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.     

Bone formation by rat bone marrow cells cultured on titanium fiber mesh: effect of in vitro culture time

The objective of this study was to examine the effect of cell culture time on bone formation by rat bone marrow cells seeded in titanium fiber mesh. As a seeding technique, a high cell suspension was used (3 x 10(6) cells/mL). Therefore, 30 meshes were repeatedly rotated in a 10 mL tube (containing 30 x 10(6) cells) on a rotation plate (2 rpm) for 3 h. Osteogenic cells were cultured for 1, 4, and 8 days on titanium fiber mesh and finally implanted subcutaneously in rats. Meshes without cells were also implanted subcutaneously in rats. DNA and scanning electron microscopy (SEM) analyses and calcium measurements determined cellular proliferation and differentiation during the in vitro incubation period of the mesh implants. Four weeks after implant insertion, the animals were sacrificed. The implants, with their surrounding tissue, were retrieved and prepared for histologic evaluation and calcium measurements. DNA analysis of the in vitro experiment showed a lag phase from day 1 through day 4, but a 42% increase in DNA between days 4 and 8. SEM and calcium measurements indicated an increase in calcium from day 1 to day 4, yet only a small but significant increase from days 4 to 8. Histologic analysis demonstrated that bone was formed in all day 1 and day 4 implants, and that the bone-like tissue was present uniformly through the meshes. The bony tissue was morphologically characterized by osteocytes embedded in a mineralized matrix, with a layer of osteoid and osteoblasts at the surface. The day 8 implants showed only calcium phosphate deposition in the titanium fiber mesh. Calcium measurements of the implants revealed that calcification in day 1 implants was significantly higher (p < 0.05) compared to day 4 and day 8 implants. No significant difference in calcium content existed between day 4 and day 8 implants. On the basis of our results, we conclude that 1) bone formation was generated more effectively in osteogenic cells by a short culture time after seeding in titanium fiber mesh; 2) dynamic cell seeding is probably more effective than static cell seeding; and 3) selection of the right cells from the heterogenous bone marrow population remains a problem.     

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.     

Scaffold mesh size affects the osteoblastic differentiation of seeded marrow stromal cells cultured in a flow perfusion bioreactor

In this study, we cultured marrow stromal cells on titanium fiber meshes in a flow perfusion bioreactor and examined the effect of altering scaffold mesh size on cell behavior in an effort to develop a bone tissue construct composed of a scaffold, osteogenic cells, and extracellular matrix. Scaffolds of differing mesh size, that is, distance between fibers, were created by altering the diameter of the mesh fibers (20 or 40 microm) while maintaining a constant porosity. These scaffolds had a porosity of 80% and mesh sizes of 65 microm (20-microm fibers) or 119 microm (40-microm fibers). Cell/scaffold constructs were grown in static culture or under flow for up to 16 days and assayed for osteoblastic differentiation. Cellularity was higher at early time points and Ca2+ deposition was higher at later time points for flow constructs over static controls. The 20-microm mesh had reduced cellularity in static culture. Under flow conditions, mass transport limitations are mitigated allowing uniform cell growth throughout the scaffold, and there was no difference in cellularity between mesh types. There was greater alkaline phosphatase (ALP) activity, osteopontin levels, and calcium under flow at 8 days for the 40-microm mesh compared to the 20-microm mesh. However, by day 16, the trend was reversed, suggesting the time course of differentiation was dependent on scaffold mesh size under flow conditions. However, this dependence was not linear with respect to time; larger mesh size was conducive to early osteoblast differentiation while smaller mesh size was conducive to later differentiation and matrix deposition.     

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.     

