An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Octacalcium Phosphate for Bone Tissue Engineering: Synthesis, Modification, and In Vitro Biocompatibility Assessment.

Octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) is known to be a possible precursor of biological hydroxyapatite formation of organic bone tissue. OCP has higher biocompatibility and osseointegration rate compared to other calcium phosphates. In this work, the synthesis of low-temperature calcium phosphate compounds and substituted forms of those at physiological temperatures is shown. Strontium is used to improve bioactive properties of the material. Strontium was inserted into the OCP structure by ionic substitution in solutions. The processes of phase formation of low-temperature OCP with theoretical substitution of strontium for calcium up to 50 at.% in conditions close to physiological, i.e., temperature 35-37 °C and normal pressure, were described. The effect of strontium substitution range on changes in the crystal lattice of materials, the microstructural features, surface morphology and biological properties in vitro has been established. The results of the study indicate the effectiveness of using strontium in OCP for improving biocompatibility of OCP based composite materials intended for bone repair.

In-vitroassessment of ß-tricalcium phosphate/bredigite-ciprofloxacin (CPFX) scaffolds for bone treatment applications.

In the present study, ß-tricalcium phosphate (ß-TCP) scaffolds with various amounts of bredigite (Bre) were fabricated by the space holder method. The effect of bredigite content on the structure, mechanical properties,in vitrobioactivity, and cell viability was investigated. The structural assessment of the composite scaffolds presented interconnected pores with diameter of 300-500 µm with around 78%-82% porosity. The results indicated that the compressive strength of the scaffolds with 20% bredigite (1.91 MPa) was improved in comparison with scaffolds with 10% bredigite (0.52 MPa), due to the reduction of the average pore and grain sizes. Also, the results showed that the bioactivity and biodegradability of ß-TCP/20Bre were better than that of ß-TCP/10Bre. Besides, in this study, the release kinetics of ciprofloxacin (CPFX) loaded ß-TCP/Bre composites as well as the ability of scaffolds to function as a sustained release drug carrier was investigated. Drug release pattern of ß-TCP/bredigite-5CPFX scaffolds exhibited the rapid burst release of 43% for 3 h along with sustained release (82%) for 32 h which is favorable for bone infection treatment. Antibacterial tests revealed that the antibacterial properties of ß-TCP/bredigite scaffolds are strongly related to the CPFX concentration, wherein the scaffold containing 5% CPFX showed the most significant zone of inhibition (33 ± 0.5 mm) againstStaphylococcus aureus. The higher specific surface areas of nanostructure ß-TCP/bredigite scaffolds containing CPFX lead to an initial rapid release followed by constant drug delivery. MTT assay showed that the cell viability of ß-TCP/bredigite scaffold loading with up to 1%-3% CPFX (95 ± 2%), is greater than for scaffolds containing 5% CPFX (84 ± 2%). In Overall, it may suggested that ß-TCP/bredigite containing 1%-3% CPFX possesses great cell viability and antibacterial activity and be employed as bactericidal biomaterials and bone infection treatment.

The Isolation and Manufacture of GMP-Grade Bone Marrow Stromal Cells from Bone Specimens.

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.

Assessment of Collagen-Based Nanostructured Biomimetic Systems with a Co-Culture of Human Bone-Derived Cells.

Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and other factors, is considered a potential alternative to conventional treatments. Innovative scaffolds need to be tested in in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs), the two main players of bone remodeling, is required to mimic their crosstalk and molecular cooperation. To this aim, two composite materials were developed, based on type I collagen, and containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. The developed nanostructured systems underwent genipin chemical crosslinking and were then tested with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors, for 2-3 weeks. The favorable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of cells, encouraging a further investigation of the developed bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

Assessment of human ovarian follicular fluid derived mesenchymal stem cells in chitosan/PCL/Zn scaffold for bone tissue regeneration.

