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-ß. 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.

Development of chimeric forms of the matrix metalloproteinase 2 collagen binding domain as artificial membrane binding proteins for targeting stem cells to cartilage lesions in osteoarthritic joints.

Targeting stem cells to cartilage lesions has the potential to enhance engraftment and chondrogenesis. Denatured type II collagen fibrils (gelatin) are exposed in lesions at the surface of osteoarthritic articular cartilage and are therefore ideal target sites. We have designed and investigated chimeric mutants of the three modules of the MMP-2 collagen binding domain (CBD) as potential ligands for stem cell targeting. We expressed full-length CBD for the first time and used it to identify the most important amino acid residues for binding to gelatin. Module 2 of CBD had the highest affinity binding to both Type I and Type II gelatin, whereas module 1 showed specificity for type II gelatin and module 3 for type I gelatin. We went on to generate chimeric forms of CBD consisting of three repeats of module 1 (111), module 2 (222) or module 3 (333). 111 lacked solubility and could not be further characterised. However 222 was found to bind to type II gelatin 14 times better than CBD, suggesting it would be optimal for attachment to cartilage lesions, whilst 333 was found to bind to type I gelatin 12 times better than CBD, suggesting it would be optimal for attachment to lesions in type I collagen-rich tissues. We coated 222 onto the external membrane of Mesenchymal Stem Cells and demonstrated higher attachment of the coated cells to type II gelatin than uncoated cells. We conclude that the three modules of CBD each have specific biological properties that can be exploited for targeting stem cells to cartilage lesions and other pathological sites.

Localization of type VI collagen in tissue-engineered cartilage on polymer scaffolds.

Together, the chondrocyte and its pericellular matrix have been collectively termed the chondron. Current opinion is that the pericellular matrix has both protective and signalling functions between chondrocyte and extracellular matrix. Formation of a native chondrocyte pericellular matrix or chondron structure might therefore be advantageous when tissue engineering a functional hyaline cartilage construct. The presence of chondrons has not been previously described in cartilage engineered on a scaffold. In this paper, we describe a modified immunochemical method to detect collagen VI, a key molecular marker for the pericellular matrix, and an investigation of type VI collagen distribution in engineered hyaline cartilage constructs. Cartilage constructs were engineered from adult human or bovine hyaline chondrocytes cultured on sponge or nonwoven fiber based HYAFF 11 scaffolds. Type VI collagen was detected in all constructs, but a distinctive, high-density, chondron-like distribution of collagen VI was present only in constructs exhibiting additional features of hyaline cartilage engineered using nonwoven HYAFF 11. Chondron structures were localized in areas of the extracellular matrix displaying strong collagen II and GAG staining of constructs where type II collagen composed a high percentage (over 65%) of the total collagen.

Quantitative analysis of repair tissue biopsies following chondrocyte implantation.

Outcome measures for cartilage repair techniques include clinical assessment of functional status, magnetic resonance imaging, mechanical indentation in situ and second-look biopsies, which are used for detailed ex vivo histological and immunohistochemical assessment. Biopsy analysis is considered an important outcome measure, despite being highly invasive, since it provides a visual record of the spatial organization of matrix proteins and cells. We propose that the value of second-look biopsies would be significantly enhanced if accurate quantification of cartilage matrix molecules could also be obtained. The goal of our work has been to develop a combined method for histological and biochemical analysis of a single biopsy. We have developed a method of cutting frozen sections of cartilage and recovering the uncut tissue for subsequent biochemical analysis. We have also developed a range of miniaturized assays that can be performed after cartilage digestion with trypsin. In this way we are now able to analyse biopsies with a wet weight as low as 5 mg using both histological and biochemical methods, so obtaining the maximum amount of information from the minimum volume of tissue. This new approach will allow a more accurate assessment of the quality of cartilage repair tissue than histological analysis alone.

Hyaluronan-based scaffolds (Hyalograft C) in the treatment of knee cartilage defects: preliminary clinical findings.

Hyalograft C is an innovative tissue-engineering approach for the treatment of knee cartilage defects involving the implantation of laboratory expanded autologous chondrocytes grown on a three-dimensional hyaluronan-based scaffold. This technique has recently been introduced into clinical practice, with more than 600 patients treated so far. Because no periosteal coverage is required to keep the graft in place, surgical time and morbidity are reduced, and handling of the graft is much simpler than currently available autologous chondrocyte implantation techniques. The safety profile of the treatment appears positive, with a limited number of adverse events reported. Here we discuss the clinical, arthroscopic and histological results from a cohort of 67 patients treated with Hyalograft C (mean follow-up time from implantation of 17.5 months). Results are reported based on four endpoints: patients' subjective evaluation of knee conditions (97% of patients improved) and quality of life (94% improved), surgeons' knee functional test (87% of patients with the best scores), arthroscopic evaluation of cartilage repair (96.7% biologically acceptable) and histological assessment of the grafted site (majority of specimens hyaline-like). The positive clinical results obtained indicate that Hyalograft C may be a viable therapeutic option for the treatment of acute cartilage lesions.

Alginate as a chondrocyte-delivery substance in combination with a non-woven scaffold for cartilage tissue engineering.

For tissue engineering of cartilage, chondrocytes can be seeded in a scaffold and stimulated to produce a cartilage-like matrix. In the present study, we investigated the effect of alginate as a chondrocyte-delivery substance for the construction of cartilage grafts. E210 (a non-woven fleece of polyglactin) was used as a scaffold. When bare' E210 (without alginate and without chondrocytes) was implanted subcutaneously in nude mice for 8 weeks. the explanted tissue consisted of fat and fibrous tissue only. When E210 with alginate but without chondrocytes was implanted in nude mice, small areas of newly formed cartilage were found. Alginate seems to stimulate chondrogenesis of ingrowing cells. When chondrocytes were seeded in E210, large amounts of cartilage were found, independent of the use of alginate. This was expressed by a high concentration of glycosaminoglycans (30 microg/mg w.w.) and the presence of collagen type II (1.5 microg/mg w.w.). Macroscopically the grafts of E210 without alginate were shrunk and warped, whereas the grafts with alginate had kept their original shape during the 8 weeks of implantation. The use of alginate did not lead to inflammatory reactions nor increased capsule formation. In conclusion, the use of alginate to seed chondrocytes in E210 does not influence the amount of cartilage matrix proteins produced per tissue wet weight. However, it provides retention of the graft shape.

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.

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.

Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach.

OBJECTIVE: To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. METHODS: Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. RESULTS: Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. CONCLUSION: Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.