Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide

Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.     

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.     

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

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

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.     

Potential use of chitosan as a cell scaffold material for cartilage tissue engineering

One of the most important factors in any tissue-engineering application is the cell substrate. The purpose of this study was the initial evaluation of chitosan, a derivative of the abundant, naturally occurring biopolymer chitin, as a cell scaffold for cartilage tissue engineering. Chitosan scaffolds having an interconnecting porous structure were easily fabricated by simple freezing and lyophilization of a chitosan solution. After rehydration of scaffolds, porcine chondrocytes were seeded onto scaffolds and cultured for up to 28 days in a rotating-wall bioreactor. Chitosan scaffolds supported cell attachment and maintenance of a rounded cell morphology. After 18 days, cells within the scaffolds had synthesized extracellular matrix in which proteoglycan and type II collagen were detected by toluidine blue staining and immunohistochemistry, respectively. Abundant extracellular matrix was found almost exclusively in the periphery of the scaffolds, as scaffold microstructure prevented cells from penetrating to interior regions. Nonetheless, the results suggest that chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage.     

Extracellular matrix protein gene expression of bovine chondrocytes cultured on resorbable scaffolds

It has been demonstrated that using cultured chondrocytes that have been seeded onto various biomatrices can enhance the quality of the articular cartilage repair tissue. As tissue-engineering becomes increasingly more complex there is a need to understand how a specific biomaterial may influence gene expression. In this study several commonly used scaffold materials for cartilage tissue engineering were evaluated with respect to their influence on matrix gene expression. Primary cultures of bovine chondrocytes were established in monolayer then seeded onto polylactic acid (PLLA), polyglycolic acid (PGA), collagen matrices. The induction of collagen type I, collagen type II, and aggrecan was observed at various time points on these biomaterials using RT-PCR. The collagen type I gene was upregulated on collagen scaffolds throughout the culture period. PLLA and PGA showed initial induction followed by downregulation. Monolayer culture did not induce collagen I message. Collagen II genes were selectively upregulated after 72 and 96 h post seeding depending the scaffold material. Monolayer culture had strong induction of collagen II. The aggrecan protein was consistently expressed in all scaffold materials cultures and monolayer.     

Incorporation of an aggrecan mimic prevents proteolytic degradation of anisotropic cartilage analogs

Biomimetic scaffolds that promote regeneration and resist proteolysis are required as a tissue engineering solution to repair or replace a broad range of diseased tissues. Native corrosive environments, such as the richly enzymatic milieu of diseased articular cartilage, degrade the local extracellular matrix structure, so an implantable replacement must both replicate the healthy structure and demonstrate substantial proteolytic immunity, yet promote regeneration, if long-term functional success is to be achieved. Here, we combine magnetically aligned collagen with peptidoglycans, biosynthetic molecules that mimic proteoglycan activity but lack core proteins susceptible to proteases, to develop cartilage scaffold analogs with tailored functionality. With the incorporation of the aggrecan mimic, we demonstrate an ability to enhance bulk mechanical properties and prevent cytokine-induced degradation. Furthermore, fiber alignment in collagen scaffolds enhanced the gene expression of aggrecan, indicating cell responsiveness to anisotropy that also better replicates the natural environment of cartilage. Finally, the expression of type II collagen is enhanced with both alignment and incorporation of the aggrecan mimic, showing synergism between fiber alignment and incorporation of the aggrecan mimic. The work presented here identified a mechanistic synergy of matrix molecules and organization to prevent proteolysis while simultaneously upregulating protein expression.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells–fibrin suspension and polymerized by dropping thrombin–sodium chloride (CaCl₂) solution. A PLGA–cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold–cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.     

In vitro cartilage tissue engineering using adipose-derived extracellular matrix scaffolds seeded with adipose-derived stem cells

Extracellular matrix (ECM) secreted from the resident cell of tissue is an ideal biomaterial evolved by nature. Cartilage is also built from well-organized ECM components in a gel-like structure with a high collagen and proteoglycan content. Here, we explored cartilage tissue engineering using ECM scaffolds seeded with stem cells. Both scaffolds and stem cells were isolated from human adipose tissue, which is abundant and easily harvested in the human body. The human ECM scaffolds contained various endogenous bioactive factors, including transforming growth factor-beta1 (TGF-β1, 8782±4989?pg/g, dry ECM), insulin growth factor-1 (13319±1388?pg/g, dry ECM), basic fibroblast growth factor (82373±9572?pg/g, dry ECM), and vascular endothelial growth factor (25647±2749?pg/g, dry ECM). A composite of ECM and stem cells was prepared and cultured in chondrogenic medium (with 10?ng/mL TGF-β1 or not) for 45 days. The volumes and weights of the composites increased during culture and the surface gradually became smooth. Cell viability remained high throughout the 45 days of in vitro culture. Composites showed the formation of cartilage-like tissue with the synthesis of cartilage-specific proteins such as collagen and glycosaminoglycan. Important chondrogenic markers were expressed including Sox-9, aggrecan, and collagen type II and XI. These results demonstrate that a cell/ECM composite containing endogenous bioactive factors could provide biochemical cues for the promotion of cartilage formation.     

