RESULTS: The regenerated area after autologous transplantation of hyaluronic acid gel sponge loaded with MSC into the osteochondral defect at 12 weeks after surgery showed well-repaired cartilage tissue, resembling the articular cartilage of the surrounding structure, of which the histologic score was significantly better than that of the untreated osteochondral defect. In the regenerated cartilage, type II collagen was found in the pericellular matrix of regenerative chondrocytes, while CD44 expression in the regenerative tissue could not be revealed. DISCUSSION: These data suggest that the autologous transplantation of MSC embedded in hyaluronan-based material may support chondrogenic differentiation and be useful for osteochondral defect repair.     

Cross-linked collagen sponges loaded with plant polyphenols with inhibitory activity towards chronic wound enzymes

Collagen sponges loaded with polyphenols from Hamamelis virginiana were investigated as active materials for chronic wound dressings, evaluating in vitro the inhibition of two major enzymes that impair the wound healing process - myeloperoxidase (MPO) and collagenase. Prior to polyphenols loading, collagen was cross-linked with genipin to improve its biostability. The effect of genipin cross-linking and polyphenol concentration in the development of mechanically and enzymatically stable sponges was studied. The tensile strength of the cross-linked collagen increased with the increase of the cross-linking degree, coupled to decrease in the elongation and the swelling capacity of the sponges. The stability of the sponges to collagenase digestion reached maximum when 1 mM genipin was used. However, the biostability decreased more than 10-fold after loading the sponges with polyphenols (0.5 mg/mL), nevertheless, this effect was partially overcome using higher concentration of polyphenols (1 and 2 mg/mL) to inhibit collagenase. Moreover, the polyphenols released from the sponges were sufficient for complete inhibition of MPO activity. No considerable cytotoxicity of the genipin cross-linked collagen loaded with polyphenols was observed evaluating the NIH 3T3 fibroblasts viability.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Bioluminescent mammalian cells grown in sponge matrices to monitor immune rejection

The growth and bioluminescence of cells seeded in collagen and gelatin sponge matrices were compared in vitro under different conditions, and immune rejection was quantified and visualized directly in situ based on loss of bioluminescence activity. Mammalian cells expressing a Renilla luciferase complementary deoxyribonucleic acid (cDNA) were used to seed collagen and gelatin sponge matrices soaked in either polylysine or gelatin to determine optimal growth conditions in vitro. The sponges were incubated in tissue culture plates for 3 weeks and received 2, 9, or 15 injections of coelenterazine. Measurements of bioluminescence activity indicated that gelatin sponges soaked in gelatin emitted the highest levels of light emission, multiple injections of coelenterazine did not affect light emission significantly, and light emission from live cells grown in sponges could be measured qualitatively but not quantitatively. Histologic analysis of sponge matrices cultured in vitro showed that cells grew best in gelatin matrices. Visualization of subcutaneously implanted sponges in mice showed accelerated loss of light emission in immunocompetent BALB/c mice compared with immunodeficient BALB/c-scid mice, which was associated with increased cell infiltration. Our results indicate that sponge matrices carrying bioluminescent mammalian cells are a valid model system to study immune rejection in situ.     

In vitro sponge fragment culture of Chondrosia reniformis (Nardo, 1847)

In vitro cultivation systems for sponges (Porifera) have to be developed to produce compounds of value in biotechnological processes. Organotypic culture attempts, which maintain or mimic the natural tissue structure, are promising ways towards a biotechnology of sponges. We used the Mediterranean species Chondrosia reniformis for sponge fragment in vitro cultivation. The species is common throughout the Mediterranean, easy to keep in aquariums and shows good recovery and regeneration after fragmentation. The regeneration process of the 50-80 mm(3) fragments lasted for several days and resulted in a rounded or ovoid body shape. The aquiferous system was reduced. Cells performed proliferation during the first weeks as we could demonstrate by 5-bromo-2'-deoxy-uridine (BrdU) incorporation. No proliferation could be demonstrated after a culture period of 3 months, but silicate uptake. Cellular density decreased with cultivation length, but collagen production increased. Fragments have been kept in culture up to 19 months. C. reniformis can be used as a model system to develop feeding strategies and evaluate the biotechnological potential of sponge fragment in vitro cultivation.     

