Enzymatic outside-in cross-linking enables single-step microcapsule production for high-throughput three-dimensional cell microaggregate formation.

Cell-laden hydrogel microcapsules enable the high-throughput production of cell aggregates, which are relevant for three-dimensional tissue engineering and drug screening applications. However, current microcapsule production strategies are limited by their throughput, multistep protocols, and limited amount of compatible biomaterials. We here present a single-step process for the controlled microfluidic production of single-core microcapsules using enzymatic outside-in cross-linking of tyramine-conjugated polymers. It was hypothesized that a physically, instead of the conventionally explored biochemically, controlled enzymatic cross-linking process would improve the reproducibility, operational window, and throughput of shell formation. Droplets were flown through a silicone delay line, which allowed for highly controlled diffusion of the enzymatic cross-linking initiator. The microcapsules' cross-linking density and shell thickness is strictly depended on the droplet's retention time in the delay line, which is predictably controlled by flow rate. The here presented hydrogel cross-linking method allows for facile and cytocompatible production of cell-laden microcapsules compatible with the formation and biorthogonal isolation of long-term viable cellular spheroids for tissue engineering and drug screening applications.

Dickkopf-related protein 1 and gremlin 1 show different response than frizzled-related protein in human synovial fluid following knee injury and in patients with osteoarthritis.

OBJECTIVE: To explore the involvement of the wingless-type MMTV integration site (WNT) and bone morphogenetic protein (BMP) antagonists dickkopf-related protein 1 (DKK1), frizzled-related protein (FRZB) and gremlin 1 (GREM1) in knee injury and osteoarthritis (OA). DESIGN: The antagonists were immunoassayed in synovial fluid from a cross-sectional cohort of nine knee healthy reference subjects, patients with recent (0-77 days, n = 158) or old (1-37 years, n = 50) knee injuries, and OA (n = 22). Cartilage (ARGS-aggrecan, cartilage oligomeric matrix protein and C2C type II collagen) and other biomarkers were assessed in synovial fluid in a subset of samples. Statistical analysis was by Kendall's tau (τ) correlation, Mann-Whitney U test, and linear regression analysis. RESULTS: Compared to references, median concentration of GREM1 (but not DKK1 and FRZB) was elevated 1.5-fold immediately after injury, and FRZB was reduced 1000-folds in OA. All three antagonists decreased with increasing time after injury as well as with increasing age, but the temporal change after injury was less accentuated for FRZB (peaked 8-22 days after injury) compared to that of DKK1 and GREM1 (peaked immediately after injury). In the recent injury group, there was a correlation between GREM1 and DKK1 (τ = 0.172); FRZB concentrations correlated with concentrations of cartilage biomarkers (τ between 0.257 and 0.369), while DKK1 and GREM1 were inversely correlated (τ between -0.177 and -0.217) with these markers. CONCLUSIONS: Our results indicate separate roles for the antagonists, where DKK1 and GREM1 had similarities in response to injury and in OA, with a different response for FRZB.

Bioinspired seeding of biomaterials using three dimensional microtissues induces chondrogenic stem cell differentiation and cartilage formation under growth factor free conditions.

Cell laden biomaterials are archetypically seeded with individual cells and steered into the desired behavior using exogenous stimuli to control growth and differentiation. In contrast, direct cell-cell contact is instructive and even essential for natural tissue formation. Namely, microaggregation and condensation of mesenchymal progenitor cells triggers chondrogenesis and thereby drives limb formation. Yet a biomimetic strategy translating this approach into a cell laden biomaterial-based therapy has remained largely unexplored. Here, we integrate the microenvironment of cellular condensation into biomaterials by encapsulating microaggregates of a hundred human periosteum-derived stem cells. This resulted in decreased stemness-related markers, up regulation of chondrogenic genes and improved in vivo cartilage tissue formation, as compared to single cell seeded biomaterials. Importantly, even in the absence of exogenous growth factors, the microaggregate laden hydrogels outperformed conventional single cell laden hydrogels containing supraphysiological levels of the chondrogenic growth factor TGFB. Overall, the bioinspired seeding strategy described herein represents an efficient and growth factor-free approach to efficiently steer cell fate and drive tissue formation for biomaterial-based tissue engineering strategies.

Cell based advanced therapeutic medicinal products for bone repair: Keep it simple?

The development of cell based advanced therapeutic medicinal products (ATMPs) for bone repair has been expected to revolutionize the health care system for the clinical treatment of bone defects. Despite this great promise, the clinical outcomes of the few cell based ATMPs that have been translated into clinical treatments have been far from impressive. In part, the clinical outcomes have been hampered because of the simplicity of the first wave of products. In response the field has set-out and amassed a plethora of complexities to alleviate the simplicity induced limitations. Many of these potential second wave products have remained "stuck" in the development pipeline. This is due to a number of reasons including the lack of a regulatory framework that has been evolving in the last years and the shortage of enabling technologies for industrial manufacturing to deal with these novel complexities. In this review, we reflect on the current ATMPs and give special attention to novel approaches that are able to provide complexity to ATMPs in a straightforward manner. Moreover, we discuss the potential tools able to produce or predict 'goldilocks' ATMPs, which are neither too simple nor too complex.

Gene expression profiling of dedifferentiated human articular chondrocytes in monolayer culture.

