Skeletal biology: Where matrix meets mineral

The skeleton is unique from all other tissues in the body because of its ability to mineralize. The incorporation of mineral into bones and teeth is essential to give them strength and structure for body support and function. For years, researchers have wondered how mineralized tissues form and repair. A major focus in this context has been on the role of the extracellular matrix, which harbors key regulators of the mineralization process. In this introductory minireview, we will review some key concepts of matrix biology as it related to mineralized tissues. Concurrently, we will highlight the subject of this special issue covering many aspects of mineralized tissues, including bones and teeth and their associated structures cartilage and tendon. Areas of emphasis are on the generation and analysis of new animal models with permutations of matrix components as well as the development of new approaches for tissue engineering for repair of damaged hard tissue. In assembling key topics on mineralized tissues written by leaders in our field, we hope the reader will get a broad view of the topic and all of its fascinating complexities.     

Tissue engineering a blood vessel: Regulation of vascular biology by mechanical stresses

Important to the tissue engineeping of a substitute blood vessel is an understanding of those faators which regulat vascular biology. A major factor in the mechanical environment imposed by the hemodynamics of the vascular system. In this the vascular endothelium play a critical role, and mver the past two deaades much has been learned about the influence of hemodynamics on vascular endothelial biology, to a large degree using cell culture to study the effects of flow and cyclic stretch. In our laboratory, such studies ape low being extended through the development of a model of the arterial wall involving the co-culture of endothelial cells and smomth muscle cells. The development of such a model and its use in the study of endothelial cells and smmooth muscle the evolution of approaaheq to tissue engileeping a blood vessel.     

Mechanical loading history and skeletal biology

A comprehensive theory which relates tissue mechanical stresses to many features of skeletal morphogenesis, growth, regeneration, maintenance and degeneration is reviewed. The theory considers the repeated or intermittent mechanical forces which constitute the loading history on the chondro-osseous skeleton. The results of numerous mechanical stress analyses indicate that the local tissue stress history plays a major role in controlling connective tissue biology. The strong influence of mechanical energy in ontogenesis implies a comparably strong influence in phylogenesis. The fact that the mechanical stress histories in skeletal tissues are directly related to the force of gravity suggests that the life forms that have evolved on Earth are closely tied to our gravitational field.     

Engineering cell alignment in vitro

Cell alignment plays a critical role in various cell behaviors including cytoskeleton reorganization, membrane protein relocation, nucleus gene expression, and ECM remodeling. Cell alignment is also known to exert significant effects on tissue regeneration (e.g., neuron) and modulate mechanical properties of tissues including skeleton, cardiac muscle and tendon. Therefore, it is essential to engineer cell alignment in vitro for biomechanics, cell biology, tissue engineering and regenerative medicine applications. With advances in nano- and micro-scale technologies, a variety of approaches have been developed to engineer cell alignment in vitro, including mechanical loading, topographical patterning, and surface chemical treatment. In this review, we first present alignments of various cell types and their functionality in different tissues in vivo including muscle and nerve tissues. Then, we provide an overview of recent approaches for engineering cell alignment in vitro. Finally, concluding remarks and perspectives are addressed for future improvement of engineering cell alignment.     

Functional tissue engineering of tendon: Establishing biological success criteria for improving tendon repair

Improving tendon repair using Functional Tissue Engineering (FTE) principles has been the focus of our laboratory over the last decade. Although our primary goals were initially focused only on mechanical outcomes, we are now carefully assessing the biological properties of our tissue-engineered tendon repairs so as to link biological influences with mechanics. However, given the complexities of tendon development and healing, it remains challenging to determine which aspects of tendon biology are the most important to focus on in the context of tissue engineering. To address this problem, we have formalized a strategy to identify, prioritize, and evaluate potential biological success criteria for tendon repair. We have defined numerous biological properties of normal tendon relative to cellular phenotype, extracellular matrix and tissue ultra-structure that we would like to reproduce in our tissue-engineered repairs and prioritized these biological criteria by examining their relative importance during both normal development and natural tendon healing. Here, we propose three specific biological criteria which we believe are essential for normal tendon function: (1) scleraxis-expressing cells; (2) well-organized and axially-aligned collagen fibrils having bimodal diameter distribution; and (3) a specialized tendon-to-bone insertion site. Moving forward, these biological success criteria will be used in conjunction with our already established mechanical success criteria to evaluate the effectiveness of our tissue-engineered tendon repairs.     

