Wael Kafienah

Three-Dimensional Tissue Engineering of Hyaline Cartilage: Comparison of Adult Nasal and Articular Chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.

Nucleostemin is a marker of proliferating stromal stem cells in adult human bone marrow

The identification of stem cell–specific proteins and the elucidation of their novel regulatory pathways may help in the development of protocols for control of their self‐renewal and differentiation for cell‐based therapies. Nucleostemin is a recently discovered nucleolar protein predominantly associated with proliferating rat neural and embryonic stem cells, and some human cancer cell lines. A comprehensive study of nucleostemin in human adult bone marrow stem cells is lacking. The aim of the study was to determine if nucleostemin is synthesized by adult bone marrow stem cells and to analyze its expression during their expansion and differentiation. Using a multipotential adherent population of stem cells, nucleostemin was localized to the nucleoli and occurred in 43.3% of the cells. There was a high level of expression of nucleostemin mRNA in bone marrow stem cells and this remained unchanged over time during cell expansion in culture. When bone marrow stem cells were stimulated to proliferate by fibroblast growth factor (FGF)‐2, nucleostemin expression increased in a dose‐dependent manner. Small interfering RNA (siRNA) knockdown of nucleostemin abolished the proliferative effect of FGF‐2. When bone marrow stem cells were differentiated into chondrocytes, adipocytes, or osteocytes, nucleostemin expression was 70%–90% lower than in the undifferentiated cells retained in monolayer culture. We conclude that nucleostemin is a marker of undifferentiated human adult bone marrow stem cells and that it is involved in the regulation of proliferation of these cells.

Repair of meniscal cartilage white zone tears using a stem cell/collagen-scaffold implant

Injuries to the avascular region of knee meniscal cartilage do not heal spontaneously. To address this problem we have developed a new stem cell/collagen-scaffold implant system in which human adult bone marrow mesenchymal stem cells are seeded onto a biodegradable scaffold that allows controlled delivery of actively dividing cells to the meniscus surface. Sandwich constructs of two white zone ovine meniscus discs with stem cell/collagen-scaffold implant in between were cultured in vitro for 40 days. Histomorphometric analysis revealed superior integration in the stem cell/collagen-scaffold groups compared to the cell-free collagen membrane or untreated controls. The addition of TGF-beta1 to differentiate stem cells to chondrocytes inhibited integration. Biomechanical testing demonstrated a significant 2-fold increase in tensile strength in all constructs using the stem cell/collagen-scaffold compared to control groups after 40 days in culture. Integration was significantly higher when collagen membranes were used that had a more open/spongy structure adjacent to both meniscal cartilage surfaces, whereas a collagen scaffold designed for osteoinduction failed to induce any integration of meniscus. In conclusion, the stem cell/collagen-scaffold implant is a potential therapeutic treatment for the repair of white zone meniscal cartilage tears.

Stem cells and cartilage development: complexities of a simple tissue.

Cartilage is considered to be a simple tissue that should be easy to engineer because it is avascular and contains just one cell type, the chondrocyte. Despite this apparent simplicity, regenerating cartilage in a form that can function effectively after implantation in the joint has proven difficult. This may be because we have not fully appreciated the importance of different structural regions of articular cartilage or of understanding the origins of chondrocytes and how this cell population is maintained in the normal tissue. This review considers what is known about different regions of cartilage and the types of stem cells in articulating joints and emphasizes the potential importance of regeneration of the lamina splendens at the joint surface and calcified cartilage at the junction with bone for long‐term survival of regenerated tissue in vivo. STEM CELLS 2010;28:1992–1996

Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.

Pharmacological Regulation of Adult Stem Cells: Chondrogenesis Can Be Induced Using a Synthetic Inhibitor of the Retinoic Acid Receptor

Conventional methods for regulating the differentiation of stem cells are largely based on the use of biological agents such as growth factors. We hypothesize that stem cell differentiation could be driven by specific synthetic molecules. If true, this would offer the possibility of screening chemical libraries to develop pharmacological agents with improved efficacy. To test our hypothesis, we have determined which, if any, of the nuclear receptor superfamily might be involved in chondrogenesis. We used fluorescence‐activated cell sorting, as well as quantitative polymerase chain reaction, to study expression of a range of nuclear receptors in the undifferentiated mesenchymal population and after growth factor‐driven differentiation of these cells to chondrocytes. In this way, we identified retinoic acid receptor β (RARβ) as a potential pharmacological target. A low molecular weight synthetic inhibitor of the RARα and RARβ receptors was able to induce chondrogenic differentiation of mesenchymal stem cells derived from osteoarthritis patients, in the absence of serum and growth factors. Furthermore, the pathway is independent of SOX9 upregulation and does not lead to hypertrophy. When mesenchymal cells were seeded on to polyglycolic acid scaffolds and cultured with LE135, there was a dose‐dependent formation of cartilage, demonstrated both histologically and by biochemical analysis of the collagen component of the extracellular matrix. These results demonstrate the feasibility of a pharmacological approach to the regulation of stem cell function.

