Inflammatory factors in the circulation of patients with active rheumatoid arthritis stimulate osteoclastogenesis via endogenous cytokine production by osteoblasts.

SUMMARY: The combination of cytokines present in the circulation of patients with active rheumatoid arthritis might contribute to the generalized bone loss that commonly occurs in these patients, by directly inhibiting osteoblast proliferation and differentiation, but especially by enhancing endogenous cytokine (i.e., receptor activator of nuclear factor-kappa B ligand (RANKL) and interleukin-6 (IL)-6) production by osteoblasts, thereby stimulating osteoclastogenesis. INTRODUCTION: Generalized bone loss, as occurs in patients with rheumatoid arthritis (RA), is related to elevated levels of circulating cytokines. Individual cytokines have deleterious effects on proliferation and differentiation of osteoblast cell lines, but little is known about the effect of the interaction between inflammatory factors in the circulation of patients with active RA on human osteoblast function, including their communication towards other bone cells. We investigated whether serum from patients with active RA enhances cytokine production by osteoblasts, thereby effectively altering osteoblast-stimulated osteoclastogenesis. METHODS: Serum was obtained from 20 patients with active RA (active RA sera) and from the same patients in clinical remission (remission RA sera). To determine osteoclastogenesis, RA serum-pretreated primary human osteoblast cultures were established in direct contact with human osteoclast precursors in the presence or absence of osteoprotegerin (OPG) or IL-6 inhibitor. RESULTS: Compared to remission RA sera, active RA sera inhibited osteoblast proliferation and differentiation in vitro as demonstrated by a reduced DNA content and gene expression of KI-67, collagen type 1, osteopontin, and osteocalcin. Active RA sera inhibited OPG expression and enhanced RANKL and IL-6 expression but did not alter IL-8 expression in osteoblasts. IL-1ß, IL-17, and tumor necrosis factor-α (TNF-α) expression were undetectable. In coculture, active RA sera treatment of osteoblasts stimulated while addition of OPG or IL-6 inhibitory antibodies significantly reduced the number of osteoclasts. CONCLUSION: Active RA sera contain circulating factors, likely cytokines and chemokines, that might contribute to bone loss by directly inhibiting osteoblast proliferation and differentiation, but especially, these factors modulate endogenous cytokine production by osteoblasts, thereby affecting osteoclastogenesis.

IL-6 alters osteocyte signaling toward osteoblasts but not osteoclasts.

Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts.

Nitric oxide signaling in mechanical adaptation of bone.

One of the most serious healthcare problems in the world is bone loss and fractures due to a lack of physical activity in elderly people as well as in bedridden patients or otherwise inactive youth. Crucial here are the osteocytes. Buried within our bones, these cells are believed to be the mechanosensors that stimulate bone formation in the presence of mechanical stimuli and bone resorption in the absence of such stimuli. Intercellular signaling is an important physiological phenomenon involved in maintaining homeostasis in all tissues. In bone, intercellular communication via chemical signals like NO plays a critical role in the dynamic process of bone remodeling. If bones are mechanically loaded, fluid flows through minute channels in the bone matrix, resulting in shear stress on the cell membrane that activates the osteocyte. Activated osteocytes produce signaling molecules like NO, which modulate the activity of the bone-forming osteoblasts and the bone-resorbing osteoclasts, thereby orchestrating bone adaptation to mechanical loading. In this review, we highlight current insights in the role of NO in the mechanical adaptation of bone mass and structure, with emphasis on its role in local bone gain and loss as well as in remodeling supervised by osteocytes. Since mechanical stimuli and NO production enhance bone strength and fracture resistance, these new insights may facilitate the development of novel osteoporosis treatments.

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, ß-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes.

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/ß-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.

Growth factor gene expression profiles of bone morphogenetic protein-2-treated human adipose stem cells seeded on calcium phosphate scaffolds in vitro.

The secretome of stem cells strongly determines the outcome of tissue engineering strategies. We investigated how the secretome of human adipose stem cells (hASCs) can be affected by substrate, BMP-2 treatment, and degree of differentiation. We hypothesized that as differentiation progresses, hASCs produce increasingly more gene products associated with processes such as angiogenesis and bone remodeling. Human ASCs were treated for 15 min with BMP-2 (10 ng/ml) to enhance osteogenic differentiation, or with vehicle. Subsequently, hASCs were seeded on plastic or on biphasic calcium phosphate (BCP) consisting of 60% hydroxyapatite and 40% ß-tricalcium phosphate. A PCR array for ~150 trophic factors and differentiation-related genes was performed at day 21 of culture. A limited set of factors was quantified by qPCR at days 0, 4, 14 and 21, and/or ELISA at day 21. Compared to plastic, BCP-cultured hASCs showed ≥2-fold higher expression of ~20 factors, e.g. cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. Expression of another ~50 genes was decreased ≥2-fold on BCP vs. plastic, even though hASCs differentiate better on BCP than on plastic. BMP-2-treatment increased the expression of ~30 factors by hASCs seeded on BCP, while it decreased the expression of only PGF, PPARG and PTN. Substrate affected hASC secretion of Activin A and seemed to affect P1NP release. No clear association between hASC osteogenic differentiation and growth factor expression pattern was observed. Considering our observed lack of association between the degree of differentiation and the expression of factors associated with angiogenesis and bone remodeling by hASCs, future bone regeneration studies should focus more on systematically orchestrating the secretome of stem cells, rather than on inducing osteogenic differentiation of stem cells only. Short incubation with BMP-2 may be a promising treatment to enhance both osteogenic differentiation and environmental modulation.

Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: a model for cell-mechanics studies.

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.

Mechanical loading and how it affects bone cells: the role of the osteocyte cytoskeleton in maintaining our skeleton.

Lack of physical activity causes bone loss and fractures not only in elderly people, but also in bedridden patients or otherwise inactive youth. This is fast becoming one of the most serious healthcare problems in the world. Osteocytes, cells buried within our bones, stimulate bone formation in the presence of mechanical stimuli, as well as bone degradation in the absence of such stimuli. As yet, we do not fully comprehend how osteocytes sense mechanical stimuli, and only know a fraction of the whole range of molecules that osteocytes subsequently produce to regulate bone formation and degradation in response to mechanical stimuli. This dramatically hampers the design of bone loss prevention strategies. In this review we will focus on the first step in the cascade of events leading to adaptation of bone mass to mechanical loading, i.e., on how osteocytes are able to perceive mechanical stimuli placed on whole bones. We will place particular emphasis on the role of the osteocyte cytoskeleton in mechanosensing. Given the crucial importance of osteocytes in maintaining a proper resistance against bone fracture, greater knowledge of the molecular mechanisms that govern the adaptive response of osteocytes to mechanical stimuli may lead to the development of new strategies towards fracture prevention and enhanced bone healing.

Tumor necrosis factor alpha and interleukin-1beta modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes.

OBJECTIVE: Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) modulate the osteocyte response to mechanical loading. METHODS: MLO-Y4 osteocytes were incubated with TNFalpha (0.5-30 ng/ml) or IL-1beta (0.1-10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean +/- amplitude 0.7 +/- 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca(2+)](i)) using Fluo-4/AM. Focal adhesions and filamentous actin (F-actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell-generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry. RESULTS: Pulsatile fluid flow increased [Ca(2+)](i) within seconds (in 13% of cells) and NO production within 5 minutes (4.7-fold). TNFalpha and IL-1beta inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow-induced NO production. TNFalpha and IL-1beta affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFalpha and IL-1beta decreased the amount of F-actin. Incubation with IL-1beta for 24 hours stimulated osteocyte apoptosis. CONCLUSION: Our results suggest that TNFalpha and IL-1beta inhibit mechanical loading-induced NO production by osteocytes via abrogation of pulsatile fluid flow-stimulated [Ca(2+)](i), and that IL-1beta stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.

Orthodontic force stimulates eNOS and iNOS in rat osteocytes.

Mechanosensitive osteocytes are essential for bone remodeling. Nitric oxide, an important regulator of bone remodeling, is produced by osteocytes through the activity of constitutive endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS). We hypothesized that these enzymes regulate the tissue response to orthodontic force, and therefore we investigated eNOS and iNOS expression in osteocytes during orthodontic force application. The upper rat molars were moved mesially by NiTi coil springs (10 cN, 120 hrs) in a split-mouth design. Immunohistochemical staining revealed that, in the tension area, eNOS-positive osteocytes increased from 24 hrs on, while iNOS-positive osteocytes remained largely constant. In the compression area, iNOS-positive osteocytes increased after 6 hrs, while eNOS- positive osteocytes increased after 24 hrs. This suggests that eNOS mediates bone formation in the tension area, while iNOS mediates inflammation-induced bone resorption in the compression area. Both eNOS and iNOS seem to be important regulators of bone remodeling during orthodontic force application.

Predictive value of femoral head heterogeneity for fracture risk.

