Fabrication and biocompatibility of collagen sponge reinforced with poly(glycolic acid) fiber

This article describes an investigation of collagen sponge mechanically reinforced through the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously dispersed at collagen:PGA weight ratios of 1.5, 0.8, 0.4, and 0.2 was freeze-dried, followed by dehydrothermal cross-linking to obtain collagen sponges incorporating PGA fiber to various extents. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. As expected, PGA fiber incorporation enabled the collagen sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly greater for any collagen sponge incorporating PGA fiber than for collagen sponge. The shrinkage of sponge after cell seeding was suppressed by fiber incorporation. It is possible that shrinkage suppression results in the superior cell attachment of sponge incorporating PGA fiber. After subcutaneous implantation into the backs of mice, the residual volume of collagen sponge incorporating PGA fiber was significant compared with that of collagen sponge and increased with a decrease in the collagen:PGA ratio. The greater number of cells infiltrated and deeper infiltration were observed for collagen sponge incorporating PGA fiber implanted subcutaneously. We conclude that the incorporation of PGA fiber is a simple and promising way to reinforce collagen sponge without impairing biocompatibility.     

Perfusion culture enhances osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with poly(glycolic Acid) fiber

The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation.     

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.     

Preparation of hybrid scaffold from fibrin and biodegradable polymer fiber

A biodegradable hybrid scaffold was prepared from fibrin and poly(glycolic acid) (PGA) fiber. Mixed fibrinogen and thrombin solution homogeneously dispersed in the presence of various amounts (0, 1.5, 3.0, and 6.0mg) of PGA fiber was freeze-dried to obtain fibrin sponges with or without PGA fiber incorporation. By scanning electron microscopy observation, the fibrin sponges had an interconnected pore structure, irrespective of the amount of PGA fiber incorporated. PGA fiber incorporation enabled the fibrin sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly larger for any fibrin sponge with PGA fiber incorporation than for the fibrin sponge without PGA fiber. The shrinkage of sponges after cell seeding was suppressed by fiber incorporation. It is possible that the shrinkage suppression of sponges maintains their intraspace, resulting in the superior cell attachment of a sponge with PGA fiber incorporation. After subcutaneous implantation into the backs of mice, the residual volume of a fibrin sponge with PGA fiber incorporation was significant compared with that of a fibrin sponge without PGA fiber. Larger number of cells infiltrated deep inside the fibrin sponges with PGA fiber incorporation implanted subcutaneously. It is concluded that the fibrin sponge reinforced by fiber incorporation is a promising three-dimensional scaffold of cells for tissue engineering.     

Proliferation of Rat Mesenchymal Stem Cells in Collagen Sponges Reinforced with Poly(Ethylene Terephthalate) Fibers by Stirring Culture Method

Abstract

The objective of this study is to investigate the effect of medium stirring conditions on the proliferation of rat mesenchymal stem cells (MSC) in collagen sponges reinforced by the incorporation of poly(ethylene terephthalate) (PET) fibers. A collagen solution with PET fibers homogeneously dispersed was freeze-dried, followed by dehydrothermal cross-linking to obtain a collagen sponge incorporating PET fibers. MSC were proliferated in the sponge by a stirring culture method. The PET fibers reinforcement significantly suppressed the sponge deformation in culture. The MSC proliferation was enhanced by the stirring culture to a significantly higher extent than that of a static one. Homogeneous distribution of cells proliferated was observed at the stirring rate of 50 rpm and compared with that at lower and higher rates. Combination of the PET fiber-reinforced sponge with the stirring culture method is a promising way to allow cells to homogeneously proliferate in the sponge.     

