Tendon healing is a complex process that often results in compromised healing of the tendon tissue. It has recently been shown that temporal changes in the expression profile and the histological tissue quality of the tendons occur during the early healing process after acute Achilles tendon rupture. Whether these changes are accompanied by an altered healing process, is not yet known and was the aim of the present study. Tendon biopsies were obtained from 24 patients with acute Achilles tendon rupture at the time of surgery (2–9 days after rupture) and examined histologically as well as on RNA level. Histologically, the tendon architecture, the amount of aligned collagen, glycosaminoglycan and fat as well as the cellularity, vascularity and immune cell infiltration were determined. On RNA level the expression of markers for the modeling/remodeling (MMPs and TIMPs), collagens (1, 3, 5), tendon markers (scleraxis, tenomodulin), pro- and anti-inflammatory markers (IL-1beta, IL6, IL10, IL33, TNFa, TGF-beta1, COX2) and immune cell markers (CD3, CD68, CD80, CD206) were analyzed by Real-Time PCR. To determine the clinical outcome, the patients were followed up 12 months after the operation and the following scores were recorded: Subjective score, Tegner score, Visual Analog Scale (VAS) pain, VAS function, Matles Test, Achilles tendon total rupture score (ATRS), Therman 100-points score, Heel rise test. Statistics: Spearman correlation analysis. Correlation analysis shows that early post-rupture surgery is associated with better clinical outcome (ATRS Score: p=0.022). Histologically, a good functional healing outcome shows a positive correlation to the amount of aligned collagen (Heel Rise Test: p = 0.009) and glycosaminoglycans in the tendon (Heel Rise Test: p = 0.026, Matles difference: p = 0.029), as well as a negative correlation to the fat content (Thermann score: p = 0.018, subjective score: p = 0.027, VAS function: p = 0.031). On RNA level, a good healing outcome correlates with increased expression of MMP13, collagen 1, 3, 5 (Heel Rise Test: p = 0.019, p = 0.048, p = 0.030), and TIMP2 (Tegner Score: p = 0.040), TGF-beta1 (Thermann Score: p = 0.032) and CD80 (ATRS: p = 0.025, Thermann score:, p = 0.032). Whereas a limited healing outcome is associated with an increased expression of MMP2 (Heel Rise Test: p = 0.033), MMP3 (Matles Test: p=0.001, Heal Rise test p = 0.017), and IL33 (Tegner Score: p = 0.047). The results of the study show a clear relationship between the tendon biology at the time of the surgery and the clinical and functional healing outcome 12 months after the operation. Especially matrix formation and remodeling play a crucial role, while the examined immunological factors seem to influence the tendon healing to a lesser extent. The modulation of matrix formation could potentially lead to improved treatment options in the future

Remodeling of the cancellous bone is more active than that of the cortical bone. It is known that the remodeling is governed by the intracancellous fluid pressure. Particularly, the lacunocanalicular pore (PLC) fluid pressure (FP) is essential for survival of the osteocyte and communication of remodeling signals between the PLC and intertrabecular pore (PIT). As a result, knowledge about the PLCFP generation of trabeculae is required to understand human cancellous bone biology. At this moment, the PLCFP measurement of human trabeculae is not reported. The purpose of this study was a direct measurement of PLCFP generation of human proximal femoral trabeculae in the direction of superior-to-fovea. Twenty one microscopic cylindrical trabecular specimens from trabeculae of five fresh human proximal femur (75 to 77 years) were fabricated using a micro-milling machine composed of the laser (Teemphotonics: 532nm), 3-dimensional PZT stage (PI Gmbh, resolution: 0.5nm), and microscope (lens: Navitar, and CCD: Hitachi) with the image processor. The fabrication resolution of the micro-milling machine was 0.4 um. Based on the trabecular trajectory of femoral head, the specimens were obtained in the direction of superior-to-fovea. The cylindrical specimen size had 120 um in diameter and 240 um in length. The test methods described in the previous study were utilized. The used undrained uniaxial strain condition could induce the maximum PLCFP within the trabecular elastic limit. The measured trabecular PLCFP (±SD) at the strain of 0.4% was 693.7±79.1 kPa. Since this experiment is equivalent to the instantaneous response of PLCFP with free flow boundaries after application of an extremely fast loading speed such an ideal step loading, a PLCFP generation in the physiological condition will be much less than the results obtained in this study. Base on the linear isotropic poroelasticity, the obtained Skempton's coefficient is almost 0. Thus, the load bearing capability by trabecular PLC fluid is negligible. The Biot coefficient is 0.35 which is higher than that of the cortical tissue (0.14). As a result, the intraosseous fluid communication through trabecular surfaces is active compared to that through Haversian canal surfaces. This imply that mass transports from the trabecular PLC into the PIT and from the PIT into the trabecular PLC could be significantly affected by the PITFP (the physiological blood systolic and diastolic pressure: 16 and 11 kPa, respectively) that acts as the FP boundary condition for the PLC flow. It is known that the PLC flow generates the electrical charges on the trabecular surface (‘+’ for being spouted into the PIT and ‘−’ for being flown into the PLC), which control differentiation and proliferation of the osteoblast and mesenchymal stem cell. Thus, significant changes in the PITFT could cause changes in the intra-trabecular PLC flow characteristics, mass transports between the PLC and PIT, and electrical charges on the trabeculae. Eventually, these could result in pathologies related to the trabecular remodeling

