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.     

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.     

In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix

The objective of this investigation was to demonstrate the effectiveness of a tissue-engineered collagen sponge as a substrate for the culture of human corneal cells. To that end, human kerotocyte, epithelial, and endothelial cells were cultured separately on collagen sponges composed of native fibrillar collagen with a pore size of approximately 0.1 mm. Co-culture experiments were also performed (epithelial/endothelial and epithelial/keratocyte cultures). Proliferation of keratocytes and matrix production was assessed. The morphology of the epithelial and endothelial cell cultures was characterized by histology and scanning electron microscopy. Keratocytes cultured on collagen sponges exhibited increased matrix synthesis over time as well as proliferation and repopulation of the matrix. Epithelial and endothelial cells showed the ability to migrate over the collagen sponge. The thickness of the epithelial layer was influenced by soluble factors produced by endothelial cells. The morphology of the bottom layer of epithelial cells was influenced by the presence of keratocytes in the culture. These studies indicate that human corneal cells exhibit normal cell phenotype when cultured individually on an engineered collagen sponge matrix and co-culture of different cell types in the cornea can influence cell behavior.     

Porosity and biological properties of polyethylene glycol-conjugated collagen materials

Collagen-based materials can be designed for use as scaffolds for connective tissue reconstruction. The goal of the present study was to evaluate the behavior of collagen materials as well as cell and tissue reactions after the conjugation of activated polyethylene glycols (PEGs) with collagen. It is known that proteins conjugated with PEGs exhibit a decrease in their biodegradation rate and their immunogenicity. Different concentrations and molecular weights of activated PEGs (PEG-750 and PEG-5000) were conjugated to collagen materials (films or sponges) which were then investigated by collagenase assay, fibroblast cell culture, and subcutaneous implantation. PEG-conjugated collagen sponge degradation by collagenase was delayed in comparison to untreated sponges. In culture, fibroblasts with a normal morphology reached confluency on PEG-conjugated collagen films. In vivo, the porous structure of non-modified sponges collapsed by day 15 with a few observable fibroblasts between the collagen fibers. In PEG-modified collagen sponges, the porous structure remained stable for 30 days. Cell infiltration was particularly enhanced in PEG-750-conjugated collagen sponges. In conclusion, PEGs conjugated onto collagen sponges stabilize the porous structure without deactivating the biological properties of collagen. These porous composite materials could function as a scaffold to organize tissue ingrowth.     

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.     

The pro-angiogenic characteristics of a cross-linked gelatin matrix

To overcome limitations on regeneration in the nervous system and other organs caused by insufficient blood supply, we have developed a gelatin sponge material which stimulates blood vessel formation, i.e. angiogenesis. Controlled chemical cross-linking was employed to slow down enzymatic degradation of the gelatin matrix. Four different in vitro assays using L929 fibroblasts and purified endothelial cells indicated that the sponge material did not release toxic components, but provided a permissive substratum for cell attachment, cell migration and pronounced cell proliferation, all of which are crucial for the formation of vasculature. Two in vivo models were employed to directly monitor the pro-angiogenic impact of the sponge material. Implantation of gelatin sponges onto the chorioallantoic membrane of fertilized chicken eggs induced robust attraction of endothelial cells and formation of blood vessels. Angiogenesis inside gelatin implants occurred more than 200 times faster than in a commercial collagen sponge. Similarly, after subcutaneous implantation of tube-like sponges into mice, an increasing immigration of cells and subsequent formation of functional vasculature became evident. Immunocytochemistry revealed no fibronection accumulation and no scarring. In summary, our matrix based on cross-linked gelatin promises to be a valuable component of future implants, improving neuronal and non-neuronal regeneration by concomitant pro-angiogenic stimulation.     