Magnesium calcium phosphate as a novel component enhances mechanical/physical properties of gelatin scaffold and osteogenic differentiation of bone marrow mesenchymal stem cells

Biodegradable gelatin sponges incorporating various amounts of magnesium calcium phosphate (MCP) were introduced and the in vitro osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs) in the sponges was investigated. The MCP was added to the gelatin sponges at 0, 25, 50, 75, and 90 wt%. The pore sizes of the gelatin sponges ranged from 143 to 154.3 μm in diameter and the porosity percentage was 34.3-50.1%. The compression modulus of the sponges and the resistance to the volume change significantly increased with increases in the amount of MCP. When seeded into the sponges by an agitating method, MSCs were distributed throughout the sponges. Following the incubation of MSCs in the gelatin sponges, a significantly higher cellular proliferation and alkaline phosphatase activity was observed in the gelatin sponges incorporating higher MCP contents. On the other hand, the osteocalcin content of MSCs seeded in the gelatin sponges incorporating no or low MCP showed a significantly higher levels in comparison with the MSCs seeded in the gelatins incorporating high MCP. These findings indicate that the MCP incorporation maintained the pore size and porosity percentage of the gelatin sponges and enabled the sponge to achieve mechanical reinforcement as well as promoting MSC proliferation and osteogenic differentiation.     

Effect of the fiber diameter and porosity of non-woven PET fabrics on the osteogenic differentiation of mesenchymal stem cells

The proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) was investigated in three-dimensional non-woven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. When seeded into the fabrics of cell scaffold, more MSC attached in the fabric of thicker PET fibers than that of thinner ones, irrespective of the fabric porosity. The morphology of cells attached became more spreaded with an increase in the fiber diameter of fabrics. The rate of MSC proliferation depended on the PET fiber diameter and porosity of fabrics: the bigger the fiber diameter of fabrics with higher porosity, the higher their proliferation rate. When the alkaline phosphatase (ALP) activity and osteocalcin content of MSC cultured in different types of fabrics was measured to evaluate the ostegenic differentiation, they became maximum for the non-woven fabrics with a fiber diameter of 9.0 microm, although the values of low-porous fabrics were significantly high compared with those of high porous fabrics. We concluded that the attachment, proliferation and bone differentiation of MSC was influenced by the fiber diameter and porosity of non-woven fabrics as the scaffold.     

Effect of the fiber diameter and porosity of non-woven PET fabrics on the osteogenic differentiation of mesenchymal stem cells

The proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) was investigated in three-dimensional non-woven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. When seeded into the fabrics of cell scaffold, more MSC attached in the fabric of thicker PET fibers than that of thinner ones, irrespective of the fabric porosity. The morphology of cells attached became more spreaded with an increase in the fiber diameter of fabrics. The rate of MSC proliferation depended on the PET fiber diameter and porosity of fabrics: the bigger the fiber diameter of fabrics with higher porosity, the higher their proliferation rate. When the alkaline phosphatase (ALP) activity and osteocalcin content of MSC cultured in different types of fabrics was measured to evaluate the ostegenic differentiation, they became maximum for the non-woven fabrics with a fiber diameter of 9.0 μm, although the values of low-porous fabrics were significantly high compared with those of high porous fabrics. We concluded that the attachment, proliferation and bone differentiation of MSC was influenced by the fiber diameter and porosity of non-woven fabrics as the scaffold.     

