Modulation of osteogenic properties of biodegradable polymer/extracellular matrix scaffolds generated with a flow perfusion bioreactor

In this study, composite scaffolds consisting of both synthetic and natural components with controllable properties were generated by incorporating mineralized extracellular matrix (ECM) and electrospun poly(ε-caprolactone) (PCL) microfiber scaffolds. Mesenchymal stem cells (MSCs) were cultured on PCL scaffolds under flow perfusion conditions with culture medium supplemented with dexamethasone to investigate the effect of culture duration on mineralized extracellular matrix deposition. MSCs differentiated down the osteogenic lineage and produced extracellular matrix with different compositions of mineral, collagen, and glycosaminoglycan with distinct morphologies at various stages of osteogenesis. To determine whether the presence and maturity of mineralized extracellular matrix influences osteogenic differentiation in vitro, PCL/ECM constructs were decellularized to yield PCL/ECM composite scaffolds that were subsequently seeded with MSCs and cultured in the absence of dexamethasone. The presence of mineralized matrix reduced cellular proliferation while stimulating alkaline phosphatase activity with increasing amounts of calcium deposition over time. PCL/ECM composite scaffolds containing the most mature mineralized matrix resulted in the most rapid increase and highest levels of alkaline phosphatase activity and calcium deposition compared to all other scaffold groups. Therefore, we demonstrate that mineralized extracellular matrix generated under controlled flow perfusion conditions can impart osteogenic properties to an osteoconductive polymer scaffold, and that the maturity of this matrix influences osteogenic differentiation in vitro, even in the absence of dexamethasone.     

A porous PCL scaffold promotes the human chondrocytes redifferentiation and hyaline-specific extracellular matrix protein synthesis

The redifferentiation, proliferation, and hyaline-specific extracellular matrix (ECM) protein synthesis of chondrocytes cultured in a polycaprolactone (PCL) scaffold were analyzed. Gene expression of the type II collagen and aggrecan was assessed by real-time PCR in cells from PCL scaffolds, monolayer, and pellet cultures. The proliferative activity was assessed using Ki-67 immunodetection, and the chondrocytic differentiation was evaluated using S-100 immunodetection. The synthesis and deposition into scaffold pores of type II collagen and glycosaminoglycan were analyzed by immunohistochemistry and Alcian blue staining, respectively. All parameters were assessed throughout 28 days of cultures maintained in either fetal bovine serum-containing medium (FCM) or Insulin-Transferrin-Selenium-containing medium (ICM). Expression of the type II collagen gene was lower in FCM cultures than in ICM cultures for all culture systems (p < 0.05). Moreover, PCL scaffolds cultured in ICM were able to induce collagen gene expression more efficiently than pellet and monolayer cultures. Aggrecan gene expression did not vary significantly between mediums and three-dimensional system cultures, but in ICM cultures, the monolayer cultures had significantly higher levels of aggrecan gene expression than did either the PCL or pellet cultures. Chondrocytes cultured in PCL scaffolds or pellets with FCM did not proliferate to a great extent but did maintain their differentiated phenotype for 28 days. Levels of cartilage ECM protein synthesis and deposition into the scaffold pores were similar among PCL and pellet cultures grown in FCM and in ICM. In conclusion, chondrocytes seeded into PCL scaffolds, cultured in ICM, efficiently maintained their differentiated phenotype and were able to synthesize cartilage-specific ECM proteins.     

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.     

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 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.     

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(?-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.     

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.     

An ectopic approach for engineering a vascularized tracheal substitute

Tissue engineering can provide alternatives to current methods for tracheal reconstruction. Here we describe an approach for ectopic engineering of vascularized trachea based on the implantation of co-cultured scaffolds surrounded by a muscle flap. Poly(L-lactic-co-glycolic acid) (PLGA) or poly(ε-caprolactone) (PCL) scaffolds were seeded with chondrocytes, bone marrow stem cells and co-cultured both cells respectively (8 groups), wrapped in a pedicled muscle flap, placed as an ectopic culture on the abdominal wall of rabbits (n = 24), and harvested after two and four weeks. Analysis of the biochemical and mechanical properties demonstrated that the PCL scaffold with co-culture cells seeding displayed the optimal chondrogenesis with adequate rigidity to maintain the cylindrical shape and luminal patency. Histological analysis confirmed that cartilage formed in the co-culture groups contained a more homogeneous and higher extracellular matrix content. The luminal surfaces appeared to support adequate epithelialization due to the formation of vascularized capsular tissue. A prefabricated neo-trachea was transferred to the defect as a tracheal replacement and yielded satisfactory results. These encouraging results indicate that our co-culture approach may enable the development of a clinically applicable neo-trachea.     