Fabrication and characterization of PLLA-chitosan hybrid scaffolds with improved cell compatibility

To combine the individual advantages of synthetic and natural polymers, poly(L-lactic acid) (PLLA)-chitosan hybrid scaffolds were fabricated. PLLA sponges were prepared by particulate-leaching, and then PLLA-chitosan hybrid scaffolds were obtained by dipping the PLLA sponges in chitosan solution and subsequently freeze-drying. Physicochemical properties of the scaffolds were characterized by scanning electron microscopy (SEM), water uptake test, and mechanical strength measurement. Moreover, cell adhesion, cell proliferation, and cell viability on the scaffolds were evaluated through osteoblast-like cell culture. The experimental results indicated that, PLLA sponges exhibited macroporous structure and the interconnected microporous structure of chitosan was formed within the macropores of PLLA sponges. The incorporation of chitosan reinforced PLLA sponges in dependence on chitosan content. The hybrid scaffolds had higher water uptake ability compared with PLLA sponges. Particularly, the hybrid scaffolds exhibited excellent cell attachment efficiency, cell proliferation, and cell viability. This study suggests that the hybrid scaffolds obtain good mechanical strength from PLLA and excellent cell compatibility from chitosan.     

Osteogenic differentiation of mesenchymal stem cells in biodegradable sponges composed of gelatin and beta-tricalcium phosphate

Biodegradable gelatin sponges incorporating various amounts of beta-tricalcium phosphate (betaTCP) (gelatin-betaTCP) were fabricated and the in vitro osteogenic differentiation of mesenchymal stem cells (MSC) isolated from the rat bone marrow in the sponges was investigated. The gelatin sponges incorporating betaTCP have an interconnected pore structure with the average size of 180-200 microm, irrespective of the betaTCP amount. The stiffness of the sponges became higher with an increase in the amount of betaTCP. When seeded into the sponges by an agitated method, MSC were homogeneously distributed throughout the sponge. The morphology of cells attached got more spreaded with the increased betaTCP amount. The rate of MSC proliferation depended on the betaTCP amount and culture method: the higher the betaTCP amount in the stirring culture, the higher the proliferation rate. The deformed extent of gelatin-betaTCP sponges was suppressed with the increased amount of betaTCP. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase activity and osteocalcin content became maximum for the sponge with a betaTCP amount of 50 wt%, although both the values were significantly high in the stirring culture compared with those in the static culture. We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by sponge composition of gelatin and betaTCP as the cell scaffold.     

Endothelial cells anchoring by functionalized yeast polypeptide

The immobilization and proliferation of endothelial cells on the surface of engineered tissues requires the development of new biomaterials that can mimic the anchoring and signaling functions of basement membrane. Here, we report a modified polypeptide from yeast translation termination factor protein that can self-assembly into nanofibers and improve endothelial cell adhesion by its functional motif. The polypeptide (YNNNLQGYQAGFQ) is a beta sheet forming sequence, but it is noninfectious in mammalian tissue because of the absence of substrate protein for propagation. The prion-derived polypeptide was extended at the amino terminal with a short sequence motif from laminin I (YIGSR), and the resultant polypeptide retained self-assembly propensity. Both circular dichroism (CD) measurement and molecular dynamics simulation suggest the assembled nanofibers consists mainly beta sheet structure. The 3D porous hydrogel formed by the modified polypeptide was evaluated as a coating material for vascular tissue engineering. In static culture system, the polypeptide scaffold improved the morphology of endothelial cells and confluency of cell monolayer. In the dynamic bioreactor (pulsatile vascular deformation at 5%), the polypeptide scaffold anchored 3-fold higher number of endothelial cells, which exhibited normal nitric oxide release function. These results suggest that prion-derived polypeptides have high self-assembling and motif integrating capacities. These unique properties can be utilized to build up biomaterials with robust porous structure as well as functionalized motifs for cell enrichment.     

Three-dimensional honeycomb-patterned chitosan/poly(L-lactic acid) scaffolds with improved mechanical and cell compatibility

Micro-size patterned surfaces trigger specific biological responses such as the promotion of cell growth, cell migration, cell differentiation, and ECM production. The aim of this work was to elaborate three-dimensional scaffolds with honeycomb patterned surfaces and large open pores, and to study the influence of surface patterning on cell behavior. In this study, we used water droplets as porogen material to prepare a novel type of chitosan sponge with large open pores on its surface. The sponges obtained were then immersed into 6 wt % Poly(L-lactic acid) chloroform solution to obtain honeycomb patterned composite porous scaffolds. The morphology and mechanical properties were characterized with SEM and compression testing. The fibroblast behaviors in scaffolds were analyzed with SEM, VG, PAS, live-dead staining, and flow cytometer. Results showed that these composite scaffolds possessed better mechanical properties and hierarchical porous structure than pure chitosan sponges. Cell culture revealed that the honeycomb patterned surface had positive influences on fibroblast behaviors, wherein the cell adhesion, proliferation, ECM secretion and viability were improved dramatically. Such a hierarchical composite scaffold would be a suitable candidate for tissue engineering purposes.     