Stem cell plasticity, cell fusion, and transdifferentiation

One of the most contentious issues in biology today concerns the existence of stem cell plasticity. The term "plasticity" refers to the capacity of tissue-derived stem cells to exhibit a phenotypic potential that extends beyond the differentiated cell phenotypes of their resident tissue. Although evidence of stem cell plasticity has been reported by multiple laboratories, other scientists have not found the data persuasive and have remained skeptical about these new findings. This review will provide an overview of the stem cell plasticity controversy. We will examine many of the major objections that have been made to challenge the stem cell plasticity data. This controversy will be placed in the context of the traditional view of stem cell potential and cell phenotypic diversification. What the implications of cell plasticity are, and how its existence may modulate our present understanding of stem cell biology, will be explored. In addition, we will examine a topic that is usually not included within a discussion of stem cell biology--the direct conversion of one differentiated cell type into another. We believe that these observations on the transdifferentiation of differentiated cells have direct bearing on the issue of stem cell plasticity, and may provide insights into how cell phenotypic diversification is realized in the adult and into the origin of cell phenotypes during evolution.     

Craniofacial sutures: Signaling centres integrating mechanosensation,cell signaling,and cell differentiation

Cranial sutures are dynamic structures in which stem cell biology, bone formation, and mechanical forces interface, influencing the shape of the skull throughout development and beyond. Over the past decade, there has been significant progress in understanding mesenchymal stromal cell (MSC) differentiation in the context of suture development and genetic control of suture pathologies, such as craniosynostosis. More recently, the mechanosensory function of sutures and the influence of mechanical signals on craniofacial development have come to the forefront. There is currently a gap in understanding of how mechanical signals integrate with MSC differentiation and ossification to ensure appropriate bone development and mediate postnatal growth surrounding sutures. In this review, we discuss the role of mechanosensation in the context of cranial sutures, and how mechanical stimuli are converted to biochemical signals influencing bone growth, suture patency, and fusion through mediation of cell differentiation. We integrate key knowledge from other paradigms where mechanosensation forms a critical component, such as bone remodeling and orthodontic tooth movement. The current state of the field regarding genetic, cellular, and physiological mechanisms of mechanotransduction will be contextualized within suture biology.     

RNAi and microRNAs: from animal models to disease therapy

The discovery of the phenomenon of RNA interference (RNAi) and its existence in mammals quickly suggested a great potential for use in disease therapy. Rapid advances have been made in the development of RNAi-based technologies and promising results have been obtained from studies on mammalian cell culture systems and animal in vivo models. However, the progress in our understanding of the RNAi pathway and the related function of microRNAs (miRNAs) have also raised concerns regarding various types of side effects that may restrict the use of this technology in human therapy. At the same time, our new knowledge about the functional roles of miRNAs as regulators of many cellular processes, including proliferation, differentiation, development, and neuronal function, is revolutionizing cell biology and will have a major impact on medical research. In this review, we focus on the discoveries that have been made in animal models and how this insight can be translated to human medicine and disease therapy. In this connection, we will particularly discuss the challenges associated with the efforts to develop RNAi-based therapeutics.     

