Maintenance of cartilaginous gene expression on extracellular matrix derived from serially passaged chondrocytes during in vitro chondrocyte expansion

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering.     

The sintered microsphere matrix for bone tissue engineering: in vitro osteoconductivity studies

A tissue engineering approach has been used to design three-dimensional synthetic matrices for bone repair. The osteoconductivity and degradation profile of a novel polymeric bone-graft substitute was evaluated in an in vitro setting. Using the copolymer poly(lactide-co-glycolide) [PLAGA], a sintering technique based on microsphere technology was used to fabricate three-dimensional porous scaffolds for bone regeneration. Osteoblasts and fibroblasts were seeded onto a 50:50 PLAGA scaffold. Morphologic evaluation through scanning electron microscopy demonstrated that both cell types attached and spread over the scaffold. Cells migrated through the matrix using cytoplasmic extensions to bridge the structure. Cross-sectional images indicated that cellular proliferation had penetrated into the matrix approximately 700 microm from the surface. Examination of the surfaces of cell/matrix constructs demonstrated that cellular proliferation had encompassed the pores of the matrix by 14 days of cell culture. With the aim of optimizing polymer composition and polymer molecular weight, a degradation study was conducted utilizing the matrix. The results demonstrate that degradation of the sintered matrix is dependent on molecular weight, copolymer ratio, and pore volume. From this data, it was determined that 75:25 PLAGA with an initial molecular weight of 100,000 has an optimal degradation profile. These studies show that the sintered microsphere matrix has an osteoconductive structure capable of functioning as a cellular scaffold with a degradation profile suitable for bone regeneration.     

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.     

Nasal chondrocytes and fibrin sealant for cartilage tissue engineering

Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage.     

Human bone marrow stromal cells: In vitro expansion and differentiation for bone engineering

Stromal cells from marrow hold a great promise for bone regeneration. Even if they are already being exploited in many clinical settings, the biological basis for the source and maintenance of their proliferation/differentiation potential after in vitro isolation and expansion needs further investigation.

Most studies on osteogenic differentiation of marrow stromal cells (MSC) have been performed using bone marrow from the iliac crest. In this study, MSC were derived from spare femoral bone marrow obtained during hip replacement surgery from 20 adult donors. After in vitro isolation the cells were grown in osteogenic medium, and their proliferation and differentiation analysed during in vitro expansion. We found that MSC isolated from the femur of adult patients consistently maintain an osteogenic potential. Using biochemical signals, these cells turn to fully differentiated osteoblasts with a predictable set of molecular and phenotypic events of in vitro bone deposition. When seeded on polycaprolactone-based scaffold or surfaces, the proliferation and mineralization of femur-derived MSC were modulated by the surface chemistry/topography. Despite remarkable differences between individual colony-forming ability, alkaline phosphatase production, and mineralization ability, these cells are a potential source for bone engineering, either by direct autologous reimplantation or by ex vivo expansion and reimplantation combined to a proper scaffold.     

Autologous extracellular matrix scaffolds for tissue engineering

Development of autologous scaffolds has been highly desired for implantation without eliciting adverse inflammatory and immune responses. However, it has been difficult to obtain autologous scaffolds by tissue decellularization because of the restricted availability of autologous donor tissues from a patient. Here we report a method to prepare autologous extracellular matrix (aECM) scaffolds by combining culture of autologous cells in a three-dimensional template, decellularization, and template removal. The aECM scaffolds showed excellent biocompatibility when implanted. We anticipate that "Full Autologous Tissue Engineering" can be realized to minimize undesirable host tissue responses by culturing the patient's own cells in an aECM scaffold.     

Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering

Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes.     

