Effects of extracellular osteoanabolic agents on the endogenous response of osteoblastic cells

The complex multidimensional skeletal organization can adapt its structure in accordance with external contexts, demonstrating excellent self-renewal capacity. Thus, optimal extracellular environmental properties are critical for bone regeneration and inextricably linked to the mechanical and biological states of bone. It is interesting to note that the microstructure of bone depends not only on genetic determinants (which control the bone remodeling loop through autocrine and paracrine signals) but also, more importantly, on the continuous response of cells to external mechanical cues. In particular, bone cells sense mechanical signals such as shear, tensile, loading and vibration, and once activated, they react by regulating bone anabolism. Although several specific surrounding conditions needed for osteoblast cells to specifically augment bone formation have been empirically discovered, most of the underlying biomechanical cellular processes underneath remain largely unknown. Nevertheless, exogenous stimuli of endogenous osteogenesis can be applied to promote the mineral apposition rate, bone formation, bone mass and bone strength, as well as expediting fracture repair and bone regeneration. The following review summarizes the latest studies related to the proliferation and differentiation of osteoblastic cells, enhanced by mechanical forces or supplemental signaling factors (such as trace metals, nutraceuticals, vitamins and exosomes), providing a thorough overview of the exogenous osteogenic agents which can be exploited to modulate and influence the mechanically induced anabolism of bone. Furthermore, this review aims to discuss the emerging role of extracellular stimuli in skeletal metabolism as well as their potential roles and provide new perspectives for the treatment of bone disorders

Modulation of bone remodeling via mechanically activated ion channels

A critical factor in the maintenance of bone mass is the physical forces imposed upon the skeleton. Removal of these forces, such as in a weightless environment, results in a rapid loss of bone, whereas application of exogenous mechanical strain has been shown to increase bone formation. Numerous flight and ground-based experiments indicate that the osteoblast is the key bone cell influenced by mechanical stimulation. Aside from early transient fluctuations in response to unloading, osteoclast number and activity seem unaffected by removal of strain. However, bone formation is drastically reduced in weightlessness and osteoblasts respond to mechanical strain with an increase in the activity of a number of second messenger pathways resulting in increased anabolic activity. Unfortunately, the mechanism by which the osteoblast converts physical stimuli into a biochemical message, a process we have termed biochemical coupling, remains elusive. Prior to the application of this grant, we had characterized a mechanosensitive, cation nonselective channel (SA-cat) in osteoblast-like osteosarcoma cells that we proposed is the initial signalling mechanism for mechanotransduction. During the execution of this grant, we have made considerable progress to further characterize this channel as well as to determine its role in the osteoblastic response to mechanical strain. To achieve these goals, we combined electrophysiologic techniques with cellular and molecular biology methods to examine the role of these channels in the normal function of the osteoblast in vitro

Mathematical modelling of bone remodelling in mechanical, electro-magnetic fields at the cellular level

The skeleton is a metabolically active organ that undergoes continuous remodelling throughout life. At the cellular level, bone remodelling is an organised process whereby osteoclasts remove old bone and osteoblasts replace them with newly formed bone. The osteoclasts and osteoblasts work together in a coupled manner within a so-called 'basic multicellular unit' (BMU). Bone remodelling helps to repair microdamages in bone matrix, preventing the accumulation of old bone. It also plays an important role in maintaining plasma calcium homeostasis. The regulation of bone remodelling is both systemic and local. The most important systemic regulator is parathyroid hormone (PTH), which has been used as a therapy to treat osteoporosis in clinics; however, the underlying mechanism by which PTH is regulated is still not clear. As far as local regulation of bone remodelling is concerned, the discovery of the RANKIRANKL/OPG pathway is significant to the understanding of the interaction between osteoclastic cells and osteoblastic cells in BMU. A large number of therapeutic drugs and other stimuli have been found to apply their effects via RANKIRANKL/OPG. Mechanical stimulus has significant influence on bone remodelling. Disuse or lack of loading causes bone remodelling with bone resorption dominating bone formation and thus a loss of bone mass or density. Conversely, overuse or increased loading causes bone mass or density to increase. Additionally, loadings with different characteristics such as frequency, number of loading cycles in a session and rest time between loading bouts affects bone remodelling differently. However, the underlying mechanisms are not fully understood despite a great deal of experimental work in this field. Pulsed electro-magnetic fields (PEMF) devices have been widely used in clinics to treat bone fracture non-union and shorten the recovery period of fracture. Despite the clinical success, it is still not clear how PEMF stimulus interacts with cells, factors or molecules that are involved in bone remodelling. This thesis will use a computational system biology approach to address the issues proposed above. Computational system biology is a systems biology approach that integrates experimental and computational research in order to understand complex biological systems such as bone remodelling. Based on the latest experimental results and mathematical advances, a mathematical model of bone remodelling at the cellular level is developed with PTH included. Building on this platform model, mechanical stimulus and PEMF are taken into account. Thus, their effects on bone remodelling and the underlying control mechanisms at the cellular level are investigated. The work in the thesis will further current understanding of bone remodelling at the cellular level. The quantitative analysis using our model will help pharmacological and non-pharmacological therapies developments, which eventually benefit patients who suffer from bone loss diseases such as osteoporosis