Reinforcement of a Porous Collagen Scaffold with Surface-Activated PLA Fibers

A hybrid porous collagen scaffold mechanically reinforced with surface-activated poly(lactic acid) (PLA) fiber was prepared. PLA fibers, 20 μm in diameter and 1 mm in length, were aminolyzed with hexanediamine to introduce free amino groups on the surfaces. After the amino groups were transferred to aldehyde groups by treatment with glutaraldehyde, different amounts (1.5, 3, 5 and 8 mg) of surface-activated PLA fibers were homogeneously mixed with 2 ml type-I collagen solution (pH 2.8, 0.6 wt%). This mixture solution was then freeze-dried and cross-linked to obtain collagen sponges with surface-activated PLA fiber. Scanning electron microscopy observation indicated that the collagen sponges had a highly interconnected porous structure with an average pore size of 170 μm, irrespective of PLA fiber incorporation. The dispersion of surface-activated PLA fibers was homogeneous in collagen sponge, in contrast to unactivated PLA fibers. The compression modulus test results showed that, compared with unactivated PLA fibers, the surface-activated PLA fibers enhanced the resistance of collagen sponge to compression more significantly. Cytotoxicity assay by MTT test showed no cytotoxicity of these collagen sponges. L929 mouse fibroblast cell-culture studies in vitro revealed that the number of L929 cells attached to the collagen sponge with surface-activated PLA fibers, both 6 h and 24 h after seeding, was higher than that in pure collagen sponge and sponge with unactivated PLA fibers. In addition, a better distribution of cells infiltrated in collagen sponge with surface-activated PLA fibers was observed by histological staining. These results indicated that the collagen sponge reinforced with surface-activated PLA fibers is a promising biocompatible scaffold for tissue engineering.     

Impregnation of plasmid DNA into three-dimensional scaffolds and medium perfusion enhance in vitro DNA expression of mesenchymal stem cells

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.     

Enhanced ectopic bone formation using a combination of plasmid DNA impregnation into 3-D scaffold and bioreactor perfusion culture

The objective of this study is to enhance in vivo ectopic bone formation by combination of plasmid DNA impregnation into three-dimensional (3-D) cell scaffolds and a developed in vitro culture method. Gelatin was cationized by introducing spermine (Sm) to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, collagen sponge reinforced by incorporation of poly(glycolic acid) (PGA) fibers was used. A complex of the cationized gelatin and plasmid DNA of BMP-2 was impregnated into the scaffold. MCS were seeded into each scaffold and cultured by a static and perfusion methods. When MSC were cultured in the PGA-reinforced collagen sponge, the level of BMP-2 expression was significantly enhanced by the perfusion culture compared with static method. When the osteoinduction activity of the PGA-reinforced collagen sponges seeded with PBS, MSC, naked plasmid DNA-BMP-2, cationized gelatin-plasmid DNA-BMP-2 complex, and transfected MSC by static and perfusion method, were studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges seeded with cationized gelatin-plasmid DNA of BMP-2 complex and transfected MSC by perfusion method, although the extent of bone formation was higher for the later one. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by perfusion method were significantly high compared with those seeded with other agents. We conclude that combination of plasmid DNA-impregnated PGA-reinforced collagen sponge and the perfusion method was promising to promote the in vitro gene expression for MSC and in vivo ectopic bone formation.     

Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate

Biodegradable gelatin sponges at different contents of beta-tricalcium phosphate (beta-TCP) were fabricated to allow bone morphogenetic protein (BMP)-2 to incorporate into them. The in vivo osteoinduction activity of the sponges incorporating BMP-2 was investigated, while their in vivo profile of BMP-2 release was evaluated. The sponges prepared had an interconnected pore structure with an average pore size of 200 microm, irrespective of the beta-TCP content. The in vivo release test revealed that BMP-2 was released in vivo at a similar time profile, irrespective of the beta-TCP content. The in vivo time period of BMP-2 retention was longer than 28 days. When the osteoinduction activity of gelatin or gelatin-beta-TCP sponges incorporating BMP-2 was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges, although the extent of bone formation was higher in the sponges with the lower contents of beta-TCP. On the other hand, the level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges decreased with an increase in the content of beta-TCP. The gelatin sponge exhibited significantly higher osteoinduction activity than that of any gelatin-beta-TCP sponge, although every sponge with or without beta-TCP showed a similar in vivo profile of BMP-2 release. In addition, the in vitro collagenase digestion experiments revealed that the gelatin-beta-TCP sponge collapsed easier than the gelatin sponge without beta-TCP incorporation. These results suggest that the maintenance of the intrasponge space necessary for the osteoinduction is one factor contributing to the osteoinduction extent of BMP-2-incorporating sponges.     

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells

The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.     