Cultured cell-derived extracellular matrix scaffolds for tissue engineering

Cell-derived extracellular matrix (ECM) scaffolds have received considerable interest for tissue engineering applications. In this study, ECM scaffolds derived from mesenchymal stem cell (MSC), chondrocyte, and fibroblast were prepared by culturing cells in a selectively removable poly(lactic-co-glycolic acid) (PLGA) template. These three types of ECM scaffolds were used for in vitro cultures of MSC and fibroblasts to examine their potential as scaffolds for cartilage and skin tissue engineering. The MSC were cultured in MSC- and chondrocyte-derived ECM scaffolds. The ECM scaffolds supported cell adhesion, promoted both cell proliferation and the production of ECM and demonstrated a stronger stimulatory effect on the chondrogenesis of MSC compared with a conventional pellet culture method. Histological and immunohistochemical staining indicated that cartilage-like tissues were regenerated after the MSC were cultured in ECM scaffolds. Fibroblasts were cultured in the fibroblast-derived ECM scaffolds. Fibroblasts proliferated and produced ECM to fill the pores and spaces in the scaffold. After 2 weeks of culture, a uniform multilayered tissue was generated with homogenously distributed fibroblasts. Cell-derived ECM scaffolds have been demonstrated to facilitate tissue regeneration and will be a useful tool for tissue engineering.     

Three-dimensional reconstituted extracellular matrix scaffolds for tissue engineering

The extracellular matrix (ECM) is a rich meshwork of proteins and proteoglycans. Besides assuming a cell adhesive and structural support role, the ECM also helps to sequester and present growth factors to cells. ECM derived from tissues has been used as biological scaffolds for tissue engineering. In contrast, it has been difficult to employ ECM derived from cell lines as scaffolds due to its lack of form and structure. We have developed a mild, aqueous-based method for incorporating cell line derived ECM into biological scaffolds based on polyelectrolyte complexation, using the example of ECM from MC-3T3, a mouse preosteoblast cell line. A DNase step was incorporated in the ECM isolation procedure to further purify it of genetic material. Immunohistochemistry of fibers incorporated with MC-3T3 ECM reveal the presence of the ECM components, collagen type I, collagen type IV, fibronectin and heparan sulfate, on their surface. Reconstituted ECM scaffolds retained the cell-adhesion characteristics of the ECM, as demonstrated by ‘reseeding’ the ECM-secreting cell on the scaffolds. Human mesenchymal stem cells (hMSCs) were seeded onto the fibrous scaffolds incorporated with MC-3T3 ECM, and implanted subcutaneously into SCID mice. After 4 weeks of implantation, histological evidence showed that the hMSC seeded ECM scaffolds had induced bone formation at the ectopic site.     

Novel fabricated matrix via electrospinning for tissue engineering

Electric field-driven fiber formation (electrospinning) is developing into a practical means for preparing novel porous filament with unusual structures and affordable mechanical properties. Polycaprolactone (PCL) was dissolved in solvent mixtures of methylene chloride/N,N-dimethyl formamide with ratios of 100/0, 75/25, and 50/50 (v/v) for electrospinning. The filament was formed by coagulation of the spinning solution following the well-known principle of phase separation in polymer solutions valid in other wet shaping processes. A strand of electrospun porous filament consisted of fibers ranging from 0.5 to 12 microm in diameter. To evaluate the feasibility of three-dimensional fabric as scaffold matrices, the plain weave, which is the simplest of the weaves and the most common, was prepared with porous PCL filament. The growth characteristics of MCF-7 mammary carcinoma cells in the woven fabrics showed the important role of matrix microstructure in proliferation. This study has shown that woven fabrics, consisting of porous filaments via electrospinning, may be suitable candidates as tissue engineering scaffolds.     