The establishment of a proper musculoskeletal system depends on the well-organized and synchronized development of muscle, tendon and cartilage/bone. In tendon biology, a great progress in identifying tendon-specific genes (Scleraxis, Mohawk, Tenomodulin) had been made in the last decade. However, there are many open questions regarding the exact function of genes in tendon development and homeostasis. The purpose of this study was to perform a systematic review of publications describing tendon-related genes, which were studied in-depth and characterized by using knockout technologies and the respectively generated transgenic mouse. Method: Literature search was carried out in Pubmed using “tendon” and “mouse knockout” and “phenotype” and was not limited to year. Results: We report in a tabular manner, that from a total of 25 tendon-related genes, in 23 of the respective knockout mouse models phenotypic changes were detected. Additionally, in some of the models it was described at which developmental stages these changes appeared and progressed. Interestingly, so far only loss of Scleraxis and TGFbeta signaling led to severe tendon developmental phenotypes, while mice deficient for various proteoglycans, Mohawk, EGR1 and 2, and Tenomodulin exhibited mild phenotypes. This suggests that in general the tendon developmental program is well backup and specifically that among the members of the proteoglycan family there are clear compensatory effects. In future, it will be of great importance to discover additional master tendon transcription factors as well as genes that play indispensable roles in tendon development