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

Evaluation of the effects of Orthodontic Tooth Movement on the Levels of Interleukin - 10 in Gingival Crevicular Fluid.

When orthodontic forces are applied to a tooth, they are transmitted to the periodontal ligament (PDL) and adjacent alveolar bone. These forces, in turn, initiate a complex cascade of events that result in the remodeling of the surrounding bone and eventual movement of the tooth2. While the sequence of events at the tissue and cellular levels of bone remodeling is well described, there remains a lack of comprehensive understanding in the coordination of biochemical events at the molecular level. While there are numerous interconnected systems in place that control the levels of mature osteoclasts and osteoblasts active during bone remodeling, one predominate pathway uses the cytokine family interleukin -10 ( IL -10) as a signal transducer . Most importantly, IL - 10 plays a critical role in the differentiation of both osteoclasts and osteoblasts. Osteoclasts are derived from hematopoietic precursors in the bone marrow, whereas osteoblasts are mesenchymal in origin5. Regardless of the cell’s origin, however, IL-10 type cytokines stimulate the differentiation of the respective precursors into osteoblasts and osteoclasts. Thus the aim of the study was to evaluate the level of INTERLEUKIN – 10 in GINGIVAL CEVICULAR FLUID (GCF) during orthodontic tooth movement with the methodology of assessment using ELISA technique

Impact of mechanical stretch on the cell behaviors of bone and surrounding tissues

Mechanical loading is recognized to play an important role in regulating the behaviors of cells in bone and surrounding tissues in vivo. Many in vitro studies have been conducted to determine the effects of mechanical loading on individual cell types of the tissues. In this review, we focus specifically on the use of the Flexercell system as a tool for studying cellular responses to mechanical stretch. We assess the literature describing the impact of mechanical stretch on different cell types from bone, muscle, tendon, ligament, and cartilage, describing individual cell phenotype responses. In addition, we review evidence regarding the mechanotransduction pathways that are activated to potentiate these phenotype responses in different cell populations

