Bone regeneration can be accelerated by utilizing mechanical stress and growth factors (GFs). However, a limited understanding exists regarding the response of preosteoblasts to tensile stress alone or with GFs. We measured cell proliferation and expression of heat-shock proteins (HSPs) and other bone-related proteins by preosteoblasts following cyclic tensile stress (1%-10% magnitude) alone or in combination with bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1). Tensile stress (3%) with GFs induced greater gene upregulation of osteoprotegerin (3.3 relative fold induction [RFI] compared to sham-treated samples), prostaglandin E synthase 2 (2.1 RFI), and vascular endothelial growth factor (VEGF) (11.5 RFI), compared with samples treated with stimuli alone or sham-treated samples. The most significant increases in messenger RNA expression occurred with GF addition to either static-cultured or tensile-loaded (1% elongation) cells for the following genes: HSP47 (RFI=2.53), cyclooxygenase-2 (RFI=72.52), bone sialoprotein (RFI=11.56), and TGF-β1 (RFI=8.05). Following 5% strain with GFs, VEGF secretion increased 64% (days 3-6) compared with GF alone and cell proliferation increased 23% compared with the sham-treated group. GF addition increased osteocalcin secretion but decreased matrix metalloproteinase-9 significantly (days 3-6). Tensile stress and GFs in combination may enhance bone regeneration by initiating angiogenic and anti-osteoclastic effects and promote cell growth.

Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, which may include regulation of transcription factors that support osteoblast differentiation and function. Flow effects on osteoblast transcription factors have generally been evaluated after short exposures. In this work, we assayed flow effects on osteogenic genes at early and late time points in a preosteoblast (CIMC-4) cell line and evaluated both steady and oscillatory flows. Four hours of steady unidirectional flow decreased the level of RANKL mRNA 53 ± 7% below that of nonflowed cells, but increases in Runx2 and osterix mRNA (44 ± 22% and 129 ± 12%, respectively) were significant only after 12-19 h of continuous flow. Late flow effects on RANKL and osterix were also induced by an intermittent flow-rest protocol (four cycles of 1 h on/1 h off + overnight rest). Four hours of oscillatory flow decreased RANKL mRNA at this early time point (63 ± 2%) but did not alter either osterix or Runx2. When oscillatory flow was delivered using the intermittent flow-rest protocol, Runx2 and osterix mRNA increased significantly (85 ± 19% and 161 ± 22%, respectively). Both β-catenin and ERK1/2, known to be involved in RANKL regulation, were rapidly activated by steady flow. Inhibition of flow-activated ERK1/2 prevented the increase in osterix mRNA but not Runx2; Runx2 phosphorylation was increased by flow, an effect which likely contributes to osterix induction. This work shows that both steady and oscillatory fluid flows can support enhancement of an osteogenic phenotype.

Communication between osteoblasts, osteoclasts, and osteocytes is integral to their ability to build and maintain the skeletal system and respond to physical signals. Various physiological mechanisms, including nerve communication, hormones, and cytokines, play an important role in this process. More recently, the important role of direct, cell-cell communication via gap junctions has been established. In this review, we demonstrate the integral role of gap junctional intercellular communication (GJIC) in skeletal physiology and bone cell mechanosensing.

Bone formation occurs in vivo in response to mechanical stimuli, but the signaling pathways involved remain unclear. The ability of bone cells to communicate with each other in the presence of an applied load may influence the overall osteogenic response. The goal of this research was to determine whether inhibiting cell-to-cell gap junctional communication between bone-forming cells would affect the ensemble cell response to an applied mechanical stimulus in vitro. In this study, we investigated the effects of controlled oscillatory fluid flow (OFF) on osteoblastic cells in the presence and the absence of a gap-junction blocker. MC3T3-E1 Clone 14 cells in a monolayer were exposed to 2 h of OFF at a rate sufficient to create a shear stress of 20 dyne/cm(2) at the cell surface, and changes in steady-state mRNA levels for a number of key proteins known to be involved in osteogenesis were measured. Of the five proteins investigated, mRNA levels for osteopontin (OPN) and osteocalcin were found to be significantly increased 24 h postflow. These experiments were repeated in the presence of 18 beta-glycyrrhetinic acid (BGA), a known gap-junction blocker, to determine whether gap-junction intercellular communication is necessary for this response. We found that the increase in OPN mRNA levels is not observed in the presence of BGA, suggesting that gap junctions are involved in the signaling process. Interestingly, enzyme linked immunosorbent assay data showed that levels of secreted OPN protein increased 48 h postflow and that this increase was unaffected by the presence of intact gap junctions.