Current perspectives on NMDA-type glutamate signalling in bone

Bone is a complex, evolving tissue, architecturally defined by the activities of osteoclasts and osteoblasts that continually resorb and replace the mineralised matrix. Numerous regulatory mechanisms exist to control bone remodelling and the maintenance of bone mass. The consequences of inappropriate or uncoupled bone resorption and formation are significant and invariably lead to different disease states, the most prevalent being osteoporosis. In recent years, much attention has focused on unravelling the systemic and local signalling interactions that influence the differentiation and function of bone cells with a view to developing our understanding of bone biology and identifying potential new targets for therapeutic intervention. Several lines of evidence indicate that neurotransmitters and neuromodulators have influential roles to play in the regulation of bone remodelling and much of this research has involved analysis of the excitatory amino acid glutamate. This review will summarise current understanding of glutamate signalling in bone cells, addressing specifically the function of N-methyl-D-aspartate (NMDA)-type glutamate receptor signalling mechanisms, and will address the functional significance and future prospects for this area of research.     

Tissue Stiffness Dictates Development,Homeostasis, and Disease Progression

Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.     

The role of glutamate in the regulation of bone mass and architecture

Communication between the cells in bone underlies the way that the tissue functions physiologically, and in nearly all pathologies, the pathogenesis of skeletal diseases. The number of molecules involved in intercellular signalling in bone grows constantly and it is perhaps unsurprising that the list includes many with functions in other tissues. In recent years, evidence has accumulated to show that molecules involved in neurotransmission have paracrine roles in the skeleton. The focus of this review is the excitatory amino acid glutamate and its role in regulating bone formation and resorption. Specifically, this article will concentrate on the functional role of the system, and the reasons why mechanisms like synaptic transmission are relevant to what might appear to be a slow responding tissue, as the sites of expression of glutamate signalling components in bone have been reviewed already. While there is strong evidence for a regulatory role for glutamate in osteoblast and osteoclast differentiation and function in vitro, in vivo data is less advanced. Preliminary data from in vivo systems does however suggest that glutamate has a physiological function in the skeleton.     

Evolutionary cell biology: functional insight from “endless forms most beautiful”

In animal and fungal model organisms, the complexities of cell biology have been analyzed in exquisite detail and much is known about how these organisms function at the cellular level. However, the model organisms cell biologists generally use include only a tiny fraction of the true diversity of eukaryotic cellular forms. The divergent cellular processes observed in these more distant lineages are still largely unknown in the general scientific community. Despite the relative obscurity of these organisms, comparative studies of them across eukaryotic diversity have had profound implications for our understanding of fundamental cell biology in all species and have revealed the evolution and origins of previously observed cellular processes. In this Perspective, we will discuss the complexity of cell biology found across the eukaryotic tree, and three specific examples of where studies of divergent cell biology have altered our understanding of key functional aspects of mitochondria, plastids, and membrane trafficking.The field of cell biology has made tremendous strides in understanding eukaryotic cells, especially animals and yeast. Concurrently, evolutionary biology has opened up a window to the origins of our species and the genes that define us. Though these fields have intersected conceptually for decades, a recent movement is explicitly uniting these two fields into the discipline of evolutionary cell biology with great success (Brodsky et al., 2012 ; Lynch et al., 2014 ) and, we argue here, potentially an even greater future. One drive behind this movement is to harness the comparative approach of evolutionary biology and apply it to questions of cellular origins and cellular function. This approach has yielded beautiful insight into animal cellular function from mitotic spindle dynamics (Helmke and Heald, 2014 ) to glycosylation machinery (Varki, 2006 ). However, expanding the scope of investigation to organisms beyond fungi and animals to span eukaryotic diversity has allowed for discoveries that force us to adjust some fundamental ideas of how eukaryotic organelles work, and why.     

The osteocyte lineage

The osteocyte resides in the lacuna/canalicular system in bone and has been hypothesized to orchestrate local bone remodeling. Certainly the identification of the osteocyte as the source of Sclerostin, a molecule that regulates osteoblast function, has supported this possibility. As our understanding of this cell increases it has become clear that it has more far reaching influence than simply local bone turnover activity. The osteocyte is also the source of DMP-1 and FGF-23, the later being a hormone that regulates kidney function in terms of phosphate uptake. We now see the osteocyte as having important roles both locally in the skeleton and also in other distant tissues. The study of osteocyte biology has reached a particularly exiting level of maturity and illustrates the value of this cell type as a drug discovery target.     