Preparation of Open Porous Hyaluronic Acid Scaffolds for Tissue Engineering Using the Ice Particulate Template Method

A novel method to fabricate highly interconnected porous hyaluronic acid (HA) scaffolds with open surface pore structures was developed by using embossed ice particulates as a template. HA sponges were cross-linked by water-soluble carbodiimide (WSC) and the optimal cross-linking condition was analyzed by infrared spectroscopy. Cross-linking with 50 mM WSC in a 90% (v/v) ethanol/water solvent mixture assured the highest degree of cross-linking and most stable structure and, therefore, was used to cross-link the HA sponges. Observation with a scanning electron microscope showed that the HA scaffolds had funnel-like porous structures. There were large, open pores on the top surfaces and inner bulk pores under the top surface of the funnel-like HA sponges. The inner bulk pores were interconnected with the large, top surface pores and extended into the whole sponge. The pore morphology and density of the large, top surface pores were dependent on the dimension and density of the ice particulates. The size of the inner bulk pores was dependent on the freezing temperature. The funnel-like pore structures of the HA sponges facilitated cell penetration into the inner pores of the sponges and resulted in homogenous cell distribution in the sponges.     

细胞共培养技术促进类前微血管结构发生

背景：三维(3D)组织化培养模型的体外构建是现代组织工程与再生医学工程技术的重要核心。如何实现所培养模型的微血管化以改善培养体系内部的营养传递并最终提高细胞的活性是组织工程研究领域所亟待解决的关键。 目的：尝试探索在3D体系内采用细胞共培养技术促进类前微血管结构发生的可行性。 方法：以蚕丝蛋白为多孔材料支架,将人骨髓间充质干细胞与血管内皮细胞进行体外共培养。通过DNA含量测定定量检测细胞的增殖;扫描电镜和激光共聚焦显微镜的图像分析表征细胞的生长形态学特征;实时定量RT-PCR方法对内皮细胞功能性标志基因的表达水平进行定量分析。 结果与结论：丝蛋白支架和人骨髓间充质干细胞能够提供理想的3D生长微环境,利于血管内皮细胞的体外增殖。微环境还能够显著提高内皮细胞功能性标志基因CD31和vWF的表达水平,促进类前微血管结构的发生。提示共培养体系有利于内皮细胞在体外的进一步分化和自组织化,可能为微血管化组织工程研究提供一定的技术基础。     

Osteoblastic response to collagen scaffolds varied in freezing temperature and glutaraldehyde crosslinking

Collagen sponges are widely used scaffolds in bone engineering. To form bone, the osteoblastic cells undergo proliferation, differentiation, and mineralization stages in the scaffold. Crosslinking and freezing temperature are two important variables in fabricating collagen sponges. The purpose of this study was to examine the osteoblastic responses to collagen sponges prepared with or without glutaraldehyde crosslinking at different freezing temperatures (-20 degrees C or -80 degrees C). MC3T3-E1 osteoblastic cells were cultured in differently prepared sponges. Osteoblastic responses examined included cell numbers, osteocalcin expression, and calcium deposition. Cell numbers were measured by DNA content. Osteocalcin expression was determined by RT-PCR and real-time RT-PCR. Calcium deposition was assayed by ortho-cresophthalein complexone method and von Kossa stain. The osteoblastic cells grown in all collagen sponges did not show apparent signs of cytotoxicity. Collagen sponges differed in freezing temperatures resulted in similar osteoblastic responses. Glutaraldehyde-crosslinked sponges demonstrated less cell-mediated contraction and more cell numbers at day 7 (p < 0.005). However, they showed lower osteocalcin expression at day 7 (p < 0.05) and less calcium deposition at day 21 (p < 0.001). In summary, different freezing temperatures played a minor role in osteoblastic responses. Glutaraldehyde crosslinking process, though improved the dimensional stability of collagen sponges, might compromise the osteoblastic differentiation and mineralization.     