Bone tissue engineering compasses the use of mesenchymal stem cells (MSCs) along with engineered biomaterial construct to augment bone regeneration. Till now, MSCs were isolated from various sources and used in cellular constructs. For the first time, in this study, MSCs were isolated from human Ovarian Follicular Fluid (OFF) and characterized by CD 44+ and CD 105+ markers via confocal microscopy and flow cytometry. Additionally, MSCs stemness, proliferation and colony-forming unit ability, multi-lineage differentiation potential were also studied. To test its suitability for bone tissue engineering applications, we grew the MSCs with the conditioned medium obtained from biocomposite scaffold by fusing a natural polymer, Chitosan (CS) and a synthetic polymer, Polycaprolactone (PCL) and the scaffold were coated with Zinc divalent ions to impart osteogenic properties. The physico-chemical characterization of scaffold, such as FTIR, XRD, and SEM studies was carried out. The biological characterization showed that the scaffolds were compatible with MSCs and promoted osteoblast differentiation which was confirmed at both cellular and molecular levels. The cellular construct increased calcium deposition, analyzed by alizarin red staining and ALP activity at cellular level. At the molecular level, the osteoblast markers expression such as Runx2 and type 1 collagen mRNAs, and osteonectin (ON) and osteocalcin (OC) secretory proteins were increased in the presence of scaffold. Overall, the current study recommends that MSCs can be easily obtained from human waste OFF, and grown in standard in vitro conditions. Successful growth of such MSCs with CS/PCL/Zn scaffold opens new avenues in utilizing the cell source for bone tissue engineering.

Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone.

In this study, we examined the immunolocalization of podoplanin/E11, CD44, actin filaments, and phosphorylated ezrin in the osteoblasts on the verge of differentiating into osteocytes in murine femora and tibiae. When observing under stimulated emission depletion microscopy, unlike podoplanin-negative osteoblasts, podoplanin-positive osteoblasts showed a rearranged assembly of actin filaments along the cell membranes which resembled that of embedded osteocytes. In the metaphysis, i.e., the bone remodeling site, CD44-bearing osteoclasts were either proximal to or in contact with podoplanin-positive osteoblasts, but the podoplanin-positive osteoblasts also localized CD44 on their own cell surface. These podoplanin-positive osteoblasts, which either possessed CD44 on their cell surface or were close to CD44-bearing osteoclasts, showed phosphorylated ezrin-positivity on the cell membranes. Therefore, the CD44/podoplanin interaction on the cell surface may be involved in the osteoblastic differentiation into osteocytes in the metaphyses, via the mediation of podoplanin-driven ezrin phosphorylation and the subsequent reorganized assembly of actin filaments. Consistently, the protein expression of phosphorylated ezrin was increased after CD44 administration in calvarial culture. Conversely, in modeling sites such as the cortical bones, podoplanin-positive osteoblasts were uniformly localized at certain intervals even without contact with CD44-positive bone marrow cells; furthermore, they also exhibited phosphorylated ezrin immunoreactivity along their cell membranes. Taken together, it seems likely that the CD44/podoplanin interaction is involved in osteoblastic differentiation into osteocytes in the bone remodeling area but not in modeling sites.

Histochemical assessment on the cellular interplay of vascular endothelial cells and septoclasts during endochondral ossification in mice.

This study was aimed to verify the cellular interplay between vascular endothelial cells and surrounding cells in the chondro-osseous junction of murine tibiae. Many CD31-positive endothelial cells accompanied with Dolichos Biflorus Agglutinin lectin-positive septoclasts invaded into the hypertrophic zone of the tibial epiphyseal cartilage. MMP9 immunoreactive cytoplasmic processes of vascular endothelial cells extended into the transverse partitions of cartilage columns. In contrast, septoclasts included several large lysosomes which indicate the incorporation of extracellular matrices despite no immunopositivity for F4/80-a hallmark of macrophage/monocyte lineage. In addition, septoclasts were observed in c-fos-/- mice but not in Rankl-/- mice. Unlike c-fos-/- mice, Rankl-/- mice showed markedly expanded hypertrophic zone and the irregular shape of the chondro-osseous junction. Immunoreactivity of platelet-derived growth factor-bb, which involved in angiogenic roles in the bone, was detected in not only osteoclasts but also septoclasts at the chondro-osseous junction. Therefore, septoclasts appear to assist the synchronous vascular invasion of endothelial cells at the chondro-osseous junction. Vascular endothelial cells adjacent to the chondro-osseous junction possess endomucin but not EphB4, whereas those slightly distant from the chondro-osseous junction were intensely positive for both endomucin and EphB4, while being accompanied with ephrinB2-positive osteoblasts. Taken together, it is likely that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. MINI-ABSTRACT: Our original article demonstrated that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. (A figure that best represents your paper is Fig. 5c).

A computational framework and sensitivity analysis for the hormonal treatment of bone.

