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

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

Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges

Various culture systems have been used for examining the anabolic and catabolic functions of isolated chondrocytes as well as for tissue engineering purposes. Perfusion or frequent medium change is beneficial for three-dimensional (3D) cultures of many cell types. In this study, bovine articular chondrocytes (bACs) were grown in 3D collagen sponges with or without medium perfusion (0.33 mL/min) for up to 15 days. The influence of medium perfusion was evaluated using markers of cartilage matrix accumulation, synthesis, and gene expression. Metachromatic matrix, collagen type II, and hyaluronan accumulated around the cells within the collagen sponges. Sulfated glycosaminoglycans (S-GAGs) that accumulated in the sponge exposed to nonperfused control were 130% of that in the perfused sponge at day 7. S-GAG accumulation after 15 days in the nonperfused control was 230% more than at day 7 (p < 0.01). (35)S-sulfate incorporation during the final 18 h of culture in the sponge exposed to nonperfusion was 180% greater than that in the perfused sponge (p < 0.01). Quantitative analyses show that at day 7, aggrecan and collagen type II gene expression were 350% and 240% greater, respectively, in the nonperfused culture than in the perfused one. These results indicate that perfused conditions that are beneficial for other cell types inhibit chondrogenesis by articular chondrocytes in 3D culture.     

Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.     

Differentiation Capacity of Human Chondrocytes Embedded in Alginate Matrix

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco’s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.     

Differentiation capacity of human chondrocytes embedded in alginate matrix

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco?s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.     

MRI and histologic analysis of collagen type II sponge on repairing the cartilage defects of rabbit knee joints

There are limited treatment options for cartilage defects in clinical practice because of the lack of suitable biomaterials. Here, we evaluated the effects of collagen type II sponge on the articular cartilage repairing process using a cartilage injury of a rabbit knee joint model. We showed that the home-made collagen type II sponges appeared to have a suitable pore size of 93.26 ± 38.4 μm for chondrocyte growth. MRI with H&E staining results demonstrated that the effusion absorption in the collagen type II sponge treated group was quicker than that of the control group. Moreover, sporadic cartilage signals first appeared at 6 weeks in the collagen type II sponge treated group. Safranin O staining and immunohistochemical analysis confirmed that the newly formed cartilage expresses glycosaminoglycan and type II collagen matrix. Using Sirius red polarized light staining, we showed that the newly formed cartilage-like areas from the collagen type II treated group are significantly greater than those of the control group. Taken together, our data demonstrated that the home-made collagen type II sponge is able to promote cartilage repair in the cartilage injury of a rabbit knee joint model.     

Effects of high molecular weight hyaluronan on chondrocytes cultured within a resorbable gelatin sponge

Freshly isolated bovine articular chondrocytes were seeded into a resorbable gelatin sponge and cultured in the absence or presence of extrinsic high molecular weight hyaluronan (HA) for up to 1 month. The gelatin sponge could be uniformly and reproducibly loaded with chondrocytes. Immunostaining demonstrated that accumulation of pericellular HA increased in the presence of extrinsic HA. However, this approach could not differentiate between extrinsic and endogenous HA. More chondrocytes were retained within the loaded sponges in the presence of HA. Both cell number and matrix synthesis were increased in the presence of high molecular weight HA throughout the time course. Proteoglycan synthesis per cell increased by 22-fold in the presence of HA at 500 microg/mL. Our model demonstrates that HA can be used as a tool not only to expand freshly isolated chondrocyte numbers but also to increase matrix synthesis and deposition within a resorbable gelatin sponge. Autologous chondrocytes for tissue engineering are always in short supply, so this could be a useful tool with which to increase the retention of cells seeded into other types of scaffold matrices before implanting them into a cartilage defect.     

TGF-beta1 immobilized tri-co-polymer for articular cartilage tissue engineering

Tri-co-polymer with composition of gelatin, hyaluronic acid and chondroitin-6-sulfate has been used to mimic the cartilage extracellular matrix as scaffold for cartilage tissue engineering. In this study, we try to immobilize TGF-beta1 onto the surface of the tri-co-polymer sponge to suppress the undesired differentiation during the cartilage growth in vitro. The scaffold was synthesized with a pore size in a range of 300-500 microm. TGF-beta1 was immobilized on the surface of the tri-co-polymer scaffold with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Tri-co-polymer scaffolds with and without TGF-beta1 were seeded with porcine chondrocytes and cultured in a spinner flask for 2, 4, and 6 weeks. The chondrocytes were characterized by the methods of immunohistochemical staining with anti-type II collagen and anti-S-100 protein monoclonal antibody, and RT-PCR. After culturing for 4 weeks, chondrocytes showed positive in S-100 protein, Alcian blue, and type II collagen for the scaffold with TGF-beta1 immobilization. There is no observed type I and type X collagen expression in the scaffolds from the observation of RT-PCR. In addition, the scaffold without TGF-beta1 immobilization, type X collagen, can be detected after cultured for 2 weeks. Type I collagen was progressively expressed after 4 weeks. These results can conclude that TGF-beta1 immobilized scaffold can suppress chondrocytes toward prehypertrophic chondrocytes and osteolineage cells. The tri-co-polymer sponge with TGF-beta1 immobilization should have a great potential in cartilage tissue engineering in the future.     

Comparative phenotypic analysis of articular chondrocytes cultured within type I or type II collagen scaffolds

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.     

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

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

Chondrogenesis from human placenta-derived mesenchymal stem cells in three-dimensional scaffolds for cartilage tissue engineering

Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.     

