Cell-cell interactions in regulating osteogenesis and osteoblast function

Endochondral bone formation requires an elaborate interplay among autocrine, paracrine, and endocrine signals, positional cues, and cell-cell contacts to mediate the complex three-dimensional architecture and function of the skeleton. Embryonic bone development occurs by migration, aggregation, and condensation of immature mesenchymal progenitor cells to form the cartilaginous anlage. Upon vascular invasion, the cartilaginous scaffold is colonized and subsequently mineralized by osteoblasts. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. This review examines the role of cell-cell interactions mediated by adherens junctions formed by cadherins and communicative gap junctions formed by connexins in regulating bone development and osteogenic function.     

Mechanical Loading Stimulates Expression of Connexin 43 in Alveolar Bone Cells in the Tooth Movement Model

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.     

Describing force-induced bone growth and adaptation by a mathematical model

This work proposes a mathematical model that qualitative describes the process of mechanically force-induced bone growth and adaptation. The mathematical model includes osteocytes as the key interfacing layer connecting tissue, cellular and molecular signaling levels. Specifically, in the presence of an increase in the mechanical stimuli, osteocytes respond by mechano-transduction releasing the local factors nitric oxide (NO) and prostaglandin E(2) (PGE(2)). These local factors act as the signaling recruitment signals for bone cells progenitors and influence the coupling activity among osteoblasts and osteoclasts during the process of bone remodeling. The model is in agreement with qualitative observations found in the literature concerning the process of bone adaptation and the cellular interactions during a local bone remodeling cycle induced by mechanical stimulation.     

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.     

Mechanical Stimulation of Gap Junctions in Bone Osteocytes is Mediated by Prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factors) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin E₂ (PGE₂) release and increase cyclooxygenase 2 (COX-2) expression. When PGE₂ was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE₂. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.     

Mechanical loading stimulates expression of connexin 43 in alveolar bone cells in the tooth movement model

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.     

Role of Gap junctions in bone tissue

Gap junctions are transmembrane channels, that connect the membranes of adjacent cells and are involved in the direct signal transmission between the cells. The intercellular communication involving this type of channels provides the proper functioning of tissues and organs. Gap junctions formation and synthesis of connexins is regulated by hormones, growth factors and signaling molecules. Gap junctions, located between osteoblasts, osteoclasts, and osteocytes, play a key role in the process of bone turnover, and therefore in the modeling and bone tissue regeneration. They also mediate the regulation of proliferation, differentiation and maturation of osteoblasts, as well as formation and activity of osteoclasts. This paper presents the current state of knowledge on the role of intercellular connections via Gap junctions in bone cells, with particular emphasis on the involvement of connexin 43, in the regulation of osteogenesis.     

Gap junctions and hemichannels in signal transmission, function and development of bone

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

Theoretical and numerical study of a bone remodeling model: The effect of osteocyte cells distribution

It is well argued that osteocytes are mechanosensory cells and are involved in the regulation of bone remodeling. In previous works, the predictions from a simulation model have suggested that both the influencing distance of osteocytes and the magnitude of the mechanical loads determine the thickness of trabeculae whereas the number of osteocytes primarily affects the rate of bone remodeling. The question that remains not completely answered is: for the same number of osteocytes, what is the effect of different distributions on the remodeling process? Based on a particular regulatory bone remodeling model, the question is addressed, in part, by performing a stability analysis in connection with numerical simulations. The results allow us to demonstrate that, on one hand, we cannot reach a conclusion about the stability of the model for a nonuniform osteocyte distribution. This implies that there is no relationship between the different parameters conveying the stability of the model. On the other hand, we show that the osteocyte cell distribution has a significant influence on the bone morphology, which seems to be confirmed by simulations with real data obtained from rat tibia.     

The turnover of mineralized growth plate cartilage into bone may be regulated by osteocytes

During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes, in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage.     

