Flow perfusion culture induces the osteoblastic differentiation of marrow stroma cell-scaffold constructs in the absence of dexamethasone

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.     

Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor

This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(epsilon-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities-- 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination with the culture conditions, may affect the functionality of tissues formed in vitro.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used.     

Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model

The aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site. Bone marrow stromal osteoblasts were seeded on titanium fiber mesh scaffolds using a cell suspension (5 x 10(5) cells per scaffold) and cultured for 1, 4, and 8 days under either static or flow perfusion conditions forming six different treatment groups. A total of 16 constructs from each one of the six treatment groups were then implanted into an 8-mm critical size calvarial defect created in the cranium of adult syngeneic male Fisher rats. Half of the constructs from each group were retrieved 7 days postimplantation, and the other half of the constructs were retrieved 30 days postimplantation and examined for new bone formation and tissue response. Constructs retrieved 7 days postimplantation were filled with fibrous tissue and capillaries, but no bone formation was observed in any of the six treatment groups. Constructs retrieved 30 days postimplantation showed bone formation (at least 7 out of 8 constructs in all treatment groups). Titanium fiber meshes seeded with bone marrow stromal osteoblasts and cultured for 1 day under flow perfusion conditions before implantation appeared to give the highest percentage of bone formation per implant (64 +/- 17%). They also showed the highest ratio of critical size cranial defects that resulted in union of the defect 30 days postimplantation (7 out of 8) together with the constructs cultured for 1 day under static conditions before implantation. There were no significant differences between the different treatment groups; this finding is most likely due to the large variability of the results and the small number of animals per group. However, these results show that titanium fiber mesh scaffolds loaded with bone marrow stromal osteoblasts can have osteoinductive properties when implanted in an orthotopic site. They also indicate the importance of the stage of the osteoblastic differentiation and the quality of the in vitro generated extracellular matrix in the observed osteoinductive potential.     

Fabrication and characterization of poly(propylene fumarate) scaffolds with controlled pore structures using 3-dimensional printing and injection molding

Poly(propylene fumarate) (PPF) is an injectable, biodegradable polymer that has been used for fabricating preformed scaffolds in tissue engineering applications because of in situ crosslinking characteristics. Aiming for understanding the effects of pore structure parameters on bone tissue ingrowth, 3-dimensional (3D) PPF scaffolds with controlled pore architecture have been produced in this study from computer-aided design (CAD) models. We have created original scaffold models with 3 pore sizes (300, 600, and 900 microm) and randomly closed 0%, 10%, 20%, or 30% of total pores from the original models in 3 planes. PPF scaffolds were fabricated by a series steps involving 3D printing of support/build constructs, dissolving build materials, injecting PPF, and dissolving support materials. To investigate the effects of controlled pore size and interconnectivity on scaffolds, we compared the porosities between the models and PPF scaffolds fabricated thereby, examined pore morphologies in surface and cross-section using scanning electron microscopy, and measured permeability using the falling head conductivity test. The thermal properties of the resulting scaffolds as well as uncrosslinked PPF were determined by differential scanning calorimetry and thermogravimetric analysis. Average pore sizes and pore shapes of PPF scaffolds with 600- and 900-microm pores were similar to those of CAD models, but they depended on directions in those with 300-microm pores. Porosity and permeability of PPF scaffolds decreased as the number of closed pores in original models increased, particularly when the pore size was 300 microm as the result of low porosity and pore occlusion. These results show that 3D printing and injection molding technique can be applied to crosslinkable polymers to fabricate 3D porous scaffolds with controlled pore structures, porosity, and permeability using their CAD models.     

Corrugated round fibers to improve cell adhesion and proliferation in tissue engineering scaffolds

Optimal cell interaction with biomaterial scaffolds is one of the important requirements for the development of successful in vitro tissue-engineered tissues. Fast, efficient and spatially uniform cell adhesion can improve the clinical potential of engineered tissue. Three-dimensional (3-D) solid free form fabrication is one widely used scaffold fabrication technique today. By means of deposition of polymer fibers, scaffolds with various porosity, 3-D architecture and mechanical properties can be prepared. These scaffolds consist mostly of solid round fibers. In this study, it was hypothesized that a corrugated fiber morphology enhances cell adhesion and proliferation and therefore leads to the development of successful in vitro tissue-engineered constructs. Corrugated round fibers were prepared and characterized by extruding poly(ethylene oxide terephthalate)-co-poly(butylene terephthalate) (300PEOT55PBT45) block co-polymer through specially designed silicon wafer inserts. Corrugated round fibers with 6 and 10 grooves on the fiber surface were compared with solid round fibers of various diameters. The culture of mouse pre-myoblast (C2C12) cells on all fibers was studied under static and dynamic conditions by means of scanning electron microscopy, cell staining and DNA quantification. After 7 days of culturing under static conditions, the DNA content on the corrugated round fibers was approximately twice as high as that on the solid round fibers. Moreover, under dynamic culture conditions, the cells on the corrugated round fibers seemed to experience lower mechanical forces and therefore adhered better than on the solid round fibers. The results of this study show that the surface architecture of fibers in a tissue engineering scaffold can be used as a tool to improve the performance of the scaffold in terms of cell adhesion and proliferation.     

