Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells

The delicately orchestrated process of bone fracture healing is not always successful and long term non union of fractured bone occurs in 5-20% of all cases. Atrophic fracture non unions have been described as the most difficult to treat and this is thought to arise through a cellular and local failure of osteogenesis. However, little is known about the presence and osteogenic proficiency of cells in the local area of non union tissue. We have examined the growth and differentiation potential of cells isolated from human non union tissues compared with normal human bone marrow mesenchymal stromal cells (BMSC). We report the isolation and culture expansion of a population of non union stromal cells (NUSC) which have a CD profile similar to that of BMSC, i.e. CD34-ve, CD45-ve and CD105+ve. The NUSC demonstrated multipotentiality and differentiated to some extent along chondrogenic, adipogenic and osteogenic lineages. However, and importantly, the NUSC showed significantly reduced osteogenic differentiation and mineralization in vitro compared to BMSC. We also found increased levels of cell senescence in NUSC compared to BMSC based on culture growth kinetics and cell positivity for senescence associated beta galactosidase (SA-beta-Gal) activity. The reduced capacity of NUSC to form osteoblasts was associated with significantly elevated secretion of Dickkopf-1 (Dkk-1) which is an important inhibitor of Wnt signalling during osteogenesis, compared to BMSC. Conversely, treating BMSC with levels of rhDkk-1 that were equivalent to those levels secreted by NUSC inhibited the capacity of BMSC to undergo osteogenesis. Treating BMSC with NUSC conditioned medium also inhibited the capacity of the BMSC to undergo osteogenic differentiation when compared to their treatment with BMSC conditioned medium. Our results suggest that the development of fracture non union is linked with a localised reduced capacity of cells to undergo osteogenesis, which in turn is associated with increased cell senescence and Dkk-1 secretion.

Sensitivity of proximal femoral stiffness and areal bone mineral density to changes in bone geometry and density

Areal bone mineral density (aBMD) is the most common surrogate measurement for assessing the bone strength of the proximal femur associated with osteoporosis. Additional factors, however, contribute to the overall strength of the proximal femur, primarily the anatomical geometry. Finite element analysis (FEA) is an effective and widely used computerbased simulation technique for modeling mechanical loading of various engineering structures, providing predictions of displacement and induced stress distribution due to the applied load. FEA is therefore inherently dependent upon both density and anatomical geometry. FEA may be performed on both three-dimensional and two-dimensional models of the proximal femur derived from radiographic images, from which the mechanical stiffness may be redicted. It is examined whether the outcome measures of two-dimensional FEA, two-dimensional, finite element analysis of X-ray images (FEXI), and three-dimensional FEA computed stiffness of the proximal femur were more sensitive than aBMD to changes in trabecular bone density and femur geometry. It is assumed that if an outcome measure follows known trends with changes in density and geometric parameters, then an increased sensitivity will be indicative of an improved prediction of bone strength. All three outcome measures increased non-linearly with trabecular bone density, increased linearly with cortical shell thickness and neck width, decreased linearly with neck length, and were relatively insensitive to neck-shaft angle. For femoral head radius, aBMD was relatively insensitive, with two-dimensional FEXI and threedimensional FEA demonstrating a non-linear increase and decrease in sensitivity, respectively. For neck anteversion, aBMD decreased non-linearly, whereas both two-dimensional FEXI and three dimensional FEA demonstrated a parabolic-type relationship, with maximum stiffness achieved at an angle of approximately 15o. Multi-parameter analysis showed that all three outcome measures demonstrated their highest sensitivity to a change in cortical thickness. When changes in all input parameters were considered simultaneously, three and twodimensional FEA had statistically equal sensitivities (0.41±0.20 and 0.42±0.16 respectively, p = ns) that were significantly higher than the sensitivity of aBMD (0.24±0.07; p = 0.014 and 0.002 for three-dimensional and two-dimensional FEA respectively). This simulation study suggests that since mechanical integrity and FEA are inherently dependent upon anatomical geometry, FEXI stiffness, being derived from conventional two-dimensional radiographic images, may provide an improvement in the prediction of bone strength of the proximal femur than currently provided by aBMD.

Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Predictions of angle dependent tortuosity and elasticity effects on sound propagation in cancellous bone

The anisotropic pore structure and elasticity of cancellous bone cause wave speeds and attenuation in cancellous bone to vary with angle. Previously published predictions of the variation in wave speed with angle are reviewed. Predictions that allow tortuosity to be angle dependent but assume isotropic elasticity compare well with available data on wave speeds at large angles but less well for small angles near the normal to the trabeculae. Claims for predictions that only include angle-dependence in elasticity are found to be misleading. Audio-frequency data obtained at audio-frequencies in air-filled bone replicas are used to derive an empirical expression for the angle-and porosity-dependence of tortuosity. Predictions that allow for either angle dependent tortuosity or angle dependent elasticity or both are compared with existing data for all angles and porosities.

Creation of a validated 3D finite element model of an ovine tibia

Introduction Ovine models are widely used in orthopaedic research. To better understand the impact of orthopaedic procedures computer simulations are necessary. 3D finite element (FE) models of bones allow implant designs to be investigated mechanically, thereby reducing mechanical testing. Hypothesis We present the development and validation of an ovine tibia FE model for use in the analysis of tibia fracture fixation plates. Material & Methods Mechanical testing of the tibia consisted of an offset 3-pt bend test with three repetitions of loading to 350N and return to 50N. Tri-axial stacked strain gauges were applied to the anterior and posterior surfaces of the bone and two rigid bodies – consisting of eight infrared active markers, were attached to the ends of the tibia. Positional measurements were taken with a FARO arm 3D digitiser. The FE model was constructed with both geometry and material properties derived from CT images of the bone. The elasticity-density relationship used for material property determination was validated separately using mechanical testing. This model was then transformed to the same coordinate system as the in vitro mechanical test and loads applied. Results Comparison between the mechanical testing and the FE model showed good correlation in surface strains (difference: anterior 2.3%, posterior 3.2%). Discussion & Conclusion This method of model creation provides a simple method for generating subject specific FE models from CT scans. The use of the CT data set for both the geometry and the material properties ensures a more accurate representation of the specific bone. This is reflected in the similarity of the surface strain results.

The feasibility of vibration analysis as a technique to detect osseointegration of transfemoral implants

One of the main causes of above knee or transfemoral amputation (TFA) in the developed world is trauma to the limb. The number of people undergoing TFA due to limb trauma, particularly due to war injuries, has been increasing. Typically the trauma amputee population, including war-related amputees, are otherwise healthy, active and desire to return to employment and their usual lifestyle. Consequently there is a growing need to restore long-term mobility and limb function to this population. Traditionally transfemoral amputees are provided with an artificial or prosthetic leg that consists of a fabricated socket, knee joint mechanism and a prosthetic foot. Amputees have reported several problems related to the socket of their prosthetic limb. These include pain in the residual limb, poor socket fit, discomfort and poor mobility. Removing the socket from the prosthetic limb could eliminate or reduce these problems. A solution to this is the direct attachment of the prosthesis to the residual bone (femur) inside the residual limb. This technique has been used on a small population of transfemoral amputees since 1990. A threaded titanium implant is screwed in to the shaft of the femur and a second component connects between the implant and the prosthesis. A period of time is required to allow the implant to become fully attached to the bone, called osseointegration (OI), and be able to withstand applied load; then the prosthesis can be attached. The advantages of transfemoral osseointegration (TFOI) over conventional prosthetic sockets include better hip mobility, sitting comfort and prosthetic retention and fewer skin problems on the residual limb. However, due to the length of time required for OI to progress and to complete the rehabilitation exercises, it can take up to twelve months after implant insertion for an amputee to be able to load bear and to walk unaided. The long rehabilitation time is a significant disadvantage of TFOI and may be impeding the wider adoption of the technique. There is a need for a non-invasive method of assessing the degree of osseointegration between the bone and the implant. If such a method was capable of determining the progression of TFOI and assessing when the implant was able to withstand physiological load it could reduce the overall rehabilitation time. Vibration analysis has been suggested as a potential technique: it is a non destructive method of assessing the dynamic properties of a structure. Changes in the physical properties of a structure can be identified from changes in its dynamic properties. Consequently vibration analysis, both experimental and computational, has been used to assess bone fracture healing, prosthetic hip loosening and dental implant OI with varying degrees of success. More recently experimental vibration analysis has been used in TFOI. However further work is needed to assess the potential of the technique and fully characterise the femur-implant system. The overall aim of this study was to develop physical and computational models of the TFOI femur-implant system and use these models to investigate the feasibility of vibration analysis to detect the process of OI. Femur-implant physical models were developed and manufactured using synthetic materials to represent four key stages of OI development (identified from a physiological model), simulated using different interface conditions between the implant and femur. Experimental vibration analysis (modal analysis) was then conducted using the physical models. The femur-implant models, representing stage one to stage four of OI development, were excited and the modal parameters obtained over the range 0-5kHz. The results indicated the technique had limited capability in distinguishing between different interface conditions. The fundamental bending mode did not alter with interfacial changes. However higher modes were able to track chronological changes in interface condition by the change in natural frequency, although no one modal parameter could uniquely distinguish between each interface condition. The importance of the model boundary condition (how the model is constrained) was the key finding; variations in the boundary condition altered the modal parameters obtained. Therefore the boundary conditions need to be held constant between tests in order for the detected modal parameter changes to be attributed to interface condition changes. A three dimensional Finite Element (FE) model of the femur-implant model was then developed and used to explore the sensitivity of the modal parameters to more subtle interfacial and boundary condition changes. The FE model was created using the synthetic femur geometry and an approximation of the implant geometry. The natural frequencies of the FE model were found to match the experimental frequencies within 20% and the FE and experimental mode shapes were similar. Therefore the FE model was shown to successfully capture the dynamic response of the physical system. As was found with the experimental modal analysis, the fundamental bending mode of the FE model did not alter due to changes in interface elastic modulus. Axial and torsional modes were identified by the FE model that were not detected experimentally; the torsional mode exhibited the largest frequency change due to interfacial changes (103% between the lower and upper limits of the interface modulus range). Therefore the FE model provided additional information on the dynamic response of the system and was complementary to the experimental model. The small changes in natural frequency over a large range of interface region elastic moduli indicated the method may only be able to distinguish between early and late OI progression. The boundary conditions applied to the FE model influenced the modal parameters to a far greater extent than the interface condition variations. Therefore the FE model, as well as the experimental modal analysis, indicated that the boundary conditions need to be held constant between tests in order for the detected changes in modal parameters to be attributed to interface condition changes alone. The results of this study suggest that in a clinical setting it is unlikely that the in vivo boundary conditions of the amputated femur could be adequately controlled or replicated over time and consequently it is unlikely that any longitudinal change in frequency detected by the modal analysis technique could be attributed exclusively to changes at the femur-implant interface. Therefore further development of the modal analysis technique would require significant consideration of the clinical boundary conditions and investigation of modes other than the bending modes.