Osteoporosis (OP) is characterized by low bone mass and weak bone structure, which results in increased fracture risk. It has been suggested that osteoporotic bone is strongly adapted to the main loading direction and less adapted to the other directions. In this study, we hypothesized that osteoporotic femoral heads have 1) an increased anisotropy; 2) a more heterogenic distribution of bone volume fraction (BV/TV) throughout the femoral head; and, 3) a more heterogenic distribution of the trabecular thickness (Tb.Th.) throughout the femoral head, as compared to non-osteoporotic bone. To test these hypotheses, we used 7 osteoporotic femoral heads from patients who fractured their femoral neck and 7 non-fractured femoral heads from patients with osteoarthrosis (OA). Bone structural parameters from the entire trabecular region were analyzed using microCT. We found that the degree of anisotropy was higher in the fractured femoral heads, i.e. 1.72, compared to a value of 1.61 in the non-fractured femoral heads. The BV/TV and Tb.Th. and their variations throughout the femoral head, however, were all significantly lower in the fractured group. Hence, the first hypothesis was confirmed, whereas the other two were rejected. Interestingly, the variation of Tb.Th. throughout the femoral head provided a 100% discrimination between the OP and OA groups, i.e. for the same BV/TV, all fractured cases had a less heterogenic distribution. In conclusion, our results suggest that bone loss in OP takes place uniformly throughout the femoral head, leading to an overall decrease in bone mass and trabecular thickness. Furthermore, the variation of Tb.Th. in the femoral head could be an interesting parameter to improve the prediction of fracture risk in the proximal femur.

Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro.

We investigated whether low-intensity pulsed ultrasound (LIPUS) stimulates chondrocyte proliferation and matrix production in explants of human articular cartilage obtained from donors suffering from unicompartimental osteoarthritis of the knee, as well as in isolated human chondrocytes in vitro. Chondrocytes and explants were exposed to LIPUS (30 mW/cm(2); 20 min/day, 6 days). Stimulation of [35S]-sulphate incorporation into proteoglycans by LIPUS was 1.3-fold higher in degenerative than in collateral monolayers as assessed biochemically and 1.9-fold higher in explants as assessed by autoradiography. LIPUS decreased the number of cell nests containing 1-3 chondrocytes by 1.5 fold in collateral and by 1.6 fold in degenerative explants. LIPUS increased the number of nests containing 4-6 chondrocytes by 4.8 fold in collateral and by 3.9 fold in degenerative explants. This suggests that LIPUS stimulates chondrocyte proliferation and matrix production in chondrocytes of human articular cartilage in vitro. LIPUS might provide a feasible tool for cartilage tissue repair in osteoarthritic patients, since it stimulates chondrocyte proliferation and matrix production.

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide.

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7+/-0.3Pa, 5Hz, 1h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.

The polymine spermine regulates osteogenic differentiation in adipose stem cells.

For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) differentiate into osteoblasts. To develop a method for differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) along the osteogenic lineage, we studied the effect of polyamines, which are organic cations implicated in bone growth and development, on differentiation of AT-MSCs. Treatment of goat-derived AT-MSCs with 1,25-dihydroxyvitamin-D3 (1,25(OH)(2)D(3)), which stimulates osteogenic differentiation, for 7 days induced gene expression of the polyamine-modulated transcription factor-1 (PMF-1) and spermidine/spermine N (1)-acetyltransferase (SSAT), which are both involved in polyamine metabolism, suggesting that polyamines are involved in osteogenic differentiation of AT-MSCs. Furthermore, treatment of AT-MSCs with the polyamine spermine-regulated gene expression of runx-2, a transcription factor involved in early stages of osteogenic differentiation, and that of osteopontin, a bone matrix protein expressed in later stages of osteogenic differentiation. Runx-2 gene expression was increased 4 and 14 days after a short 30 min. treatment with spermine, while osteopontin gene expression was only increased 4 days after spermine treatment. Finally, alkaline phosphatase activity, which is intimately involved in the formation of extracellular matrix of bone, was increased 4 weeks after the 30 min.-spermine treatment of AT-MSCs. In conclusion, this study shows for the first time that the polyamine spermine regulates differentiation of AT-MSCs along the osteogenic lineage, which can be used as a new method for differentiation of AT-MSCs along the osteogenic lineage. Therefore, polyamines may constitute a promising tool for bone tissue engineering approaches using AT-MSCs, such as a one-step surgical procedure for spinal interbody fusion.

Paxillin localisation in osteocytes--is it determined by the direction of loading?

External mechanical loading of cells aligns cytoskeletal stress fibres in the direction of principle strains and localises paxillin to the mechanosensing region. If the osteocyte cell body can indeed directly sense matrix strains, then cytoskeletal alignment and distribution of paxillin in osteocytes in situ will bear alignment to the different mechanical loading patterns in fibulae and calvariae. We used confocal microscopy to visualise the immunofluorescence-labelled actin cytoskeleton in viable osteocytes and paxillin distribution in fixated osteocytes in situ. In fibular osteocyte cell bodies, actin cytoskeleton and nuclei were elongated and aligned parallel to the principal (longitudinal) mechanical loading direction. Paxillin was localised to the 'poles' of elongated osteocyte cell bodies. In calvarial osteocyte cell bodies, actin cytoskeleton and nuclei were relatively more round. Paxillin was distributed evenly in the osteocyte cell bodies. Thus in osteocyte cell bodies in situ, the external mechanical loading pattern likely determines the orientation of the actin cytoskeleton, and focal adhesions mediate direct mechanosensation of matrix strains.