Mechanical signals are major regulators of skeletal homeostasis as the addition of exogenous load is followed by enhanced bone formation and the removal of normal loads is followed by net bone loss. The mechanism by which bone cells perceive and respond to changes in their biophysical environment are still poorly understood, but it is widely accepted that the detection of interstitial fluid flow is an initiating cue. In this chapter, we describe two in vitro systems designed to examine the effects of fluid flow on bone cell behavior and to elucidate the signaling cascades activated by this stimulus. The first utilizes a parallel plate flow chamber designed to stimulate a single bone cell type grown on glass slides. The second employs a rotating disk fluid flow apparatus. Commercially-available cell culture inserts allow one type of bone cell to be exposed to fluid flow and signals to be communicated to a second bone cell model not exposed to fluid flow.

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.

Osteoblast biology is influenced in vivo by a 3-dimensional (3D) extracellular matrix that mediates their adhesion and interaction and by a constant state of compressive and tensile forces. To study the role of mechanical stress on osteoblasts in vitro, these parameters must be addressed. Therefore, this study describes the use of a novel, in vitro system that subjects cells to distractive and compressive forces in a 3D environment. This system, termed a microdistractor system, was used to apply linear forces to 3D collagen type I gels containing preosteoblasts. Gels were induced for up to 16 days in osteogenic medium and subjected to either constant linear distraction (distraction gels) or to repeating cycles of distraction and compression (oscillation gels). The effect of these stresses was evaluated over time by measuring proliferation rates, protein synthesis (i.e., cellular activity), and osteogenic differentiation levels. While linear forces in general appeared to increase protein synthesis, force-specific effects on proliferation and differentiation were observed. Specifically, distraction forces appeared to enhance MC3T3 proliferation while distraction/compressive forces appeared to accelerate their osteogenic differentiation program. Therefore, these results suggest that the microdistraction system may be an appropriate in vitro system for the study of mechanobiology in osteoblast phenotype.

To evaluate effects of zoledronate on markers of bone metabolism in dogs after transection of the cranial cruciate ligament (CrCL).

21 adult dogs.

Unilateral CrCL transection was performed arthroscopically. Dogs were allocated to 3 groups (control group, low-dose zoledronate [10 microg/kg, SC, q 90 d for 12 months], and high-dose zoledronate [25 microg/kg, SC, q 90 d for 12 months]). Serum osteocalcin (OC), serum bone-specific alkaline phosphatase (BAP), and urine pyridinoline and deoxypyridinoline concentrations were measured at 0, 1, 3, 6, 9, and 12 months after surgery. Bone mineral density (BMD) was determined in the distal portion of the femur and proximal portion of the tibia via computed tomography at each time point. Data were analyzed by a repeated-measures ANOVA.

oledronate inhibited OC in the high-dose group at 9 and 12 months and at 12 months in the low-dose group, compared with the control group. High-dose zoledronate decreased BAP concentrations 3 and 9 months after surgery. In the control group, BMD was decreased in the femoral condyle and caudal tibial plateau. Zoledronate prevented significant BMD decreases starting 1 month after transection, compared with control dogs. In the caudomedial aspect of the tibial plateau, both zoledronate groups had significant increases in BMD after 3 months, compared with control dogs.

Zoledronate may reduce subchondral bone loss and effect markers of bone metabolism in dogs with experimentally induced instability of the stifle joint and subsequent development of osteoarthritis.

Bone tissue has the capacity to adapt to its functional environment such that its morphology is "optimized" for the mechanical demand. The adaptive nature of the skeleton poses an interesting set of biological questions (e.g., how does bone sense mechanical signals, what cells are the sensing system, what are the mechanical signals that drive the system, what receptors are responsible for transducing the mechanical signal, what are the molecular responses to the mechanical stimuli). Studies of the characteristics of the mechanical environment at the cellular level, the forces that bone cells recognize, and the integrated cellular responses are providing new information at an accelerating speed. This review first considers the mechanical factors that are generated by loading in the skeleton, including strain, stress and pressure. Mechanosensitive cells placed to recognize these forces in the skeleton, osteoblasts, osteoclasts, osteocytes and cells of the vasculature are reviewed. The identity of the mechanoreceptor(s) is approached, with consideration of ion channels, integrins, connexins, the lipid membrane including caveolar and non-caveolar lipid rafts and the possibility that altering cell shape at the membrane or cytoskeleton alters integral signaling protein associations. The distal intracellular signaling systems on-line after the mechanoreceptor is activated are reviewed, including those emanating from G-proteins (e.g., intracellular calcium shifts), MAPKs, and nitric oxide. The ability to harness mechanical signals to improve bone health through devices and exercise is broached. Increased appreciation of the importance of the mechanical environment in regulating and determining the structural efficacy of the skeleton makes this an exciting time for further exploration of this area.