Fused deposition modeling of novel scaffold architectures for tissue engineering applications.

Fused deposition modeling, a rapid prototyping technology, was used to produce novel scaffolds with honeycomb-like pattern, fully interconnected channel network, and controllable porosity and channel size. A bioresorbable polymer poly(epsilon-caprolactone) (PCL) was developed as a filament modeling material to produce porous scaffolds, made of layers of directionally aligned microfilaments, using this computer-controlled extrusion and deposition process. The PCL scaffolds were produced with a range of channel size 160-700 microm, filament diameter 260-370 microm and porosity 48-77%, and regular geometrical honeycomb pores, depending on the processing parameters. The scaffolds of different porosity also exhibited a pattern of compressive stress-strain behavior characteristic of porous solids under such loading. The compressive stiffness ranged from 4 to 77 MPa, yield strength from 0.4 to 3.6 MPa and yield strain from 4% to 28%. Analysis of the measured data shows a high correlation between the scaffold porosity and the compressive properties based on a power-law relationship.     

The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.     

Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds with Different Architectures

Engineered bone grafts have been generated in static and dynamic systems by seeding and culturing osteoblastic cells on 3-D scaffolds. Seeding determines initial cellularity and cell spatial distribution throughout the scaffold, and affects cell–matrix interactions. Static seeding often yields low seeding efficiencies and poor cell distributions; thus creating a need for techniques that can improve these parameters. We have evaluated the effect of oscillating flow perfusion on seeding efficiency and spatial distribution of MC3T3-E1 pre-osteoblastic cells in fibrous polystyrene matrices (20, 35 and 50-μm fibers) and foams prepared by salt leaching, using as controls statically seeded scaffolds. An additional control was investigated where static seeding was followed by unidirectional perfusion. Oscillating perfusion resulted in the most efficient technique by yielding higher seeding efficiencies, more homogeneous distribution and stronger cell–matrix interactions. Cell surface density increased with inoculation cell number and then reached a maximum, but significant detachment occurred at greater flow rates. Oxygen plasma treatment of the fibers greatly improved seeding efficiency. Having similar porosity and dimensions, fibrous matrices yielded higher cell surface densities than foams. Fluorescence microscopy and histological analyses in polystyrene and PLLA scaffolds demonstrated that perfusion seeding produced more homogeneous cell distribution, with fibrous matrices presenting greater uniformity than the foams.     

Utilizing acid pretreatment and electrospinning to improve biocompatibility of poly(glycolic acid) for tissue engineering

Poly(glycolic acid) (PGA) has a long history as a bioresorbable polymer. Its biocompatibility is widely accepted, yet PGA is often rejected as a soft-tissue scaffold because of fibrous encapsulation. The goal of this study was to improve the soft-tissue biocompatibility of PGA by producing scaffolds composed of small-diameter fibers through electrospinning and subjecting these scaffolds to a concentrated hydrochloric acid (HCL) pretreatment. The theory is that small-diameter fibers will elicit a reduced immune response and HCl treatment will improve cellular interactions. Scaffolds were characterized in terms of fiber diameter and pore area via image-analysis software. Biocompatibility was assessed through a WST-1 cell-proliferation assay (in vitro) with the use of rat cardiac fibroblasts and rat intramuscular implantations (in vivo). Fibers produced ranged in diameter from 0.22 to 0.88 microm with pore areas from 1.84 to 13.22 microm(2). The untreated scaffold composed of 0.88-microm fibers was encapsulated in vivo and supported the lowest rates of cell proliferation. On the contrary, the acid pretreated scaffold with 0.22-microm fibers was incorporated into the surrounding tissue and exhibited proliferation rates that exceeded the control populations on tissue-culture plastic. In conclusion, this study has shown the ability to improve the biocompatibility of PGA through acid pretreatment of scaffolds comprised of submicron fiber diameters.     

