Raman microspectroscopy: A non-invasive analysis tool for monitoring of collagen-containing extracellular matrix formation in a medium-throughput culture system

The three-dimensional environment is known to play an important role in promoting cell–matrix interactions. We have investigated the possibility of using Raman microspectroscopy—which has the great advantage of noninvasive sensing—for in vitro monitoring of extracellular matrix (ECM) formation in a medium-throughput pellet (3D) culture system with soft-litography, agarose-microwell arrays. Chondrocytes were seeded in the agarose microwells in basic or chondrocyte medium. After 3, 7, and 14 days of culture, samples were analyzed for ECM formation by Raman microspectroscopy, histology, and immunofluorescence. ECM formation in the chondrocyte medium-cultured samples was detected by histology and immunofluorescence, and also noninvasively by Raman microspectroscopy. The Raman band of collagen found at 937 cm−1 can be used as a Raman marker for collagen-containing ECM formation over time in the chondrocyte pellets. This culture system can be implemented as a medium-throughput platform for Raman applications and screening microtissue formation, since with these agarose-microwell arrays relatively large numbers of cell pellets could be screened in a short time in situ, without having to transfer the pellets onto microscopic slides. Moreover, in this manner the culture system is suitable for long-term, real-time live-cell measurements

Identification of novel SHOX target genes in the developing limb using a transgenic mouse model

Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development

Modeling Biological Pathway Dynamics With Timed Automata

Living cells are constantly subjected to a plethora of environmental stimuli that require integration into an appropriate cellular response. This integration takes place through signal transduction events that form tightly interconnected networks. The understanding of these networks requires to capture their dynamics through computational support and models. ANIMO (Analysis of Networks with Interactive MOdelling) is a tool that enables construction and exploration of executable models of biological networks, helping to derive hypotheses and to plan wet-lab experiments. The tool is based on the formalism of Timed Automata, which can be analysed via the UPPAAL model checker. Thanks to Timed Automata, we can provide a formal semantics for the domain-specific language used to represent signalling networks. This enforces precision and uniformity in the definition of signalling pathways, contributing to the integration of isolated signalling events into complex network models. We propose an approach to discretization of reaction kinetics that allows us to efficiently use UPPAAL as the computational engine to explore the dynamic behaviour of the network of interest. A user-friendly interface hides the use of Timed Automata from the user, while keeping the expressive power intact. Abstraction to single-parameter kinetics speeds up construction of models that remain faithful enough to provide meaningful insight. The resulting dynamic behaviour of the network components is displayed graphically, allowing for an intuitive and interactive modelling experience

Defining the baseline transcriptional fingerprint of rabbit hamstring autograft

Anterior cruciate ligament (ACL) injuries are common and of high relevance given their significant effects on patient function, quality of life, and posttraumatic arthritis. To date, investigators have reported on the expression of genes classically associated with tendon and ligament reconstruction, including decorin (DCN) and collagen type 1 (COL1A1 and COL1A2). However, the transcriptional fingerprint for hamstring tendons, one of the most common autografts used for ACLR, remains to be determined. The purpose of this study was to characterize the baseline transcriptional state of semitendinosus autografts in a rabbit model for ACLR and to employ such characterization to guide scientifically-driven target gene selection for future analyses. Next generation RNA sequencing was performed on whole semitendinosus autografts from four New Zealand White rabbits (mean age: 193 ± 0 days, mean weight: 2.78 kg ± 0.15 kg) and subsequently analyzed using gene enrichment and protein-protein interaction network analysis. Decorin, Secreted Protein Acidic and Cysteine Rich (SPARC), Collagen type 1, and Proline and Arginine Rich End Leucine Rich Repeat Protein (PRELP) and were determined to be the highest expressed genes with tendon-associated ontology. These results strengthen the association between genes such as DCN, COL1A1, and COL1A2 and tendon tissues as well as provide the novel addition of further high-expression, tendon characteristic genes such as SPARC and PRELP to provide guidance as to which molecules serve as high-signal candidates for future ACL research. In addition, this paper provides open-access to the expression fingerprint of hamstring autograft for ACLR in New Zealand White rabbits, thus providing a readily-accessible collaborative reference, in alignment with ethical animal research principles