Viability and proliferation of rat MSCs on adhesion protein-modified PET and PU scaffolds

In 2011, the first in-man successful transplantation of a tissue engineered trachea-bronchial graft, using a synthetic POSS-PCU nanocomposite construct seeded with autologous stem cells, was performed. To further improve this technology, we investigated the feasibility of using polymers with a three dimensional structure more closely mimicking the morphology and size scale of native extracellular matrix (ECM) fibers. We therefore investigated the in?vitro biocompatibility of electrospun polyethylene terephthalate (PET) and polyurethane (PU) scaffolds, and determined the effects on cell attachment by conditioning the fibers with adhesion proteins. Rat mesenchymal stromal cells (MSCs) were seeded on either PET or PU fiber-layered culture plates coated with laminin, collagen I, fibronectin, poly-d-lysine or gelatin. Cell density, proliferation, viability, morphology and mRNA expression were evaluated. MSC cultures on PET and PU resulted in similar cell densities and amounts of proliferating cells, with retained MSC phenotype compared to data obtained from tissue culture plate cultures. Coating the scaffolds with adhesion proteins did not increase cell density or cell proliferation. Our data suggest that both PET and PU mats, matching the dimensions of ECM fibers, are biomimetic scaffolds and, because of their high surface area-to-volume provided by the electrospinning procedure, makes them per se suitable for cell attachment and proliferation without any additional coating.     

Ectopic bone formation in collagen sponge self-assembled peptide-amphiphile nanofibers hybrid scaffold in a perfusion culture bioreactor

The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide-amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture method compared with static culture method. When the osteoinduction activity of hybrid scaffold was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds when perfusion culture was used compared with static culture method. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture method. We conclude that combination of MSC-seeded hybrid scaffold and the perfusion method was promising to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.     

Osteogenic Differentiation of Bone-Marrow-Derived Stem Cells Cultured with Mixed Gelatin and Chitooligosaccharide Scaffolds

This study investigates the effect of scaffolds prepared from gelatin (G) and chitooligosaccharide (COS) on the osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (MSC). The sponge scaffolds at G/COS mixing ratios of 100:0, 70:30 and 50:50 were fabricated by freeze-drying, followed by glutaraldehyde cross-linking. The pore size of the G/COS scaffolds ranged from 70 to 105 μm. MSC cultured in the scaffolds in the osteogenic medium were differentiated into osteogenic cells for all G/COS scaffolds. Calcium nodules were homogeneously formed on the surface of scaffolds cultured with MSC. A Fourier transform infrared (FT-IR) analysis demonstrated the formation of hydroxyapatite spectroscopically. Among all G/COS scaffolds, the highest ALP activity and calcium content were observed for MSC cultured in the G/COS 70:30 scaffolds. The G/COS 70:30 scaffolds were then pre-cultured with MSC in the osteogenic medium for 28 days and subcutaneously implanted into nude mice to evaluate ectopic bone formation. Enhanced vascularization, cell infiltration, collagen formation and calcium deposition around the scaffolds implanted were histologically observed at 2 and 8 weeks after implantation. It was concluded that the G/COS scaffold with the mixing ratio of 70:30 was a promising organic material to induce osteogenic differentiation of MSC.     

Natural marine sponge fiber skeleton: a biomimetic scaffold for human osteoprogenitor cell attachment, growth, and differentiation

Identification of suitable scaffolds onto which human stem cells can be seeded to generate functional three-dimensional tissues is a major research goal. A natural marine sponge skeleton was selected as a potential scaffold on the basis of the hydration potential of the fiber, the presence of open interconnected channels created by the fiber network, the collagenous composition of the fiber, and the structural diversity of fiber architecture. The skeleton of an undetermined species of Spongia (Class Demospongiae: Order Dictyoceratida: Family Spongiidae), composed of spongin, supported growth of human osteoprogenitor cells. Cell attachment and invasion into the framework were observed within 16 h, followed by development into membranous sheets between the sponge fibers by concentric infilling. Histochemical staining for alkaline phosphatase and type I collagen indicated formation of bone matrix as confirmed by birefringence. At 9 and 14 days alkaline phosphatase-specific activity in sponge fiber-osteoprogenitor cell cultures was significantly greater than in control cultures on cell culture plastic. Adsorption with recombinant human bone morphogenetic protein 2 confirmed the potential of this sponge skeleton as a delivery scaffold for osteogenic factors. The abundance and structural diversity of natural marine sponge skeletons and their potential as multifunctional, cell conductive and inductive frameworks indicate a promising new source of scaffold for tissue regeneration.     

