Effects of Interleukin-1ß Inhibition on Incident Hip and Knee Replacement : Exploratory Analyses From a Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Osteoarthritis is a common inflammatory disorder with no disease-modifying therapies. Whether inhibition of interleukin-1ß (IL-1ß) can reduce the consequences of large joint osteoarthritis is unclear. OBJECTIVE: To determine whether IL-1ß inhibition with canakinumab reduces incident total hip or knee replacement (THR/TKR). DESIGN: Exploratory analysis of a randomized trial. (ClinicalTrials.gov: NCT01327846). SETTING: 1091 clinical sites in 39 countries. PARTICIPANTS: 10 061 CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) participants. INTERVENTION: Random allocation to placebo or canakinumab (50, 150, or 300 mg) subcutaneously once every 3 months. MEASUREMENTS: The primary and secondary outcomes were time to first incident THR/TKR and time to first occurrence of an osteoarthritis-related adverse event (AE). Data were obtained through blinded ascertainment of trial clinical and safety databases. RESULTS: Median follow-up was 3.7 years. For the individual canakinumab dose groups, compared with placebo, hazard ratios (HRs) for incident THR/TKR during follow-up were 0.60 (95% CI, 0.38 to 0.95) for the 50-mg group, 0.53 (CI, 0.33 to 0.84) for the 150-mg group, and 0.60 (CI, 0.38 to 0.93) for the 300-mg group. Thus, in the pooled canakinumab groups, compared with the placebo group, incidence rates for THR/TKR were 0.31 and 0.54 events per 100 person-years (HR, 0.58 [CI, 0.42 to 0.80]; P = 0.001), respectively. The HR for the secondary end point of osteoarthritis-related AEs was 0.73 (CI, 0.61 to 0.87). Similar findings were observed in analyses restricted to participants with a history of osteoarthritis. LIMITATION: Because the parent trial was not designed to examine the efficacy of IL-1ß inhibitors in osteoarthritis, information on structural joint outcomes was not collected. CONCLUSION: Findings from this exploratory analysis of a randomized controlled trial support further investigation of IL-1ß inhibition for treatment of large joint osteoarthritis. PRIMARY FUNDING SOURCE: Novartis Pharmaceuticals.

Tenomodulin regulates matrix remodeling of mouse tendon stem/progenitor cells in an ex vivo collagen I gel model.

Tenomodulin (Tnmd) is predominantly expressed in tendon and ligament tissues. Loss of Tnmd in mice leads to a profound phenotype in vitro, characterized by reduced self-renewal but increased senescence of mouse tendon stem/progenitor cells (mTSPCs), as well as in vivo, by significantly impaired early tendon healing. Interestingly, injuried Achilles tendons from Tnmd-deficient mice showed inferior tendon repair, which was characterized by less contracted fibrovascular scars with disorganized matrix composition in comparison to wild type (WT) mice at day 8 after injury. To better understand Tnmd role in tendon repair, here we implemented an ex vivo three-dimensional (3D) collagen gel model and investigated whether Tnmd knockout affects the collagen contraction of mTSPCs. TSPCs were isolated from WT and Tnmd knockout (KO) tendons at 6, 9, 12, and 18 months of age. Adhesion assay demonstrated that loss of Tnmd in mTSPCs resulted in reduced adhesion to collagen type I. Quantitative time-dependent analysis revealed that Tnmd-deficient mTSPCs of all ages have significantly reduced capacity to contract collagen matrix in comparison to WT cells. Furthermore, 18 months old mTSPCs of both genotypes showed lower collagen contractility than cells obtained from 6, 9, and 12 months old animals, demonstrating an overall effect of organismal aging on matrix remodeling. Nevertheless, both cell types had a similar survival rate for the 5 days of cultivation within the gels. Lastly, quantitative PCR for 48 different genes revealed that the knockout of Tnmd majorly affected the gene expression profile of mTSPCs, as several transcription factors, tendon matrix, collagen cross-linking, and lineage maker genes were down-regulated. Taken together, our results clearly demonstrated that loss of Tnmd in mTSPCs led to profoundly altered gene expression profile, insufficient adhesion to collagen type I, and impaired ability to contract the extracellular matrix.

