ADAMs, Osteoclastogenesis and Bone Destruction in the Loosening of the Totally Replaced Hip

Total hip replacement is the golden standard treatment for severe osteoarthritis refractory for conservative treatment. Aseptic loosening and osteolysis are the major long-term complications after total hip replacement. Foreign body giant cells and osteoclasts are locally formed around aseptically loosening implants from precursor cells by cell fusion. When the foreign body response is fully developed, it mediates inflammatory and destructive host responses, such as collagen degradation. In the present study, it was hypothesized that the wear debris and foreign body inflammation are the forces driving local osteoclast formation, peri-implant bone resorption and enhanced tissue remodeling. Therefore the object was to characterize the eventual expression and the role of fusion molecules, ADAMs (an abbreviation for A Disintegrin And Metalloproteinase, ADAM9 and ADAM12) in the fusion of progenitor cells into multinuclear giant cells. For generation of such cells, activated macrophages trying to respond to foreign debris play an important role. Matured osteoclasts together with activated macrophages mediate bone destruction by secreting protons and proteinases, including matrix metalloproteinases (MMPs) and cathepsin K. Thus this study also assessed collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. ADAMs were found in the interface tissues of revision total hip replacement patients. Increased expression of ADAMs at both transcriptional and translational levels was found in synovial membrane-like interface tissue of revision total hip replacement (THR) samples compared with that in primary THR samples. These studies also demonstrate that multinucleate cell formation from monocytes by stimulation with macrophage-colony stimiulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) is characterized by time dependent changes of the proportion of ADAMs positive cells. This was observed both in the interface membrane in patients and in two different in vitro models. In addition to an already established MCS-F and RANKL driven model, a new virally (parainfluenza 2) driven model (of human salivary adenocarcinoma (HSY) cells or green monkey kidney (GMK) cells) was developed to study various fusion molecules and their role in cell fusion in general. In interface membranes, collagen was highly degraded and collagen degradation significantly correlated with the number of local cells containing collagenolytic enzymes, particularly cathepsin K. As a conclusion, fusion molecules ADAM9 and ADAM12 seem to be dynamically involved in cell-cell fusion processes and multinucleate cell formation. The highly significant correlation between collagen degradation and collagenolytic enzymes, particularly cathepsin K, indicates that the local acidity of the interface membrane in the pathologic bone and soft tissue destruction. This study provides profound knowledge about cell fusion and mechanism responsible for aseptic loosening as well as increases knowledge helpful for prevention and treatment.

Conceptual design of three-dimensional scaffolds of powder-based materials for bone tissue engineering applications

Purpose - The purpose of this paper is to investigate the possibility to construct tissue-engineered bone repair scaffolds with pore size distributions using rapid prototyping techniques. Design/methodology/approach - The fabrication of porous scaffolds with complex porous architectures represents a major challenge in tissue engineering and the design aspects to mimic complex pore shape as well as spatial distribution of pore sizes of natural hard tissue remain unexplored. In this context, this work aims to evaluate the three-dimensional printing process to study its potential for scaffold fabrication as well as some innovative design of homogeneously porous or gradient porous scaffolds is described and such design has wider implication in the field of bone tissue engineering. Findings - The present work discusses biomedically relevant various design strategies with spatial/radial gradient in pore sizes as well as with different pore sizes and with different pore geometries. Originality/value - One of the important implications of the proposed novel design scheme would be the development of porous bioactive/biodegradable composites with gradient pore size, porosity, composition and with spatially distributed biochemical stimuli so that stem cells loaded into scaffolds would develop into complex tissues such as those at the bone-cartilage interface.

Biomimetic bone-like composites fabricated through an automated alternate soaking process.

Hydroxyapatite-gelatin composites have been proposed as suitable scaffolds for bone and dentin tissue regeneration. There is considerable interest in producing these scaffolds using biomimetic methods due to their low energy costs and potential to create composites similar to the tissues they are intended to replace. Here an existing process used to coat a surface with hydroxyapatite under near physiological conditions, the alternate soaking process, is modified and automated using an inexpensive "off the shelf" robotics kit. The process is initially used to precipitate calcium phosphate coatings. Then, in contrast to previous utilizations of the alternate soaking process, gelatin was added directly to the solutions in order to co-precipitate hydroxyapatite-gelatin composites. Samples were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and nanoindentation. Calcium phosphate coatings formed by the alternate soaking process exhibited different calcium to phosphate ratios, with correspondingly distinct structural morphologies. The coatings demonstrated an interconnected structure with measurable mechanical properties, even though they were 95% porous. In contrast, hydroxyapatite-gelatin composite coatings over 2mm thick could be formed with little visible porosity. The hydroxyapatite-gelatin composites demonstrate a composition and mechanical properties similar to those of cortical bone.