Mesoporous bioglass/silk composite scaffolds for bone tissue engineering

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regí et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

The effects of bioactive akermanite on physiochemical, drug-delivery and biological properties of poly (lactide-co-glycolide) beads

Poly(lactide-co-glycolide) (PLGA) beads have been widely studied as a potential drug/protein carrier. The main shortcomings of PLGA beads are that they lack bioactivity and controllable drug-delivery ability, and their acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Akermanite (AK) (Ca(2) MgSi(2) O(7) ) is a novel bioactive ceramic which has shown excellent bioactivity and degradation in vivo. This study aimed to incorporate AK to PLGA beads to improve the physiochemical, drug-delivery, and biological properties of PLGA beads. The microstructure of beads was characterized by SEM. The effect of AK incorporating into PLGA beads on the mechanical strength, apatite-formation ability, the loading and release of BSA, and the proliferation, and differentiation of bone marrow stromal cells (BMSCs) was investigated. The results showed that the incorporation of AK into PLGA beads altered the anisotropic microporous structure into homogenous one and improved their compressive strength and apatite-formation ability in simulated body fluids (SBF). AK neutralized the acidic products from PLGA beads, leading to stable pH value of 7.4 in biological environment. AK led to a sustainable and controllable release of bovine serum albumin (BSA) in PLGA beads. The incorporation of AK into PLGA beads enhanced the proliferation and alkaline phosphatase activity of BMSCs. This study implies that the incorporation of AK into PLGA beads is a promising method to enhance their physiochemical and biological property. AK/PLGA composite beads are a potential bioactive drug-delivery system for bone tissue repair.