Effect of inflammation on bone : biological, structural and mechanical behavior

Tese de doutoramento, Ciências Biomédicas (Ciências Biopatalógicas), Universidade de Lisboa, Faculdade de Medicina, 2011The term osteoimmunology was used for the first time in 2000 to describe the interaction of cells from the immune system and bone These two systems have several regulatory factors in common, such as cytokines, transcription factors and receptors. Consequently, they interact with each other both in physiological and pathological conditions. The aim of this dissertation thesis was to understand the effect of immune mediated inflammation on bone structural, mechanical and biological behaviour, using rheumatoid arthritis (RA) and fracture healing as a model. The first study of this thesis focused on the analysis of the effect of arthritis on bone biomechanical behavior. An animal model of arthritis, the SKG mice, was used and bone behavior was evaluated by mechanical three-point bending tests. Scanning electron microscopy (SEM) and multiphoton microscopy (MPM) were applied to study, respectively, bone structure and the collagen network organization in trabecular bone. Results from this study have shown that arthritic bones had poor biomechanical quality compared to control bones. MPM and SEM observations disclosed signs of impaired collagen organization and poor trabecular architecture. In this work we verified that chronic inflammation per se leads to impairment of bone biomechanics in terms of stiffness, ductility and ultimate strength. In the same model, we then proceed to study the effect of inflammation on collagen metabolism and organization. The evaluation of bone included mechanical tests, SEM, MPM and serum bone turnover markers, specifically procollagen type - amino-terminal peptide (P1NP) and carboxy-terminal crosslinked telopeptide of type I 6 collagen (CTX). Femoral bones of SKG mice revealed increased fragility expressed by deterioration of mechanical properties. In accordance, as observed by SEM, intertrabecular distance was increased and trabecular thickness decreased. MPM depicted a disorganized matrix and loose collagen structure compared to controls. Moreover, the collagen metabolism assessed in the serum was also highly increased in arthritic mice. In this work we found that the bone weakening effect of arthritis was due to high bone turnover and disorganized collagen type I matrix. Following these initial animal model data, the first to describe a direct effect of arthritis on collagen structure and bone biomechanics, we confirmed and further detailed these observations in human RA bone. Patients with RA submitted to hip replacement surgery were recruited. Trabecular bone microarchitecture was assessed by microcomputed tomography (microCT) and mechanical behavior by compression tests of a bone cylinder extracted from the femoral epiphysis. Bone cell activity was analyzed by studying gene expression in the bone microenvironment. Genes that code for proinflammatory cytokines were upregulated in RA patients, particularly IL-17, which plays an important role in stimulating osteoclastogenesis. RA bone microenvironment had a gene expression profile characterized by upregulated pro-osteoclastogenic cytokines and dickkopf homolog 1 (DKK1) and increased RANKL/OPG ratio. This was paralleled by raised expression of factors that promote osteoblastic activity, such as IGF-I, FGF2 and PDGF, but with low type I collagen expression. DKK1 is a negative regulator of the WNT/β- catenin signaling, which is a key pathway in the stimulation of osteoblast differentiation, meaning that its upregulation in RA patients is limiting the effects of pro-osteoblastic factors. Bone loss in a chronic inflammatory disease, such as RA, thus appears to result from enhanced bone resorption and impaired bone formation, which 7 constitutes a detrimental imbalance of bone remodeling and precipitates the rapid loss of bone mass. Indeed, differences were observed in gene expression between RA and primary osteoporosis (OP) bone in spite of the fact that these two patient groups had similar bone microarchitecture and biomechanical properties. These observations might indicate that the differences in gene expression reflect biologically specific mechanisms responsible for bone fragility in RA and that the inhibition of DKK1 can be a possible treatment strategy for tackling the effects of RA on bone. In the final work of this thesis, we have used the post-fracture inflammatory reaction as a model for characterizing the kinetics of inflammatory and bone remodeling related genes. Unlike the chronic inflammation seen in RA, fracture healing is a highly regulated and brief process. Patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. Patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture; between the 4th and 7th day; and one week after fracture. Inflammation and bone metabolism related genes were assessed at the fracture site. Our results indicate that the expression of inflammation related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. Sclerostin expression, an inhibitor of osteoblast differentiation, has an initial high expression level that is diminished after the reduction of inflammatory gene expression. We propose the existence of a two step process in bone healing, dependent on an initial inflammatory stimulus and a latter decrease in sclerostin-related effects, with a consequent proosteoblastic effect. Therefore, local promotion of these events might constitute a promising medical intervention to accelerate fracture healing. 