BACKGROUND: Osteoporosis is a bone disease identified by disordering of bone formation and resorption cells. It increases the risk of bone fragility and fracture. Autocrine and paracrine signalling of osteoclasts and osteoblasts plays an important role in the regulation of bone remodelling. Calcitonin is an approved pharmacologic agent for the treatment of osteoporosis. METHODS: A novel mathematical model comprising the interaction among osteoclasts and osteoblasts cells with intermittent administration of calcitonin has been presented to study the dynamics of osteoporotic bone. The stability of model and sensitivity of parameters are also discussed. FINDINGS: The population of osteoclastic and osteoblastic cells has been predicted via numerical simulation. The results of Monte Carlo sensitivity analysis are shown via tornado diagram. INTERPRETATION: It is concluded that intermittent administration of calcitonin is an effective therapy for the treatment of osteoporosis.

Brillouin spectroscopy and radiography for assessment of viscoelastic and regenerative properties of mammalian bones.

Biomechanical properties of mammalian bones, such as strength, toughness, and plasticity, are essential for understanding how microscopic-scale mechanical features can link to macroscale bones' strength and fracture resistance. We employ Brillouin light scattering (BLS) microspectroscopy for local assessment of elastic properties of bones under compression and the efficacy of the tissue engineering approach based on heparin-conjugated fibrin (HCF) hydrogels, bone morphogenic proteins, and osteogenic stem cells in the regeneration of the bone tissues. BLS is noninvasive and label-free modality for probing viscoelastic properties of tissues that can give information on structure-function properties of normal and pathological tissues. Results showed that MCS and BPMs are critically important for regeneration of elastic and viscous properties, respectively, HCF gels containing combination of all factors had the best effect with complete defect regeneration at week nine after the implantation of bone grafts and that the bones with fully consolidated fractures have higher values of elastic moduli compared with defective bones.

Rheological, biocompatibility and osteogenesis assessment of fish collagen scaffold for bone tissue engineering.

In the present investigation, an attempt was made to find an alternative to mammalian collagen with better osteogenesis ability. Three types of collagen scaffolds - collagen, collagen-chitosan (CCH), and collagen-hydroxyapatite (CHA) - were prepared from the cartilage of Blue shark and investigated for their physico-functional and mechanical properties in relation to biocompatibility and osteogenesis. CCH scaffold was superior with pH 4.5-4.9 and viscosity 9.7-10.9cP. Notably, addition of chitosan and HA (hydroxyapatite) improved the stiffness (11-23MPa) and degradation rate but lowered the water binding capacity and porosity of the scaffold. Interestingly, CCH scaffolds remained for 3days before complete in-vitro biodegradation. The decreased amount of viable T-cells and higher level of FAS/APO-1 were substantiated the biocompatibility properties of prepared collagen scaffolds. Osteogenesis study revealed that the addition of CH and HA in both fish and mammalian collagen scaffolds could efficiently promote osteoblast cell formation. The ALP activity was significantly high in CHA scaffold-treated osteoblast cells, which suggests an enhanced bone-healing process. Therefore, the present study concludes that the composite scaffolds prepared from fish collagen with higher stiffness, lower biodegradation rate, better biocompatible, and osteogenesis properties were suitable biomaterial for a bone tissue engineering application as an alternative to mammalian collagen scaffolds.

3D ultrasound biomicroscopy for assessment of cartilage repair tissue: volumetric characterisation and correlation to established classification systems.

Objective and sensitive assessment of cartilage repair outcomes lacks suitable methods. This study investigated the feasibility of 3D ultrasound biomicroscopy (UBM) to quantify cartilage repair outcomes volumetrically and their correlation with established classification systems. 32 sheep underwent bilateral treatment of a focal cartilage defect. One or two years post-operatively the repair outcomes were assessed and scored macroscopically (Outerbridge, ICRS-CRA), by magnetic resonance imaging (MRI, MOCART), and histopathology (O'Driscoll, ICRS-I and ICRS-II). The UBM data were acquired after MRI and used to reconstruct the shape of the initial cartilage layer, enabling the estimation of the initial cartilage thickness and defect volume as well as volumetric parameters for defect filling, repair tissue, bone loss and bone overgrowth. The quantification of the repair outcomes revealed high variations in the initial thickness of the cartilage layer, indicating the need for cartilage thickness estimation before creating a defect. Furthermore, highly significant correlations were found for the defect filling estimated from UBM to the established classification systems. 3D visualisation of the repair regions showed highly variable morphology within single samples. This raises the question as to whether macroscopic, MRI and histopathological scoring provide sufficient reliability. The biases of the individual methods will be discussed within this context. UBM was shown to be a feasible tool to evaluate cartilage repair outcomes, whereby the most important objective parameter is the defect filling. Translation of UBM into arthroscopic or transcutaneous ultrasound examinations would allow non-destructive and objective follow-up of individual patients and better comparison between the results of clinical trials.

