Can Dynamic Compression in the Absence of Growth Factors Induce Chondrogenic Differentiation of Bone Marrow Derived MSCs Encapsulated in Agarose Hydrogels?

The objectives of this study were twofold; to determine if cartilage specific matrix synthesis by mesenchymal stem cells (MSCs) is regulated by the magnitude and/or duration of dynamic compression in the absence of growth factors, and to investigate if expanding MSCs in the presence of both fibroblast growth factor-2 (FGF-2) and transforming growth factor β-3 (TGF-β3) would influence their subsequent response to dynamic compression following encapsulation in agarose hydrogels. Porcine bone marrow derived MSCs were suspended in agarose and cast to produce cylinders (Ø5×3mm). Constructs were maintained in a chemically defined medium. Dynamic compression was applied at 1 Hz at strain amplitudes of 5%, 10% and 5% superimposed upon a 5% pre-strain for durations of 1, 3 and 12 hours. MSCs were also expanded in the presence of FGF-2 and TGF-β3. The biochemical constituents of constructs were analyzed. Under strain magnitudes of 5% and 10% and durations of 1 and 3 hours small increases in sGAG accumulation relative to unloaded controls were observed. However this was orders of magnitude lower than that induced by TGF-β3 stimulation. Expansion in FGF-2 and TGF-β3 did not positively modulate chondrogenesis of MSCs in either unloaded or loaded culture

Which prosthetic foot to prescribe?

Introduction: Clinicians typically use findings from cohort studies to objectively inform judgements regarding the potential (dis)advantages of prescribing a new prosthetic device. However, before finalising prescription a clinician will typically ask a patient to 'try out' a change of prosthetic device while the patient is at the clinic. Observed differences in gait when using the new device should be the result of the device’s mechanical function, but could also conceivably be due to patient related factors which can change from day-to-day and can thus make device comparisons unreliable. To determine whether a device’s mechanical function consistently has a more meaningful impact on gait than patient-related factors, the present study undertook quantitative gait analyses of a trans-tibial amputee walking using two different foot-ankle devices on two occasions over a year apart. If the observed differences present between devices, established using quantitative gait analysis, were in the same direction and of similar magnitude on each of the two occasions, this would indicate that device-related factors were more important than patient-related factors. Methods: One adult male with a unilateral trans-tibial amputation completed repeated walking trials using two different prosthetic foot devices on two separate occasions, 14 months apart. Walking speed and sagittal plane joint kinematics and kinetics for both limbs were assessed on each occasion. Clinically meaningful differences in these biomechanical outcome variables were defined as those with an effect size difference (d) between prosthetic conditions of at least 0.4 (i.e. 'medium' effect size). Results: Eight variables namely, walking speed, prosthetic 'ankle' peak plantar- and dorsi-flexion and peak positive power, and residual knee loading response flexion, peak stance-phase extension and flexion moments and peak negative power, displayed clinically meaningful differences (d > 0.4) between foot devices during the first session. All eight of these showed similar effect size differences during the second session despite the participant being heavier and older. Conclusions: Findings suggest that a prosthetic device's mechanical function consistently has a more meaningful impact on gait than patient-related factors. These findings support the current clinical practice of making decisions regarding prosthetic prescription for an individual, based on a single session evaluation of their gait using two different devices. However, to confirm this conclusion, a case series using the same approach as the present study could be undertaken

Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells

Funding was provided by Science Foundation Ireland (07-RFP-ENMF142) and Enterprise Ireland (PC/2006/384)

Cell-matrix interactions regulate mesenchymal stem cell response to hydrostatic pressure.

Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-β3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs

The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells.

Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important role in the development of phenotypically stable constructs.Funding was provided by the European Research Council Starter Grant (StemRepair—Project number 258463) and a SFI President of Ireland Young Researcher Award (08/Y15/B1336)

European Society of Biomechanics S.M. Perren Award 2012: the external mechanical environment can override the influence of local substrate in determining stem cell fate.

The aim of this study was to explore how cell-matrix interactions and extrinsic mechanical signals interact to determine stem cell fate in response to transforming growth factor-β3 (TGF-β3). Bone marrow derived mesenchymal stem cells (MSCs) were seeded in agarose and fibrin hydrogels and subjected to dynamic compression in the presence of different concentrations of TGF-β3. Markers of chondrogenic, myogenic and endochondral differentiation were assessed. MSCs embedded within agarose hydrogels adopted a spherical cell morphology, while cells directly adhered to the fibrin matrix and took on a spread morphology. Free-swelling agarose constructs stained positively for chondrogenic markers, with MSCs appearing to progress towards terminal differentiation as indicated by mineral staining. MSC seeded fibrin constructs progressed along an alternative myogenic pathway in long-term free-swelling culture. Dynamic compression suppressed differentiation towards any investigated lineage in both fibrin and agarose hydrogels in the short-term. Given that fibrin clots have been shown to support a chondrogenic phenotype in vivo within mechanically loaded joint defect environments, we next explored the influence of long term (42 days) dynamic compression on MSC differentiation. Mechanical signals generated by this extrinsic loading ultimately governed MSC fate, directing MSCs along a chondrogenic pathway as opposed to the default myogenic phenotype supported within unloaded fibrin clots. In conclusion, this study demonstrates that external cues such as the mechanical environment can override the influence specific substrates, scaffolds or hydrogels have on determining mesenchymal stem cell fate. The temporal data presented in this study highlights the importance of considering how MSCs respond to extrinsic mechanical signals in the long term