Light inside sponges

Sponges are the most basal metazoan organisms. As sessile filter feeders in marine or freshwater habitats, they often live in close association with phototrophic microorganisms. Active photosynthesis by the associated microorganisms has been believed to be restricted to the outer tissue portion of the sponge hosts. However, phototrophic microorganisms have also been detected in deeper tissue regions. In many cases they are found around spicules, siliceous skelettal elements of demosponges and hexactinellids. The finding of phototrophic organisms seemingly assembled around spicules led to the hypothesis of a siliceous light transmission system in sponges. The principle ability to conduct light was already shown for sponge derived, explanted spicules. However it was not shown until now, that in deed sponges have a light transmission system, and can harbour photosynthetically active microorganisms in deeper tissue regions.Here we show for the first time, that, as hypothesized 13 year ago, sponge spicules in living specimens transmit light into deeper tissue regions. Our results demonstrate that in opposite to the actual opinion, photosynthetically active microorganisms can also live in deeper tissue regions, and not only directly beneath the surface, when a light transmission system (spicules) is present.Our results show the possibility of massive or globular sponges being supplied with photosynthetic products or pathways throughout their whole body, implying not only a more important role of these endobioses. Our findings also elucidate the in-situ function of a recently more and more interesting biomaterial, which is unique not only for its mechanical, electrical and optical properties. Biosilica is of special interest for the possibility to produce it enzymatically under environmental conditions.     

Silencing the silencer: strategies to inhibit microRNA activity

Plants and animals microRNAs (miRNAs) have been proposed to be key regulators of many fundamental processes. However defining miRNAs function has been problematic due to the paucity of miRNA loss-of-function mutants. This is likely due to their small gene size and redundancy as most miRNA have highly related family members. Consequently, the analysis of miRNA function has been primarily based on predictive bioinformatic or transgenic gain-of-function approaches. However, a number of new methodologies have been developed able to result in loss-of-function phenotypes. This includes miRNA sponges in animals and target mimicry in plants, both of which sequesters the mature miRNAs, disrupting endogenous miRNA:mRNA target relationships. Furthermore, artificial miRNAs and RNA interference in plants have been shown to be potent silencers of MIRNA genes. We will discuss the strengths and weaknesses of these methodologies which are potentially of great biotechnological use in medicine and agriculture.     

Effects of interleukin-8 on granulation tissue maturation

The inflammatory alpha-chemokine, interleukin-8 (IL-8), affects the function and recruitment of various inflammatory cells, fibroblasts, and keratinocytes. Gap junctions are anatomical channels that facilitate the direct passage of small molecules between cells. The hypothesis is that IL-8 enhances gap junctional intercellular communication (GJIC) between fibroblasts in granulation tissue, which increases the rate of granulation tissue maturation. In vitro, human dermal fibroblasts were incubated with IL-8 prior to scrape loading, a technique that quantifies GJIC. Polyvinyl alcohol (PVA) sponges were implanted within subcutaneous pockets in rats and received local injections of either IL-8 or saline and were harvested on day 11. In vitro, IL-8 treated fibroblasts demonstrated an increase in GJIC by scrape loading compared to saline treated controls. In vivo, IL-8 treated PVA sponges demonstrated a decrease in cell density and an increase in vascularization compared to saline controls by H&E staining. Polarized light viewed Sirius red-stained specimens demonstrated greater collagen birefringence intensity, indicating thicker, more-mature collagen fibers. IL-8 increases GJIC in cultured fibroblasts and induces a more rapid maturation of granulation tissue.     