Proliferation and differentiation of rat bone marrow stromal cells on poly(glycolic acid)-collagen sponge

We studied the effects of dexamethasone (Dex) and basic fibroblast growth factor (bFGF) on proliferation and differentiation of rat bone marrow stromal cells (RBMSCs), using three scaffolds: collagen sponge, poly(glycolic acid) (PGA)-collagen sponge, and PGA-collagen (UV) sponge. RBMSCs were seeded into the sponges, and cultured in primary medium, primary medium with Dex, and primary medium with bFGF and Dex. Three weeks after cultivation, we examined alkaline phosphatase (ALP) activity and cell number in the sponges, and also performed macroscopic, light microscopic, and scanning electron microscopic (SEM) observations. Collagen sponge shrank considerably, but PGA-collagen and PGA-collagen (UV) sponges maintained most of their original shape. PGA-collagen (UV) sponge supplemented with bFGF and Dex together had the highest ALP activity and cell number, followed by PGA-collagen sponge. Although collagen sponge showed cell proliferation only on the surface, the other two sponges showed cell proliferation in the interior. SEM showed the best cell attachment to PGA-collagen (UV) sponge in the presence of bFGF and Dex, followed by PGA-collagen sponge. In conclusion, PGA-collagen (UV) and PGA-collagen sponges proved to be much more useful as scaffolding for bone regeneration when combined with bFGF and Dex.     

Co-culture of bone marrow fibroblasts and endothelial cells on modified polycaprolactone substrates for enhanced potentials in bone tissue engineering

The creation of a vascularized bed makes the survival of seeded cells on 3-dimensional scaffolds much more likely. However, relying purely on random capillary ingrowth into the porous scaffolds from the host may compromise vascularization of a scaffold. One solution is to transplant cells capable of differentiating into new blood vessels into the scaffolds to accelerate the creation of a vascularized scaffold. Because endothelial cells are the key cells involved in blood vessel formation, the present study was designed to investigate the development of a biomaterial surface that supports endothelial cell attachment and proliferation. The subsequent effects of the material surface modifications on the differentiation and proliferation of human bone marrow-derived fibroblasts (HBMFs) when grown in co-culture with a human bone marrow endothelial cell line (HBMEC-60) were studied. Endothelialization studies showed that the gelatin-coated and hydroxyapatite-coated substrates were superior for HBMEC-60 attachment and proliferation to hydrolyzed-only or untreated polycaprolactone substrates. Co-culture studies showed that the presence of the HBMEC-60 specifically enhanced HBMF cell proliferation and differentiation and that this effect was not observed with co-culture with skin fibroblasts. It is concluded that the co-culture of endothelial cells with HBMFs could be a promising culture system for bone tissue- engineering applications.     

Three-dimensional culture of human osteoblastic cells in chitosan sponges: the effect of the degree of acetylation

In this investigation, the effect of the degree of acetylation (DA) of chitosan on the behavior of human osteoblastic MG-63 cells cultured in three-dimensional chitosan matrices was assessed. Chitosan sponges with DAs in the range of 4 to 49% were prepared and characterized in terms of microstructure, porosity, and pore size. Collagen sponges were used as 3D control. Cell proliferation was determined using the MTT assay while the retention of the osteoblastic phenotype was monitored by assaying alkaline phosphatase activity. Cell morphology, cytoskeletal organization, and viability were assessed using different microscopy techniques. Chitosan sponges showed a similar microstructure regardless the DA, except for the highest DA used, where a more heterogeneous pore distribution was observed. In terms of cell proliferation, alkaline phosphatase activity and cell viability, cells cultured in chitosan scaffolds performed as well as in the 3D control regardless the DA, except for the highest DA used, where an inhibitory effect on cell proliferation was found. However, while in sponges with DAs < or = 13% cells attached and spread displaying long cell filopodia and numerous cell-to-cell contacts, in sponges with higher DAs cells tended to remain spherical and grow into spheroid-like cellular aggregates. In the present study, the DA played a key role in determining the affinity of osteoblastic cells towards the substrates, possibly by influencing the nature of the initial adsorbed protein layer.     