In vitro co-culture and ex vivo organ culture assessment of primed and cryopreserved stromal cell microcapsules for intervertebral disc regeneration

Priming towards a discogenic phenotype and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs) may offer an attractive therapeutic approach for disc repair. It potentially obviates the need for in vivo administration of exogenous growth factors, otherwise required to promote matrix synthesis, in addition to providing 'off-the-shelf' availability. Cryopreserved and primed BMSC microcapsules were evaluated in an in vitro surrogate co-culture model system with nucleus pulposus (NP) cells under intervertebral disc (IVD)-like culture conditions and in an ex vivo bovine organ culture disc model. BMSCs were microencapsulated in alginate microcapsules and primed for 14 d with transforming growth factor beta-3 (TGF-β3) under low oxygen conditions prior to cryopreservation. For the in vitro phase, BMSC microcapsules (unprimed or primed) were cultured for 28 d in a surrogate co-culture model system mimicking that of the IVD. For the ex vivo phase, microcapsules (unprimed or primed) were injected into the NP of bovine discs that underwent nucleotomy. In vitro results revealed that although NP cells produced significantly more matrix components in co-culture with BMSC microcapsules regardless of the differentiation state, unprimed microcapsules were inadequate at synthesising matrix as compared to primed microcapsules. However, this difference was diminished when evaluated in the ex vivo organ culture model,withboth unprimed and primed BMSC microcapsules accumulating large amounts of sulphated glycosaminoglycan (sGAG) and collagen and filling the defect cavity. Both models demonstrated that cryopreservation of BMSC microcapsules may offer a feasible strategy for predesigned delivery through cryobanking for on-demand regeneration of the IVD

The application of plastic compression to modulate fibrin hydrogel mechanical properties.

The inherent biocompatibility of fibrin hydrogels makes them an attractive material for use in a wide range of tissue engineering applications. Despite this, their relatively low stiffness and high compliance limits their potential for certain orthopaedic applications. Enhanced mechanical properties are desirable so as to withstand surgical handling and in vivo loading after implantation and additionally, can provide important cues to cells seeded within the hydrogel. Standard methods used to enhance the mechanical properties of biological scaffolds such as chemical or thermal crosslinking cannot be used with fibrin hydrogels as cell seeding and gel formation occurs simultaneously. The objective of this study was to investigate the use of plastic compression as a means to improve the mechanical properties of chondrocyte-seeded fibrin hydrogels and to determine the influence of such compression on cell viability within these constructs. It was found that the application of 80% strain to fibrin hydrogels for 30 min (which resulted in a permanent strain of 47.4%) produced a 2.1-fold increase in the subsequent compressive modulus. Additionally, chondrocyte viability was maintained in the plastically compressed gels with significant cellular proliferation and extracellular matrix accumulation observed over 28 days of culture. In conclusion, plastic compression can be used to modulate the density and mechanical properties of cell-seeded fibrin hydrogels and represents a useful tool for both in theatre and in vitro tissue engineering applications

Multi-Photon Signals from Composite Models at LHC

We analyze the collider signals of composite scalars that emerge in certain little Higgs models and models of vectorlike confinement. Similar to the decay of the pion into photon pairs, these scalars mainly decay through anomaly-induced interactions into electroweak gauge bosons, leading to a distinct signal with three or more photons in the final state. We study the standard model backgrounds for these signals, and find that the LHC can discover these models over a large range of parameter space with 30 fb$^{-1}$ at 14 TeV. An early discovery at the current 7 TeV run is possible in some regions of parameter space. We also discuss possibilities to measure the spin of the particles in the $\gamma \gamma$ and $Z\gamma$ decay channels.Comment: 18 pages, LaTe

Spring-mass behavioural adaptations to acute changes in prosthetic blade stiffness during submaximal running in unilateral transtibial prosthesis users

Background: Individuals with lower-limb amputation can use running specific prostheses (RSP) that store and then return elastic energy during stance. However, it is unclear whether varying the stiffness category of the same RSP affects spring-mass behaviour during self-selected, submaximal speed running in individuals with unilateral transtibial amputation. Research question: The current study investigates how varying RSP stiffness affects limb stiffness, running performance, and associated joint kinetics in individuals with a unilateral transtibial amputation. Methods: Kinematic and ground reaction force data were collected from eight males with unilateral transtibial amputation who ran at self-selected submaximal speeds along a 15 m runway in three RSP stiffness conditions; recommended habitual stiffness (HAB) and, following 10-minutes of familiarisation, stiffness categories above (+1) and below (-1) the HAB. Stance-phase centre of mass velocity, contact time, limb stiffness’ and joint/RSP work were computed for each limb across RSP stiffness conditions. Results: With increased RSP stiffness, prosthetic limb stiffness increased, whilst intact limb stiffness decreased slightly (p<0.03). Centre of mass forward velocity during stance-phase (p<0.02) and contact time (p<0.04) were higher in the intact limb and lower in the prosthetic limb but were unaffected by RSP stiffness. Intact limb hip joint positive work increased for both the +1 and -1 conditions but remained unchanged across conditions in the prosthetic limb (p<0.02). Significance: In response to changes in RSP stiffness, there were acute increased mechanical demands on the intact limb, reflecting a reliance on the intact limb during running. However, overall running speed was unaffected, suggesting participants acutely adapted to an RSP of a non-prescribed stiffness