Mechanical forces influence bone form and function. Although the adaptive capabilities of bone are well known, the nuances of the mechanical stimuli regulating adaptation remain elusive. Recently, it was suggested that strain rate influences bone adaptation, and impact exercises with high strain rates during growth may be more osteogenic than low impact aerobic exercises. Building on those findings, we hypothesized that higher rates of mechanical loading would evoke greater adaptive responses than lower rates of loading in mature bone. To test that hypothesis, skeletally mature (16 weeks) female C57BL/6 mice underwent non-invasive exogenous cantilever bending of the right tibia with a 1 Hz trapezoidal waveform for 60 s, 5 days per week, for 4 weeks. Loading was calibrated (strain gauge) to induce peak magnitudes of 1000 microepsilon on the lateral tibial middiaphysis. Mice were randomly assigned to three groups based on strain rate of the applied load: low (0.004 s(-1); n = 14), medium (0.020 s(-1); n = 15), and high (0.100 s(-1); n = 14). Calcein injections (i.p., 10 mg kg(-1)) permitted histomorphometric analyses of bone formation. Loading significantly enhanced periosteal mineral apposition rate (MAR), mineralizing surface (MS), and bone formation rate (BFR BS(-1)) in all three strain rate groups, relative to control tibiae. Furthermore, a graded dose-response relation was observed between the applied strain rate and periosteal BFR BS(-1). These increases in MAR, MS, and BFR BS(-1) were not seen on the endosteal surface. Endosteal adaptation was not statistically different between loaded and control tibiae in most endosteal indices of bone adaptation. Moreover, endosteal adaptation did not increase with strain rate. Understanding the nature of the stimuli to which bone cells respond to may underpin the development of non-pharmacological treatments devised to enhance bone mass.

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.

Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Recent reports on orthopaedic surgery focus on mechanical stimulation of the regenerate during distraction therapy of non-unions in long-bone-surgery. In the field of maxillofacial surgery, callus stimulating techniques are rarely reported. The case of a 65-year-old man with a radiogenic mandibular non-union after ablative tumour therapy and pre-operative radiation therapy presented with a non-union. Vertical distraction in combination with subsequent repeated, stepwise compression and distraction (=massage) had a positive effect on the consolidation of the regenerate.

The aim of this review is to characterise the biological and biophysical background of in vitro bone tissue engineering. The paper focuses on basic principles in extracorporal engineering of bone-like tissues, considering parameters such as scaffold design, tissue construction, bioreactors, and cell stimulation in vivo and in vitro. Scaffolds have a key function concerning cellular invasion and bone formation. The intra-architectural scaffold geometry, as well as the scaffold material, play an important role in the process of bone regeneration. Various types of bioreactors have been tested for their utility in bone substitute fabrication that is clinically effective and reproducible. Sophisticated bioreactor systems are those that mimic the three-dimensional morphology and the mechanical situation of bones. Mechanical stimulation as well as other biophysical stimuli appear to be critical factors for proliferation and differentiation of bone cells and for bone mineral and structure formation. Furthermore an enhancement of bone regeneration by application of chemical stimulation factors is discussed.

Bone undergoes a constant process of remodeling in which mass is retained or lost in response to the relative activity of osteoblasts and osteoclasts. Weight-bearing exercise-which is critical for retaining skeletal integrity-promotes osteoblast function, whereas a lack of mechanical stimulation, as seen during spaceflight or prolonged bed rest, can lead to osteoporosis. Thus, understanding mechanotransduction at the cellular level is key to understanding basic bone biology and devising new treatments for osteoporosis. Various mechanical stimuli have been studied as in vitro model systems and have been shown to act through numerous signaling pathways to promote osteoblast activity. Here, we examine the various types of stress and the sequential response of transduction pathways that result in changes in gene expression and the ensuing proliferation of osteoblasts.