Control of pore structure and size in freeze-dried collagen sponges.

Because of many suitable properties, collagen sponges are used as an acellular implant or a biomaterial in the field of tissue engineering. Generally, the inner three-dimensional structure of the sponges influences the behavior of cells. To investigate this influence, it is necessary to develop a process to produce sponges with a defined, adjustable, and homogeneous pore structure. Collagen sponges can be produced by freeze-drying of collagen suspensions. The pore structure of the freeze-dried sponges mirrors the ice-crystal morphology after freezing. In industrial production, the collagen suspensions are solidified under time- and space-dependent freezing conditions, resulting in an inhomogeneous pore structure. In this investigation, unidirectional solidification was applied during the freezing process to produce collagen sponges with a homogeneous pore structure. Using this technique the entire sample can be solidified under thermally constant freezing conditions. The ice-crystal morphology and size can be adjusted by varying the solute concentration in the collagen suspension. Collagen sponges with a very uniform and defined pore structure can be produced. Furthermore, the pore size can be adjusted between 20-40 microm. The thickness of the sponges prepared during this research was 10 mm.     

The Effect of Hybridization of Hydrogels and Poly(L-lactide-co-ε-caprolactone) Scaffolds on Cartilage Tissue Engineering

For repairing cartilage defects by cartilage tissue engineering, it is important that engineered cartilage that is fabricated with scaffolds and cells can maintain the biological and physiological functions of cartilage, and also can induce three-dimensional spatial organization of chondrocytes. In this sense, hydrogels such as fibrin gels (FG) and hyaluronan (HA) are widely used for application in cartilage treatment. However, the use of hydrogels alone as a scaffold has a physical weakness; the mechanical properties of hydrogels are too weak to endure complex loading in the body. In this study, for mimicking a native cartilage microenvironment, we made cell–hybrid scaffold constructs with poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds and hydrogels to guide three-dimensional spatial organization of cells and extracellular matrix. A highly elastic scaffold was fabricated from PLCL with 85% porosity and 300–500 μm pore size using a gel-pressing method. The mixture of rabbit chondrocytes and hydrogels was seeded on PLCL scaffolds, and was subcutaneously implanted into nude mice for up to eight weeks. The cell seeding efficiency of the hybrid scaffolds with FG or HA was higher than that of the PLCL scaffolds. From in vivo studies, the accumulation of cartilaginous extracellular matrices of constructs, which was increased by hybridization of hydrogels and PLCL scaffolds, showed that the cell–hybrid scaffold constructs formed mature and well-developed cartilaginous tissue. In conclusion, the hybridization of hydrogels and PLCL scaffold for three-dimensional spatial organization of cells would provide a biomimetic environment where cartilage tissue growth is enhanced and facilitated. It can enhance the production of cartilaginous extracellular matrices and, consequently, improve the quality of the cartilaginous tissue formed.     

Tissue engineering of cartilage using a hybrid scaffold of synthetic polymer and collagen

A biodegradable hybrid scaffold of synthetic polymer, poly (DL-lactic-co-glycolic acid) (PLGA), and naturally derived polymer, collagen, was prepared by forming collagen microsponges in the pores of PLGA sponge. This was then used as the three-dimensional scaffold for tissue engineering of bovine articular cartilage, both in vitro and in vivo. In vitro studies show that hybridization with collagen facilitated cell seeding in the sponge and raised seeding efficiency. Chondrocytes adhered to the collagen microsponges, where they proliferated and secreted extracellular matrices with time, filling the space within the sponge. Hematoxylin and eosin staining revealed that most of the chondrocytes after 4 weeks of culture, and almost all cell types after 6 weeks of culture, maintained their phenotypically rounded morphology. While new tissue formed, the scaffold degraded and lost almost 36.9% of its original weight after 10 weeks. Subcutaneous implantation studies in nude mice demonstrated more homogeneous tissue formation in hybrid sponge than in PLGA sponge. The new tissue formed maintained the original shape of the hybrid sponge. The synthetic PLGA sponge, serving as a skeleton, facilitated easy formation into desired shapes and provided appropriate mechanical strength to define the ultimate shape of engineered tissue. Incorporation of collagen microsponges facilitated cell seeding and homogeneous cell distribution and created a favorable environment for cellular differentiation. The hybrid sponge could therefore represent a promising candidate as a three-dimensional scaffold for articular cartilage tissue engineering.     

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

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

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

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

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

The modulation of integrin expression by the extracellular matrix in articular chondrocytes

Normal articular cartilage is composed of chondrocytes embedded within an extracellular matrix (ECM). The patterns of integrin expression determine the adhesive properties of cells by modulating interactions with specific ECMs. Our hypothesis is that chondrocyte integrin expression changes in response to changes in their microenvironment. Porcine articular chondrocytes were encapsulated in alginate beads with several ECMs (collagen type I, collagen type II and fibronectin) for 7 days, subjected to RT-PCR, western blot analysis and immunofluorescence staining. It was found that chondrocytes in different ECMs showed different patterns of integrin expression. Integrin alpha5 and beta1 were strongly expressed in all groups, but integrin alpha1 was strongly expressed only in collagen type I and fibronectin conjugated alginate beads, and integrin alpha2 was strongly expressed only in collagen type II conjugated alginate beads. These findings suggest that the addition of different ECMs to chondrocytes can modulate the patterns and levels of integrin expression possibly through a feedback mechanism. These finding suggest that the modulation of ECM interactions may play a critical role in the pathogenesis of osteoarthritis.