Moss systems biology en route: phytohormones in Physcomitrella development

The moss Physcomitrella patens has become a powerful model system in modern plant biology. Highly standardized cell culture techniques, as well as the necessary tools for computational biology, functional genomics and proteomics have been established. Large EST collections are available and the complete moss genome will be released soon. A simple body plan and the small number of different cell types in Physcomitrella facilitate the study of developmental processes. In the filamentous juvenile moss tissue, developmental decisions rely on the differentiation of single cells. Developmental steps are controlled by distinct phytohormones and integration of environmental signals. Especially the phytohormones auxin, cytokinin, and abscisic acid have distinct effects on early moss development. In this article, we review current knowledge about phytohormone influences on early moss development in an attempt to fully unravel the complex regulatory signal transduction networks underlying the developmental decisions of single plant cells in a holistic systems biology approach.     

Adipocyte biology: It is time to upgrade to a new model

Globally, the obesity pandemic is profoundly affecting quality of life and economic productivity, but efforts to address this, especially on a pharmacological level, have generally proven unsuccessful to date, serving as a stark demonstration that our understanding of adipocyte biology and pathophysiology is incomplete. To deliver better insight into adipocyte function and obesity, we need improved adipocyte models with a high degree of fidelity in representing the in vivo state and with a diverse range of experimental applications. Adipocyte cell lines, especially 3T3-L1 cells, have been used extensively over many years, but these are limited in terms of relevance and versatility. In this review, I propose that primary adipose-derived stromal/stem cells (ASCs) present a superior model with which to study adipocyte biology ex vivo. In particular, ASCs afford us the opportunity to study adipocytes from different, functionally distinct, adipose depots and to investigate, by means of in vivo/ex vivo studies, the effects of many different physiological and pathophysiological factors, such as age, body weight, hormonal status, diet and nutraceuticals, as well as disease and pharmacological treatments, on the biology of adipocytes and their precursors. This study will give an overview of the characteristics of ASCs and published studies utilizing ASCs, to highlight the areas where our knowledge is lacking. More comprehensive studies in primary ASCs will contribute to an improved understanding of adipose tissue, in healthy and dysfunctional states, which will enhance our efforts to more successfully manage and treat obesity.     

The mechanics of myeloid cells

The effects of cell size, shape and deformability on cellular function have long been a topic of interest. Recently, mechanical phenotyping technologies capable of analysing large numbers of cells in real time have become available. This has important implications for biology and medicine, especially haemato-oncology and immunology, as immune cell mechanical phenotyping, immunologic function, and malignant cell transformation are closely linked and potentially exploitable to develop new diagnostics and therapeutics. In this review, we introduce the technologies used to analyse cellular mechanical properties and review emerging findings following the advent of high throughput deformability cytometry. We largely focus on cells from the myeloid lineage, which are derived from the bone marrow and include macrophages, granulocytes and erythrocytes. We highlight advances in mechanical phenotyping of cells in suspension that are revealing novel signatures of human blood diseases and providing new insights into pathogenesis of these diseases. The contributions of mechanical phenotyping of cells in suspension to our understanding of drug mechanisms, identification of novel therapeutics and monitoring of treatment efficacy particularly in instances of haematologic diseases are reviewed, and we suggest emerging topics of study to explore as high throughput deformability cytometers become prevalent in laboratories across the globe.     