The effects of mechanical strains on cellular activities were assessed in an in vitro model using human osteoblastic MG-63 cells grown on titanium alloy discs coated with porous alumina and exposed to chronic intermittent loading. Strain was applied with a Dynacell device for three 15-min sequences per day for several days with a magnitude of 600 microepsilon strain and a frequency of 0.25 Hz. We have previously demonstrated that this regimen increased alkaline phosphatase activity in confluent cultures on ceramic coated titanium (alumina and hydroxyapatite) (Biomaterials 24 (2003) 3139). In this study, we analysed the production of bone matrix proteins. Osteocalcin secretion quantified by ELISA between day 5 and 11 was not affected by mechanical strain. Strain had even no quantifiable effect on collagen production from day 1 to 5 as measured by carboxy terminal collagen type I propeptide release. On the other hand, stress stimulation resulted in increased expression of fibronectin (FN) measured by Western blot after 1 day stretching. This upregulation of FN production was followed by reorganisation of the FN network after 5 days stretching observed by immunostaining. The receptors for collagen and FN, alpha2beta1, alpha5beta1 and beta1 integrins were not quantitatively affected by the strains as measured by flow cytometry. A modification of cell morphology was seen after 5 days of loading that appeared to increase cell spreading, implying consequences on intercellular contacts. For this reason, N, C11 and E-adherins were examined. We noted a selective effect characterised by increased expression of N-cadherin using both RT-PCR and Western blot analyses. We concluded that reinforcement of cell-cell adhesion and remodelling of the FN network are important adaptive responses to physiological strains for human osteoblasts grown on alumina-coated biomaterials.

Mechanical forces regulate the function of bone cells. In this paper, the effects of cyclic stretching on osteoblasts derived from rat calvaria were studied at a magnitude occurring in physiological loaded bone tissue. A four-point bending apparatus was used to apply cyclic stretching on osteoblasts. Stretching at 500 microepsilon for 2-24 h resulted in an increase in matrix synthesis(P<0.01). In contrast, the cyclic stretching at 1000 and 1500 microepsilon for 2-24 h inhibited osteoblast collagen production (P<0.01). We also described our new loading method to increase strain magnitude step-by-step. The strain magnitude increased by 500 microepsilon increments from 500 to 1500 microepsilon every 2 or 12 h, respectively. Results showed that osteoblasts could absorb large amount of proline for collagen synthesis when stretched at 500 microepsilon. However, not all the absorbed proline was used to synthesize collagen. Some of it was stored in cells. When the suitable signal (500 microepsilon) was changed to an inhibiting signal (1000 microepsilon), cells responded to it accordingly and released proline to medium. These results demonstrate that the response of osteoblasts is dependent on the magnitude of the strain applied and cells can adjust their bio-chemical response to adapt to the changing environmental stimulation.

Bone represents a porous tissue containing a fluid phase, a solid matrix, and cells. Movement of the fluid phase within the pores or spaces of the solid matrix translates endogenous and exogenous mechanobiological, biochemical and electromechanical signals from the system that is exposed to the dynamic external environment to the cells that have the machinery to remodel the tissue from within. Hence, bone fluid serves as a coupling medium, providing an elegant feedback mechanism for functional adaptation. Until recently relatively little has been known about bone fluid per se or the influences governing the characteristics of its flow. This work is designed to review the current state of this emerging field. The structure of bone, as an environment for fluid flow, is discussed in terms of the properties of the spaces and channel walls through which the fluid flows and the influences on flow under physiological conditions. In particular, the development of the bone cell syncytium and lacunocanalicular system are presented, and pathways for fluid flow are described from the systemic to the organ, tissue, cellular and subcellular levels. Finally, exogenous and endogenous mechanisms for pressure-induced fluid movement through bone, including mechanical loading, vascular derived pressure gradients, and osmotic pressure gradients are discussed. The objective of this review is to survey the current understanding of the means by which fluid flow in bone is regulated, from the level of the skeletal system down to the level of osteocyte, and to provide impetus for future research in this area of signal transduction and coupling. An understanding of this important aspect of bone physiology has profound implications for restoration of function through innovative treatment modalities on Earth and in space, as well as for engineering of biomimetic replacement tissue.