A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas

Bovine Colostrum Supplementation and Bone Health: a Pilot Study

Research has shown the positive effects of some bovine colostrum components in bone cells; for instance, lactoferrin is reported to stimulate osteoblast proliferation and inhibit osteoclast activity in cell cultures. However, whether bovine colostrum as a whole can induce bone mass gains in osteoporotic bones is relatively unclear. The aim of this study was to investigate the effects of bovine colostrum supplementation in ovariectomized-induced bone loss (OVX) rats. Methods: Twenty-seven-month-old female Wister rats (n=16) were randomly assigned to the following two groups: 1) a healthy control (non-OVX) with no supplementation, and 2) a OVX with bovine colostrum supplementation (0.5g/day; oral consumption). After 5 months supplementation, bone microstructure was scanned using micro-CT (right tibia). Bone formation markers (serum: pre-and post supplementation) were analysed (alkaline phosphatase and osteocalcin) by ECLIA. The study was approved by the National Ethics Committee for the Use of Animals in Research (ORBEA). Results: No significant differences were found between groups in serum alkaline phosphatase either before or after supplementation (p>0.05). Serum osteocalcin significantly increased post-supple-mentation in the OVX compared to pre-supplementation (pre: 11.32+/-1.61; post: 12.45+/-1.21μg/L, p0.05). Trabecular bone mineral content (BMC), trabecular thickness, cortical bone mineral density (BMD) and cortical BMC were similar between groups after supplementation (p>0.05). However, OVX group revealed significantly higher trabecular porosity (5.6%, p<0.01), trabecular separation (36.3%, p<0.01), and cortical porosity (8.0%, p<0.01) compared to the healthy control post-supplementation. Conclusion: Bovine colostrum seems to preserve bone mass of OVX by stimulating bone formation. However, these positive effects seem not to be sufficient to restore bone micro-architecture in the OVX group, possibly because the administrated dose of bovine colostrum was not sufficient for OVX to catch-up healthy rats in terms of trabecular and cortical porosity. The potential therapeutic use of bovine colostrum for osteoporosis deserves further investigation

Tethering Cells via Enzymatic Oxidative Crosslinking Enables Mechanotransduction in Non-Cell-Adhesive Materials

Cell–matrix interactions govern cell behavior and tissue function by facilitating transduction of biomechanical cues. Engineered tissues often incorporate these interactions by employing cell-adhesive materials. However, using constitutively active cell-adhesive materials impedes control over cell fate and elicits inflammatory responses upon implantation. Here, an alternative cell–material interaction strategy that provides mechanotransducive properties via discrete inducible on-cell crosslinking (DOCKING) of materials, including those that are inherently non-cell-adhesive, is introduced. Specifically, tyramine-functionalized materials are tethered to tyrosines that are naturally present in extracellular protein domains via enzyme-mediated oxidative crosslinking. Temporal control over the stiffness of on-cell tethered 3D microniches reveals that DOCKING uniquely enables lineage programming of stem cells by targeting adhesome-related mechanotransduction pathways acting independently of cell volume changes and spreading. In short, DOCKING represents a bioinspired and cytocompatible cell-tethering strategy that offers new routes to study and engineer cell–material interactions, thereby advancing applications ranging from drug delivery, to cell-based therapy, and cultured meat

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

Background: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). Methods: In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. Results: We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Conclusions: Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affectcartilage development

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the roleof Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre,Osx1-Cre, and Col2a1-Cre drivers, respectively. Wild-type and conditional knockout mice were phenotypically assessed by grossmorphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated usingRNA-sequencing, histologic evaluation, and western blotting. Aged mice with Ezh2 deficiency were also evaluated for prematuredevelopment of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age, although caused no other gross developmentaleffects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3lysine 27 (H3K27me3) and altered differentiation in vitro. RNA-seq analysis revealed enrichment of an osteogenic gene expressionprofile in Ezh2 deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes withoutinducing excessive hypertrophy or premature osteoarthritis in vivo. In summary, loss of Ezh2 reduced H3K27me3 levels, increased expression of osteogenic genes in chondrocytes, and resulted ina transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondralossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage-commitment