OBJECTIVE: When primary chondrocytes are cultured in monolayer, they undergo dedifferentiation during which they lose their phenotype and their capacity to form cartilage. Dedifferentiation is an obstacle for cell therapy for cartilage degeneration. In this study, we aimed to systemically evaluate the changes in gene expression during dedifferentiation of human articular chondrocytes to identify underlying mechanisms. METHODS: RNA was isolated from monolayer-cultured primary human articular chondrocytes at serial passages. Gene expression was analyzed by microarray. Based on the microarray analysis, relevant genes and pathways were identified. Their functions in chondrocyte dedifferentiation were further investigated. RESULTS: In vitro expanded human chondrocytes showed progressive changes in gene expression. Strikingly, an overall decrease in total gene expression was detected, which was both gradual and cumulative. DNA methylation was in part responsible for the expression downregulation of a number of genes. Genes involved in many pathways such as the extracellular-signal-regulated kinase (ERK) and Bone morphogenetic protein (BMP) pathways exhibited significant changes in expression. Inhibition of ERK pathway did not show dramatic effects in counteracting dedifferentiation process. BMP-2 was able to decelerate the dedifferentiation and reinforce the maintenance of chondrocyte phenotype in monolayer culture. CONCLUSION: Our study not only improves our knowledge of the intricate signaling network regulating maintenance of chondrocyte phenotype, but also contributes to improved chondrocyte expansion and chondrogenic performance for cell therapy.

High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo.

Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

Gremlin 1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis.

OBJECTIVE: The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation. METHODS: Whole-genome gene-expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA. RESULTS: Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled-related protein (FRP), and Dkk-1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long-bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single-nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population-based cohorts. CONCLUSION: Taken together, our study identified Gremlin 1, FRP, and Dkk-1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

Short-term in vitro drug testing of mamma tumors: relation to estrogen and progesterone receptor levels, age, and proliferation rate.

The correlation between age, proliferation rate of tumors, estrogen and progesterone receptors, and in vitro chemosensitivity to Adriamycin was studied on 43 primary mamma tumors. From an univariate statistical analysis of the results, it appeared that sensitive tumors, unlike resistant tumors, have fewer estrogen receptors and show a higher proliferation rate. And in addition, they are blocked to a greater extent by Adriamycin. In both groups age and progesterone receptors were not significantly different. A multivariate statistical analysis showed that in the classification into sensitive and resistant tumors, the percentage remaining incorporation after addition of Adriamycin and the proliferation rate contributed 94 and 5%, respectively. The first variable was the best measure for in vitro chemosensitivity. The classification of the tumors with the aid of a discriminant function proved to be successful in 91% of all the cases. No significant difference was observed between the in vitro sensitivity to Adriamycin when patients were divided into two groups according to age (less than or equal to 50 and greater than 50 years; 64 and 45% sensitive, respectively). This indicates that all patients benefit from the treatment. It also appeared that 85% of the estrogen negative tumors were sensitive to Adriamycin. So a chemotherapeutic instead of a hormonal therapy has to be considered for all ages, particularly in the case of estrogen negative receptors.

Rapid assay for the in vitro chemosensitivity testing of human breast tumours. Control of assay conditions and quality assurance.

This study has been initiated by the definite need of a rapid, in vitro and patient-specific test to examine oncolytic drug effects on tumour cells. Therefore, we applied a test in which we monitored the incorporation of labelled nucleosides in isolated tumour cells, as a measure of nucleus activity (i.e. the Volm assay). A novel technique of erythrocytes co-precipitation has been developed and this enabled us to use a small number of tumour cells per test (200,000 cells/tube). During the assay, a strict pH control and a high starting viability have been introduced. A cytotoxic control and a t-test at two levels deal with the technical errors and the imprecision. For the evaluation of a specific drug a number of 1.8 million cells proved to be sufficient. Time-course studies of the incorporation of labelled nucleosides into isolated tumour cells have shown the optimal incubation time to be 2 h. The entire assay is completed within one day. HeLa cell cultures were employed as quality control material and criteria for interpretation have been developed. Preliminary results, based upon the evaluation of 43 human breast tumours with doxorubicin, indicate the correctness of the proposed procedures. In conclusion we can state that this assay not only provides useful information but above all that the results are made available in such a short time that they can be used directly in the medical management of the individual patient.

The classification of subjects with joint complaints on incomplete biochemical and haematological datasets.

We performed a retrospective study on 163 subjects suffering from rheumatic fever (16), rheumatoid arthritis (36), lupus erythematosus (17), gout (21), arthrosis (50) and osteomyelitis (23). The number of variables evaluated was 39. These were all of a general biochemical and haematological nature. A feature reduction resulted in sixteen variables that matched well with those known from the literature. Linear discriminant analysis yielded poor results in classifying the six disease categories (with 18 variables 61.8%). A reduction to three disease categories improved the classification results remarkably. This, and the excellent discriminating power between patients and the reference group, shows that the selected variables are illustrative only for general clinical pictures, such as infection, and not for the desired differential diagnosis.

The costing of tests in a laboratory for clinical chemistry and haematology.

The total cost of a haematology and clinical chemistry laboratory, including external and hospital costs, was studied in a district general hospital in The Netherlands. The main conclusion is that the number of samples analysed is the most important cost-setting factor, but that reduction in the number of requests does not result in the proportional diminution of costs.