A nondestructive, acousto-optical method for characterizing the mechanical loss factor of biological tissues and tissue scaffolds is presented and applied to the characterization of an elastin tissue scaffold derived from bovine nuchal ligament. The method relies on launching guided surface acoustic waves into the tissue scaffold with a small speaker and simultaneously illuminating a small region of the scaffold distant from the speaker with a low-power HeNe laser. The phase lag between the driving acoustic wave and the shift in the backscattered laser speckle pattern is determined as a measure of the mechanical loss factor of the scaffold, tan delta. Measurements of tan delta and elastic modulus were also made by traditional dynamic mechanical loading techniques. Through the central portion of the loading cycle, the elastic modulus of the elastin scaffold was 1.2 x 10(6) +/- 1 x 10(5) N x m(-2) (parallel to fiber orientation). The estimated value of tan delta in the direction parallel to the elastin fibers was 0.03 +/- 0.017 by traditional methods and 0.029 +/- 0.03 when using the acousto-optical method. In the direction perpendicular to fiber orientation, tan delta was measured as 0.14 +/- 0.056 by the acousto-optical method. Because of a lack of mechanical integrity, it was not possible to measure tan delta in the direction perpendicular to fiber orientation by traditional methods. The acousto-optical method may prove to be useful in the mechanical characterization of developing engineered tissues.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments, and mechanical stress has been suggested to play an important role in the progression of OPLL. To identify the genes that participate in OPLL, the differential display reverse transcription-polymerase chain reaction (RT-PCR) method was used. A 283-base pair cDNA fragment corresponding to prostaglandin I2 (PGI2) synthase was highly expressed in OPLL cells compared with non-OPLL cells. To examine the effect of mechanical stress on the expression of PGI2 synthase, cells were subjected to uniaxial cyclic stretch (0.5 Hz, 20% stretch), and PGI2 synthase mRNA expression was assessed by quantitative RT-PCR. Cyclic stretch induced an increase in PGI2 synthase in OPLL cells in a time-dependent manner, whereas no change was observed in non-OPLL cells. Cyclic stretch for 9 h also induced a 2.86x increase in PGI2 production. Beraprost (a stable PGI2 analog) and dibutyryl cAMP (a membrane-permeable cAMP analog) increased the mRNA expression of alkaline phosphatase (ALP) as a marker for osteogenic differentiation up to 240 and 200%, respectively, in OPLL cells, whereas no change was observed in non-OPLL cells. The increases in ALP mRNA induced by beraprost and cyclic stretch were both inhibited by SQ22536, a potent adenylate cyclase inhibitor. These data suggest that the increase in PGI2 synthase induced by mechanical stress plays a key role in the progression of OPLL, at least in part through the induction of osteogenic differentiation in spinal ligament cells via the PGI2/cAMP system.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.

Differentiation of chondrocytes to cells of osteoblastic phenotype occurs during an interim period of bone development, fracture repair and distraction osteogenesis. To study the relationship between tension-stress and chondrogenesis, uniaxial strains (0 microstrains, 2000 microstrains, 20000 microstrains, 200000 microstrains, 300000 microstrains) were applied in a rabbit model of mandibular distraction osteogenesis. The results demonstrated that cell differentiation, apoptosis and tissue development in the newly formed gap tissue showed a correlation to the applied strain magnitudes. Only strains of 20000 microstrains resulted in a statistically significant (P<0.05) formation of cartilage struts with embedded chondrocyte-like cells. However, chondrocyte-like cells were rarely detected in samples distracted at lower or higher strain magnitudes. Osteoblasts appeared to replace cartilaginous matrix by mineralized bone matrix. The phenotypic change from chondrocytes to osteoblasts was accompanied by a decreased proteoglycan synthesis. a change in the expression from type II collagen towards type I and involved asymmetric cell divisions and apoptotic cell death. Therefore, we suggest that mechanical strain is an external stimulus responsible for phenotypic cell alterations.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.

Physical signals, in particular mechanical loading, are clearly important regulators of bone turnover. Indeed, the structural success of the skeleton is due in large part to the bone's capacity to recognize some aspect of its functional environment as a stimulus for achievement and retention of a structurally adequate morphology. However, while the skeleton's ability to respond to its mechanical environment is widely accepted, identification of a reasonable mechanism through which a mechanical "load" could be transformed to a signal relevant to the bone cell population has been elusive. In addition, the downstream response of bone cells to load-induced signals is unclear. In this work, we review evidence suggesting that gap junctional intercellular communication (GJIC) contributes to mechanotransduction in bone and, in so doing, contributes to the regulation of bone cell differentiation by biophysical signals. In this context, mechanotransduction is defined as transduction of a load-induced biophysical signal, such as fluid flow, substrate deformation, or electrokinetic effects, to a cell and ultimately throughout a cellular network. Thus, mechanotransduction would include interactions of extracellular signals with cellular membranes, generation of intracellular second messengers, and the propagation of these messengers, or signals they induce, through a cellular network. We propose that gap junctions contribute largely to the propagation of intracellular signals.