Molecular assessment of osseointegration in vitro: a review of current literature.

This paper presents the results of a structured review of the literature concerning in vitro molecular assessment of osseointegration at the level of cell-surface topography interactions. A search of the electronic databases was performed up to and including November 2010, with 320 articles meeting the inclusion criteria. Characteristics of the included in vitro reports were model systems used, genes examined, techniques used for molecular assessment of the osseointegration process, and wide gene expression profiling studies. There exists a growing body of in vitro evidence to support a role for surface topography in the direct influence of cellular phenotypes as related to the process of osseointegration. Most recently, functional or mechanistic studies have provided evidence that particular topographic cues can be specifically integrated among the many extracellular signals received by the cell in its signal transduction network. Such investigations begin to define linkages between the character of the implant surface and adherent cellular responses, including cells from extravasated blood (eg, platelets) and of the immune system (eg, monocytes). In vitro studies involving cell culture on endosseous implant-related biomaterials offer important and beneficial insight into the clinical control of the implant-bone interface.

[Intravital optical imaging of bone and cartilage tissues].

Intravital optical imaging technique is a promising method that allows us to investigate complex vital phenomena in vivo . In particular, discovery and development of unique fluorescent proteins and smart fluorescent dyes in conjunction with appropriate equipment such as two-photon microscopy and image processing software allow visualization of the behavior and function of bone and cartilage-related cells as well as the microenvironment of the cells in bone and cartilage in living animals. Here we show recent technological development and issues of the intravital optical imaging and the application of the fluorescent imaging approaches to bone and cartilage biology.

Direct action of radiation on mummified cells: modeling of computed tomography by Monte Carlo algorithms.

X-ray imaging is a nondestructive and preferred method in paleopathology to reconstruct the history of ancient diseases. Sophisticated imaging technologies such as computed tomography (CT) have become common for the investigation of skeletal disorders in human remains. Researchers have investigated the impact of ionizing radiation on living cells, but never on ancient cells in dry tissue. The effects of CT exposure on ancient cells have not been examined in the past and may be important for subsequent genetic analysis. To remedy this shortcoming, we developed different Monte Carlo models to simulate X-ray irradiation on ancient cells. Effects of mummification were considered by using two sizes of cells and three different phantom tissues, which enclosed the investigated cell cluster. This cluster was positioned at the isocenter of a CT scanner model, where the cell hit probabilities P(0,1,…, n) were calculated according to the Poisson distribution. To study the impact of the dominant physics process, CT scans for X-ray spectra of 80 and 120 kVp were simulated. Comparison between normal and dry tissue phantoms revealed that the probability of unaffected cells increased by 21 % following cell shrinkage for 80 kVp, while for 120 kVp, a further increase of unaffected cells of 23 % was observed. Consequently, cell shrinkage caused by dehydration decreased the impact of X-ray radiation on mummified cells significantly. Moreover, backscattered electrons in cortical bone protected deeper-lying ancient cells from radiation damage at 80 kVp X-rays.

[Recent progression in assessment of bone microstructure].

In vivo quantitative techniques for assessing the microstructure of trabecular bone noninvasively and non-destructively include high-resolution CT and magnetic resonance (MR). Compared with MR imaging, CT-based techniques have the advantage of directly visualizing the bone in the axial skeleton, with high spatial resolution, but the disadvantage of delivering a considerable radiation dose. The evaluation of microstructure is useful to investigate the biomechanical property, and to assess the efficacy of anti-osteoporotic agents. Recently, cortical microstructure has been recognized important in relation to biomechanics. Further progress in bone imaging technology is promising to bring new aspects of bone structure in the every day clinical setting.

Viability assessment of osteocytes using histological lactate dehydrogenase activity staining on human cancellous bone sections.