Ectopic bone formation in rat marrow stromal cell/titanium fiber mesh scaffold constructs: effect of initial cell phenotype

Titanium fiber mesh scaffolds have been shown to be a suitable material for culture of primary marrow stromal cells in an effort to create tissue engineered constructs for bone tissue replacement. In native bone tissue, these cells are known to attach to extracellular matrix molecules via integrin receptors for specific peptide sequences, and these attachments can be a source of cell signaling, affecting cell behaviors such as differentiation. In this study, we examined the ability of primary rat marrow stromal cells at two different stages of osteoblastic differentiation to further differentiate into osteoblasts both in vitro and in vivo when seeded on titanium fiber mesh scaffolds either with or without RGD peptide tethered to the surface. In vitro, the tethered RGD peptide resulted in reduced initial cell proliferation. In vivo, there was no effect of tethered RGD peptide on ectopic bone formation in a rat subcutaneous implant model. Scaffold/cell constructs exposed to dexamethasone for 4 days prior to implantation (+dex constructs) resulted in significant bone formation whereas no bone formation was observed in--dex constructs. These results show that the osteoblastic differentiation of marrow stromal cells was not dependent on surface tethered RGD peptide, and that the initial differentiation stage of implanted cells plays an important role in bone formation in titanium fiber mesh bone tissue engineering constructs.     

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.     

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.     

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 fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.     

Influence of rhBMP-2 on rat bone marrow stromal cells cultured on titanium fiber mesh

Titanium (Ti) fiber mesh is a candidate scaffold material for the creation of bone graft substitutes (BGS). Two densities (3.54 x 10(4) cells/cm(2) [LD or low density] and 3.54 x 10(5) cells/cm(2) [HD or high density]) of rat bone marrow stromal cells were seeded on Ti-fiber mesh discs. Cells were cultured for up to 16 days, 7 days of which the cells were in the presence of various concentrations of rhBMP-2 (0, 10, 100, and 1,000 ng/mL) in order to evaluate osteogenic expression. Scanning electron microscopy (SEM), light microscopy (LM), energy dispersive spectroscopy (EDS), DNA and calcium (Ca) content measurements, and x-ray diffraction (XRD) analysis were performed. SEM and EDS evaluation showed that a confluent layer of cells was present on top of the meshes together with collagen bundles and calcified globular accretions. Light microscopical evaluation showed a densely stained layer in the upper part of the mesh. SEM and Ca content measurement showed that calcification starts at 8 days. In addition, it was demonstrated that DNA content peaked at 8 days. LM, SEM, and Ca content evaluation revealed positive effects of increasing the cell seeding density, the rhBMP-2 concentration and the culture time on mineralization. Increasing the cell seeding density also showed a positive effect on DNA content. No effects of rhBMP-2 concentration were seen on DNA content. Finally, XRD revealed that the deposited matrix contained a precipitate of a stable calcium phosphate phase. We conclude that (1) titanium fiber mesh sustains excellent osteogenic expression in vitro, (2) increasing the cell seeding density has a positive effect on osteogenic expression in titanium mesh in vitro, and (3) in high density specimens, rhBMP-2 concentrations of 100 ng/mL and 1,000 ng/mL stimulate extracellular matrix calcification in a dose-responsive manner.     

Osteoblast differentiation with titania and titania-silica-coated titanium fiber meshes

Two surface-reactive sol-gel coatings, namely titania (TiO2) and a mixture of titania and silica (TiSi), were applied to titanium fiber meshes. Differentiation of rat bone marrow stromal cells toward an osteogenic phenotype with coated and uncoated (cpTi) substrates was compared. The amount of DNA in cpTi and TiSi matrices did not increase after day 3, but with TiO2 matrices the amount increased for 7 days. The prolonged period of proliferation with TiO2 scaffolds resulted in a delay in alkaline phosphatase induction. However, osteocalcin incorporation into extracellular matrix by day 14 was greater with TiO2 scaffolds than with cpTi scaffolds. Calcium deposition was also greater with TiO2-coated substrates than with uncoated substrates. With the TiSi scaffolds osteocalcin production and mineralization were lower than with the cpTi scaffolds. The current study confirms our previous findings that titanium fiber mesh supports attachment, growth, and differentiation of rat bone marrow stromal cells. Furthermore, the osteogenic capacities of cell-scaffold constructs under cell culture conditions were increased with a sol-gel-derived titania coating, but not with a titania-silica coating.     