The development of tissue engineering in the field of orthopaedic surgery is now booming. Two fields of research in particular are emerging: the association of osteo-inductive factors with implantable materials; and the association of osteogenic stem cells with these materials (hybrid materials). In both cases, an understanding of the phenomena of cell adhesion and, in particular, understanding of the proteins involved in osteoblast adhesion on contact with the materials is of crucial importance. The proteins involved in osteoblast adhesion are described in this review (extracellular matrix proteins, cytoskeletal proteins, integrins, cadherins, etc.). During osteoblast/material interactions, their expression is modified according to the surface characteristics of materials. Their involvement in osteoblastic response to mechanical stimulation highlights the significance of taking them into consideration during development of future biomaterials. Finally, an understanding of the proteins involved in osteoblast adhesion opens up new possibilities for the grafting of these proteins (or synthesized peptide) onto vector materials, to increase their in vivo bioactivity or to promote cell integration within the vector material during the development of hybrid materials.

Load-induced fluid flow has been postulated to provide a mechanism for the transmission of mechanical signals (e.g. via shear stresses, enhancement of molecular transport, and/or electrical effects) and the subsequent elicitation of a functional adaptation response (e.g. modeling, remodeling, homeostasis) in bone. Although indirect evidence for such fluid flow phenomena can be found in the literature pertaining to strain generated potentials, actual measurement of fluid displacements in cortical bone is inherently difficult. This problem motivated us to develop and introduce an ex vivo perfusion model for the study of transport processes and fluid flow within bone under controlled mechanical loading conditions. To this end, a closed-loop system of perfusion was established in the explanted forelimb of the adult Swiss alpine sheep. Immediately prior to mechanical loading, a bolus of tracer was introduced intraarterially into the system. Thereafter, the forelimb of the left or right side (randomized) was loaded cyclically, via Schanz screws inserted through the metaphyses, producing a peak compressive strain of 0.2% at the middiaphysis of the anterior metacarpal cortex. In paired experiments with perfusion times totalling 2, 4, 8 and 16 min, the concentration of tracer measured at the middiaphysis of the cortex in cross section was significantly higher in the loaded bone than in the unloaded contralateral control. Fluorometric measurements of procion red concentration in the anterior aspect alone showed an enhancement in transport at early stages of loading (8 cycles, 2 min) but no effect in transport after higher number of cycles or increased perfusion times, respectively. This reflects both the small size of the molecular tracer, which would be expected to be transported rapidly by way of diffusive mechanisms alone, as well as the loading mode to which the anterior aspect was exposed. Thus, using our new model it could be shown that load-induced fluid flow represents a powerful mechanism to enhance molecular transport within the lacunocanalicular system of compact bone tissue. Based on these as well as previous studies, it appears that the degree of this effect is dependent on tracer size as well as the mechanical loading mode to which a given area of tissue is exposed.

Bone regeneration is believed to be partially controlled by the applied local mechanical strain. To test whether the magnitude or frequency of discontinuous traction regulates the tissue response, defined daily strains were applied on mandibular osteotomies using an implanted mechanical distractor.

Unilateral mandibular osteotomies were performed in skeletally immature rabbits (n = 36). and distraction was done by applying 2,000, 20,000, 200,000, or 300,000 microstrains once or 10 times (2,000, 20,000 microstrains) per day, respectively. Sham-operated animals (n = 6), serving as controls, underwent frame application and osteotomy without distraction. At the end of the distraction process, the newly formed tissue was evaluated histomorphometrically by the use of a well-defined scoring system of bone-forming indices.

The highest bone-forming indices were detected in the osteotomized, nondistracted group and in samples exposed to a physiologic strain (2,000 microstrains). Application of hyperphysiologic strains (200,000 and 300,000 microstrains) resulted in the formation of fibrous tissue and decreased bone-forming indices. Using Kruskal-Wallis tests, a statistically significant relationship was found between the bone-forming indices and the applied strain magnitudes. Scanning and transmission electron microscopic examinations showed osteoblastic differentiation and early mineral deposition in samples distracted up to 20,000 microstrains, whereas higher strain magnitudes led to the formation of fibroblast-like cells surrounded by collagen fibrils and only slight mineralization. Multiple strain applications (10 cycles/d vs 1 cycle/d) did not alter the histomorphometric indices or ultrastructural morphology significantly but increased the amount of newly formed tissue.