The assessment of viable osteocytes within bone tissue is of crucial importance. Osteocytes are the most abundant cells in bone. Due to their interconnectivity in the bone matrix they are hypothesised to play an important role in the maintenance of the extracellular matrix of bone. The death of osteocytes and the resulting disturbance of the osteocyte-canalicular network are responsible for the "loss of function" seen in several bone diseases. The lactate dehydrogenase (LDH) assay is a popular method to detect cell viability in bone sections. The major advantage of the LDH assay is the stability of the LDH enzyme for up to 36 h after cell death, eliminating any false negative viability results due to processing of the tissue. Here, we present a quick, reliable, and easy modification of the LDH assay using non-decalcified, thick, unfixed cancellous bone sections for the quantification of osteocyte viability.

Looking beyond bone mineral density : Imaging assessment of bone quality.

Bone fracture is related to bone strength. In current clinical practice, bone mineral density (BMD) is used as the prime indicator of bone strength, not infrequently at the neglect of an even more pertinent measure of reduced bone strength, namely the radiographic presence of an insufficiency fracture. Bone strength depends not just on BMD but also on bone quality, which relates to such factors as bone architecture, turnover, mineralization, and cellularity. The high resolution available from current imaging techniques, particularly computed tomography and magnetic resonance imaging, along with advanced analytical software has greatly enhanced evaluation of bone architecture and strength. This has improved our knowledge of the pathophysiological processes behind osteoporosis and its treatment beyond that provided by BMD measurement alone. Although still in the experimental stage, these techniques will no doubt be incorporated into clinical practice, leading to a more tailored approach to the screening, monitoring, and treatment of osteoporosis.

Methodological assessment of acid-etching for visualizing the osteocyte lacunar-canalicular networks using scanning electron microscopy.

Osteocytes are the most abundant of the bone cells. Each osteocyte is contained within its own lacuna and connected to adjacent osteocytes via fillipodial processes, which form an intricate network of canaliculi within the matrix. Studying this intricate network of cells and their processes is difficult, because it exists embedded within a densely mineralized matrix. Scanning electron microscopy (SEM) has been shown to be a useful tool for visualizing this cellular network, yet the techniques involved for preparing specimens has not been systematically explored. The goal of this study was to investigate how variations in acid-etching, both etching media and etching duration, affect SEM-based visualization of the osteocyte lacunar-canalicular network. Bone samples were embedded in plastic and then acid etched in either 9% (10, 20, 40, and 60 s durations) or 37% (5, 10, and 15 s) phosphoric acid. Specimens were imaged using SEM, and qualitative evaluation of the lacunar-canalicular network was undertaken. Our findings show acid etchingwith a 9% phosphoric acid solution for 20 s provided the most favorable visualization of the osteocyte lacunar-canalicular network.

Skeletal dosimetry in cone beam computed tomography.

Cone beam computed tomography (CBCT) is a relatively new patient imaging technique that has proved invaluable for treatment target verification and patient positioning during image-guided radiotherapy (IGRT). It has been shown that CBCT results in additional dose to bone that may amount to 10% of the prescribed dose. In this study, voxelized human phantoms, FAX06 (adult female) and MAX06 (adult male), are used together with phase-space data collected from a realistic model of a CBCT imager to calculate dose in the red bone marrow (RBM) and bone surface cells (BSCs), the two organs at risk within the bone spongiosa, during simulated head and neck, chest and pelvis CBCT scans. The FAX06/MAX06 phantoms model spongiosa based on micro-CT images, filling the relevant phantom voxels, which are 0.12 x 0.12 x 0.12 cm3, with 17 x 17 x 17 microm3 microvoxels to form a micromatrix of trabecular bone and bone marrow. FAX06/ MAX06 have already been implemented in an EGSnrc-based Monte Carlo code to simulate radiation transport in the phantoms; however, this study required significant modifications of the code to allow use of phase-space data from a simulated CBCT imager as a source and to allow scoring of total dose, RBM dose and BSC dose on a voxel-by-voxel basis. In simulated CBCT scans, the BSC dose is significantly greater than the dose to other organs at risk. For example, in a simulated head and neck scan, the average BSC dose is 25% higher than the average dose to eye lens (approximately 8.3 cGy), and 80% greater than the average dose to brain (5.7 cGy). Average dose to RBM, on the other hand, is typically only approximately 50% of the average BSC dose and less than the dose to other organs at risk (54% of the dose to eye lens and 76% of dose to brain in a head and neck scan). Thus, elevated dose in bone due to CBCT results in elevated BSC dose. This is potentially of concern when using CBCT in conjunction with radiotherapy treatment.