Mesenchymal stem cells in arthritic diseases

Mesenchymal stem cells (MSCs), the nonhematopoietic progenitor cells found in various adult tissues, are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive culture expansion to obtain large quantities suitable for therapeutic use. These properties make MSCs an ideal candidate cell type as building blocks for tissue engineering efforts to regenerate replacement tissues and repair damaged structures as encountered in various arthritic conditions. Osteoarthritis (OA) is the most common arthritic condition and, like rheumatoid arthritis (RA), presents an inflammatory environment with immunological involvement and this has been an enduring obstacle that can potentially limit the use of cartilage tissue engineering. Recent advances in our understanding of the functions of MSCs have shown that MSCs also possess potent immunosuppression and anti-inflammation effects. In addition, through secretion of various soluble factors, MSCs can influence the local tissue environment and exert protective effects with an end result of effectively stimulating regeneration in situ. This function of MSCs can be exploited for their therapeutic application in degenerative joint diseases such as RA and OA. This review surveys the advances made in the past decade which have led to our current understanding of stem cell biology as relevant to diseases of the joint. The potential involvement of MSCs in the pathophysiology of degenerative joint diseases will also be discussed. Specifically, we will explore the potential of MSC-based cell therapy of OA and RA by means of functional replacement of damaged cartilage via tissue engineering as well as their anti-inflammatory and immunosuppressive activities.     

Physiologically based microenvironment for in vitro neural differentiation of adipose-derived stem cells

The limited capacity of nervous system to promote a spontaneous regeneration and the high rate of neurodegenerative diseases appearance are keys factors that stimulate researches both for defining the molecular mechanisms of pathophysiology and for evaluating putative strategies to induce neural tissue regeneration. In this latter aspect, the application of stem cells seems to be a promising approach, even if the control of their differentiation and the maintaining of a safe state of proliferation should be troubled. Here, we focus on adipose tissue-derived stem cells and we seek out the recent advances on the promotion of their neural differentiation, performing a critical integration of the basic biology and physiology of adipose tissuederived stem cells with the functional modifications that the biophysical, biomechanical and biochemical microenvironment induces to cell phenotype. The pre-clinical studies showed that the neural differentiation by cell stimulation with growth factors benefits from the integration with biomaterials and biophysical interaction like microgravity. All these elements have been reported as furnisher of microenvironments with desirable biological, physical and mechanical properties. A critical review of current knowledge is here proposed, underscoring that a real advance toward a stable, safe and controllable adipose stem cells clinical application will derive from a synergic multidisciplinary approach that involves material engineer, basic cell biology, cell and tissue physiology.     

One mechanostat or many? Modifications of the site-specific response of bone to mechanical loading by nature and nurture

The concept of the mechanostat was not new in 1983, when Harold Frost coined the term to describe a mechanism by which bone responded to habitual exercise and changes in loading with structurally appropriate alterations in bone architecture. However, the word "mechanostat" has a meaning that is immediately apparent, and its adoption has led to a much wider appreciation of the process of functional adaptation by other scientists than those whose primary research focus is in the biology of adaptation. One problem exists though: it is widely thought that in a single individual, there is a setting for the mechanostat, just as a single thermostat might set the temperature for a whole house, and this is reflected in the idea that bones throughout the skeleton require a specific strain magnitude for maintenance. Increases in loading above that threshold are expected to induce bone formation and a stiffer structure that then experiences again the habitual strain magnitude. Reductions in strain magnitude supposedly induce resorption to reduce tissue mass and architectural properties so that the lower loading restores habitual strain magnitude. That widely held belief of a single unifying number of strain is fundamentally flawed. The purpose of this article is to explain the real basis of the mechanostat; that the skeleton responds to a complex strain stimulus, made up of numerous different parameters, of which peak magnitude is only one, and that the strain stimulus is different in different parts of the skeleton, so there is no universal number to describe a tissue strain magnitude that underlies the mechanostat's setting. Furthermore, males and females have different responses to loading, and those responses change in response to many factors including genetic constitution, age, concomitant disease, nutrient availability, and exposure to drugs or biochemicals. In summary then, there is not a single mechanostat controlling the skeleton of each of us. At a fundamental tissue level, small functional units of bone each have their own multifactorial threshold target strain stimuli for a given set of dynamic modifying influences. Understanding the biology behind the way that each of these mechanostats functions independently is likely to have pervasive consequences on our ability to control bone mass by manipulation of loading, either directly through different exercise regimens, or in a targeted manner using tailored site and individual specific pharmaceuticals.