In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold

The objective of this study was to assess bone formation from mesenchymal stem cells (MSCs) on a novel nanofibrous scaffold in a rat model. A highly porous, degradable poly(epsilon-caprolactone) (PCL) scaffold with an extracellular matrix-like topography was produced by electrostatic fiber spinning. MSCs derived from the bone marrow of neonatal rats were cultured, expanded, and seeded on the scaffolds. The cell-polymer constructs were cultured with osteogenic supplements in a rotating bioreactor for 4 weeks, and subsequently implanted in the omenta of rats for 4 weeks. The constructs were explanted and characterized by histology, immunohistochemistry, and scanning electron microscopy. The constructs maintained the size and shape of the original scaffolds. Morphologically, the constructs were rigid and had a bone-like appearance. Cells and extracellular matrix (ECM) formation were observed throughout the constructs. In addition, mineralization and type I collagen were also detected. This study establishes the ability to develop bone grafts on electrospun nanofibrous scaffolds in a well-vascularized site using MSCs.     

Application of porous glycosaminoglycan-based scaffolds for expansion of human cord blood stem cells in perfusion culture

In vitro expansion of hematopoietic stem cells (HSCs) has been employed to obtain sufficient numbers of stem cells for successful engraftment after HSC transplantation. A three-dimensional perfusion bioreactor system with a heparin-chitosan scaffold was designed and evaluated for its capability to support maintenance and expansion of HSCs. Porous chitosan scaffolds were fabricated by a freeze-drying technique and N-desulfated heparin was covalently immobilized within the scaffolds using carbodiimide chemistry. CD34+ HSCs isolated from umbilical cord blood by immunomagnetic separation were cultured within the porous scaffold in a perfusion bioreactor system. Control cultures were maintained on dishes coated with similar heparin-chitosan films. Oxygen uptake was measured during the culture period. After 7 days of culture, scaffolds were harvested for analysis. Cellular phenotype and HSC characteristics were evaluated via flow cytometry and colony forming unit assays. The results indicate good cell retention and proliferation within the perfused scaffolds. Oxygen consumption in the perfusion bioreactor system increased continuously during the culture, indicating steady cell growth. Cells from the perfused scaffold cultures showed higher percentages of primitive progenitors and exhibited superior colony forming unit performance as compared to cells from static cultures. In addition, perfusion culture at low oxygen (5%) enhanced the expansion of CD34+ cells and colony-forming activity compared to high oxygen (19%) cultures. The results suggest that perfusion culture of cord blood CD34+ cells under bone marrow-like conditions enhances HSC expansion compared to static cultures.     

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.     

Biodegradable PCL scaffolds with an interconnected spherical pore network for tissue engineering

A technique for producing controlled interconnected porous structures for application as a tissue engineering scaffold is presented in this article. The technique is based on the fabrication of a template of interconnected poly(ethyl methacrylate) (PEMA) microspheres, the introduction of a biodegradable polymer, poly-epsilon-caprolactone (PCL), and the elimination of the template by a selective solvent. A series of PCL scaffolds with a porosity of 70% and pore sizes up to 200 microm were produced and characterized (both thermally and mechanically). Human chondrocytes were cultured in monolayer on bulk PCL disks or seeded into porous PCL scaffolds. Cell adhesion, viability, proliferation, and proteoglycan (PG) synthesis were tested and compared with monolayer cultures on tissue-treated polystyrene or pellet cultures as reference controls. Cells cultured on PCL disks showed an adhesion similar to that of the polystyrene control (which allowed high levels of proliferation). Stained scaffold sections showed round-shaped chondrocyte aggregates embedded into porous PCL. PG production was similar to that of the pellet cultures and higher than that obtained with monolayer postconfluence cultures. This shows that the cells are capable of attaching themselves to PCL. Furthermore, in porous PCL, cells maintain the same phenotype as the chondrocytes within the native cartilage. These results suggest that PCL scaffolds may be a suitable candidate for chondrocyte culture.     

Evaluation of extracellular matrix formation in polycaprolactone and starch-compounded polycaprolactone nanofiber meshes when seeded with bovine articular chondrocytes

Cartilage defects are a major health problem. Tissue engineering has developed different strategies and several biomaterial morphologies, including natural-based ones, for repairing these defects. We used electrospun polycaprolactone (PCL) and starch-compounded PCL (SPCL) nanofiber meshes to evaluate extracellular matrix (ECM) formation by bovine articular chondrocytes (BACs). The main aim of this work was to evaluate the suitability of PCL and SPCL nanofiber meshes in chondrocyte cultures, and their capability to produce ECM when seeded onto these nanostructured materials. The effect of culture conditions (static vs dynamic) on ECM formation was also assessed. BACs were seeded onto PCL and SPCL nanofiber meshes using a dynamic cell-seeding procedure and cultured under static or dynamic conditions for 4 weeks. Constructs were characterized using scanning electron microscopy, histology, immunolocalization of collagen types I and II, and glycosaminoglycan (GAG) quantification. Results show an extensive cell colonization of the entire nanofiber mesh, for both materials, and that chondrocytes presented typical spherical morphology. Some degree of cell infiltration inside the nanofiber meshes was noticeable for both materials. ECM formation and GAG were detected throughout the materials, evidencing typical construct maturation. PCL and SPCL nanofiber meshes are suitable as supports for ECM formation and therefore are adequate for cartilage tissue-engineering approaches.     