Effect of fibronectin- and collagen I-coated titanium fiber mesh on proliferation and differentiation of osteogenic cells

The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes coated with fibronectin, meshes coated with collagen I, and meshes coated first with collagen I and then subsequently with fibronectin. Rat bone marrow cells were cultured for 1, 4, 8, and 16 days in plain and coated titanium fiber meshes. In addition, a portion of each of these coating treatment groups was cultured in the presence of antibodies against fibronectin and collagen I integrins. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, osteocalcin, and calcium content and alkaline phosphatase activity. There were no significant effects of the coatings on cellular proliferation as indicated by the DNA quantification analysis. When antibodies against fibronectin and collagen I integrins were used, a significant reduction (p < 0.05) in cell proliferation was observed for the uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. The different coatings also did not affect the alkaline phosphatase activity of the cells seeded on the coated meshes. However, the presence of antibodies against fibronectin or collagen I integrins resulted in significantly delayed expression of alkaline phosphatase activity for uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. Calcium measurements did not reveal a significant effect of fibronectin or collagen I coating on calcium deposition in the meshes. Also, no difference in calcium content was observed in the uncoated titanium meshes and meshes coated with fibronectin when antibodies against fibronectin or collagen I integrins were present. Meshes coated with both collagen I and fibronectin showed significantly higher calcium content when cultured in the presence of antibodies to collagen and fibronectin integrins. A similar phenomenon was also observed for collagen-coated meshes cultured in the presence of antibodies to fibronectin integrins. No significant differences in osteocalcin content were observed between the treatment groups. However, all groups exposed to antibodies against fibronectin integrins showed a significant decrease in osteocalcin content on day 16. These results show that a fibronectin or collagen I coating does not stimulate the differentiation of rat bone marrow cells seeded in a titanium fiber mesh.     

Analysis of the osteoinductive capacity and angiogenicity of an in vitro generated extracellular matrix

In this study, the osteoinductive potential of an in vitro generated extracellular matrix (ECM) deposited by marrow stromal cells seeded onto titanium fiber mesh scaffolds and cultured in a flow perfusion bioreactor was investigated. Culture periods of 8, 12, and 16 days were selected to allow for different amounts of ECM deposition by the cells as well as ECM with varying degrees of maturity (Ti/ECM/d8, Ti/ECM/d12, and Ti/ECM/d16, respectively). These ECM-containing constructs were implanted intramuscularly in a rat animal model. After 56 days, histologic analysis of retrieved constructs revealed no bone formation in any of the implants. Surrounding many of the implants was a fibrous capsule, which was often interspersed with fat cells. Within the pore spaces, the predominant tissue response was the presence of blood vessels and young fibroblasts or fat cells. The number of blood vessels on a per area basis calculated from a histomorphometric analysis increased as a function of the amount of ECM within the implanted constructs, with a significant difference between Ti/ECM/d16 and plain Ti constructs. These results indicate that although an in vitro generated ECM alone may not induce bone formation at an ectopic site, its use may enhance the vascularization of implanted constructs.     

Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds

In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 m fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 × 10⁵ cells in 300 l, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.     

Evaluation of various seeding techniques for culturing osteogenic cells on titanium fiber mesh