These results suggest that the magnitude and not the frequency of mechanical loading controls the differentiation of bone cells and the subsequent formation of bone tissue.

The periodontal ligament may play an important role in tooth eruption, root development and resorption. The tissue physiologically receives mechanical force during mastication. We focused on the effects of intermittent mechanical strain on the cytokine synthesis of periodontal ligament (PDL) fibroblasts in vitro. The cells were derived from human periodontal ligament of deciduous teeth (HPLF-Y) and permanent teeth (HPLF). The two kinds of PDL cells and human gingival fibroblasts (HGF) were cultured in flexible bottomed culture plates. The cells were mechanically stretched at 5% elongation, 3-cycles/min for 24 h on d 7 in culture using a Flexercell strain unit. After the stretching, we measured DNA content and alkaline phosphatase activity in the cell layer, transforming growth factor beta 1 (TGF-beta 1) and macrophage colony stimulating factor (M-CSF) contents in the conditioned medium. The TGF-beta 1 level in the conditioned medium of HPLF was significantly higher than that of HPLF-Y and HGF. It was stimulated by mechanical stretching only on HPLF, whereas no significant effect was observed on HPLF-Y and HGF. M-CSF secretion was inhibited by the stretching on all of HPLF, HPLF-Y and HGF. 1 alpha, 25 dihydroxy vitamin D3 (D3) stimulated M-CSF secretion into the culture medium of both HPLF and HPLF-Y, but the stretching inhibited M-CSF secretion and completely blocked the enhancement by D3. These data suggest that periodontal ligament cells synthesize and secrete the molecules as autocrine or paracrine factors that affect bone remodelling and root resorption and the level of those factors change in response to mechanical stress.

The aim of this study is to evaluate the regulation of the osteopontin (OPN) gene expression by non-hormonal stimuli, such as calcium flux and mechanical strain during the daily egg cycle in the oviduct of the laying hen. After the egg enters the eggshell gland (ESG), the OPN gene is expressed by the epithelium cells in two waves: first by the basal cells and only then by the apical cells of the epithelium. A reduction in OPN gene expression was observed 1 h prior to laying. The calbindin gene, which marks the onset of calcification, was found to be expressed in the glandular epithelium starting 2 h after OPN gene expression. In addition, the formation of soft shells was accompanied by a reduction in calbindin, but not in OPN, gene expression. The application of a mechanical strain comparable to that induced by an egg led to induction of OPN gene expression at a normally quiescent phase in the cyclical expression of this gene. The induction of the gene was time- and strain-dependent and temporally similar to that induced by the entry of the egg into the ESG. In contrast, the calbindin gene was not affected by mechanical strain. The ESG of the laying hen provides a system to study the effect of a mechanical strain on matrix protein production in vivo, in a relevant physiological setting. The finding suggests that, in contrast to calbindin, OPN gene expression is not regulated by calcium flux but rather by the mechanical strain imposed by the resident egg.

Several researchers have developed theories implicating some manifestation of mechanical forces such as stress, strain, and strain energy density for the initiation of cellular processes associated with functional adaptation. The mechanisms underlying dynamic bone growth and repair in response to mechanical stimuli, however, are not fully understood. Load-induced fluid flow has been postulated to provide a mechanism for the transmission of mechanical signals (eg, via shear stresses, enhancement of molecular transport, or electrical effects) and the subsequent elicitation of a functional adaptation response in bone. Although indirect evidence for such fluid flow phenomena can be found in the literature pertaining to strain-generated potentials, experimental studies are inherently difficult. This motivated the authors to develop theoretical as well as ex vivo, in vitro, and in vivo experimental methods for the study of transport processes and fluid flow within bone under well-controlled mechanical loading conditions. By introducing tracer substances such as disulphine blue, procion red, and microperoxidase into the experimental system, transport and fluid flow could be visualized at tissue, cellular, and subcellular levels, respectively. Based on these studies, it could be shown that load-induced fluid flow represents a powerful mechanism to enhance molecular transport within compact bone tissue. Furthermore, the distribution of transport-elucidating tracers is a function of mechanical loading parameters as well as the location within the cross-section of the bone cortex.

