The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues

Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced microtissues fall into common microwells. Herein we describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells; the microwell-mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells. To demonstrate the utility of this platform, we used the microwell-mesh to manufacture hundreds of cartilage microtissues, each formed from 5 × 10(3) bone marrow-derived mesenchymal stem/stromal cells (MSC). The microwell-mesh enabled reliable microtissue retention over 21-day cultures that included multiple full medium exchanges. Cartilage-like matrix formation was more rapid and homogeneous in microtissues than in conventional large diameter control cartilage pellets formed from 2 × 10(5) MSC each. The microwell-mesh platform offers an elegant mechanism to retain microtissues in microwells, and we believe that this improvement will make this platform useful in 3D culture protocols that require multiple medium exchanges, such as those that mimic specific developmental processes or complex sequential drug exposures.

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

High connectivity in rastrelliger kanagurta: Influence of historical signatures and migratory behaviour inferred from mtDNA Cytochrome b

Phylogeographic patterns and population structure of the pelagic Indian mackerel, Rastrelliger kanagurta were examined in 23 populations collected from the Indonesian-Malaysian Archipelago (IMA) and the West Indian Ocean (WIO). Despite the vast expanse of the IMA and neighbouring seas, no evidence for geographical structure was evident. An indication that R. kanagurta populations across this region are essentially panmictic. This study also revealed that historical isolation was insufficient for R. kanagurta to attain migration drift equilibrium. Two distinct subpopulations were detected between the WIO and the IMA (and adjacent populations); interpopulation genetic variation was high. A plausible explanation for the genetic differentiation observed between the IMA and WIO regions suggest historical isolation as a result of fluctuations in sea levels during the late Pleistocene. This occurrence resulted in the evolution of a phylogeographic break for this species to the north of the Andaman Sea.

Pro-angiogenic and anti-angiogenic factors in the degradation of osteoarthritic cartilage

This project has determined angiogenic and anti-angiogenic factors in osteoarthritis cartilage. The work has expanded our knowledge and understanding of the importance of anti-angiogenic factors in maintaining cartilage homeostasis. This study also tested the concept of topical application of anti-angiogenic treatment strategy for osteoarthritis.

Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5′-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate

Objective Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. Methods ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. Results Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5′-untranslated region (5′-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. Conclusion A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5′-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

Genetic studies of chondrocalcinosis

Purpose of review Our understanding of the causation of the chondrocalcinosis and other disorders characterized by ectopic mineralization is rapidly increasing, and genetic studies have contributed substantially to recent major advances in the field. This review will discuss what is known about the genetics of chondrocalcinosis and what we have learned from genetic studies to date. Recent findings: Chondrocalcinosis is one of a family of conditions associated with ectopic mineralization. This family also includes disorders of mineralization of bone and spinal and other ligaments, and vascular calcification. There has been increasing evidence of the key role of transport and metabolism of inorganic pyrophosphate (PPi) in control of mineralization, and as the likely explanation for the association of a variety of genetic variants with chondrocalcinosis and ectopic mineralization elsewhere. This may be an overly simplistic view of this family of conditions, with recent evidence suggesting that, for example, ANKH variants may not all predispose to chondrocalcinosis by effects on PPi transport, but may also influence chondrocyte maturation. Summary: Understanding the control of the process of mineralization and its tissue specificity are important steps in the search for rational therapies for these conditions.

Groucho homologue Grg5 interacts with the transcription factor Runx2-Cbfa1 and modulates its activity during postnatal growth in mice

Runx2-Cbfal, a Runt transcription factor, plays important roles during skeletal development. It is required for differentiation and function of osteoblasts. In its absence, chondrocyte hypertrophy is severely impaired and there is no vascularization of cartilage templates during skeletal development. These tissue-specific functions of Runx2 are likely to be dependent on its interaction with other proteins. We have therefore searched for proteins that may modulate the activity of Runx2. The yeast two-hybrid system was used to identify a groucho homologue, Grg5, as a Runx2-interacting protein. Grg5 enhances Runx2 activity in a cell culture-based assay and by analyses of postnatal growth in mice we demonstrate that Grg5 and Runx2 interact genetically. We also show that Runx2 haploinsufficiency in the absence of Grg5 results in a more severe delay in ossification of cranial sutures and fontanels than occurs with Runx2 haploinsufficiency on a wild-type background. Finally, we find shortening of the proliferative and hypertrophic zones, and expansion of the resting zone in the growth plates of Runx2(+/-)Grg5(-/-) mice that are associated with reduced Ihh expression and Indian hedgehog (Ihh) signaling. We therefore conclude that Grg5 enhances Runx2 activity in vivo.