Evolution of collagen-based adhesion systems

Collagens are large, triple-helical proteins that form fibrils and network-like structures in the extracellular matrix. The collagens may have participated in the evolution of the metazoans from their very earliest origins. Cell adhesion receptors, such as the integrins, are at least as old as the collagens. Still, the early metazoan cells might not have been able to anchor directly to collagen fibrils, since the integrin-type collagen receptors have only been identified in vertebrates. Instead, the early metazoans may have used integrin-type receptors in the recognition of collagen-binding glycoproteins. It is possible that specialized, high-avidity collagen-receptor integrins have become instrumental for the evolution of bone, cartilage, circulatory and immune systems in the chordates. In vertebrates, specific collagen-binding receptor tyrosine kinases send signals into cells after adhesion to collagen. These receptors are members of the discoidin domain receptor (DDR) group. The evolutionary history of DDRs is poorly known at this time. DDR orthologs have been found in many invertebrates, but their ability to function as collagen receptors has not yet been tested. The two main categories of collagens, fibrillar and non-fibrillar, already exist in the most primitive metazoans, such as the sponges. Interestingly, both integrin and DDR families seem to have members that favor either one or the other of these two groups of collagens.     

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds^1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick^1,2.Each dead space model has its own limitations based on its material''s composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins¹. Later stages of wound healing are best analyzed using the ePTFE model^1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid². The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal⁵. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis^2,5.In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process^1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment^1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies^1,2.Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.     

The molecular and fibrillar structure of collagen and its relationship to the mechanical properties of connective tissue

The conformation of type I collagen molecules has been refined using a linked-atom least-squares procedure in conjunction with high-quality X-ray diffraction data. In many tendons these molecules pack in crystalline arrays and a careful measurement of the positions of the Bragg reflections allows the unit cell to be determined with high precision. From a further analysis of the X-ray data it can be shown that the highly ordered overlap region of the collagen fibrils consists of a crystalline array of molecular segments inclined by a small angle with respect to the fibril axis. In contrast, the gap region is less well ordered and contains molecular segments that are likely to be inclined by a similar angle but in a different vertical plane to that found in the overlap region. The collagen molecule thus has a D-periodic crimp in addition to the macroscopic crimp observed visually in the collagen fibres of many connective tissues. The growth and development of collagen fibrils have been studied by electron microscopy for a diverse range of connective tissues and the general pattern of fibril growth has been established as a function of age. In particular, relationships between fibril size distribution, the content and composition of the glycosaminoglycans in the matrix and the mechanical role played by the fibrils in the tissue have been formulated and these now seem capable of explaining many new facets of connective tissue structure and function.     

Fabricating tissues: Analysis of farming versus engineering strategies

Tissue Engineering has expanded rapidly towards target applications of tissue repair and regeneration, whilst generating surprisingly novel models to study tissue modelling. However, clinical success in producing effective engineered tissues such as bone, skin, cartilage, and tendon, have been rare and limited. Problems tend to focus on how to stimulate the replacement of initial scaffold with mechanically functional, native extracellular matrix (principally collagen). Typical approaches have been to develop perfused and mechanically active bioreactors, with the use of native collagen itself as the initial scaffold, though the idea remains that cells do the fabrication (i.e. a cultivation process). We have developed a new, engineering approach, in which the final collagen template is fabricatedwithout cell involvement. The first part of this biomimetic engineering involves a plastic compression of cellular native collagen gels to form dense, strong, collagenous neotissues (in minutes). Further steps can be used to orientate and increase collagen fibril diameter, again by non-cell dependent engineering. This allows operator control of cell or matrix density and material properties (influencing biological half life and fate). In addition, this (non-cultivation) approach can incorporate techniques to generate localised 3D structures and zones at a meso-scale. In conclusion, the use of biomimetic engineering based on native collagen, rather than cell-cultivation approaches for bulk matrix fabrication, produces huge benefits. These include speed of fabrication (minutes instead of weeks and months), possibility of fine control of composition and 3D nano-micro scale structure and biomimetic complexity.     