The process of cartilage-to-bone transition (CBT) is a key event for the achievement of rigid bone healing during fracture repair. Since mineralization of cartilaginous matrix is a prerequisite for the initiation of CBT, the genetic localization of mineralization-related bone matrix proteins in CBT was examined in this study. An in situ hybridization method used on decalcified sections with digoxigenin-11-UTP labelled probes identified the cellular localizations of these genes in CBT. Cessation of osteonectin mRNA together with induction of osteopontin mRNA in chondrocyte maturation was observed during the process of CBT in the fracture callus on day 12 after fracture; osteocalcin mRNA was absent in chondrocytes of the CBT area. Induction of osteopontin mRNA in maturated chondrocytes was followed by the expression of mRNAs for osteonectin, osteopontin and osteocalcin in osteogenic cells in the ossification front of CBT. The data suggest that the switch from osteonectin to osteopontin mRNA expression in chondrocyte maturation is one of the key events during CBT. Transcriptional disorders of the expression of these molecules may be linked to the failure of fracture repair, i.e. delayed or prevented hypertrophic osteosynthesis.

Intercellular gap junctions permit bone cells to intercellularly transmit, and subsequently process, periosteal functional matrix information, after its initial intracellular mechanotransduction. In addition, gap junctions, as electrical synapses, underlie the organization of bone tissue as a connected cellular network, and the fact that all bone adaptation processes are multicellular. The structural and operational characteristics of such biologic networks are outlined and their specific bone cell attributes described. Specifically, bone is "tuned" to the precise frequencies of skeletal muscle activity. The inclusion of the concepts and databases that are related to the intracellular and intercellular bone cell mechanisms and processes of mechanotransduction and the organization of bone as a biologic connected cellular network permit revision of the functional matrix hypothesis, which offers an explanatory chain, extending from the epigenetic event of muscle contraction hierarchically downward to the regulation of the bone cell genome.

To determine the phenotypic expression of cementoblasts responsible for acellular cementum, an immunohistochemical study was performed using a polyclonal antibody raised against the aminoterminal peptide of rat osteocalcin (OC). Maxillary first molars of Wistar male rats aged 2 and 3 wk were used for observations. Serial sections of decalcified paraffin embedded specimens were stained either with hematoxylin and eosin or with the anti-OC antibody. In 2-wk-old rats, apical roots were lined with the epithelial root sheath. A thin layer of acellular cementum was seen at most of the root surface, but was not seen near to root apex. In 3-wk-old rats, cellular cementum began to be formed at root apex, and acellular cementum became more thick than in 2-wk-old rats. Acellular and cellular cementum were lined with the fibroblast-like cells. Osteocalcin staining was detected in cells lining root surface in both 2- and 3-wk-old rats. Almost all cells lining cellular cementum were positive for OC. In contrast OC positive cells lining acellular cementum and root surface devoid of cementum appeared at a specific site of the root. The cells at the interradicular area of root surface were positive but the cells at the outer area (the opposite side of the interradicular area) were negative for OC. Osteoblasts and odontoblasts were positive with the antibody. The present results suggest that the OC expression of cementoblasts forming acellular cementum is similar to that of cells forming cellular cementum as well as osteoblasts and odontoblasts, and has a role for calcification of acellular cementum.

In this study we tested the hypothesis that exercise induces an adaptive response in the developing skeleton which may be monitored in vivo by measuring biochemical markers of bone metabolism. The effects of exercise on two biochemical markers of bone formation were determined; the carboxy-terminal propeptide of type I procollagen (PICP), and the bone-specific isoenzyme of alkaline phosphatase (BAP), and one putative marker of resorption, the pyridinoline crosslinked telopeptide domain of type I collagen (ICTP). All three markers were measured for a year in 2-year-old thoroughbred horses exercised three times a week on a treadmill, and values compared to a control group of age-matched animals. Levels of all three markers fell in both exercised and control groups over the 12-month period reflecting normal age changes. However, there were differences between groups in the pattern of this decrease. When expressed as a percentage of baseline values, BAP was higher (p < 0.05) at 2 months and both BAP and the PICP were higher at 4 months (p < 0.01 and p < 0.05, respectively) in the exercised group, reflecting an increase in bone turnover in this group in the early stages of training. PICP levels were also elevated in the exercised group at 10 months and this result indicates an increase in bone turnover at this time. The changes in ICTP were different; at 2 months, levels were higher in exercised animals than in controls, but there was no significant difference between the two groups at 4 and 6 months. After 8 months, ICTP levels in the exercised group increased returning to near baseline values at 10 months.(ABSTRACT TRUNCATED AT 250 WORDS)