The mechanism of transduction of mechanical strains into biological signals at the bone cellular level

As appears from the literature, the majority of bone researchers consider osteoblasts and osteoclasts the only very important bony cells. In the present report we provide evidence, based on personal morphofunctional investigations, that such a view is incorrect and misleading. Indeed osteoblasts and osteoclasts undoubtedly are the only bone forming and bone reabsorbing cells, but they are transient cells, thus they cannot be the first to be involved in sensing both mechanical and non-mechanical agents which control bone modeling and remodeling processes. Briefly, according to our view, osteoblasts and osteoclasts represent the arms of a worker; the actual operation center is constituted by the cells of the osteogenic lineage in the resting state. Such a resting phase is characterized by osteocytes, bone lining cells and stromal cells, all connected in a functional syncytium by gap junctions, which extends from the bone to the vessels. We named this syncytium the Bone Basic Cellular System (BBCS), because it represents the only permanent cellular background capable first of sensing mechanical strains and biochemical factors and then of triggering and driving both processes of bone formation and bone resorption. As shown by our studies, signalling throughout BBCS can occur by volume transmission (VT) and/or wiring transmission (WT). VT corresponds to the routes followed by soluble substances (hormones, cytokines etc.), whereas WT represents the diffusion of ionic currents along cytoplasmic processes in a neuron-like manner. It is likely that non-mechanical agents first affect stromal cells and diffuse by VT to reach the other cells of BBCS, whereas mechanical agents are first sensed by osteocytes and then issued throughout     

Mechanical stimulation of gap junctions in bone osteocytes is mediated by prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factor(s) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin F2 (PGE2) release and increase cyclooxygenase 2 (COX-2) expression. When PGE2 was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE2. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.     

Extracellular NO signalling from a mechanically stimulated osteocyte

Bone remodelling is a dynamic process that requires the coordinated interaction of osteocytes, osteoblasts, and osteoclasts, collaborating in basic multicellular units (BMUs). Communication between these cells can be by extracellular soluble molecules as well as directly propagating intercellular signalling molecules. Key to the understanding of bone remodelling is osteocyte mechanosensing and chemical signalling to the surrounding cells, since osteocytes are believed to be the mechanosensors of bone, responding to mechanical stresses. Nitric oxide (NO) is an important parameter to study osteocyte activation following mechanical loading. It is a small short-lived molecule, which makes its real-time, quantitative monitoring difficult. However, recently we demonstrated that DAR-4M AM chromophore can be used for real-time quantitative monitoring of intracellular NO production in individual cells following mechanical loading. Here we studied if a single mechanically stimulated osteocyte communicates with, and thus activates its surrounding cells via extracellular soluble factors. We monitored quantitatively intracellular NO production in the stimulated osteocyte and in its surrounding osteocytes, which were not interconnected. Mechanical stimulation by microneedle of a single-MLO-Y4 osteocyte-like cell upregulated the average intracellular NO production by 94% in the stimulated cell, and by 31-150% in the surrounding osteocytes. In conclusion, a single osteocyte can disseminate a mechanical stimulus to its surrounding osteocytes via extracellular soluble signalling factors. This reinforces the putative mechanosensory role of osteocytes, and demonstrates a possible mechanism by which a single mechanically stimulated osteocyte can communicate with other cells in a BMU, which might help to better understand the intricacies of intercellular interactions in BMUs and thus bone remodelling.     

Gap junctions in skeletal development and function

Gap junctions play a critical role in the coordinated function and activity of nearly all of the skeletal cells. This is not surprising, given the elaborate orchestration of skeletal patterning, bone modeling and subsequent remodeling, as well as the mechanical stresses, strains and adaptive responses that the skeleton must accommodate. Much remains to be learned regarding the role of gap junctions and hemichannels in these processes. A common theme is that without connexins none of the cells of bone function properly. Thus, connexins play an important role in skeletal form and function.     

Osteocyte lacunae tissue strain in cortical bone

Current theories suggest that bone modeling and remodeling are controlled at the cellular level through signals mediated by osteocytes. However, the specific signals to which bone cells respond are still unknown. Two primary theories are: (1) osteocytes are stimulated via the mechanical deformation of the perilacunar bone matrix and (2) osteocytes are stimulated via fluid flow generated shear stresses acting on osteocyte cell processes within canaliculi. Recently, much focus has been placed on fluid flow theories since in vitro experiments have shown that bone cells are more responsive to analytically estimated levels of fluid shear stress than to direct mechanical stretching using macroscopic strain levels measured on bone in vivo. However, due to the complex microstructural organization of bone, local perilacunar bone tissue strains potentially acting on osteocytes cannot be reliably estimated from macroscopic bone strain measurements. Thus, the objective of this study was to quantify local perilacunar bone matrix strains due to macroscopically applied bone strains similar in magnitude to those that occur in vivo. Using a digital image correlation strain measurement technique, experimentally measured bone matrix strains around osteocyte lacunae resulting from macroscopic strains of approximately 2000 microstrain are significantly greater than macroscopic strain on average and can reach peak levels of over 30,000 microstrain locally. Average strain concentration factors ranged from 1.1 to 3.8, which is consistent with analytical and numerical estimates. This information should lead to a better understanding of how bone cells are affected by whole bone functional loading.     