A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells

The utilization of adult stem cells in tissue engineering is a promising solution to the problem of tissue or organ shortage. Adult bone marrow derived mesenchymal stem cells (MSCs) are undifferentiated, multipotential cells which are capable of giving rise to chondrocytes when maintained in a three-dimensional culture and treated with members of the transforming growth factor-beta (TGF-beta) family of growth factors. In this study, we fabricated a nanofibrous scaffold (NFS) made of a synthetic biodegradable polymer, poly(-caprolactone) (PCL), and examined its ability to support in vitro chondrogenesis of MSCs. The electrospun PCL porous scaffold was constructed of uniform, randomly oriented nanofibers with a diameter of 700 nm, and structural integrity of this scaffold was maintained over a 21-day culture period. MSCs cultured in NFSs in the presence of TGF-beta1 differentiated to a chondrocytic phenotype, as evidenced by chondrocyte-specific gene expression and synthesis of cartilage-associated extracellular matrix (ECM) proteins. The level of chondrogenesis observed in MSCs seeded within NFSs was comparable to that observed for MSCs maintained as cell aggregates or pellets, a widely used culture protocol for studying chondrogenesis of MSCs in vitro. Due to the physical nature and improved mechanical properties of NFSs, particularly in comparison to cell pellets, the findings reported here suggest that the PCL NFS is a practical carrier for MSC transplantation, and represents a candidate scaffold for cell-based tissue engineering approaches to cartilage repair.     

Online measurement of oxygen consumption by goat bone marrow stromal cells in a combined cell-seeding and proliferation perfusion bioreactor

In an effort to produce clinically useful volumes of tissue engineered bone products, a direct perfusion bioreactor system was developed. Perfusion flow rate, flow direction, and the position of the bioreactor are factors that influenced the amounts and homogeneity of the cells seeded on the scaffold surface. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cm(3)) of small-sized macroporous biphasic calcium phosphate (BCP) scaffolds (2-6 mm). Cell load and cell distribution were shown using Methylene Blue block staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate the viability of the cells. Although cells were not distributed homogenously after cell seeding, the scaffolds were covered with a viable, homogeneous cell layer after 25 days of cultivation. The hybrid structures became interconnected, and a dense layer of extracellular matrix formed on and in the scaffolds. Online oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption could possibly be used to estimate GBMSC population doubling times during growth in this bioreactor system. On the basis of our results, we conclude that a direct perfusion bioreactor system is capable of seeding and proliferating GBMSCs on BCP ceramic scaffolds that can be monitored online during cultivation.     

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.     

三维环境下软骨细胞诱导滑膜间充质干细胞向软骨样细胞的分化

BACKGROUND: The co-culture of chondrocytes and synovial mesenchymal stem cells can induce the cartilage differentiation of synovial mesenchymal stem cells in vitro, but the cell differentiation induced by co-culture in vivo is rarely reported. OBJECTIVE: To investigate the chondrogenic differentiation of synovial mesenchymal stem cells co-cultured with chondrocytes on the chitosan/type I collagen composite scaffolds after being transplanted into the subcutaneous layer of Sprague-Dawley rats. METHODS: The synovial mesenchymal stem cells and chondrocytes harvested from the synovial membrane and articular cartilage of Sprague-Dawley rats were obtained by enzyme digestion method and cultured respectively. Passage 3 synovial mesenchymal stem cells and passage 2 chondrocytes, which were divided into four groups: group A (chondrocytes alone), group B (synovial mesenchymal stem cells alone), group C (ratio of synovial mesenchymal stem cells:chondrocytes=1:2) and group D (scaffold material without cells), were cultured on chitosan/type I collagen composite scaffolds and transplanted into the subcutaneous layer of rats followed by morphological observation and immunohistochemical staining at 4 and 8 weeks.   . RESULTS AND CONCLUSION: After 4 and 8 weeks, the discoid-like scaffold was visible. The immunohistochemical staining of type II collagen and the toluidine blue staining of aggrecan were significantly positive in groups A and C. These results show that the co-culture of synovial mesenchymal stem cells and chondrocytes on the scaffold in vivo can form cartilage-like tissues.      

Interaction of mesenchymal stem cells and osteoblasts for in vitro osteogenesis

It has recently been reported that bone marrow-derived mesenchymal stem cells (MSCs), which are systemically administrated to different species, undergo site-specific differentiation. This suggests that the tissue specific cells may cause or promote the differentiation of the MSCs toward their cell type via a cell-to-cell interaction that is mediated not only by hormones and cytokines, but also by direct cell-to-cell contact. In this study, in order to assess the possible synergistic interactions for osteogenesis between the two types of cells, the MSCs derived from rabbit bone marrow were co-cultured with rat calvarial osteoblasts in direct cell-to-cell contact in a control medium (CM) and in an osteogenic medium (OM). The cell number, alkaline phosphatase activity, and amount of calcium deposition were assayed in the cultures of MSCs, osteoblasts, and co-cultures of them in either OM or CM for up to 40 days. The cell numbers and the alkaline phosphatase activities in the co-culture were somewhere in between those of the osteoblast cultures and the MSC cultures. The amounts of deposited calcium were lower in the co-culture compared to those of the other cultures. This suggests that there are little synergistic interactions during osteogenesis in vitro between the rat osteoblasts and rabbit MSCs.     

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.