A bone remodelling model coupling micro-damage growth and repair by 3D BMU-activity.

Bone as most of living tissues is able, during its entire lifetime, to adapt its internal microstructure and subsequently its associated mechanical properties to its specific mechanical and physiological environment in a process commonly known as bone remodelling. Bone is therefore continuously renewed and micro-damage, accumulated by fatigue or creep, is removed minimizing the risk of fracture. Nevertheless, bone is not always able to repair itself completely. Actually, if bone repairing function is slower than micro-damage accumulation, a type of bone fracture, usually known as "stress fracture", can finally evolve. In this paper, we propose a bone remodelling continuous model able to simulate micro-damage growth and repair in a coupled way and able therefore to predict the occurrence of "stress fractures". The biological bone remodelling process is modelled in terms of equations that describe the activity of basic multicellular units. The predicted results show a good correspondence with experimental and clinical data. For example, in disuse, bone porosity increases until an equilibrium situation is achieved. In overloading, bone porosity decreases unless the damage rate is so high that causes resorption or "stress fracture".

Mechanobiology of bone remodelling

Bone as most of living tissues is able, during its entire lifetime, to adapt its internal microstructure and subsequently its associated mechanical properties to the specific mechanical and physiological environment in a process commonly known as bone remodelling. Bone is therefore continuously renewed and microdamage removed minimizing the risk of fracture. Bone remodelling is controlled by mechanical and metabolical stimuli. In this paper, we introduce a new model of bone remodelling that takes into account both types of influences. The predicted results show a good correspondence with experimental and clinical data. For example, in disuse, bone porosity increases until an equilibrium situation, while, in overloading, decreases unless the damage rate is so high that causes resorption and "stress fracture". This model has been employed to predict bone adaptation in the proximal femur after total hip replacement proving its consistence and good correspondence with well-known clinical experiences.

Poroelastic indentation analysis for hydrated biological tissues

Indentation techniques are employed for the measurement of mechanical properties of a wide range of materials. In particular, techniques focused at small length-scales, such as nanoindentation and AFM indentation, allow for local characterization of material properties in heterogeneous materials including natural tissues and biomimetic materials. Typical elastic analysis for spherical indentation is applicable in the absence of time-dependent deformation, but is inappropriate for materials with time-dependent responses. Recent analyses for the viscoelastic indentation problem, based on elastic-viscoelastic correspondence, have begun to address the issue of time-dependent deformation during an indentation test. The viscoelastic analysis has been shown to fit experimental indentation data well, and has been demonstrated as useful for characterization of viscoelasticity in polymeric materials and in hydrated mineralized tissues. However, a viscoelastic analysis is not necessarily sufficient for multi-phase materials with fluid flow. In the current work, a poroelastic analysis-based on fluid motion through a porous elastic network-is used to examine spherical indentation creep responses of hydrated biological materials. Both analytical and finite element approaches are considered for the poroelastic Hertzian indentation problem. Modeling results are compared with experimental data from nanoindentation of hydrated bone immersed in water and polar solvents (ethanol, methanol, acetone). Baseline (water-immersed) bone responses are characterized using the poroelastic model and numerical results are compared with altered hydration states due to polar solvents. © 2007 Materials Research Society.

Ultrastructural mechanical and material characterization of fossilized bone

Bone plays a key role in the paleontological and archeological records and can provide insight into the biology, ecology and the environment of ancient vertebrates. Examination of bone at the tissue level reveals a definitive relationship between nanomechanical properties and the local organic content, mineral content, and microstructural organization. However, it is unclear as to how these properties change following fossilization, or diagenesis, where the organic phase is rapidly removed and the remaining mineral phase is reinforced by the deposition of apatites, calcites, and other minerals. While the process of diagenesis is poorly understood, its outcome clearly results in the potential for dramatic alteration of the mechanical response of biological tissues. In this study, fossilized specimens of mammalian long bones, collected from Colorado and Wyoming, were studied for mechanical variations. Nanoindentation performed in both longitudinal and transverse directions revealed preservation of bone's natural anisotropy as transverse modulus values were consistently smaller than longitudinal values. Additionally, modulus values of fossilized bone from 35.0 to 89.1 GPa increased linearly with logarithm of the sample's age. Future studies will aim to clarify what mechanical and material elements of bone are retained during diagenesis as bone becomes part of the geologic milieu. © 2007 Materials Research Society.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Multicenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone

Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated.

Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR-based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB.

Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis.

Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV.

Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR-based techniques.

Participation of adult mouse bone marrow cells in reconstitution of skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells.

Marine Biosilica as a Tool to Understand the Therapeutic Benefit of Silica in Bone Repair

It is widely accepted that silicon-substituted materials enhance bone formation, yet the mechanism by which this occurs is poorly understood. This work investigates the potential of using diatom frustules to answer on fundamental questions surrounding the role of silica in bone healing. Biosilica with frustules 20m were isolated from Cyclotella meneghiniana a unicellular microalgae that was sourced from the Mississippi River, USA. Silanisation chemistry was used to modify the surface of C. meneghiniana with amine (–NH2) and thiol (–SH) terminated silanes. Untreated frustules and both functionalised groups were soaked in culture medium for 24hrs. Following the culture period, frustules were separated from the conditioned medium by centrifugation and both were tested separately in vitro for cytotoxicity using murine-monocyte macrophage (J774) cell line. Cytotoxicity was measured using LDH release to measure damage to cell membrane, MTS to measure cell viability and live-dead staining. The expression and release of pro-inflammatory cytokines (IL-6 and TNF) were measured using ELISA. Our results found that diatom frustules and those functionalised with amino groups showed no cytotoxicity or elevated cytokine release. Diatom frustules functionalised with thiol groups showed higher levels of cytotoxicity. Diatom frustules and those functionalised with amino groups were taken forward to an in vivo mouse toxicity model, whereby the immunological response, organ toxicity and route of metabolism/excretion of silica were investigated. Histological results showed no organ toxicity in any of the groups relative to control. Analysis of blood Si levels suggests that modified frustules are metabolised quicker than functionalised frustules, suggesting that physiochemical attributes influence their biodistribution. Our results show that diatom frustules are non cytotoxic and are promising materials to better understand the role of silica in bone healing.

Chimaerism following allogeneic bone marrow transplantation:detection of residual host cells using the polymerase chain reaction

Chimaerism was assessed in five recipients following sex mismatched allogeneic bone marrow transplantation. Techniques included karyotyping of bone marrow cells, dot blot DNA analysis of blood and bone marrow suspensions, and in vitro amplification of DNA by the polymerase chain reaction (PCR) using blood and bone marrow suspensions and stored bone marrow slides. Results of karyotypic analysis suggested complete chimaerism in four patients, while in one patient mixed chimaerism was detected. Mixed chimaerism was also detected, however, in a second patient using PCR and confirmed by dot blot analysis on all tissues examined. PCR is a sensitive tool for investigation of chimaerism following bone marrow transplantation. Since this technique does not require radioactivity, it is an attractive method for use in a clinical laboratory. This technique represents a further development in the use of DNA methodologies in the assessment of haematological disease.

Alkali-free bioactive glasses for bone regeneration

Bioactive glasses and glass-ceramics are a class of third generation biomaterials which elicit a special response on their surface when in contact with biological fluids, leading to strong bonding to living tissues. The purpose of the present study was to develop diopside based alkali-free bioactive glasses in order to achieve good sintering behaviour, high bioactivity, and a dissolution/ degradation rates compatible with the target applications in bone regeneration and tissue engineering. Another aim was to understand the structure-property relationships in the investigated bioactive glasses. In this quest, various glass compositions within the Diopside (CaMgSi2O6) – Fluorapatite (Ca5(PO4)3F) – Tricalcium phosphate (3CaO•P2O5) system have been investigated. All the glasses were prepared by melt-quenching technique and characterized by a wide array of complementary characterization techniques. The glass-ceramics were produced by sintering of glass powders compacts followed by a suitable heat treatment to promote the nucleation and crystallization phenomena. Furthermore, selected parent glass compositions were doped with several functional ions and an attempt to understand their effects on the glass structure, sintering ability and on the in vitro bio-degradation and biomineralization behaviours of the glasses was made. The effects of the same variables on the devitrification (nucleation and crystallization) behaviour of glasses to form bioactive glass-ceramics were also investigated. Some of the glasses exhibited high bio-mineralization rates, expressed by the formation of a surface hydroxyapatite layer within 1–12 h of immersion in a simulated body fluid (SBF) solution. All the glasses showed relatively lower degradation rates in comparison to that of 45S5 Bioglass®. Some of the glasses showed very good in vitro behaviour and the glasses co-doped with zinc and strontium showed an in vitro dose dependent behaviour. The as-designed bioactive glasses and glass–ceramic materials are excellent candidates for applications in bone regeneration and for the fabrication of scaffolds for tissue engineering.

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008