Summary Statement. Dickkopf-3 is upregulated in OA cartilage and synovial tissue. In vitro studies show Dkk3 can prevent cartilage degradation and antagonise Wnt signaling. We hypothesis that Dkk3 can protect against OA-related cartilage destruction. Introduction. Our group has previously shown that Dkk3, a member of the Dkk family of Wnt antagonists, is upregulated in OA cartilage and synovium. Levels of Dkk3 in synovial fluid are also increased in individuals with tricompartmental OA and after arthroscopy. The role of Dkk3 in cartilage or the factors regulating its expression are not currently understood. Correct regulation of cell signalling pathways is integral to cartilage homeostasis and thus the prevention of OA pathogenesis. Dkk3 is a member of the Dkk family of Wnt antagonists and therefore may impact on chondrocyte biology through interaction with the Wnt pathway. Dkk3 has also been found to influence TGFβ signalling in other cell systems. Methods. Expression of Dkk3 was assessed in primary human articular chondrocytes (HAC) following treatment with interleukin-1,-6 (IL1, IL6), TNFα, FGF2 and oncostatin-M (OSM). Dkk3 expression was assessed following ex vivo injury of murine cartilage explants. The effect of Dkk3 on IL1/OSM-induced proteoglycan and collagen release from explants of bovine nasal (BNC)- and primary human-cartilage was assessed. SW1353 chondrosarcoma cells were treated with Dkk3+/−Wnt3a, TGFβ and Activin and TOPFlash and CAGA luciferase reporters used to measure Wnt and Smad signalling. RNA was extracted from primary HAC treated with Dkk3+/−TGFβ or Wnt3a. ADAM12 and TIMP3 expression were measured to assess TGFβ signalling and AXIN2 to assess Wnt signalling. Micromass HAC were treated with Wnt3a +/− Dkk3 and proteoglycan output assessed using alcian blue staining. β-catenin was silenced in primary HAC prior to TGFβ and Activin treatment. Dkk3 was silenced in primary HAC for microarray analysis. Results. Dkk3 expression was decreased in primary HAC following IL1/OSM treatment but increased by TNFα. Dkk3 expression was decreased immediately following injury to murine explants. In BNC explants, IL1/OSM-induced proteoglycan release was inhibited by Dkk3. Dkk3 antagonised chondrocyte Wnt signalling and Wnt3a-induced reductions in proteoglycan production in micromass cultures. Interestingly, Dkk3 enhanced TGFβ signalling, increasing TGFβ-induced TIMP3 and ADAM12 expression and TGFβ-induced luciferase from the CAGA-luc reporter. In contrast Dkk3 antagonised Activin-induced CAGA-luc activity, TIMP3 and ADAM12 expression. β-catenin knockdown did not significantly alter TGFβ- or Activin-induced expression of TIMP3 or ADAM12, suggesting that Dkk3-effects on these pathways is not mediated solely by inhibition of Wnt signalling. Conclusions. Dkk3 expression is increased in OA and can be regulated injury and inflammatory cytokines. This suggests a balance of Dkk3 effects depending upon the biological stimuli within the cartilage. Dkk3 may act in a protective role in the presence of inflammatory cytokines as exemplified by its ability to inhibit matrix loss. Dkk3 knockdown decreases DICER expression and thus changes in Dkk3 expression in OA may alter chondrocyte phenotype through alterations in miRNA activity. The ability of Dkk3 to antagonise Wnt, enhance TGFβ and antagonise Activin signalling would have multiple effects on chondrocyte activity. These results imply that Dkk3 could influence multiple OA-relevant processes, protect cartilage from degradation and be important in cartilage development and homeostasis

Introduction

Intervertebral disc degeneration has been associated with low back pain (LBP) which is a major cause of long-term disability worldwide. Observed mechanical and biological modifications have been related to decreased water content.

Clinical traction protocols as part of LBP management have shown positive outcomes. However, the underlying mechanical and biological processes are still unknown.

The study purpose was to evaluate the impact of unloading through traction on the mechanobiology of healthy bovine tail discs in culture.

Cigarette smoking has a negative impact on the skeletal system by reducing bone mass and increasing the risk of fractures through its direct or indirect effects on bone remodeling. Recent evidence shows that smoking causes an imbalance in bone turnover, making bone vulnerable to osteoporosis and fragility fractures. In addition, cigarette smoking is known to have deleterious effects on fracture healing, as a positive correlation has been shown between the daily number of cigarettes smoked and years of exposure to smoking, although the underlying mechanisms are not fully understood. Smoking is also known to cause several medical and surgical complications responsible for longer hospital stays and a consequent increase in resource consumption. Smoking cessation is, therefore, highly advisable to prevent the onset of metabolic bone disease. However, some of the consequences appear to continue for decades. Based on this evidence, the aim of our work was to assess the impact of smoking on the skeletal system, particularly bone fractures, and to identify the pathophysiological mechanisms responsible for the impairment of fracture healing. Because smoking represents a major public health problem, understanding the association between cigarette smoking and the occurrence of bone disease is necessary in order to identify potential new targets for intervention.