Growth defect in Grg5 null mice is associated with reduced Ihh signaling in growth plates

Gene-targeted disruption of Grg5, a mouse homologue of Drosophila groucho (gro), results in postnatal growth retardation in mice. The growth defect, most striking in approximately half of the Grg5 null mice, occurs during the first 4-5 weeks of age, but most mice recover retarded growth later. We used the nonlinear mixed-effects model to fit the growth data of wild-type, heterozygous, and Grg5 null mice. On the basis of preliminary evidence suggesting an interaction between Grg5 and the transcription factor Cbfa1/Runx2, critical for skeletal development, we further investigated the skeleton in the mice. A long bone growth plate defect was identified, which included shorter zones of proliferative and hypertrophic chondrocytes and decreased trabecular bone formation. This decreased trabecular bone formation is likely caused by a reduced recruitment of osteoblasts into the growth plate region of Grg5 null mice. Like the growth defect, the growth plate and trabecular bone abnormality improved as the mice grew older. The growth plate defect was associated with reduced Indian hedgehog expression and signaling. We suggest that Grg5, a transcriptional coregulator, modulates the activities of transcription factors, such as Cbfa1/Runx2 in vivo to affect Ihh expression and the function of long bone growth plates.

Modelling the movement of interacting cell populations: A moment dynamics approach

Mathematical models describing the movement of multiple interacting subpopulations are relevant to many biological and ecological processes. Standard mean-field partial differential equation descriptions of these processes suffer from the limitation that they implicitly neglect to incorporate the impact of spatial correlations and clustering. To overcome this, we derive a moment dynamics description of a discrete stochastic process which describes the spreading of distinct interacting subpopulations. In particular, we motivate our model by mimicking the geometry of two typical cell biology experiments. Comparing the performance of the moment dynamics model with a traditional mean-field model confirms that the moment dynamics approach always outperforms the traditional mean-field approach. To provide more general insight we summarise the performance of the moment dynamics model and the traditional mean-field model over a wide range of parameter regimes. These results help distinguish between those situations where spatial correlation effects are sufficiently strong, such that a moment dynamics model is required, from other situations where spatial correlation effects are sufficiently weak, such that a traditional mean-field model is adequate.

The study of genetic diversity and population structure of the Korean fleshy shrimp, Fenneropenaeus chinensis, using newly developed microsatellite markers

The fleshy shrimp, Fenneropenaeus chinensis, is the family of Penaeidae and one of the most economically important marine culture species in Korea. However, its genetic characteristics have never been studied. In this study, a total of 240 wild F. chinensis individuals were collected from four locations as follows: Narodo (NRD, n = 60), Beopseongpo (BSP, n = 60), Chaesukpo (CSP, n = 60), and Cheonsuman (CSM, n = 60). Genetic variability and the relationships among four wild F. chinensis populations were analyzed using 13 newly developed microsatellite loci. Relatively high levels of genetic variability (mean allelic richness = 16.87; mean heterozygosity = 0.845) were found among localities. Among the 52 population loci, 13 showed significant deviation from the Hardy–Weinberg equilibrium. Neighbor-joining, principal coordinate, and molecular variance analyses revealed the presence of three subpopulations (NRD, CSM, BSP and CSP), which was consistent with clustering based on genetic distance. The mean observed heterozygosity values of the NRD, CSM, BSP, and CSP populations were 0.724, 0.821, 0.814, and 0.785 over all loci, respectively. These genetic variability and differentiation results of the four wild populations can be applied for future genetic improvement using selective breeding and to design suitable management guidelines for Korean F. chinensis culture.

Short and long term mortality rates after a lower limb amputation

Objective To determine mortality rates after a first lower limb amputation and explore the rates for different subpopulations. Methods Retrospective cohort study of all people who underwent a first amputation at or proximal to transtibial level, in an area of 1.7 million people. Analysis with Kaplan-Meier curves and Log Rank tests for univariate associations of psycho-social and health variables. Logistic regression for odds of death at 30-days, 1-year and 5-years. Results 299 people were included. Median time to death was 20.3 months (95%CI: 13.1; 27.5). 30-day mortality = 22%; odds of death 2.3 times higher in people with history of cerebrovascular disease (95%CI: 1.2; 4.7, P = 0.016). 1 year mortality = 44%; odds of death 3.5 times higher for people with renal disease (95%CI: 1.8; 7.0, P < 0.001). 5-years mortality = 77%; odds of death 5.4 times higher for people with renal disease (95%CI: 1.8; 16.0,P = 0.003). Variation in mortality rates was most apparent in different age groups; people 75–84 years having better short term outcomes than those younger and older. Conclusions Mortality rates demonstrated the frailty of this population, with almost one quarter of people dying within 30-days, and almost half at 1 year. People with cerebrovascular had higher odds of death at 30 days, and those with renal disease and 1 and 5 years, respectively.

Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: Sequential not parallel processes

Background Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. Methods The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Results Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. Conclusions The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Vα14Jα18 TCR α-chain specific for glycolipid Ags presented by CD1d. Murine Vα14Jα18+ account for 30–50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Vα24Vβ11+ NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3+ cells) and blood (0.02%). In contrast to those in blood, most hepatic Vα24+ NKT cells express the Vβ11 chain. They include CD4+, CD8+, and CD4−CD8− cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Vα24+ T cells are potent producers of IFN-γ and TNF-α, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, α-galactosylceramide. Vα24+Vβ11+ cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-γ in response to α-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.