The objective of the present study was to learn more about the effect of seeding and loading techniques on the osteogenic differentiation in vitro of rat bone marrow cells into titanium fiber mesh. This material was used as received or subjected to glow discharge treatment (RFGD). The seeding methods that were used included a so-called droplet, cell suspension (high and low cell density), and rotating plate method. Osteogenic cells were cultured for 4, 8, and 16 days into titanium fiber mesh. DNA, osteocalcin, scanning electron microscopy (SEM) analysis, and calcium measurements were used to determine cellular proliferation and differentiation. DNA analysis of the differently seeded specimens showed that proliferation proceeded faster in the first versus second run for droplet and cell suspension samples. No clear and distinct additional effect was found when RFGD treatment was used. Statistical analyses revealed that high cell density and low rotational speed resulted always in a significantly higher DNA content. Calcium measurements and osteocalcin analysis showed that using high cell densities during inoculation of the scaffolds prevented the occurrence of differences between experimental runs. SEM examination showed that for droplet and cell suspension samples cells were present at only one side of the mesh. The mesh side where the cell sheet was observed depended on the additional use of glow discharge treatment. On these materials, the cells had penetrated through the meshes and formed a cell sheet at the bottom side. When rotation was used, no cell sheet was formed and cells had invaded the meshes and were growing around the titanium fibers. On the basis of our results, we conclude that (1). titanium fiber mesh is indeed suitable to support the osteogenic expression of bone marrow cells, and (2). changing the initial cell density as well as the use of dynamic seeding methods can influence the osteogenic capacity of the scaffold.     

In vitro localization of bone growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and cultured in a flow perfusion bioreactor

Tissue engineering strategies aim at controlling the behavior of individual cells to stimulate tissue formation. This control is achieved by mimicking signals that manage natural tissue development or repair. Flow perfusion bioreactors that create culture environments with minimal diffusion constraints and provide cells with mechanical stimulation may closely resemble in vivo conditions for bone formation. Therefore, these culturing systems, in conjunction with an appropriate scaffold and cell type, may provide significant insight towards the development of in vitro tissue engineering models leading to improved strategies for the construction of bone tissue substitutes. The objective of this study was to investigate the in vitro localization of several bone growth factors that are usually associated with bone formation in vivo by culturing rat bone marrow stromal cells seeded onto starch-based biodegradable fiber meshes in a flow perfusion bioreactor. The localization of several bone-related growth factors-namely, transforming growth factor-beta1, platelet-derived growth factor- A, fibroblast growth factor-2, vascular endothelial growth factor, and bone morphogenetic protein- 2-was determined at two different time points in scaffolds cultured under perfusion conditions at two different flow rates using an immunohistochemistry technique. The results show the presence of regions positively stained for all the growth factors considered, except platelet-derived growth factor-A. Furthermore, the images obtained from the positively stained sections suggest an increase in the immunohistochemically stained area at the higher flow rate and culture time. These observations demonstrate that flow perfusion augments the functionality of scaffold/cell constructs grown in vitro as it combines both biological and mechanical factors to enhance cell differentiation and cell organization within the construct. This study also shows that flow perfusion bioreactor culture of marrow stromal cells, combined with the use of appropriate biodegradable fiber meshes, may constitute a useful model to study bone formation and assess bone tissue engineering strategies in vitro.     

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.     

Three-dimensional cell culture and tissue engineering in a T-CUP (tissue culture under perfusion)

The aim of this study was to develop and validate a simple and compact bioreactor system for perfusion cell seeding and culture through 3-dimensional porous scaffolds. The developed Tissue Culture Under Perfusion (T-CUP) bioreactor is based on the concept of controlled and confined alternating motion of scaffolds through a cell suspension or culture medium, as opposed to pumping of the fluid through the scaffolds. Via the T-CUP, articular chondrocytes and bone marrow stromal cells could be seeded into porous scaffolds of different compositions and architectures (chronOS, Hyaff-11, and Polyactive) at high efficiency (greater than 75%), uniformity (cells were well distributed throughout the scaffold pores), and viability (greater than 97%). Culture of articular chondrocytes seeded into 4-mm thick Polyactive scaffolds for 2 weeks in the T-CUP resulted in uniform deposition of cartilaginous matrix. Cultivation of freshly isolated human bone marrow nucleated cells seeded into ENGipore ceramic scaffolds for 19 days in the T-CUP resulted in stromal cell-populated constructs capable of inducing ectopic bone formation in nude mice. The T-CUP bioreactor represents an innovative approach to simple, efficient, and reliable 3D cell culture, and could be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system in the context of regenerative medicine.