Osteogenic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(epsilon-caprolactone) fabricated via co-extrusion and gas foaming

A novel scaffold fabrication method utilizing both polymer blend extrusion and gas foaming techniques to control pore size distribution is presented. Seventy-five per cent of all pores produced using polymer blend extrusion alone were less than 50mum. Introducing a gas technique provided better control of pore size distribution, expanding the range from 0-50 to 0-350mum. Varying sintering time, annealing temperature and foaming pressure also helped to reduce the percentage of pore sizes below 50mum. Scaffolds chosen for in vitro cellular studies had a pore size distribution of 0-300mum, average pore size 66+/-17mum, 0.54+/-0.02% porosity and 98% interconnectivity, measured by micro-computed tomography (microCT) analysis. The ability of the scaffolds to support osteogenic differentiation for subsequent cranial defect repair was evaluated by static and dynamic (0.035+/-0.006ms(-1) terminal velocity) cultivation with dura mater stem cells (DSCs). In vitro studies showed minimal increases in proliferation over 28 days in culture in osteogenic media. Alkaline phosphatase expression remained constant throughout the study. Moderate increases in matrix deposition, as assessed by histochemical staining and microCT analysis, occurred at later time points, days 21 and 28. Although constructs cultured dynamically showed greater mineralization than static conditions, these trends were not significant. It remains unclear whether bioreactor culture of DSCs is advantageous for bone tissue engineering applications. However, these studies show that polycaprolactone (PCL) scaffolds alone, without the addition of other co-polymers or ceramics, support long-term attachment and mineralization of DSCs throughout the entire porous scaffold.     

Optimization of bone-tissue engineering in goats

Successful bone-tissue engineering (TE) has been reported for various strategies to combine cells with a porous scaffold. In particular, the period after seeding until implantation of the constructs may vary between hours and several weeks. Differences between these strategies can be reduced to (a) the presence of extracellular matrix, (b) the differentiation status of the cells, and (c) the presence of residual potentially immunogenic serum proteins. These parameters are investigated in two types of calcium phosphate scaffolds in a goat model of ectopic bone formation. Culture-expanded bone-marrow stromal cells from eight goats were seeded onto two types of hydroxyapatite granules: HA60/400 (60% porosity, 400-microm average pore size) and HA70/800. Scaffolds seeded with cells and control scaffolds were cultured for 6 days in medium containing autologous or semisynthetic serum, in the presence or absence of dexamethasone. Other scaffolds were seeded with cells just before implantation in medium with or without serum. All conditions were implanted autologously in the paraspinal muscles. After 12 weeks, bone had formed in 87% of all TE constructs, as demonstrated by histology. Histomorphometry indicated significantly more bone in the HA70/800 scaffolds. Furthermore, a significant advantage in bone formation was found when the constructs had been cultured for 6 days. In conclusion, both scaffold characteristics (porosity) and TE strategy (culturing of the constructs) were demonstrated to be important for bone TE.     

Fiber density of electrospun gelatin scaffolds regulates morphogenesis of dermal-epidermal skin substitutes

Porous, nowoven fibrous gelatin scaffolds were prepared using electrospinning. Electrospun scaffolds with varying fiber diameter, interfiber distance, and porosity were fabricated by altering the concentration of the electrospinning solution. Solution concentration was a significant predictor of fiber diameter, interfiber distance, and porosity with higher solution concentration correlated with larger fiber diameters and interfiber distances. The potential of electrospun gelatin as a scaffolding material for dermal and epidermal tissue regeneration was also evaluated. Interfiber distances >5.5 microm allowed deeper penetration of human dermal fibroblasts into the scaffold, whereas cells in scaffolds with more densely packed fibers were able to infiltrate only into the upper regions. Scaffolds with interfiber distances 相似文献   

Structural features and mechanical properties of in situ-bonded meshes of segmented polyurethane electrospun from mixed solvents

The relationships between the structural features and mechanical properties of electrospun segmented polyurethane (SPU) meshes and electrospinning parameters such as formulation (e.g., polymer concentration and solvent mixing ratio) and operation parameters (e.g., applied voltage, air gap, and flow rate) were studied with the use of a mixed-solvent system of tetrahydrofuran (THF) and N,N-dimethylacrylamide (DMF). After the relationships between the structure of electrospun SPU and the operation parameters under a fixed SPU concentration of single THF solution were established, SPU was electrospun from the mixed solvent of THF and DMF with different mixing ratios [DMF content: 5, 10, and 30% (v/v)]. Scanning electron microscopy showed that an increase in DMF ratio significantly enhances the degree of bonding between SPU fibers at contact sites and decreases the diameter of fibers formed. The porosimetric characterization showed the following: (1) The porosity of the electrospun SPU meshes decreased with an increase of DMF ratio. (2) The pore size distribution exhibited three representative peaks of different void sizes (i.e., approximately 5, 20, and 70 microm). (3) The proportion of the 20-microm void markedly decreased with an increase in DMF ratio. A tensile test on the meshes showed that an increase in DMF ratio induces an increase in elasticity of the mesh. Such a regulation of the structural features and mechanical properties of electrospun SPU meshes using a mixed-solvent system with low- and high-boiling-point solvents may be useful in the engineering of SPU-fiber based matrices or scaffolds.     

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.     

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.     

The role of lipase and alpha-amylase in the degradation of starch/poly(epsilon-caprolactone) fiber meshes and the osteogenic differentiation of cultured marrow stromal cells

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.