The precision structural regulation of PLLA porous scaffold and its influence on the proliferation and differentiation of MC3T3-E1 cells

A thermal-induced phase separation combined sugar template method was used to fabricate the Poly (L-lactide) acid (PLLA) scaffolds with precisely regulated porous structure. The effect of tuned porous structure of scaffolds on osteoblasts proliferation and differentiation was investigated. The results showed that the pore diameters (200–300, 300–400, 400–500 μm), porosity and interconnectivity of PLLA scaffolds can be accurately controlled indicated by scanning electron microscope. The results of cell experiments showed that the porous structure including the pore size and interconnectivity of scaffolds dramatically influence the cell proliferation and differentiation. The scaffold with pore diameter of 400–500 μm exhibited the highest cell viability and alkaline phosphatase activity among all the scaffolds for the MC3T3-E1 cells. The higher cell proliferation and biocompatibility observed in the 400–500 μm scaffold indicated the high selectivity for MC3T3-E1cells on the pore size of scaffold in tissue engineering. The precise control of the porous structure of scaffold may better guide the cell–matrix interaction in the future research.     

Disulfide-crosslinked hyaluronan-gelatin sponge: growth of fibrous tissue in vivo

The modification of hyaluronan (HA) and gelatin using dithiobis(propanoic dihydrazide) (DTP) has provided two thiolated macromolecular components of the extracellular matrix (ECM), specifically HA-DTPH and gelatin-DTPH. Blends of these thiolated ECM components were crosslinked in air to form hydrogels that were interpenetrating disulfide-crosslinked networks. Lyophilization of the hydrogels afforded sponge-like macroporous scaffolds suitable for cell attachment and proliferation. Increasing percentages of gelatin-DTPH (0, 25, 50, and 75%) were blended with HA-DTPH, and the resulting sponges were evaluated in vitro and in vivo as scaffolds for tissue engineering by seeding with human tracheal scar (HTS) fibroblasts. While cells failed to attach and grow in HA-only sponges, the gelatin-modified HA sponges promoted cell adhesion, proliferation, and spreading in vitro. Optimal attachment and growth was observed with 50% gelatin-HA sponges. Cell attachment to the gelatin-HA sponge could be blocked by preincubation of cells with a soluble fibronectin peptide Gly-Arg-Gly-Asp (GRGD). Finally, HTS fibroblast-seeded gelatin-HA sponges were implanted into the flanks of nude mice and evaluated at 2 and 8 weeks postimplantation. The sponges were fully biocompatible and new fibrous tissue formed, gradually replacing the sponge-like scaffold. The gelatin-HA sponges act as synthetic, macroporous, covalent mimics of the ECM and constitute novel scaffolds for cell growth and tissue augmentation.     

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.     

Enhanced proliferation and osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with different poly(ethylene terephthalate) fibers

The objective of this study was to investigate the feasibility of collagen sponges mechanically reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers in stem cell culture. A collagen solution with homogeneously dispersed PET fibers was freeze-dried, followed by dehydrothermal cross-linking to obtain the collagen sponge incorporating PET fibers. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 200 microm, irrespective of PET fiber incorporation. As expected, PET fibers incorporation significantly enhanced the compression strength of collagen sponge. When used for rat mesenchymal stem cells (MSC), the collagen sponge incorporating PET fibers was superior to the original collagen sponge without PET fibers incorporation in terms of the initial attachment, proliferation and osteogenic differentiation of cells, irrespective of the amount and diameter of fibers incorporated. The shrinkage of sponges during cell culture was significantly suppressed by the fiber incorporation. It is possible that the shrinkage suppression maintains the three-dimensional inner pore structure of collagen sponges without impairing the cell compatibility, resulting in the superior MSC attachment and the subsequent osteogenic differentiation in the sponge incorporating PET fiber.