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.     

Nanofibrous architecture of silk fibroin scaffolds prepared with a mild self-assembly process

Besides excellent biocompatibility and biodegradability, a useful tissue engineering scaffold should provide suitable macropores and nanofibrous structure, similar to extracellular matrix (ECM), to induce desired cellular activities and to guide tissue regeneration. In the present study, a mild process to prepare porous and nanofibrous silk-based scaffolds from aqueous solution is described. Using collagen to control the self-assembly of silk, nanofibrous silk scaffolds were firstly achieved through lyophilization. Water annealing was used to generate insolubility in the silk-based scaffolds, thereby avoiding the use of organic solvents. The nano-fibrils formed in the silk-collagen scaffolds had diameters of 20-100 nm, similar with native collagen in ECM. The silk-collagen scaffolds dissolved slowly in PBS solution, with about a 28% mass lost after 4 weeks. Following the dissolution or degradation, the nanofibrous structure inside the macropore walls emerged and interacted with cells directly. During in vitro cell culture, the nanofibrous silk-collagen scaffolds containing 7.4% collagen demonstrated significantly improved cell compatibility when compared with salt-leached silk scaffolds and silk-collagen scaffolds containing 20% collagen that emerged less nano-fibrils. Therefore, this new process provides useful scaffolds for tissue engineering applications. Furthermore, the process involves all-aqueous, room temperature and pressure processing without the use of toxic chemicals or solvents, offering new green chemistry approaches, as well as options to load bioactive drugs or growth factors into process.     

Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.     

Effect of 3D-microstructure of bioabsorbable PGA:TMC scaffolds on the growth of chondrogenic cells

Various biomaterial scaffolds have been investigated for cartilage tissue engineering, although little attention has been paid to the effect of scaffold microstructure on tissue growth. Non-woven, fibrous, bioabsorbable scaffolds constructed from a copolymer of glycolide and trimethylene carbonate with varying levels of porosity and pore size were seeded with mesenchymal stroma cells with a chondrogenic lineage. Scaffolds and media were evaluated for both cell and extracellular matrix organization and content after up to 28 days of culture in a spinner flask. Analysis of DNA and glycosaminoglycan contents showed that the most porous of the three scaffold types, with a porosity of 81% and a porometry determined mean flow pore diameter of 54 microm, supported the most rapid proliferation of cells and accumulation of extracellular matrix. Analysis of the high porosity scaffold system, using Western Blot and immunohistochemistry confirmed the presence of collagen type II and absence of collagen type I, and demonstrated cells with a chondrocyte morphology with aggrecan and collagen II accumulation attached to the scaffolds. It was concluded that the 3D-microstructural characteristics of the scaffold (interconnecting porosity and pore size) play an important role in proliferation and phenotype of chondrogenic cells and accumulation of extracellular matrix molecules.     

Effect of an avidin-biotin binding system on chondrocyte adhesion, growth and gene expression

Cell adhesion to synthetic biomaterials is a prerequisite for anchorage cell culture and tissue engineering. The current study investigated utilization of an avidin-biotin binding system in enhancing chondrocyte adhesion to tissue culture polystyrene (TCPS). Biotinylated chondrocytes adhered to avidin-coated TCPS more quickly than untreated chondrocytes to bare TCPS. Also the avidin-biotin binding system enhanced cell initial spreading. However, the effects were only transient. The growth of biotinylated chondrocytes was first decreased during the first 3 days but increased afterwards. The progeny of biotinylated chondrocytes still maintained the ability in expressing cartilage extracellular matrix proteins such as type II collagen, type IX collagen and aggrecan. These results show potential for the application of the avidin-biotin binding system to cell culture and tissue engineering.     

Biomimetic nanofibrous scaffolds for bone tissue engineering

Bone tissue engineering is a highly interdisciplinary field that seeks to tackle the most challenging bone-related clinical issues. The major components of bone tissue engineering are the scaffold, cells, and growth factors. This review will focus on the scaffold and recent advancements in developing scaffolds that can mimic the natural extracellular matrix of bone. Specifically, these novel scaffolds mirror the nanofibrous collagen network that comprises the majority of the non-mineral portion of bone matrix. Using two main fabrication techniques, electrospinning and thermally-induced phase separation, and incorporating bone-like minerals, such as hydroxyapatite, composite nanofibrous scaffolds can improve cell adhesion, stem cell differentiation, and tissue formation. This review will cover the two main processing techniques and how they are being applied to fabricate scaffolds for bone tissue engineering. It will then cover how these scaffolds can enhance the osteogenic capabilities of a variety of cell types and survey the ability of the constructs to support the growth of clinically relevant bone tissue.