8 The work herein discussed clearly shows that inflammation has a complex role in bone regulation. The identification of key regulators of this system will be crucial both for RA and fracture healing future management.O termo osteoimunologia foi usado pela primeira vez em 2000 para descrever a interacção entre células do sistema imunitário e do osso. Estes dois sistemas têm vários factores em comum, tais como citocinas, factores de transcrição e receptores. Consequentemente, a sua interacção ocorre, não só em condições fisiológicas, mas também patológicas. O objectivo desta dissertação de doutoramento é o de compreender o efeito da inflamação mediada pelo sistema imunitário no comportamento estrutural, mecânico e biológico do osso usando a artrite reumatóide e a regeneração de fracturas como modelos. O primeiro estudo desta tese foca a análise do efeito da artrite no comportamento biomecânico do osso. Foi utilizado um modelo animal de artrite, o ratinho SKG, para avaliar o comportamento biomecânico do osso por testes de flexão de 3 pontos. A microscopia electrónica de varrimento e a microscopia multifotão foram utilizadas, respectivamente, para o estudo da estrutura óssea e da organização da rede de colagénio no osso trabecular. Os resultados deste estudo mostraram que o osso de ratinhos com artrite apresenta pior qualidade biomecânica comparando com os controlos. Observações por microscopia multifotão e microscopia electrónica de varrimento demonstraram alteração da organização do colagénio e da estrutura trabecular. Neste trabalho verificámos que a inflamação crónica per se conduz à degradação das propriedades biomecânicas, particularmente da rigidez, ductilidade e resistência. No mesmo modelo animal, prosseguimos com o estudo do efeito da inflamação no metabolismo e organização do colagénio. A avaliação do osso incluiu ensaios mecânicos, microscopia electrónica de varrimento, microscopia multifotão e doseamento de marcadores 2 séricos de remodelação óssea, especificamente o péptido aminoterminal do procolagénio tipo I (P1NP) e o carboxi-terminal do telopéptido do colagénio tipo I (CTX). O osso dos ratinhos SKG revelou aumento da fragilidade óssea expresso pela deterioração das propriedades mecânicas. Em concordância, por microscopia electrónica de varrimento foi observado que a distância intertrabecular aumentou e a espessura trabecular reduziu e a microscopia multifotão demonstrou uma matriz desorganizada, com baixa densidade de colagénio. O metabolismo do colagénio foi ainda estudado no soro, encontrando-se aumentado nos ratinhos com artrite. Neste trabalho determinámos que o efeito de fragilidade óssea induzido pela artrite se deve à elevada remodelação óssea e desorganização da matriz de colagénio tipo I. Seguindo os dados iniciais obtidos em modelo animal, os primeiros a descrever o efeito directo da artrite na estrutura do colagénio e na biomecânica do osso, confirmámos e detalhámos estas observações em osso humano com artrite reumatóide. Foram recrutados doentes com AR submetidos a artroplastia da anca. A microarquitectura do osso trabecular foi avaliada por microtomografia computadorizada e o comportamento biomecânico por teste de compressão de um cilindro de osso trabecular extraído da epífise femoral. A actividade das células ósseas foi analisada através do estudo da expressão génica no microambiente ósseo. Observou-se um aumento da expressão dos genes que codificam citocinas pró-inflamatórias, particularmente a IL- 17, a qual desempenha um importante papel na estimulação da osteoclastogénese. O microambiente do osso com AR apresenta um perfil de expressão génico caracterizado pelo aumento das citocinas pró-osteoclastogénicas e de DKK1 e ainda do rácio de expressão RANKL/OPG. Paralelamente, verificou-se um aumento da expressão de factores indutores da actividade osteoblástica, tais como o IGF-I, FGF2 e PDGF, apesar da baixa expressão de colagénio tipo I. O DKK1 3 é um regulador negativo da via de sinalização WNT/β-catenina, sendo esta uma via chave na estimulação da diferenciação do osteoblasto. Assim, o aumento deste inibidor nos doentes com artrite reumatóide limita o efeito pró-osteoblastogénico do ambiente molecular do osso artrítico. A perda óssea associada às doenças crónicas inflamatórias, tal como a artrite reumatóide, parece, desta forma, resultar do aumento da reabsorção óssea e de uma diminuição na formação, conduzindo ao desequilíbrio da remodelação e precipitando a rápida perda de massa óssea. De facto, foram observadas diferenças na expressão génica entre o osso de doentes com artrite reumatóide e com osteoporose primária, apesar destes dois grupos de doentes terem microestrutura óssea e propriedades biomecânicas semelhantes. Estas observações parecem indicar que as diferenças encontradas a nível da expressão génica reflectem mecanismos biológicos específicos responsáveis pela fragilidade óssea na artrite reumatóide e a inibição do DKK1 poderá vir a ser uma possível estratégia terapêutica para diminuir os efeitos da artrite reumatóide sobre o osso. No último trabalho desta tese, utilizámos a reacção inflamatória pósfractura como modelo para caracterizar a cinética de expressão dos genes relacionados com a inflamação e remodelação óssea. Ao contrário da inflamação crónica, característica da artrite reumatóide, a regeneração de fracturas é um processo breve e cuidadosamente regulado. Foram incluídos neste estudo doentes submetidos a artroplastia da anca devido a fracturas de baixo impacto. Os doentes foram agrupados de acordo com o tempo decorrido entre a fractura e a cirurgia: osso colhido nos 3 dias após fractura, entre o 4º e o 7º dias, e uma semana após fractura. Um conjunto de genes relacionados com a inflamação e o metabolismo ósseo foram estudados no local de fractura. Os resultados obtidos indicam que a expressão dos genes relacionados com a inflamação, especialmente a 4 IL-6, estão aumentados nos primeiros dias após fractura; contudo, a partir do dia 4 verifica-se um desvio para os genes de remodelação óssea, sugerindo que a fase inflamatória activa a regeneração da fractura. A expressão da esclerostina, um inibidor da diferenciação do osteoblasto, está aumentada durante os primeiros dias após fractura e diminui após a redução da expressão dos genes pró-inflamatórios. Desta forma, propomos a existência de um processo em duas fases que conduz à regeneração de fracturas, dependente de um estímulo inicial inflamatório e uma diminuição subsequente dos efeitos relacionados com a esclerostina, com consequente estímulo osteoblástico. Assim, a indução local destes eventos poderá constituir uma intervenção promissora para acelerar a regeneração óssea após fractura. O trabalho aqui discutido claramente demonstra o complexo papel da inflamação na regulação óssea. A identificação de factores reguladores chave deste sistema será crucial para o futuro tratamento da artrite reumatóide e das fracturas