Extracellular matrix networks in bone remodeling

Bones are constantly remodeled throughout life to maintain robust structure and function. Dysfunctional remodeling can result in pathological conditions such as osteoporosis (bone loss) or osteosclerosis (bone gain). Bone contains 100s of extracellular matrix (ECM) proteins and the ECM of the various bone tissue compartments plays essential roles directing the remodeling of bone through the coupled activity of osteoclasts (which resorb bone) and osteoblasts (which produce new bone). One important role for the ECM is to serve as a scaffold upon which mineral is deposited. This scaffold is primarily type I collagen, but other ECM components are involved in binding of mineral components. In addition to providing a mineral scaffolding role, the ECM components provide structural flexibility for a tissue that would otherwise be overly rigid. Although primarily secreted by osteoblast-lineage cells, the ECM regulates cells of both the osteoblast-lineage (such as progenitors, mature osteoblasts, and osteocytes) and osteoclast-lineage (including precursors and mature osteoclasts), and it also influences the cross-talk that occurs between these two oppositional cells. ECM influences the differentiation process of mesenchymal stem cells to become osteoblasts by both direct cell-ECM interactions as well as by modulating growth factor activity. Similarly, the ECM can influence the development of osteoclasts from undifferentiated macrophage precursor cells, and influence osteoclast function through direct osteoclast cell binding to matrix components. This comprehensive review will focus on how networks of ECM proteins function to regulate osteoclast- and osteoblast-mediated bone remodeling. The clinical significance of these networks on normal bone and as they relate to pathologies of bone mass and geometry will be considered. A better understanding of the dynamic role of ECM networks in regulating tissue function and cell behavior is essential for the development of new treatment approaches for bone loss.     

The cytokine interleukin-11 crucially links bone formation,remodeling and resorption

Bone development is a complex process that requires the activity of several different signaling pathways and cell types. It involves the coordinated action of osteoclasts (cells that are capable of resorbing bone), osteoblasts (cells that are able to form bone), osteocytes (cells that form a syncytial network within the bone), skeletal muscle cells and the bone marrow. In recent years, the cytokine interleukin-11 (IL-11), a member of the IL-6 family of cytokines, has emerged as an important regulatory protein for bone formation, remodeling and resorption. Furthermore, coding missense mutations in the IL11RA gene, which encodes the IL-11 receptor (IL-11R), have recently been linked to craniosynostosis, a human disease in which the sutures that line the head bones close prematurely. This review summarizes current knowledge about IL-11 and highlights its role in bone development and homeostasis. It further discusses the specificity and redundancy provided by the other members of the IL-6 cytokine family and how they facilitate signaling and cross-talk between skeletal muscle cells, bone cells and the bone marrow. We describe their actions in physiological and in pathological states and discuss how this knowledge could be translated into therapy.     

Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen

Osteocytes are the most abundant cells in bone and distributed throughout the bone matrix. They are connected to the each other and to the cells on the bone surface. Thus, they may also secrete some regulatory factors controlling bone remodeling. Using a newly established osteocyte-like cell line MLO-Y4, we have studied the interactions between osteocytes and osteoclasts. We collected the conditioned medium (CM) from MLO-Y4 cells, and added it into the rat osteoclast cultures. The conditioned medium had no effect on osteoclast number in 24-h cultures, but it dramatically inhibited resorption. With 5, 10, and 20% CM, there was 25, 39, and 42% inhibition of resorption, respectively. Interestingly, the inhibitory effect was even more pronounced, when MLO-Y4 cells were pretreated with 10(-8) M 17-beta-estradiol. With 5, 10, and 20% CM, there was 46, 51, and 58% of inhibition. When the conditioned medium was treated with neutralizing antibody against transforming growth factor-beta (TGF-beta), the inhibitory effect was abolished. This suggests that osteocytes secrete significant amounts of TGF-beta, which inhibits bone resorption and is modulated by estrogen. RT-PCR and Western blot analysis show that in MLO-Y4 cells, the prevalent TGF-beta isoform is TGF-beta3. We conclude that osteocytes have an active, inhibitory role in the regulation of bone resorption. Our results further suggest a novel role for TGF-beta in the regulation of communication between different bone cells and suggest that at least part of the antiresorptive effect of estrogen in bone could be mediated via osteocytes.