Human Fetal and Adult Bone Marrow-Derived Mesenchymal Stem Cells Use Different Signaling Pathways for the Initiation of Chondrogenesis

Cartilage injuries and osteoarthritis are leading causes of disability in developed countries. The regeneration of damaged articular cartilage using cell transplantation or tissue engineering holds much promise but requires the identification of an appropriate cell source with a high proliferative propensity and consistent chondrogenic capacity. Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues, including first-trimester bone marrow, and have demonstrated enhanced expansion and differentiation potential. However, their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here, we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering, they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signaling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signaling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modeling chondrogenic differentiation and skeletal development studies.

Directing chondrogenesis of stem cells with specific blends of cellulose and silk

Biomaterials that can stimulate stem cell differentiation without growth factor supplementation provide potent and cost-effective scaffolds for regenerative medicine. We hypothesize that a scaffold prepared from cellulose and silk blends can direct stem cell chondrogenic fate. We systematically prepared cellulose blends with silk at different compositions using an environmentally benign processing method based on ionic liquids as a common solvent. We tested the effect of blend compositions on the physical properties of the materials as well as on their ability to support mesenchymal stem cell (MSC) growth and chondrogenic differentiation. The stiffness and tensile strength of cellulose was significantly reduced by blending with silk. The characterized materials were tested using MSCs derived from four different patients. Growing MSCs on a specific blend combination of cellulose and silk in a 75:25 ratio significantly upregulated the chondrogenic marker genes SOX9, aggrecan, and type II collagen in the absence of specific growth factors. This chondrogenic effect was neither found with neat cellulose nor the cellulose/silk 50:50 blend composition. No adipogenic or osteogenic differentiation was detected on the blends, suggesting that the cellulose/silk 75:25 blend induced specific stem cell differentiation into the chondrogenic lineage without addition of the soluble growth factor TGF-β. The cellulose/silk blend we identified can be used both for in vitro tissue engineering and as an implantable device for stimulating endogenous stem cells to initiate cartilage repair.

Lumican inhibits collagen deposition in tissue engineered cartilage

Lumican is a glycoprotein that is found in the extracellular matrix of many connective tissues, including cartilage. It is a member of the small leucine-rich repeat proteoglycans family and along with two others, decorin and fibromodulin, has the capacity to bind to fibrillar collagens and limit their growth. Cartilage tissue engineering provides a potential method for the production of three-dimensional tissue for implantation into eroded joints. Many studies have demonstrated the growth of cartilage in vitro. However in all cases, biochemical analysis of the tissue revealed a significant deficit in the collagen content. We have now tested the hypothesis that the reduced collagen accumulation in engineered cartilage is a result of over-expression of decorin, fibromodulin or lumican. We have found that the lumican gene and protein are both over-expressed in engineered compared to natural cartilage whereas this is not the case for decorin or fibromodulin. Using a small hairpin lumican antisense sequence we were able to knockdown the lumican gene and protein expression in chondrocytes being used for tissue engineering. This resulted in increased accumulation of type II collagen (the major collagen of cartilage) whilst there was no significant alteration in the proteoglycan content. Furthermore, the antisense knockdown of lumican resulted in an increase in the average collagen fibril diameter measured by transmission electron microscopy. These results suggest that lumican plays a pivotal role in the development of tissue engineered cartilage and that regulation of this protein may be important for the production of high-quality implants.

Ionic liquids-based processing of electrically conducting chitin nanocomposite scaffolds for stem cell growth

In the present study, we have successfully combined the biocompatible properties of chitin with the high electrical conductivity of carbon nanotubes (CNTs) by mixing them using an imidazolium-based ionic liquid as a common solvent/dispersion medium. The resulting nanocomposites demonstrated uniform distribution of CNTs, as shown by scanning electron microscopy (SEM) and optical microscopy. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction confirmed the α-crystal structure of chitin in the regenerated chitin nanocomposite scaffolds. Increased CNT concentration in the chitin matrix resulted in higher conductivity of the scaffolds. Human mesenchymal stem cells adhered to, and proliferated on, chitin–CNT nanocomposites with different ratios. Cell growth in the first 3 days was similar on all composites at a range of (0.01 to 0.07) weight fraction of CNT. However, composites at a 0.1 weight fraction of CNTs showed reduced cell attachment. There was a significant increase in cell proliferation using 0.07 weight fraction CNT composites, suggesting a stem cell enhancing function for CNTs at this concentration. In conclusion, the ionic liquid allowed the uniform dispersion of CNTs and dissolution of chitin to create a biocompatible, electrically conducting scaffold permissive for mesenchymal stem cell function. This method will enable the fabrication of chitin-based advanced multifunctional biocompatible scaffolds where electrical conduction is critical for tissue function.