Vibration stimuli and the differentiation of musculoskeletal progenitor cells: Review of results in vitro and in vivo

Due to the increasing burden on healthcare budgets of musculoskeletal system disease and injury, there is a growing need for safe, effective and simple therapies. Conditions such as osteoporosis severely impact on quality of life and result in hundreds of hours of hospital time and resources. There is growing interest in the use of low magnitude, high frequency vibration (LMHFV) to improve bone structure and muscle performance in a variety of different patient groups. The technique has shown promise in a number of different diseases, but is poorly understood in terms of the mechanism of action. Scientific papers concerning both the in vivo and in vitro use of LMHFV are growing fast, but they cover a wide range of study types, outcomes measured and regimens tested. This paper aims to provide an overview of some effects of LMHFV found during in vivo studies. Furthermore we will review research concerning the effects of vibration on the cellular responses, in particular for cells within the musculoskeletal system. This includes both osteogenesis and adipogenesis, as well as the interaction between MSCs and other cell types within bone tissue

Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes

Background During endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes. Results Using a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition. Conclusion Our results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate

Effect of cell sample size in atomic force microscopy nanoindentation

Single-cell technologies are powerful tools to evaluate cell characteristics. In particular, Atomic Force Microscopy (AFM) nanoindentation experiments have been widely used to study single cell mechanical properties. One important aspect related to single cell techniques is the need for sufficient statistical power to obtain reliable results. This aspect is often overlooked in AFM experiments were sample sizes are arbitrarily set. The aim of the present work was to propose a tool for sample size estimation in the context of AFM nanoindentation experiments of single cell. To this aim, a retrospective approach was used by acquiring a large dataset of experimental measurements on four bone cell types and by building saturation curves for increasing sample sizes with a bootstrap resampling method. It was observed that the coefficient of variation (CV%) decayed with a function of the form y = axb with similar parameters for all samples tested and that sample sizes of 21 and 83 cells were needed for the specific cells and protocol employed if setting a maximum threshold on CV% of 10% or 5%, respectively. The developed tool is made available as an open-source repository and guidelines are provided for its use for AFM nanoindentation experimental design

Boosting the osteogenic and angiogenic performance of multiscale porous polycaprolactone scaffolds by In vitro generated extracellular matrix decoration

Tissue engineering (TE)-based bone grafts are favorable alternatives to autografts and allografts. Both biochemical properties and the architectural features of TE scaffolds are crucial in their design process. Synthetic polymers are attractive biomaterials to be used in the manufacturing of TE scaffolds, due to various advantages, such as being relatively inexpensive, enabling precise reproducibility, possessing tunable mechanical/chemical properties, and ease of processing. However, such scaffolds need modifications to improve their limited interaction with biological tissues. Structurally, multiscale porosity is advantageous over single-scale porosity; therefore, in this study, we have considered two key points in the design of a bone repair material; (i) manufacture of multiscale porous scaffolds made of photocurable polycaprolactone (PCL) by a combination of emulsion templating and three-dimensional (3D) printing and (ii) decoration of these scaffolds with the in vitro generated bone-like extracellular matrix (ECM) to create biohybrid scaffolds that have improved biological performance compared to PCL-only scaffolds. Multiscale porous scaffolds were fabricated, bone cells were cultured on them, and then they were decellularized. The biological performance of these constructs was tested in vitro and in vivo. Mesenchymal progenitors were seeded on PCL-only and biohybrid scaffolds. Cells not only showed improved attachment on biohybrid scaffolds but also exhibited a significantly higher rate of cell growth and osteogenic activity. The chick chorioallantoic membrane (CAM) assay was used to explore the angiogenic potential of the biohybrid scaffolds. The CAM assay indicated that the presence of the in vitro generated ECM on polymeric scaffolds resulted in higher angiogenic potential and a high degree of tissue infiltration. This study demonstrated that multiscale porous biohybrid scaffolds present a promising approach to improve bioactivity, encourage precursors to differentiate into mature bones, and to induce angiogenesis

In Vitro Bone Cell Models: Impact of Fluid Shear Stress on Bone Formation.

This review describes the role of bone cells and their surrounding matrix in maintaining bone strength through the process of bone remodeling. Subsequently, this work focusses on how bone formation is guided by mechanical forces and fluid shear stress in particular. It has been demonstrated that mechanical stimulation is an important regulator of bone metabolism. Shear stress generated by interstitial fluid flow in the lacunar-canalicular network influences maintenance and healing of bone tissue. Fluid flow is primarily caused by compressive loading of bone as a result of physical activity. Changes in loading, e.g., due to extended periods of bed rest or microgravity in space are associated with altered bone remodeling and formation in vivo. In vitro, it has been reported that bone cells respond to fluid shear stress by releasing osteogenic signaling factors, such as nitric oxide, and prostaglandins. This work focusses on the application of in vitro models to study the effects of fluid flow on bone cell signaling, collagen deposition, and matrix mineralization. Particular attention is given to in vitro set-ups, which allow long-term cell culture and the application of low fluid shear stress. In addition, this review explores what mechanisms influence the orientation of collagen fibers, which determine the anisotropic properties of bone. A better understanding of these mechanisms could facilitate the design of improved tissue-engineered bone implants or more effective bone disease models