CaSiO3 microstructure modulating the in vitro and in vivo bioactivity of poly (lactide-co-glycolide) microspheres

Poly (lactide-co-glycolide) (PLGA) microspheres have been used for regenerative medicine due to their ability for drug delivery and generally good biocompatibility, but they lack adequate bioactivity for bone repair application. CaSiO3 (CS) has been proposed as a new class of material suitable for bone tissue repair due to its excellent bioactivity. In this study, we set out to incorporate CS into PLGA microspheres to investigate how the phase structure (amorphous and crystal) of CS influences the in vitro and in vivo bioactivity of the composite microspheres, with a view to the application for bone regeneration. X-ray diffraction (XRD), N2 adsorption-desorption analysis and scanning electron microscopy (SEM) were used to analyze the phase structure, surface area/pore volume, and microstructure of amorphous CS (aCS) and crystal CS (cCS), as well as their composite microspheres. The in vitro bioactivity of aCS and cCS – PLGA microspheres was evaluated by investigating their apatite-mineralization ability in simulated body fluids (SBF) and the viability of human bone mesenchymal stem cells (BMSCs). The in vivo bioactivity was investigated by measuring their de novo bone-formation ability. The results showed that the incorporation of both aCS and cCS enhanced the in vitro and in vivo bioactivity of PLGA microspheres. cCS/PLGA microspheres improved better in vitro BMSC viability and de novo bone-formation ability in vivo, compared to aCS/PLGA microspheres. Our study indicates that controlling the phase structure of CS is a promising method to modulate the bioactivity of polymer microsphere system for potential bone tissue regeneration.

The effect of friction on indenter force and pile-up in numerical simulations of bone nanoindentation

Nanoindentation is a useful technique for probing the mechanical properties of bone, and finite element (FE) modeling of the indentation allows inverse determination of elasto-plastic constitutive properties. However, all but one FE study to date have assumed frictionless contact between indenter and bone. The aim of this study was to explore the effect of friction in simulations of bone nanoindentation. Two dimensional axisymmetric FE simulations were performed using a spheroconical indenter of tip radius 0.6 m and angle 90°. The coefficient of friction between indenter and bone was varied between 0.0 (frictionless) and 0.3. Isotropic linear elasticity was used in all simulations, with bone elastic modulus E=13.56GPa and Poisson‟s ratio f 0.3. Plasticity was incorporated using both Drucker-Prager and von Mises yield surfaces. Friction had a modest effect on the predicted force-indentation curve for both von Mises and Drucker-Prager plasticity, reducing maximum indenter displacement by 10% and 20% respectively as friction coefficient was increased from zero to 0.3 (at a maximum indenter force of 5mN). However, friction has a much greater effect on predicted pile-up after indentation, reducing predicted pile-up from 0.27 to 0.11 m with a von Mises model, and from 0.09 to 0.02 m with Drucker-Prager plasticity. We conclude that it is potentially important to include friction in nanoindentation simulations of bone if pile-up is used to compare simulation results with experiment.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

Orthopedic bone plates : evolution in structure implementation technique and biomaterial

With many important developments over the last century, nowadays orthopedic bone plate now excels over other types of internal fixators in bone fracture fixation. The developments involve the design, material and implementation techniques of the plates. This paper aims to review the evolution in implementation technique and biomaterial of the orthopedic bone plates. Plates were initially used to fix the underlying bones firmly. Accordingly, Compression plate (CP), Dynamic compression plate (DCP), Limited contact dynamic compression plate (LC-DCP) and Point contact fixator (PC-Fix) were developed. Later, the implementation approach was changed to locking, and the Less Invasive Stabilization System (LISS) plate was introduced as a result. Finally, a combination of both of these approaches has been used by introducing the Locking Compression Plate (LCP). Currently, precontoured LCPs are mainly used for bone fracture fixation. In parallel with structure and implementation techniques, numerous advances have occurred in biomaterials of the plates. Titanium and stainless steel alloys are now the most common biomaterials in production of orthopedic bone plates. However, regarding the biocompatibility, bioactivity and biodegradability characteristics of Mg alloys, Ta alloys, SMAs, carbon fiber composites and bioceramics, these materials are considered as potentially suitable for plates. However, due to poor mechanical properties, they have very limited applications. Therefore, further studies are required in future to solve these problems and make them feasible for heavy-duty bone plates.

Quantifying alterations in cell migration : tracking fluorescently-tagged migrating cells by FACs and live-imaging

Cell migration is fundamental to many different physiological processes including embryonic development, inflammation and wound healing. Given the range and importance cell migration plays a number of assays have been developed to measure different aspects of cell migration. Here we describe two different methods to analyze cell migration. The first method analyzes the migration of fluorescently tagged cells using Boyden chambers and FACs and the second looks at migration properties using time-lapse microscopy.