Preparation of stem cell aggregates with gelatin microspheres to enhance biological functions

The objective of this study is to improve the viability and osteogenic differentiation of cultured rat bone marrow-derived mesenchymal stem cells (MSC) by the use of gelatin hydrogel microspheres. Gelatin was dehydrothermally crosslinked at 140° C for 48 h in a water in oil emulsion state. When cultured with the gelatin hydrogel microspheres in round, U-bottomed wells of 96-well plates coated with poly(vinyl alcohol) MSC formed aggregates homogeneously incorporating the microspheres. The viability of the cell aggregates was significantly higher compared with that of aggregates formed without microspheres. MSC proliferation in the aggregates depended on the number and diameter of the incorporated microspheres. Higher MSC proliferation was observed for aggregates incorporating a greater number of larger gelatin microspheres. When evaluated as a measure of aerobic glycolysis the ratio of l-lactic acid production/glucose consumption in MSC was significantly lower for MSC cultured with gelatin microspheres than those without microspheres. MSC production of alkaline phosphatase (ALP) and sulfated glycosaminaglycan (sGAG) was examined to evaluate their potential osteogenic and chondrogenic differentiation. The amount of ALP produced was significantly higher for MSC aggregates cultured with gelatin microspheres than that of MSC cultured without microspheres. On the other hand, the amount of sGAG produced was significantly lower for MSC aggregates containing microspheres. It is concluded that the incorporation of gelatin hydrogel microspheres prevents the aggregated MSC suffering from a lack of oxygen, resulting in enhanced MSC aggregation and cell proliferation and osteogenic differentiation.     

Collagen-calcium phosphate cement scaffolds seeded with umbilical cord stem cells for bone tissue engineering

Human umbilical cord mesenchymal stem cells (hUCMSCs) avoid the invasive procedure required to harvest bone marrow MSCs. The addition of collagen fibers into self-setting calcium phosphate cement (CPC) may increase the scaffold strength, and enhance cell attachment and differentiation. The objectives of this study were to develop a novel class of collagen-CPC composite scaffolds, and to investigate hUCMSC attachment, proliferation, and osteogenic differentiation on collagen-CPC scaffolds for the first time. Collagen fibers in CPC improved the load-bearing capability. Flow cytometry showed that the hUCMSCs expressed cell surface markers characteristic of MSCs, and were negative for hematopoietic and endothelial cell markers. hUCMSCs proliferated rapidly in all CPC composite scaffolds, with cell number increasing by sevenfold in 8 days. Cellular function was enhanced with collagen fibers in CPC scaffolds. Cell density increased from (645±60) cells/mm(2) on CPC with 0% collagen, to (1056±65) cells/mm(2) on CPC with 8% collagen (p<0.05). The actin stress fibers inside the hUCMSCs were stained, and the fluorescence intensity was doubled when the collagen in CPC was increased by 0% to 8%. RT-PCR showed that hUCMSCs on CPC with collagen had higher osteogenic expression than those on CPC without collagen. Alizarin Red S staining revealed a great increase in mineralization by hUCMSCs on CPC with collagen than that without collagen. In conclusion, hUCMSCs showed excellent proliferation, differentiation, and synthesis of bone minerals in collagen-CPC composite scaffolds for the first time. The novel hUCMSC-seeded collagen-CPC construct with superior cell function and load-bearing capability is promising to enhance bone regeneration in a wide range of orthopedic and craniofacial applications.