Prostaglandins differentially affect osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are currently used for bone tissue engineering. AT-MSCs undergoing osteogenic differentiation respond to mechanical loading with increased cyclooxygenase-2 gene expression, a key enzyme in prostaglandin (PG) synthesis. PGs are potent multifunctional regulators in bone, exhibiting stimulatory and inhibitory effects on bone formation and resorption. PGE(2), but not PGI(2) or PGF(2), recruits osteoprogenitors from the bone marrow space and influences their differentiation. We hypothesize that PGE(2), PGI(2), and PGF(2) may differentially regulate osteogenic differentiation of human AT-MSCs. PGE(2), PGI(2), and PGF(2) (0.01-10 microM) affected osteogenic differentiation, but not proliferation of AT-MSCs after 4-14 days. Only PGF(2) (0.01-10 microM) increased alkaline phosphatase (ALP) activity at day 4. PGE(2) (10 microM), PGI(2) (0.01-10 microM), and PGF(2) (10 microM) decreased ALP activity, whereas PGF(2) (0.1 microM) increased ALP activity at day 14. PGF(2) (0.01-0.1 microM) and PGI(2) (0.01 microM) upregulated osteopontin gene expression, and PGF(2) (0.01 microM) upregulated alpha1(I)procollagen gene expression at day 4. PGE(2) and PGF(2) (10 microM) at day 4 and PGF(2) (1 microM) at day 14 downregulated runt-related transcription factor-2 gene expression. We conclude that PGE(2), PGI(2), and PGF(2) differentially affect osteogenic differentiation of AT-MSCs, with PGF(2) being the most potent. Thus, locally produced PGF(2) might be most beneficial in promoting osteogenic differentiation of AT-MSCs, resulting in enhanced bone formation for bone tissue engineering.

Fluid shear stress inhibits TNFalpha-induced osteocyte apoptosis.

Bone tissue can adapt to orthodontic load. Mechanosensing in bone is primarily a task for the osteocytes, which translate the canalicular flow resulting from bone loading into osteoclast and osteoblast recruiting signals. Apoptotic osteocytes attract osteoclasts, and inhibition of osteocyte apoptosis can therefore affect bone remodeling. Since TNF-alpha is a pro-inflammatory cytokine with apoptotic potency, and elevated levels are found in the gingival sulcus during orthodontic tooth movement, we investigated if mechanical loading by pulsating fluid flow affects TNF-alpha-induced apoptosis in chicken osteocytes, osteoblasts, and periosteal fibroblasts. During fluid stasis, TNF-alpha increased apoptosis by more than two-fold in both osteocytes and osteoblasts, but not in periosteal fibroblasts. One-hour pulsating fluid flow (0.70 +/- 0.30 Pa, 5 Hz) inhibited (-25%) TNF-alpha-induced apoptosis in osteocytes, but not in osteoblasts or periosteal fibroblasts, suggesting a key regulatory role for osteocyte apoptosis in bone remodeling after the application of an orthodontic load.

Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.

BACKGROUND: Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS: Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS: The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION: We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.

Different responsiveness to mechanical stress of bone cells from osteoporotic versus osteoarthritic donors.

INTRODUCTION: Osteoporosis (OP) and osteoarthritis (OA) are both common diseases in the elderly, but remarkably seldom coexist. The bone defects that are related to both diseases develop with increasing age, which suggests that they are related to some form of imperfect bone remodeling. Current opinion holds that the bone remodeling process is supervised by bone cells that respond to mechanical stimuli. An imperfect response of bone cells to mechanical stimuli might thus relate to imperfect bone remodeling, which could eventually lead to a lack bone mass and strength, such as in OP patients. MATERIALS: To investigate whether the cellular response to mechanical stress differs between OP and OA patients, we compared the response of bone cells from both groups to fluid shear stress of increasing magnitude. Bone cells from 9 female OP donors (age 60-90 year) and 9 female age-matched OA donors were subjected to pulsating fluid flow (PFF) of low (0.4+/-0.1 Pa at 3 Hz), medium (0.6+/-0.3 Pa at 5 Hz), or high shear stress (1.2+/-0.4 at 9Hz), or were kept under static culture conditions. RESULTS: We found subtle differences in the shear-stress response of the two groups, measured as nitric oxide (NO) and prostaglandin E2 (PGE2) production. The NO-response to shear stress was higher in the OP than the OA cells, while the PGE2-response was higher in the OA cells. CONCLUSIONS: Assuming that NO and PGE2 play a role in cell-cell communication during remodeling, these results suggest that slight differences in mechanotransduction might relate to the opposite bone defects in osteoporosis and osteoarthritis.