The effect of dietary fatty acids on osteocyte-mediated mechanotransduction : a thesis presented in partial fulfilment of the requirements for the degree of Master of Health Science at Massey University, Manawatū, Palmerston North, New Zealand

Figures are re-used with permission.The bones in our skeleton are subjected to mechanical loading every day and are continuously remodelled in a process called “bone remodelling”. The capacity of the skeleton to adapt its mass and structure in response to mechanical loading has been intensively studied. Over the last few decades, much focus has been given to bone cells such as osteoblasts and osteoclasts but less so to osteocytes, even though these comprise almost 90% of the cellular space in bone. The detailed way these cells function in bone mechanotransduction and therefore control bone remodelling is not well understood. Diet and especially dietary fatty acids (DFA) are an important aspect of the regulation of bone health. Strong evidence has been presented in the scientific literature to support the benefits of DFA in bone health, but the exact mechanism of how they benefit bone health is still unclear. It has been shown that bone cells secrete various osteogenic molecules in response to fluid shear stress. The goal of this project was to explore osteocyte cellular mechanisms involved in their response to mechanical loading and dietary fatty acids. This project specifically focused on the osteocytic secretion of ATP in response to dietary fatty acids and fluid shear stress in vitro. This study showed for the first time that the increased ATP secretion induced by fluid shear stress due to DFA treatment possibly explore a new understanding of how DFA might benefit bone health and could be used in future experiments to help us understand their possible effects. This study might be set as a model experimental design to study the cell response to DFA treatment exposed to fluid shear stress

Nanoscale vibration to modulate osteoclastogenesis

Mechanical factors have been shown to significantly influence stem cell differentiation and fate. Researchers have demonstrated that nanoscale vibration can promote osteogenesis in isolated mesenchymal stem cell (MSC) cultures. In the bone marrow niche, there is a co-dependent existence between MSCs and cells from the haematopoietic lineage (HSCs), particularly osteoclasts. While MSC derived osteoblasts stimulate new bone formation, osteoclasts resorb bone. Given the close overlap between these two cells types, an investigation in to the effect of nanoscale vibration on osteoclasts was required. Two culture methods were used: an isolated culture of osteoclasts and osteoclast-precursors, and a co-culture of bone marrow stromal cells and bone marrow haematopoietic cells. Vibration was produced with the Nanokick bioreactor – a recently developed technology that facilitates the delivery of accurate and reproducible nanoscale vertical displacements. This bioreactor allows otherwise standard cell culture techniques to be used. A range of experiments was used to investigate the effect of nanoscale vibration, including immunostaining, resorption analysis, RT-qPCR, ELISA and metabolomics. Nanoscale vibration was found to influence osteoclast differentiation and function. A reduction in osteoclast numbers was observed in both culture conditions. Furthermore, less resorption occurred in the nanokick group. There was no significant impairment in osteoblast development or function when osteoclasts were present, with evidence of increased cytoskeleton tension and mineralisation following stimulation. A number of changes in gene, protein and regulations were observed, suggesting a state of lower inflammation in the nanokick group. It is hoped that these results will provide further evidence to validate the use of the Nanokick bioreactor as a method of producing tissue-engineered bone graft for clinical applications