Mining the extracellular matrix for tissue engineering applications

Tissue engineering is a rapidly evolving interdisciplinary field that aims to regenerate new tissue to replace damaged tissues or organs. The extracellular matrix (ECM) of animal tissues is a complex mixture of macromolecules that play an essential instructional role in the development of tissues and organs. Therefore, tissue engineering approaches rely on the need to present the correct cues to cells, to guide them to maintain tissue-specific functions. Recent research efforts have allowed us to mine various sequences and motifs, which play key roles in these guidance functions, from the ECM. Small conserved peptide sequences mined from ECM molecules can mimic some of the biological functions of their large parent molecules. In addition, these peptide sequences can be linked to various biomaterial scaffolds that can provide the cells with mechanical support to ensure appropriate cell growth and aid the formation of the correct tissue structure. The tissue engineering field will continue to benefit from the advent of these mined ECM sequences which have two major advantages over recombinant ECM molecules: material consistency and scalability.     

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.     

Rabbit articular chondrocytes seeded on collagen-chitosan-GAG scaffold for cartilage tissue engineering in vivo

In this study, we prepared a tri-copolymer porous matrices by natural polymer, collagen (Col), Chitosan (Chi) and Chondroitin (CS). Rabbit articular chondrocytes were isolated from the shoulder articular joints of a rabbit, seeded in Col-Chi-CS scaffold, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. In vitro studies show that Chondrocytes adhered to the scaffold, where they proliferated and secreted extracellular matrices with time, filling the space within the scaffold. The results of hematoxylin and eosin staining scanning electron microscopy revealed that most of the chondrocytes maintained their typically rounded morphology. After 28 days of culture within Col-Chi-CS scaffold in vitro, the results of histological staining showed forming of cartilage-specific morphological appearance and structural characteristics such as lacunae. Subcutaneous implantation studies in nude mice demonstrated that a homogeneous cartilaginous tissue, which was similar to those of natural cartilage, formed when chondrocytes were seeded in Col-Chi-CS matrix after implant 12 weeks. The tri-copolymer matrix could therefore have potential applications as a three-dimensional scaffold for cartilage tissue engineering.     

Cartilage tissue formation using redifferentiated passaged chondrocytes in vitro

Articular cartilage has limited ability for repair when damaged by trauma or degenerative disease, such as osteoarthritis, which can result in pain and compromised quality of life. Biological surface replacements developed using tissue engineering methods are a promising approach for cartilage repair, which would avoid the need for total joint replacement with the synthetic implants used currently. A basic requirement of in vitro tissue generation is a supply of sufficient number of cells, which are difficult to acquire from sparsely cellular cartilage tissue. Previously, we have shown that coculture of in vitro-expanded dedifferentiated chondrocytes (P2) with small numbers of primary chondrocytes (P0) induces redifferentiation in passaged (P2) cells. In this study we show that this redifferentiation is not a transient change. After 4 weeks of coculture, the P0 and P2 cells were separated by flow-associated cell sorting, and the redifferentiated P2 (dP2) were cultured alone for a further 4 weeks. The redifferentiated dP2 cells formed thicker cartilage tissue compared to the tissue generated by P2 cells. The newly formed tissue contained type II collagen as demonstrated by immunohistochemical staining and accumulated more proteoglycan per cell than the tissue formed by P2 cells. The dP2 cells also exhibited higher type II collagen and lower type I collagen gene expression than the P2 cells. Interestingly, dP2 cells were able to exert the same effect as P0 cells when cocultured with P2 cells. In conclusion, under proper culture conditions, redifferentiated passaged chondrocytes behave similarly to primary chondrocytes. This coculture system approach can be used to increase the number of differentiated chondrocytes that can be obtained by classical monolayer cell expansion and represents a novel way to acquire sufficient cell numbers for cartilage tissue engineering.     

