Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering

Articular cartilage injuries and degeneration affect a large proportion of the population in developed countries world wide. Stem cells can be differentiated into chondrocytes by adding transforming growth factor-beta1 and dexamethasone to a pellet culture, which are unfeasible for tissue engineering purposes. We attempted to achieve stable chondrogenesis without any requirement for exogenous growth factors. Human mesenchymal stem cells were transduced with an adenoviral vector containing the SRY-related HMG-box gene 9 (SOX9), and were cultured in a three-dimensional (3D) hydrogel scaffold composite. As an additional treatment, mechanical stimulation was applied in a custom-made bioreactor. SOX9 increased the expression level of its known target genes, as well as its cofactors: the long form of SOX5 and SOX6. However, it was unable to increase the synthesis of sulfated glycosaminoglycans (GAGs). Mechanical stimulation slightly enhanced collagen type X and increased lubricin expression. The combination of SOX9 and mechanical load boosted GAG synthesis as shown by (35)S incorporation. GAG production rate corresponded well with the amount of (endogenous) transforming growth factor-beta1. Finally, cartilage oligomeric matrix protein expression was increased by both treatments. These findings provide insight into the mechanotransduction of mesenchymal stem cells and demonstrate the potential of a transcription factor in stem cell therapy.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

The Peri-Implant Sulcus Compared with Internal Implant and Suprastructure Components: A Microbiological Analysis

Purpose: A recent in vivo study has shown considerable contamination of internal implant and suprastructure components with great biodiversity, indicating bacterial leakage along the implant-abutment interface, abutment-prosthesis interface, and restorative margins. The goal of the present study was to compare microbiologically the peri-implant sulcus to these internal components on implants with no clinical signs of peri-implantitis and in function for many years. Checkerboard DNA-DNA hybridization was used to identify and quantify 40 species. Material and Methods: Fifty-eight turned titanium Brånemark implants in eight systemically healthy patients (seven women, one man) under regular supportive care were examined. All implants had been placed in the maxilla and loaded with a screw-retained full-arch bridge for an average of 9.6 years. Gingival fluid samples were collected from the deepest sulcus per implant for microbiological analysis. As all fixed restorations were removed, the cotton pellet enclosed in the intra-coronal compartment and the abutment screw were retrieved and microbiologically evaluated. Results: The pellet enclosed in the suprastructure was very similar to the peri-implant sulcus in terms of bacterial detection frequencies and levels for practically all the species included in the panel. Yet, there was virtually no microbial link between these compartments. When comparing the abutment screw to the peri-implant sulcus, the majority of the species were less frequently found, and in lower numbers at the former. However, a relevant link in counts for a lot of bacteria was described between these compartments. Even though all implants in the present study showed no clinical signs of peri-implantitis, the high prevalence of numerous species associated with pathology was striking. Conclusions: Intra-coronal compartments of screw-retained fixed restorations were heavily contaminated. The restorative margin may have been the principal pathway for bacterial leakage. Contamination of abutment screws most likely occurred from the peri-implant sulcus via the implant-abutment interface and abutment-prosthesis interface.

Does delaying the first mowing date benefit biodiversity in meadowland?

Background Meadows are regularly mown in order to provide fodder or litter for livestock and to prevent vegetation succession. However, the time of year at which meadows should be first mown in order to maximize biological diversity remains controversial and may vary with respect to context and focal taxa. We carried out a systematic review and meta-analysis on the effects of delaying the first mowing date upon plants and invertebrates in European meadowlands. Methods Following a CEE protocol, ISI Web of Science, Science Direct, JSTOR, Google and Google Scholar were searched. We recorded all studies that compared the species richness of plants, or the species richness or abundance of invertebrates, between grassland plots mown at a postponed date (treatment) vs plots mown earlier (control). In order to be included in the meta-analysis, compared plots had to be similar in all management respects, except the date of the first cut that was (mostly experimentally) manipulated. They were also to be located in the same meadow type. Meta-analyses applying Hedges’d statistic were performed. Results Plant species richness responded differently to the date to which mowing was postponed. Delaying mowing from spring to summer had a positive effect, while delaying either from spring to fall, or from early summer to later in the season had a negative effect. Invertebrates were expected to show a strong response to delayed mowing due to their dependence on sward structure, but only species richness showed a clearly significant positive response. Invertebrate abundance was positively influenced in only a few studies. Conclusions The present meta-analysis shows that in general delaying the first mowing date in European meadowlands has either positive or neutral effects on plant and invertebrate biodiversity (except for plant species richness when delaying from spring to fall or from early summer to later). Overall, there was also strong between-study heterogeneity, pointing to other major confounding factors, the elucidation of which requires further field experiments with both larger sample sizes and a distinction between taxon-specific and meadow-type-specific responses.

Monitoring Butterfly Abundance: Beyond Pollard Walks

Most butterfly monitoring protocols rely on counts along transects (Pollard walks) to generate species abundance indices and track population trends. It is still too often ignored that a population count results from two processes: the biological process (true abundance) and the statistical process (our ability to properly quantify abundance). Because individual detectability tends to vary in space (e.g., among sites) and time (e.g., among years), it remains unclear whether index counts truly reflect population sizes and trends. This study compares capture-mark-recapture (absolute abundance) and count-index (relative abundance) monitoring methods in three species (Maculinea nausithous and Iolana iolas: Lycaenidae; Minois dryas: Satyridae) in contrasted habitat types. We demonstrate that intraspecific variability in individual detectability under standard monitoring conditions is probably the rule rather than the exception, which questions the reliability of count-based indices to estimate and compare specific population abundance. Our results suggest that the accuracy of count-based methods depends heavily on the ecology and behavior of the target species, as well as on the type of habitat in which surveys take place. Monitoring programs designed to assess the abundance and trends in butterfly populations should incorporate a measure of detectability. We discuss the relative advantages and inconveniences of current monitoring methods and analytical approaches with respect to the characteristics of the species under scrutiny and resources availability.

Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

Visual histological grading system for the evaluation of in vitro-generated neocartilage

Here we present the development of a visual evaluation system for routine assessment of in vitro-engineered cartilaginous tissue. Neocartilage was produced by culturing human articular chondrocytes in pellet culture systems or in a scaffold-free bioreactor system. All engineered tissues were embedded in paraffin and were sectioned and stained with Safranin O-fast green. The evaluation of each sample was broken into 3 categories (uniformity and intensity of Safranin O stain, distance between cells/amount of matrix produced, and cell morphology), and each category had 4 components with a score ranging from 0 to 3. Three observers evaluated each sample, and the new system was independently tested against an objective computer-based histomorphometry system. Pellets were also assessed biochemically for glycosaminoglycan (GAG) content. Pellet histology scores correlated significantly with GAG contents and were in agreement with the computer-based histomorphometry system. This system allows a valid and rapid assessment of in vitro-generated cartilaginous tissue that has a relevant association with objective parameters indicative of cartilage quality.

Cartilage tissue engineering by expanded goat articular chondrocytes

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

Tissue neogenesis and STRO-1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO-1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin-O, for type II collagen and STRO-1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO-1 expression patterns. Results show that immature explants contained more STRO-1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO-1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO-1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO-1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO-1+ cells in immature cartilage changes with joint maturation. STRO-1+ cells have the potential to form new cartilage spontaneously and after tissue injury. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.