Protective treatments against endothelial glycocalyx degradation in surgery : a systematic review and meta-analysis

The aim was to explore the body of literature focusing on protective treatments against endothelial glycocalyx degradation in surgery. A comprehensive systematic review of relevant articles was conducted across databases. Inclusion criteria: (1) treatments for the protection of the endothelial glycocalyx in surgery; (2) syndecan-1 used as a biomarker for endothelial glycocalyx degradation. Outcomes analysed: (1) mean difference of syndecan-1 (2) correlation between glycocalyx degradation and inflammation; (3) correlation between glycocalyx degradation and extravasation. A meta-analysis was used to present mean differences and 95% confidence intervals. Seven articles with eight randomised controlled trials were included. The greatest change from baseline values in syndecan-1 concentrations was generally from the first timepoint measured post-operatively. Interventions looked to either dampen the inflammatory response or fluid therapy. Methylprednisolone had the highest mean difference in plasma syndecan-1 concentrations. Ulinastatin showed correlations between alleviation of degradation and preserving vascular permeability. In this systematic review of 385 patients, those treated were more likely than those treated with placebo to exhibit less shedding of the endothelial glycocalyx. Methylprednisolone has been shown to specifically target the transient increase of glycocalyx degradation immediately post-operation and has displayed anti-inflammatory effects. We have proposed suggestions for improved uniformity and enhanced confidence for future randomised controlled trials

Characterization of a composite polylactic acid-hydroxyapatite 3D-printing filament for bone-regeneration

Autologous cancellous-bone grafts are the current gold standard for therapeutic interventions in which bone-regeneration is desired. The main limitations of these implants are the need for a secondary surgical site, creating a wound on the patient, the limited availability of harvest-safe bone, and the lack of structural integrity of the grafts. Synthetic, resorbable, bone-regeneration materials could pose a viable treatment alternative, that could be implemented through 3D-printing. We present here the development of a polylactic acid-hydroxyapatite (PLA-HAp) composite that can be processed through a commercial-grade 3D-printer. We have shown that this material could be a viable option for the development of therapeutic implants for bone regeneration. Biocompatibility in vitro was demonstrated through cell viability studies using the osteoblastic MG63 cell-line, and we have also provided evidence that the presence of HAp in the polymer matrix enhances cell attachment and osteogenicity of the material. We have also provided guidelines for the optimal PLA-HAp ratio for this application, as well as further characterisation of the mechanical and thermal properties of the composite. This study encompasses the base for further research on the possibilities and safety of 3D-printable, polymer-based, resorbable composites for bone regeneration

Osteocytes and primary cilia

Purpose of Review The purpose of this review is to provide a background on osteocytes and the primary cilium, discussing the role it plays in osteocyte mechanosensing. Recent Findings Osteocytes are thought to be the primary mechanosensing cells in bone tissue, regulating bone adaptation in response to exercise, with the primary cilium suggested to be a key mechanosensing mechanism in bone. More recent work has suggested that, rather than being direct mechanosensors themselves, primary cilia in bone may instead form a key chemo-signalling nexus for processing mechanoregulated signalling pathways. Recent evidence suggests that pharmacologically induced lengthening of the primary cilium in osteocytes may potentiate greater mechanotransduction, rather than greater mechanosensing. Summary While more research is required to delineate the specific osteocyte mechanobiological molecular mechanisms governed by the primary cilium, it is clear from the literature that the primary cilium has significant potential as a therapeutic target to treat mechanoregulated bone diseases, such as osteoporosis

A new mode of contrast in biological second harmonic generation microscopy

Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature

A simple rocker-induced mechanical stimulus upregulates mineralization by human osteoprogenitor cells in fibrous scaffolds

Biodegradable electrospun polycaprolactone scaffolds can be used to support bone-forming cells and could fill a thin bony defect, such as in cleft palate. Oscillatory fluid flow has been shown to stimulate bone production in human progenitor cells in monolayer culture. The aim of this study was to examine whether bone matrix production by primary human mesenchymal stem cells from bone marrow or jaw periosteal tissue could be stimulated using oscillatory fluid flow supplied by a standard see-saw rocker. This was investigated for cells in two-dimensional culture and within electrospun polycaprolactone scaffolds. From day 4 of culture onwards, samples were rocked at 45 cycles/min for 1 h/day, 5 days/week (rocking group). Cell viability, calcium deposition, collagen production, alkaline phosphatase activity and vascular endothelial growth factor secretion were evaluated to assess the ability of the cells to undergo bone differentiation and induce vascularisation. Both cell types produced more mineralized tissue when subjected to rocking and supplemented with dexamethasone. Mesenchymal progenitors and primary human mesenchymal stem cells from bone marrow in three-dimensional scaffolds upregulated mineral deposition after rocking culture as assessed by micro-computed tomography and alizarin red staining. Interestingly, vascular endothelial growth factor secretion, which has previously been shown to be mechanically sensitive, was not altered by rocking in this system and was inhibited by dexamethasone. Rocker culture may be a cost effective, simple pretreatment for bone tissue engineering for small defects such as cleft palate