Sponge cell culture: a comparative evaluation of adhesion to a native tissue extract and other culture substrates

Hexactinellids are deep water sponges that possess syncytial rather than cellular tissues. In order to investigate the syncytial character of the tissue of these unusual sponges, primary cultures were developed using a substrate of acellular tissue extract (ATE) that promotes the adhesion and spreading of sponge tissues. Primary cultures of the hexactinellid sponge Rhabdocalyptus dawsoni, grown on this substrate, form thinly spread, multinucleate, confluent tissue masses which exhibit active cytoplasmic streaming. Sponge tissue adhered equally well to commercial substrates of concanavalin A and poly-l-lysine, but did not adhere to chicken collagen. Acellular tissue extracts prepared from demosponges, which are known to be cellular, also promoted adhesion and spreading of cells from those sponges. Scanning electron microscopy showed adherent Rhabdocalyptus tissue to have an uninterrupted, smooth membrane covering the entire culture, unlike primary cultures of the cellular demosponge, Haliclona sp., which consisted of numerous individual cells. Tissue from freshly collected sponges adhered preferentially to ATE from a conspecific. However, after continued wounding, tissue adhered indescriminately to any substrate. The tissue extract congealed if added to sea water or 10 mM CaCl(2), forming a white, cloudy solid, which could be fixed and sectioned for transmission electron microscopy. Thin sections of the congealed extract showed it to contain membranes but no visible collagen fibrils.     

Mesenchymal progenitor cells derived from chorionic villi of human placenta for cartilage tissue engineering

Human mesenchymal stem cells are currently being studied extensively because of their capability for self-renewal and differentiation to various connective tissues, which makes them attractive as cell sources for regenerative medicine. Herein we report the isolation of human placenta-derived mesenchymal cells (hPDMCs) that have the potential to differentiate into various lineages to explore the possibility of using these cells for regeneration of cartilage. We first evaluated the chondrogenesis of hPDMCs in vitro and then embedded the hPDMCs into an atelocollagen gel to make a cartilage-like tissue with chondrogenic induction media. For in vivo assay, preinduced hPDMCs embedded in collagen sponges were subcutaneously implanted into nude mice and also into nude rats with osteochondral defect. The results of these in vivo and in vitro studies suggested that hPDMCs can be one of the possible allogeneic cell sources for tissue engineering of cartilage.     

Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.     

In vitro comparison of six different matrix systems for the cultivation of human chondrocytes

Summary In recent years, a great variety of different matrix systems for the cultivation of chondrocytes have been developed. Although some of these scaffolds show promising experimental results in vitro, the potential clinical value remains unclear. In this comparative study, we propagated human articular chondrocytes precultivated in monolayer culture on six different scaffolds (collagen gels, membranes and sponges) under standardized in vitro conditions. Mechanical properties of the matrix systems were not improved significantly by cultivation of human chondrocytes under the given in vitro conditions. The gel systems (CaReS, Ars Artho, Germany and Atelocollagen, Koken, Japan) showed a homogeneous cell distribution; chondrocytes propagated on Chondro-Gide (Geistlich Biomaterials, Switzerland) and Integra membranes (Integra, USA) were building multilayers. Only few cells penetrated the two Atelocollagen honeycomb sponges (Koken, Japan). During cultivation, chondrocytes propagated on all systems showed a partial morphological redifferentiation, which was best with regard to the gel systems. In general, only small amounts of collagen type-II protein could be detected in the pericellular region and chondrocytes failed to build a territorial matrix. During the first two weeks of cultivation, the two gel systems showed a significantly higher collagen type-II gene expression and a lower collagen type-I gene expression than the other investigated matrix systems. Although collagen gels seem to be superior when dealing with deep cartilage defects, membrane systems might rather be useful in improving conventional autologous chondrocyte transplantation or in combination with gel systems.