In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair.

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

Oxygen mapping: Probing a novel seeding strategy for bone tissue engineering.

Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen-imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell-seeding strategy. Immortalized hMSCs, pre-cultured in a monolayer for 30-40% or 70-80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real-time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle-type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30-40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70-80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell-seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two-dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894-902. © 2016 Wiley Periodicals, Inc.

Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.

BACKGROUND: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies. RESULTS: Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC. CONCLUSIONS: Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

Efficacy and safety of extracorporeal shock wave therapy for orthopedic conditions: a systematic review on studies listed in the PEDro database.

BACKGROUND: Extracorporeal shock wave therapy (ESWT) is an effective and safe non-invasive treatment option for tendon and other pathologies of the musculoskeletal system. SOURCES OF DATA: This systematic review used data derived from the Physiotherapy Evidence Database (PEDro; www.pedro.org.au, 23 October 2015, date last accessed). AREAS OF AGREEMENT: ESWT is effective and safe. An optimum treatment protocol for ESWT appears to be three treatment sessions at 1-week intervals, with 2000 impulses per session and the highest energy flux density the patient can tolerate. AREAS OF CONTROVERSY: The distinction between radial ESWT as 'low-energy ESWT' and focused ESWT as 'high-energy ESWT' is not correct and should be abandoned. GROWING POINTS: There is no scientific evidence in favour of either radial ESWT or focused ESWT with respect to treatment outcome. AREAS TIMELY FOR DEVELOPING RESEARCH: Future randomized controlled trials should primarily address systematic tests of the aforementioned optimum treatment protocol and direct comparisons between radial and focused ESWT.

MiR-134-mediated ß1 integrin expression and function in mesenchymal stem cells.

The composition of the hematopoietic stem cell (HSC) niche within the bone marrow is highly dynamic, tightly regulated, and of importance for various HSC properties. Integrins are important molecules within this niche that influence those properties through the interactions of HSCs and mesenchymal stem cells (MSCs). Here we investigated the function of miR-134 in integrin regulation in MSCs. In MSCs, miR-134 post-transcriptionally regulated ß1 integrin expression. This negative regulation of ß1 integrin was mediated by the binding of miR-134 to its 3' untranslated region, which contains two conserved binding sites for miR-134. The miR-134-mediated silencing of ß1 integrin in MSCs was shown by atomic force microscopy to decrease the adhesion of 32D cells to MSCs transfected with miR-134. Furthermore, the adhesion of MSCs to fibronectin was reduced after transfection with miR-134. MSCs from patients with myelodysplastic syndrome (MDS) revealed highly significant miR-134 overexpression compared with MSCs from healthy bone marrow donors. MSCs from MDS patients showed lower ß1 integrin protein, but not lower mRNA, expression, suggesting post-transcriptional regulation. The present study demonstrates miR-134-mediated negative regulation of ß1 integrin that influences cell adhesion to and of MSCs. These results further contribute to our understanding of the complexity of MDS.

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7.

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression. HMSC were isolated from 18 healthy or osteoporotic donors. Migration was assessed using a collagen IV coated micro-slide linear gradient chamber and time-lapse microscopy. Invasion was analyzed utilizing an ECM coated transmembrane invasion assay. Quantitative real-time RT PCR was performed for the ECM binding integrins α1, α2, α3, α4, α5, α11, αv and ß1. HMSC from osteoporotic patients showed a significant increase of migration upon BMP-2 or FCS stimulation, as well as a significant increase of invasion upon BMP-2, BMP-7 or FCS stimulation. Nevertheless, the migration and invasion capacity was significantly decreased compared to healthy controls. Out of all integrins analyzed, collagen binding integrin α2 was significantly downregulated in hMSC from osteoporotic patients. In conclusion, we here demonstrate for the first time osteoporosis associated alterations in BMP mediated hMSC recruitment. These findings may underlie the reduced healing of osteoporotic fractures. Nevertheless, the maintained migration and invasion response upon BMP stimulation illustrates the therapeutic potential of these clinically approved substances in the treatment of osteoporotic fractures. Another therapeutic target may be the downregulation of the collagen binding integrin α2 in hMSC from osteoporotic patients.

MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells.

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3'UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3'UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodys-plastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche.

Influence of osteogenic stimulation and VEGF treatment on in vivo bone formation in hMSC-seeded cancellous bone scaffolds.

BACKGROUND: Tissue engineering approaches for reconstruction of large bone defects are still technically immature, especially in regard to sufficient blood supply. Therefore, the aim of the present study was to investigate the influence of osteogenic stimulation and treatment with VEGF on new bone formation and neovascularization in hMSC-loaded cancellous bone scaffolds in vivo. METHODS: Cubic scaffolds were seeded with hMSC and either cultured in stem cell medium or osteogenic stimulation medium. One osteogenically stimulated group was additionally treated with 0.8 µg VEGF prior to subcutaneous implantation in athymic mice. After 2 and 12 weeks in vivo, constructs and selected organs were harvested for histological and molecular analysis. RESULTS: Histological analysis revealed similar vascularization of the constructs with and without VEGF treatment and absence of new bone formation in any group. Human DNA was detected in all inoculated scaffolds, but a significant decrease in cells was observed after 2 weeks with no further decrease after 12 weeks in vivo. CONCLUSION: Under the chosen conditions, osteogenic stimulation and treatment with VEGF does not have any influence on the new bone formation and neovascularization in hMSC-seeded cancellous bone scaffolds.

Overexpression of dnIKK in mesenchymal stem cells leads to increased migration and decreased invasion upon TNFα stimulation.

IκB kinase 2 (IKK-2) mediates tumor necrosis-factor α (TNFα) induced invasion of human mesenchymal stem cell (hMSC) to sites of tissue injury. Suppressing IKK-2 activity leads to reduced expression of proteolytic enzymes and impaired invasive capacity. In order to further reveal mechanisms of hMSC recruitment, we here aimed to analyse the impact of IKK-2 on two-dimensional migration upon TNFα stimulation in contrast to three-dimensional invasion. An immortalized hMSC line (SCP-1) was transduced with a dominant-negative mutant of IκB kinase 2 (SCP-1 dnIKK). Migration was assessed using a linear-gradient chemotaxis chambers by time-lapse analysis. Invasive capacity through human extracellular matrix was analysed using transwell invasion assays. RT-PCR confirmed increased IKK-2 expression levels in SCP-1 dnIKK cells, while TNFα receptor I and II expression was not altered. Invasion upon TNFα stimulation was significantly reduced by 78% in SCP-1 dnIKK. In contrast, migration was significantly increased, represented by a 60% elevated forward migration index and a 2.1-fold higher mean dislocation of the center of mass towards TNFα. In conclusion, our data confirms the impact of IKK-2 in TNFα dependent hMSC recruitment. Interestingly, reducing IKK-2 function increases two-dimensional migration towards TNFα, while invasive capacity is impaired. These findings contribute to a deeper understanding of MSC's biological properties orchestrating the complex processes of stem cell recruitment and homing.

Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained osteoinduction in response to BMP-2 stimulation.

Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing. Therefore, MSC were isolated from femoral heads of healthy and osteoporotic patients. Grouping was carried out by bone mineral densitometry in an age-matched manner. MSC were stimulated with BMP-2. Signal transduction was assessed by western blotting of pSMAD1/5/8 and pERK1/2 as well as by quantitative RT-PCR of Runx-2, Dlx5, and Osteocalcin. Osteogenic differentiation was assessed by quantifying Alizarin Red staining. Osteoporotic MSC featured an accurate phosphorylation pattern of SMAD1/5/8 but a significantly reduced activation of ERK1/2 by BMP-2 stimulation. Furthermore, osteoporotic MSC showed significantly reduced basal expression levels of Runx-2 and Dlx5. However, Runx-2, Dlx5, and Osteocalcin expression showed adequate up-regulation due to BMP-2 stimulation. The global osteogenic differentiation in standard osteogenic differentiation media was reduced in osteoporotic MSC. Nevertheless, osteoporotic MSC were shown to feature an adequate induction of osteogenic differentiation due to BMP-2 stimulation. Taken together, we here demonstrate osteoporosis associated alterations in BMP-2 signaling but sustained specific osteogenic differentiation capacity in response to BMP-2. Therefore, BMP-2 may represent a promising therapeutic agent for the treatment of fractures in osteoporotic patients.