Self-assembly of fibrochondrocytes and chondrocytes for tissue engineering of the knee meniscus

Chondrocyte self-assembly in high-density scaffoldless culture has shown success in producing articular cartilage constructs, and a similar process could be applied to fibrocartilage tissue engineering. Three cell combinations were compared in self-assembly culture-100% chondrocytes, 100% meniscal fibrochondrocytes, and 50:50 co-cultures of fibrochondrocytes and chondrocytes with the goal of creating a proteoglycan, collagen I, and collagen II matrix similar to native meniscus. Two culture surfaces were also compared for self-assembly: agarose-coated wells and tissue culture plastic. After 4 weeks, the resulting self-assembled chondrocyte constructs were 10.24+/-0.63 mm in diameter and 0.96+/-0.14 mm thick, weighing 84.5+/-7.2 mg. Co-culture constructs were smaller and weighed 22.5+/-1.0 mg. In contrast, the fibrochondrocyte constructs contracted into spheres weighing 1.3+/-0.3 mg. Immunostaining showed collagen II in the chondrocyte constructs, both collagen I and collagen II in the co-cultures, and only collagen I in the fibrochondrocyte constructs. Collagen densities for chondrocyte, co-culture, and fibrochondrocyte constructs were 41+/-3, 38+/-3, and 20+/-2 microg/mg dry weight, and glycosaminoglycan densities were 230+/-2, 80+/-6, and 10+/-1 microg/mg dry weight, respectively. Self-assembled co-cultures, with their mixed collagen I and II matrix and robust gross characteristics, appear promising for tissue engineering of the knee meniscus.     

Lactoyl-poloxamine/collagen matrix for cell-containing tissue engineering modules

Collagen-containing crosslinked, remodelable poloxamine derivatives were produced by introducing very short oligo(lactic acid) segments through the reaction of poloxamine with L-lactide and the later addition of unsaturated bonds by the reaction of modified poloxamine with methacryloyl chloride. Degradation studies on discs indicated a faster weight loss in comparison to the stability of lactoyl-free samples. Cell-containing modules (both HepG2 cells and two different umbilical vein smooth muscle cell (UVSMC) cell-types) were produced. Live/Dead assay showed high survival levels for both HepG2 and UVSMC cell types after crosslinking. While nondegradable modules did not change shape over time, lactoyl-poloxamine matrices showed a gradual shrinkage and size decrease and an increase in the roughness of the surface. These findings were consistent with the expected degradability of the lactoyl derivative. A UVSMC cell line (CRL-2481) embedded in a LA-poloxamine/collagen matrix showed the characteristic elongated shape at day 9. UVSMC primary cells behaved in a manner similar to that seen in collagen gels: these cells formed isolated clusters through the matrix that gradually lost viability. On tissue culture polystyrene the same cells aggregated and did not reach confluence. Modules with embedded CRL-2481 UVSMC led to a better initial adhesion of endothelial cells and a higher extent of surface coverage than seen with the UVSMC-free system. With embedded primary UVSMC, some EC attachment and formation of gap junctions was seen. The pattern was not well organized. With further improvement (and characterization), the lactoyl poloxamine derivative is potentially useful as a scaffold for modular tissue engineering, when tissue remodeling is an important consideration.     

Development of photosynthetic biomaterials for in vitro tissue engineering

Tissue engineering has opened a new therapeutic avenue that promises a revolution in regenerative medicine. To date, however, the translation of engineered tissues into clinical settings has been highly limited and the clinical results are often disappointing. Despite decades of research, the appropriate delivery of oxygen into three-dimensional cultures still remains one of the biggest unresolved problems for in vitro tissue engineering. In this work, we propose an alternative source of oxygen delivery by introducing photosynthetic scaffolds. Here we demonstrate that the unicellular and photosynthetic microalga Chlamydomonas reinhardtii can be cultured in scaffolds for tissue repair; this microalga shows high biocompatibility and photosynthetic activity. Moreover, Chlamydomonas can be co-cultured with fibroblasts, decreasing the hypoxic response under low oxygen culture conditions. Finally, results showed that photosynthetic scaffolds are capable of producing enough oxygen to be independent of external supply in vitro. The results of this study represent the first step towards engineering photosynthetic autotrophic tissues.     

A comparison of human umbilical cord matrix stem cells and temporomandibular joint condylar chondrocytes for tissue engineering temporomandibular joint condylar cartilage

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.