RNA-Seq or whole transcriptome shotgun sequencing has been adopted in the last years as a reference technique to determine the presence and the quantity of different species of RNA in determined biological samples, thanks to it allows the identification every single RNA species transcribed from a reference genome. Meta-profiling takes advantage of the public availability of an increasing set of RNA-Seq data produced by different laboratories to summarize the expression levels of the different RNA species of many samples according to their biological context, giving the opportunity to perform comparisons on the gene expression profiles of different tissues by integrating data derived from a high number of studies. By using Genevestigator™; a platform which integrates RNA-Seq data into meta-profiles, we have performed a comparison between the gene expression profiles of bone, cartilage, muscle tendon and skin by means of interrogating its database with different gene sets and families with relevance to the function of the tissues of the musculoskeletal system. The collagen gene family and genes coding for proteoglycans, matrix metalloproteinases and tissue inhibitors of metalloproteinases, mechanotransduction-related proteins and signalling pathways involved in tissue development and differentiation have been analysed. Hierarchical clustering for every gene set was performed for the understanding the differences and similarities between the different tissues included in the analyses. The results of this study will help to improve our understanding of the musculoskeletal system, and will help to identify new biomarkers and signalling pathways of specific relevance for the bone, cartilage, muscle and tendon.

After initial hesitance and failures, with growing knowledge about advanced products and their characteristics, increasingly more medtech and also pharma companies enter the advanced therapies market. However, due to the specifics of the biology and regulation of advanced therapy products, a lot of new know-how is necessary to be successful in this highly promising field

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration. Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors. Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering

While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome. Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses. In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism. Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression

Abstract. OBJECTIVE. Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity. 1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain. 2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively. 3. and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion. 4. Sema3A is also differentially expressed in human OA bone. 5. HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. METHODS. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model. 6. of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed. RESULTS. PC1 (32.94% of variation) and PC2 (25.79% of variation) from PCA analysis and correlation matrices separated patients according to correlated clusters of established inflammatory markers of OA pain and progression (IL6/IL8, r=0.754, p<0.001) and anti-inflammatory mediators (IL4/IL10, r=0.469, p=0.005). Bone turnover marker ALP was positively associated with KL grade (r=0.815, p=0.002) and negatively associated with IL10 (r=−0.402, p=0.018) and first peak knee loading pressures (r=−0.688, p=0.019). RANKL was positively associated with IL4 (r=0.489, p=0.003). Synovial fluid Sema3A concentrations showed separate clustering from all OA progression markers and was inversely correlated with TNF-α (r=−0.423, p=0.022) in HTO patients. Sema3A was significantly inversely correlated with total predicted force in the medial joint compartment (r=−0.621, p=0.041), mean (r=−0.63, p=0.038) and maximum (r=−0.613, p=0.045) calculated medial compartment joint pressures during the first phase and mean (r=−0.618, p=0.043) and maximum (r=−0.641, p=0.034) medial compartment joint pressures during midstance outputs of patient-specific musculoskeletal model. CONCLUSIONS. This study shows joint inflammatory status and mechanical overloading influence subchondral bone-remodelling. Synovial Sema3A concentrations are inversely correlated to patient-specific musculoskeletal model estimations of pathological medial overloading. This study reveals Sema3A as a biological mediator with capacity to induce OA pain and disease progression that is directly regulated by gait mechanical loading. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Biomaterials are no longer considered innate structures and using functionalisation and biofabrication strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding of the host response will enable us to design appropriate strategies. The input from the host response needs to be weighed in depending on the host disease condition. Our current inputs have been through a thorough understanding of glyco-proteomics based tools which we are developing in our laboratory. In addition, biomaterials themselves provide immense therapeutic benefits which needs to be accounted in the design paradigm. Using functionalisation strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of scaffolds. Recent design efforts have utilized a glycobiology and developmental biology approach toward both understanding and engineering supramolecular protein and sugar assemblies

Abstract. Objectives. The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. This study investigated the response of a 3D in vitro bone cell model to mechanical load. Methods. Human MSC cells (Y201) embedded in 3D type I collagen gels were differentiated in osteogenic media for 7-days in deformable, silicone plates. Gels were loaded once (5000 µstrain, 10Hz, 3000 cycles), RNA extracted 1-hr post load and assessed by RT-qPCR and RNAseq analysis (n=5/treatment). Cell shape and phenotype were assessed by immunocytochemistry and phalloidin staining. Data was analysed by Minitab. Results. RTqPCR revealed cells expressed markers of mature osteocytes (E11, sclerostin, DMP-1) and osteoprotegerin (OPG), alkaline phosphatase and type I collagen (COL1A1). Immunolocalisation of sclerostin and DMP-1 protein along with phalloidin staining confirmed a dendritic osteocyte phenotype. Load almost abolished sclerostin gene expression (p=0.05) and reduced E11 (2-fold p=0.03); COL1A1 was unchanged (p=0.349). Using DEseq2 analysis, of the 981 genes differentially regulated more than 2-fold at FDR p<0.05, 159 were downregulated and 821 upregulated by load. These were involved in processes important in bone biology including the inflammatory response (56 genes), ECM organisation (27), ageing (30), response to mechanical load (23), ER stress (34), regulation of ossification (26), bone morphogenesis (14), cartilage development (14), programmed cell death (161), and positive regulation of bone mineralisation (6). Discussion. Y201 cells were successfully differentiated to osteocytes. The osteocytes’ mechanical response revealed regulation of factors that contribute to bone remodelling and inflammation. Since the biological mechanisms underlying mechanically induced joint degeneration are unclear, there is a need for humanised, cell models to delineate molecular pathways activated by mechanical load. Such pathways may reveal the molecular basis for genetic predispositions to osteoarthritis and identify new therapeutic targets. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Calcium is an important element for a wide range of physiological functions including muscle contraction, neuronal activity, exocytosis, blood coagulation and cell communication. In the musculoskeletal system calcium is crucial for the structural integrity of bones, teeth, intervertebral disc and articular cartilage. At the cellular level calcium acts as a second messenger. Calcium signalling uses intracellular calcium ions to drive intracellular communication and signal transduction processes. When calcium enters the cell it exerts allosteric regulatory effects on many enzymes and proteins. Examining the role of calcium in chondrocyte biology is important for understanding the role for this divalent ion in the metabolic modulation of chondrocyte function in health and disease. This includes the study of calcium transport systems such as channels, transporters and pumps involved in calcium homeostasis in chondrocytes and how existing pharmacological drugs act on these transport systems. L-type calcium channel blockers are drugs used as cardiac antiarrhythmics or antihypertensives, depending on whether the drugs have higher affinity for the heart (the phenylalkylamines, like verapamil), or for the blood vessels (the dihydropyridines, like nifedipine). L-type calcium channels are present in many musculoskeletal tissues including skeletal muscle, smooth muscle, bone and cartilage. L-type calcium channel inhibitors like nifedipine used for the treatment of some forms of hypertension modulate calcium-mediated events in chondrocytes under dynamic loading, thus affecting metabolism, osmotic responses and extracellular matrix turnover in cartilage. The aim of our work is to understand the impact of L-type calcium channel inhibitors used for the treatment of hypertension on chondrocytes and on the chondrogenic differentiation of bone marrow derived mesenchymal stem cells (MSCs). This knowledge will enhance our understanding of the development of osteoarthritis (OA) and may lead to new opportunities for chondroprotection and regenerative medicine for OA. We have used electrophysiology to demonstrate L-type calcium currents in chondrocytes immediately after pharmacological activation with the calcium channel opener Bay-K8644. We have also used immunohistochemistry to demonstrate expression of the a1C subunit Ca. v. 1.2 (CACNA1C) in human chondrocytes and MSCs. Inhibitors of L-type calcium channels such as nifedipine downregulate mitochondrial respiration and ATP production in MSCs but not in chondrocytes. Nifedipine inhibits proliferation of chondrocytes and enhances glycolytic capacity in chondrocytes, promoting glycolytic reserve in both MSCs and chondrocytes. Nifedipine can also stimulate chondrogenic differentiation in MSCs (with or without growth factors). Metabolic responses to nifedipine differs in mesenchymal stem cells and chondrocytes highlighting important metabolic differences between these cells. In summary, antihypertensive drugs such as nifedipine can affect the biological function of chondrocytes and MSCs and may modulate the course of OA progression and impact on cartilage repair

Introduction. The two-dimensional (2D) monolayer culture paradigm has limited translational potential to physiological systems; chondrocytes and tenocytes in monolayer lose expression of hallmarks of differentiated status (dedifferentiation). Qualitative assessment of three-dimensional (3D) cultures in musculoskeletal biology relative to native tissues has been limited. An understanding of prevailing gene regulatory networks is required to define whether 3D culture systems faithfully restitute the native tissue phenotype (redifferentiation). Using a systems biology approach to explore the gene networks associated with de- and re-differentiation may define targetable regulators associated with phenotypic plasticity of adult musculoskeletal cells. Materials and Methods. Global transcriptomic and proteomic profiling of matrix-depleted chondrocytes and tenocytes from the rat was performed for each of three conditions (native tissue, monolayer at passage three, or tissue-appropriate 3D cultures). Differential analysis of mRNA and protein abundance, gene ontology annotation, pathway topology impact analysis, and derivation of common mechanistic networks was undertaken to define consensus expression profiles, signalling pathways, and upstream regulators for de- and re-differentiation in each cell type. Results. Principal component analysis demonstrated a convergence of gene expression profiles in monolayer, including the expression of musculoskeletal progenitor markers scleraxis (Scx) and Mohawk (Mkx). Three-dimensional culture systems failed to demonstrate parity with native tissue and incited the expression of Il-6 and Ptgs2 (COX2). The CCN-family member Ctgf (CCN2), and the marker of skeletal differentiation Grem1 (gremlin 1), were consistently differentially abundant in de- and re-differentiation at both the mRNA and protein level. Pathway topology impact analysis defined PI-3K/Akt as the common signalling pathway in de- and re-differentiation. Discussion. Historically, the terms de- and re-differentiation have been used with no mechanistic definition. Additionally, there is no standardised phenotype for 3D cultures to benchmark novel progress in bioengineering. Consensus upstream regulators yielded a unified mechanistic network for chondrocyte and tenocyte phenotypes in three conditions. The PI-3K/Akt signalling pathway has been implicated in a range of physiological activities including dedifferentiation, proliferation, matrix synthesis, and cell survival. Pathway analysis suggests that the PI-3K/Akt signalling pathway may contribute to the de- and re-differentiation phenotypes for both chondrocytes and tenocytes and represents a rational target for further network-level analysis

Introduction. Adult tendon injuries occur very frequently, but injured tendon heals very slowly and the mechanisms of the slow-healing response to injury are still largely unknown. Currently, the main barrier is our insufficient understanding of the mechanisms responsible for homeostasis, regeneration and repair of adult tendon. This gap in knowledge translates to a lack of experimental models. Therefore, using the combination of state-of-the-art genetic approaches, we have established novel cell biological tools to advance the understanding of tendon biology. Materials and Methods. Adult mouse tendon progenitor lines and Adult mouse tenocyte lines: Primary adult tenocytes were isolated from Achilles tendon in Scleraxis(fl/fl)/Scleraxis-GFP/p21(−/−) mice, then CD90.2- and subsequent Sca1-positive cells were sorted by Flow Cytometry. Then Scleraxis-null progenitor lines were generated by the treatment of those cells with adenovirus-Cre. Adult Scleraxis(+/+) and Scleraxis-null tenocyte lines were also generated from Scleraxis(fl/fl)/Scleraxis-GFP/p21(−/−) mice. To establish Scleraxis-Flag overexpressing tenocyte lines, Scleraxis and Flag-tag fusion-protein expression construct was generated and transfected into Scleraxis-null tenocytes (Scleraxis transgenic mouse strains were provided by Dr Ronen Schweitzer). Scleraxis antibody: DNA coding mouse Scleraxis residues were obtained by PCR, then the recombinant protein was expressed, immunized in rabbits, and an affinity-purified antibody was generated. Results. Established parental progenitor lines highly expressed Sca1 (98.9%), CD90.2 (97.3%), and CD44 (99.8%) and were almost negative for ScxGFP (2.3%). Interestingly, Scleraxis-null progenitors showed significantly increased clonogenicity. Furthermore, when stimulated toward mesenchymal lineages, Scleraxis-null progenitors enhanced differentiation into chondrocytes. Our Scleraxis antibody reacted with lysates from cells expressing Scleraxis-Flag fusion proteins (∼30 kDa), whereas it did not react with Scleraxis-null cells by Western analysis. Immunofluorescence analysis of adult mouse Achilles tendons further confirmed intense Scleraxis protein expression in wild-type tenocytes, whereas considerably decreased expression of Scleraxis was evident in Cre-treated Scleraxis(fl/fl) tenocytes. Discussion. These novel tools will be the promising resources to get an insight into molecular framework for Scleraxis in adult tendons. It is anticipated that the establishment of experimental models using these resources will fill major gaps in the current knowledge of adult tendon biology and will facilitate development of novel strategies to treat adult tendon injury

We define regenerative engineering as a convergence of advanced materials science, stem cell science, physics, developmental biology, and clinical translation. Stem cells play an important role. Work in the area of musculoskeletal tissue regeneration has focused on a number of paradigms. Polymer and polymer-ceramic systems can be utilized for the regeneration of bone. Direct induction can be controlled through material characteristics. Through the use of inducerons, small molecules fostering induction, the design of regeneration-inducing materials can be realized. We believe the medicinal use of stem cells will be of critical importance in the design of next generation systems answering grand challenges to musculoskeletal regeneration

Cells directly probe and respond to the physicomechanical properties of their extracellular environment, a dynamic process which has been shown to play a key role in regulating both cellular adhesive processes and differential function. Recent studies indicate that stem cells show lineage-specific differentiation when cultured on substrates approximating the stiffness profiles of specific tissues. Although tissues are associated with ranging Young's modulus values for bulk rigidity, at the sub-cellular level, and particularly at the micro- and nanoscales, tissues are comprised of heterogeneous distributions of rigidity. Lithographic processes have been widely explored in cell biology for the generation of analytical substrates to probe cellular physicomechanical responses. In this work, we show for the first time that that direct-write e-beam exposure can significantly alter the rigidity of elastomeric PDMS substrates and develop a new class of two-dimensional elastomeric substrates with controlled patterned rigidity ranging from the micron to the nanoscale. The mechano-response of human mesenchymal stem cells to e-beam patterned substrates was subsequently probed in vitro and significant modulation of focal adhesion formation and osteochondral lineage commitment was observed as a function of both feature diameter and rigidity, establishing the groundwork for a new generation of biomimetic material interfaces

Advances in our understanding of skeletal stem cells and their role in bone development and repair, offer the potential to open new frontiers in bone regeneration. However, the ability to harness these cells to replace or restore the function of traumatised or lost skeletal tissue as a consequence of age or disease remains a significant challenge. We have developed protocols for the isolation, expansion and translational application of skeletal cell populations with cues from developmental biology informed by in vitro and ex vivo models as well as, nanoscale architecture and biomimetic niche development informing our skeletal tissue engineering approaches. We demonstrate the importance of biomimetic cues and delivery strategies to directly modulate differentiation of human adult skeletal cells and, central to clinical application, translational studies to examine the efficacy of skeletal stem and cell populations in innovative scaffold compositions for orthopaedics. While a number of challenges remain multidisciplinary approaches that integrate developmental and engineering processes as well as cell, molecular and clinical techniques for skeletal tissue engineering offer significant promise. Harnessing such approaches across the hard tissue interface will ultimately improve the quality of life of an increasing ageing population

Robust repair relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with extracellular matrix proteins to create a natural environment for the tissue- specific cells. Tropoelastin, the precursor of the elastin, is the extracellular matrix protein responsible for elasticity in diverse tissues, including robust blood vessels. We find that tropoelastin contributes a physical role in elasticity and also substantially to the biology of repairing tissue. The emerging model from a range of our in vivo studies is that tropoelastin encodes direct biological effects and has the versatility to promote repair. We have discovered that tropoelastin substantially improves healing by halving the time to repair bone in small animals and large animal preclinical models; tropoelastin elicits this response with early stage neo-angiogenesis, recruitment of endogenous cells with consistently accelerated repair. This potency is marked by the concerted appearance of blood vessels, tissue and phased cellular contributions that work together to accelerate repair