First inducible transgene expression in porcine large animal models.

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken ß-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-κB ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.

Polypropylene meshes coated with a polysaccharide based bioadhesive for intra-abdominal mesh fixation in a rabbit model.

BACKGROUND: In this study, we evaluate a new bioadhesive for intra-abdominal onlay mesh fixation of a polypropylene-polyvinylchloride graft. METHODS: Three pieces of a commercially available polypropylene/polyvinylfluoride mesh, each 3 × 3 cm in size, and three pieces of the same mesh coated with a polysaccharide bioadhesive were fixated to the surface of the anterior abdominal wall of 30 New Zealand white rabbits. The fixation was performed either by using four transabdominal Prolene(®) 4/0 sutures, four spiral tacks (Protack 5 mm Tyco), or cyanoacrylate glue (Glubran(®) GEM, Viareggio, Italy). Each mesh position and the according kind of fixation were randomized before implantation. The animals were sacrificed 12 weeks postoperatively. After determining the extent of intra-abdominal adhesions, the meshes were excised en bloc with the anterior abdominal wall for tensile strength measurements and histological analysis. RESULTS: All meshes coated with the bioadhesive adhered to the intact peritoneum without extra fixation. Irrespective of the fixation technique coated meshes led to more and stronger adhesions. Mesh shrinkage by scarring was increased in coated meshes fixed with glue and low in uncoated meshes fixed with tacks. Testing the tensile strength, coated meshes fixed with transfascial sutures achieved the best results (16.14 ± 6.1 N), whereas coated meshes fixed with glue showed the lowest strength (10.39 ± 4.81 N). The foreign body reaction was considerably more distinctive using coated mesh. The mesh ingrowth was not influenced by this reaction. CONCLUSIONS: All meshes coated with the new bioadhesive were self-adhesive in that way; they stayed in position when attached to the peritoneum. Although this may facilitate intra-operative mesh fixation, the bioadhesive displayed several disadvantages, such as stronger adhesions and an increased shrinkage of the implant. The tensile strength was not influenced by the use of the bioadhesive. At present, we see no major advantage for polysaccharide bioadhesive applied in this study.

Study protocol: the effect of whole body vibration on acute unilateral unstable lateral ankle sprain- a biphasic randomized controlled trial.

BACKGROUND: Ankle sprains often result in ankle instability, which is most likely caused by damage to passive structures and neuromuscular impairment. Whole body vibration (WBV) is a neuromuscular training method improving those impaired neurologic parameters. The aim of this study is to compare the current gold standard functional treatment to functional treatment plus WBV in patients with acute unilateral unstable inversion ankle sprains. METHODS/DESIGN: 60 patients, aged 18-40 years, presenting with an isolated, unilateral, acute unstable inversion ankle sprain will be included in this bicentric, biphasic, randomized controlled trial. Samples will be randomized by envelope drawing. All patients will be allowed early mobilization and pain-dependent weight bearing, limited functional immobilization by orthosis, PRICE, NSARDs as well as home and supervised physiotherapy. Supervised physical therapy will take place twice a week, for 30 minutes for a period of 6 weeks, following a standardized intervention protocol. During supervised physical therapy, the intervention group will perform exercises similar to those of the control group, on a side-alternating sinusoidal vibration platform. Two time-dependent primary outcome parameters will be assessed: short-term outcome after six weeks will be postural control quantified by the sway index; mid-term outcome after one year will be assessed by subjective instability, defined by the presence of giving-way attacks. Secondary outcome parameters include: return to pre-injury level of activities, residual pain, recurrence, objective instability, energy/coordination, Foot and Ankle Disability Index and EQ 5D. DISCUSSION: This is the first trial investigating the effects of WBV in patients with acute soft tissue injury. Inversion ankle sprains often result in ankle instability, which is most likely due to damage of neurological structures. Due to its unique, frequency dependent, influence on various neuromuscular parameters, WBV is a promising treatment method for patients with acute unstable inversion ankle sprains. TRIAL REGISTRATION: NCT01702597.

Poloxamer-based hydrogels hardening at body core temperature as carriers for cell based therapies: in vitro and in vivo analysis.

Cell-based regenerative therapies for bone defects usually employ bone precursor cells seeded on solid scaffolds. Thermosensitive hydrogels that harden at body core temperature are promising alternative cell carriers as they are applicable minimally invasively. We modified Pluronic® P123 with different chain extenders and assessed rheology and biocompatibility of the resulting hydrogels. The best candidate was tested in a rat's femoral defect model. All gels hardened above 25 °C with butane-diisocyanate-hydrogels (BDI-gels) displaying the highest storage moduli. BDI-gels showed the most favourable biocompatibility and did not affect cellular adipogenic or osteogenic differentiation in vitro. Implantation of BDI-hydrogel into femoral defects did not impede bone healing in vivo as evidenced by µCT and histological analysis. We conclude that thermosensitive BDI-gels are promising alternative cell carriers. The gels harden upon injection in vivo without interfering with bone metabolism. Further experiments will assess the gels' capacity to effectively transport living cells into bone defects.

Unsupervised machine-learning algorithms for the identification of clinical phenotypes in the osteoarthritis initiative database.

OBJECTIVES: Osteoarthritis (OA) is a complex disease comprising diverse underlying patho-mechanisms. To enable the development of effective therapies, segmentation of the heterogenous patient population is critical. This study aimed at identifying such patient clusters using two different machine learning algorithms. METHODS: Using the progression and incident cohorts of the Osteoarthritis Initiative (OAI) dataset, deep embedded clustering (DEC) and multiple factor analysis with clustering (MFAC) approaches, including 157 input-variables at baseline, were employed to differentiate specific patient profiles. RESULTS: DEC resulted in 5 and MFAC in 3 distinct patient phenotypes. Both identified a "comorbid" cluster with higher body mass index (BMI), relevant burden of comorbidity and low levels of physical activity. Both methods also identified a younger and physically more active cluster and an elderly cluster with functional limitations, but low disease impact. The additional two clusters identified with DEC were subgroups of the young/physically active and the elderly/physically inactive clusters. Overall pain trajectories over 9 years were stable, only the numeric rating scale (NRS) for pain showed distinct increase, while physical activity decreased in all clusters. Clusters showed different (though non-significant) trajectories of joint space changes over the follow-up period of 8 years. CONCLUSION: Two different clustering approaches yielded similar patient allocations primarily separating complex "comorbid" patients from healthier subjects, the latter divided in young/physically active vs elderly/physically inactive subjects. The observed association to clinical (pain/physical activity) and structural progression could be helpful for early trial design as strategy to enrich for patients who may specifically benefit from disease-modifying treatments.

Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression.

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), ß(1) and ß(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression.

In situ guided tissue regeneration in musculoskeletal diseases and aging : Implementing pathology into tailored tissue engineering strategies.

In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide "minimal invasive" applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.

Collagen type I and decorin expression in tenocytes depend on the cell isolation method.

BACKGROUND: The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE) might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB) can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED) and cell migration (CM), are investigated on differences in gene expression and cell morphology. METHODS: Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan. RESULTS: The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC) using ED as isolation method (p < 0.05), however decorin expression was higher in TLCC using CM as isolation method (p < 0.05). Dedifferentiation potential seemed to be similar for both isolation techniques. CONCLUSION: In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM) from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved.