Simon C.F. Rawlinson

Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?

The structural competence of the skeleton is maintained by an adaptive mechanism in which resident bone cells respond to load‐induced strains. To investigate the possible role of the messenger molecule nitric oxide (NO) in this response, we studied NO production in well‐characterized organ culture systems, rat long bone‐derived osteoblast‐like (LOBs) cells, and embryonic chick osteocytes (LOCYs) in monolayer culture. In superfused cancellous bone cores, loading (for 15 min) produces increases in NO2‐ (stable NO metabolite) release during the loading period, which paralleled those in PGI2 and PGE2‐ Loading of rat vertebrae and ulnae produces increases in NO2‐ release, and in ulnae NO synthase inhibitors diminish these responses. Transient rapid increases in NO release are stimidated by strain in both LOBs and LOCYs. Polymerase chain reaction amplification of extracted mRNA shows that rat ulnae, LOBs, and LOCYs express both the inducible and neuronal (constitutive) isoforms of NO synthase. Adaptability to mechanical strain relies on assessment of the strain environment followed by modification of bone architecture. Immediate increases in NO production induced by loading suggest the involvement of NO in strain measurement and cellular communication to establish strain distribution, as well as potentially in adaptive changes in bone cell behavior.—Pitsillides, A. A., Rawlinson, S. C. F., Suswillo, R. F. L., Bourrin, S., Zaman, G., Lanyon, L. E. Mechanical strain‐induced NO production by bone cells: a possible role in adaptive bone (re)modeling? FASEB J. 9, 1614‐1622(1995)

Mechanical Strain Stimulates Nitric Oxide Production by Rapid Activation of Endothelial Nitric Oxide Synthase in Osteocytes

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast‐like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading‐induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non–load‐bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain‐induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast‐like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.

Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways

Extracellular regulated kinases (ERKs)-1 and -2 are members of the MAPK family of protein kinases involved in the proliferation, differentiation, and apoptosis of bone cells. We have shown previously that ROS 17/2.8 cells show increased activation of ERK-1 or -2, which is sustained for 24 h, when the strips onto which they are seeded are subjected to a 10 min period of cyclic four point bending that produces physiological levels of mechanical strain along with associated fluid movement of the medium. Movement of the strips through the medium without bending causes fluid movement without strain. This also increases ERK-1/2 activation, but in a biphasic manner over the same time period. Our present study investigates the role of components of signaling pathways in the activation of ERK-1/2 in ROS 17/2.8 cells in response to these stimuli. Using a range of inhibitors we show specific differences by which ERK-1 and ERK-2 are activated in response to fluid movement alone, compared with those induced in response to strain plus its associated fluid movement. ERK-1 activation induced by fluid movement was markedly reduced by nifedipine, and therefore appears to involve L-type calcium channels, but was unaffected by either L-NAME or indomethacin. This suggests independence from prostacyclin (PGI(2)) and nitric oxide (NO) production. In contrast, ERK-1 activation induced by application of strain (and its associated fluid disturbance) was abrogated by TMB-8 hydrochloride, L-NAME, and indomethacin. This suggests that strain-induced ERK-1 activation is dependent upon calcium mobilization from intracellular stores and production of NO and PGI(2). ERK-2 activation appears to be mediated by a separate mechanism in these cells. Its activation by fluid movement alone involved both PGI(2) and NO production, but its activation by strain was not affected by any of the inhibitors used. The G protein inhibitor, pertussis toxin, did not cause a reduction in the activation of ERK-1 or -2 in response to either stimulus. These results are consistent with earlier observations of ERK activation in bone cells in response to both strain (with fluid movement) and fluid movement alone, and further demonstrate that these phenomena stimulate distinct signaling pathways.

Human Osteoblasts' Proliferative Responses to Strain and 17β-Estradiol Are Mediated by the Estrogen Receptor and the Receptor for Insulin-Like Growth Factor I†

The mechanism by which mechanical strain and estrogen stimulate bone cell proliferation was investigated using monolayer cultures of human osteoblastic TE85 cells and female human primary (first‐passage) osteoblasts (fHOBs). Both cell types showed small but statistically significant dose‐dependent increases in [3H]thymidine incorporation in response to 17β‐estradiol and to a single 10‐minute period of uniaxial cyclic strain (1 Hz). In both cell types, the peak response to 17β‐estradiol occurred at 10−8‐10−7 M and the peak response to strain occurred at 3500 microstrain (μϵ). Both strain‐related and 17β‐estradiol‐related increases in [3H]thymidine incorporation were abolished by the estrogen receptor (ER) modulator ICI 182,780 (10−8 M). Tamoxifen (10−9‐10−8 M) increased [3H]thymidine incorporation in both cell types but had no effect on their response to strain. In TE85 cells, tamoxifen reduced the increase in [3H]thymidine incorporation associated with 17β‐estradiol to that of tamoxifen alone but had no such effect in fHOBs. In TE85 cells, strain increased medium concentrations of insulin‐like growth factor (IGF) II but not IGF‐I, whereas 17β‐estradiol increased medium concentrations of IGF‐I but not IGF‐II. Neutralizing monoclonal antibody (MNAb) to IGF‐I (3 μg/ml) blocked the effects of 17β‐estradiol and exogenous truncated IGF‐I (tIGF‐I; 50 ng/ml) but not those of strain or tIGF‐II (50 ng/ml). Neutralizing antibody to IGF‐II (3 μg/ml) blocked the effects of strain and tIGF‐II but not those of 17β‐estradiol or tIGF‐I. MAb αIR‐3 (100 ng/ml) to the IGF‐I receptor blocked the effects on [3H]thymidine incorporation of strain, tIGF‐II, 17β‐estradiol, and tIGF‐I. HOBs and TE85 cells, act similarly to rat primary osteoblasts and ROS 17/2.8 cells in their dose‐related proliferative responses to strain and 17β‐estradiol, both of which can be blocked by the ER modulator ICI 182,780. In TE85 cells (as in rat primaries and ROS 17/2.8 cells), the response to 17β‐estradiol is mediated by IGF‐I, and the response to strain is mediated by IGF‐II. Human cells differ from rat cells in that tamoxifen does not block their response to strain and reduces the response to 17β‐estradiol in TE85s but not primaries. In both human cell types (unlike rat cells) the effects of strain and IGF‐II as well as estradiol and IGF‐I can be blocked at the IGF‐I receptor.

Exogenous prostacyclin, but not prostaglandin E2, produces similar responses in both G6PD activity and RNA production as mechanical loading, and increases IGF-II release, in adult cancellous bone in culture

SummaryCyclic mechanical loadingin vivo that leads to new bone formation is also associated in osteocytes and surface bone cells with almost immediate increases in G6PD activity, and later increases in RNA production. Both these early, loading-related, responses can be reproduced in organ culture of adult cancellous bone, and both are abolished by the presence of indomethacin in the culture medium at the time of loading. The implication that prostaglandins (PGs) are involved in the control of loading-related osteogenesis is supported by increases in prostacyclin (PGIZ) and PGE2 release from cores of cancellous bone during loading. In the experiments reported here, PGE2 and PGI2 were added exogenously (10−6 M) to perfusable cores of adult canine cancellous bone to determine whether they would simulate the loading-related responses in G6PD activity and RNA synthesis. PGE2 increased GOD activity in surface cells and osteocytes within 8 minutes but had no effect on [3H]-uridine incorporation at 6 hours. PGI2 stimulated both G6PD activity and [3H]-uridine incorporation equally in osteocytes and surface cells. Neither PG produced any significant change in medium concentrations of IGF-I, and PGE2 had no effect on IGF-II. In contrast, PGI2 elevated the medium concentration of IGF-II threefold. IGF-I and IGF-II were localized immunocytochemically to osteocytes and surface cells in both treated and untreated cores. Prostacyclin, but not PGE2, appears to imitate the early loading-related increases in G6PD activity and RNA synthesis in bone cellsin situ. Prostacyclin, but not PGE2, also stimulates the early release of IGF-II.

Mechanical strain stimulates ROS cell proliferation through IGF-II and estrogen through IGF-I

The mechanism by which mechanical strain stimulates bone cell proliferation was investigated and compared with that of estrogen in ROS 17/2 .8 cells. Similarity of strain‐related responses between ROS cells and osteoblasts was established by demonstrating that ROS cells respond to a short single period of strain in their substrate (1000–3500 μϵ, 600 cycles, 1 Hz) by a similar strain magnitude‐related increase in glucose 6‐phosphate dehydrogenase activity as rat osteoblasts and osteocytes in explants in situ. ROS17/2 .8 cells also showed similar proliferative responses to strain and 17β‐estradiol, as assessed by [3H]thymidine incorporation and cell counting, as primary cultures of long bone‐derived osteoblast‐like cells. Strain‐related increase in proliferation in ROS cells was accompanied by a 4‐fold increase in levels of insulin‐like growth factor‐II (IGF‐II) in conditioned medium. Neither strain nor estrogen had an effect on the conditioned medium levels of IGF‐I. Exogenous truncated IGFs tIGF‐I and tIGF‐II both increased proliferation in a dose‐dependent manner. The neutralizing monoclonal antibody (nMAb) to IGF‐I blocked proliferation stimulated by tIGF‐I but not that due to tIGF‐II and vice versa. IGF‐I receptor blocking antibody (IGF‐IRBAb) blocked the proliferative effect of tIGF‐I but not that to tIGF‐II. The proliferative effect of estrogen was abolished by IGF‐I nMAb and IGF‐IRBAb, but these antibodies had no effect on the proliferative response to strain. In contrast IGF‐II nMAb abolished the proliferative effect of strain but had no effect on that of estrogen. These data show that ROS17/2 .8 cells have similar responses to strain and estrogen qualitatively and quantitatively as rat osteoblasts in situ and rat long bone–derived osteoblast‐like cells in primary culture. Estrogen‐related proliferation in ROS17/2 .8 cells appears to be mediated by IGF‐I acting through the IGF‐I receptor and does not involve IGF‐II. In contrast, strain‐related proliferation appears to be mediated by IGF‐II and does not involve either IGF‐I or the IGF‐I receptor.

Enhancement by Sex Hormones of the Osteoregulatory Effects of Mechanical Loading and Prostaglandins in Explants of Rat Ulnae

Explants of ulnae from 5‐week‐old male and female rats were cleaned of marrow and soft tissue and, in the presence and absence of 10−8 M 17β‐estradiol (E2) or 5α‐dihydrotestosterone (DHT), mechanically loaded or treated with exogenous prostanoids previously shown to be produced during loading. Over an 18‐h period, mechanical loading (peak strain 1300 μϵ, 1 Hz, 8 minutes, maximum strain rate 25,000 μϵ/s), prostaglandin E2 (PGE2) and prostacyclin (PGI2) (10−6 M), each separately produced quantitatively similar increases in cell proliferation and matrix production in bones from males and females, as indicated by incorporation of [3H]thymidine into DNA and [3H]proline into collagen. E2 and DHT both increased [3H]thymidine and [3H]proline incorporations, E2 producing greater increases in females than in males. Indomethacin abrogated the effects of loading, but had no effects on those of sex hormones. Loading, or prostanoids, together with sex hormones, produced responses generally equal to or greater than the addition of the individual influences acting independently. In females there was a synergistic response in [3H]thymidine incorporation between loading and E2, which was quantitatively similar to the interaction between E2 and PGE2 or PGI2. The interaction between loading and E2 for [3H]proline incorporation was not mimicked by these prostanoids. In males the synergism in [3H]proline incorporation seen between loading and DHT was mimicked by that between PGI2 and DHT. We conclude that loading stimulates increased bone cell proliferation and matrix production in situ through a prostanoid‐dependent mechanism. This response is equal in size in males and females. Estrogen and testosterone increase proliferation and matrix production through a mechanism independent of prostanoid production. The interactions between loading and hormones are reproduced in some but not all cases by E2 and prostaglandins. E2 with loading and prostaglandins has greater effects in female bones, while DHT with loading and prostaglandins has greater effects in males.

Adult rat bones maintain distinct regionalized expression of markers associated with their development.

The incidence of limb bone fracture and subsequent morbidity and mortality due to excessive bone loss is increasing in the progressively ageing populations of both men and women. In contrast to bone loss in the weight-bearing limb, bone mass in the protective skull vault is maintained. One explanation for this could be anatomically diverse bone matrix characteristics generated by heterogeneous osteoblast populations. We have tested the hypothesis that adult bones demonstrate site-specific characteristics, and report differences at the organ, cell and transcriptome levels. Limb bones contain greater amounts of polysulphated glycosaminoglycan stained with Alcian Blue and have significantly higher osteocyte densities than skull bone. Site-specific patterns persist in cultured adult bone-derived cells both phenotypically (proliferation rate, response to estrogen and cell volumes), and at the level of specific gene expression (collagen triple helix repeat containing 1, reelin and ras-like and estrogen-regulated growth inhibitor). Based on genome-wide mRNA expression and cluster analysis, we demonstrate that bones and cultured adult bone-derived cells segregate according to site of derivation. We also find the differential expression of genes associated with embryological development (Skull: Zic, Dlx, Irx, Twist1 and Cart1; Limb: Hox, Shox2, and Tbx genes) in both adult bones and isolated adult bone-derived cells. Together, these site-specific differences support the view that, analogous to different muscle types (cardiac, smooth and skeletal), skull and limb bones represent separate classes of bone. We assign these differences, not to mode of primary ossification, but to the embryological cell lineage; the basis and implications of this division are discussed.

Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants

Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA composite-coated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-α and IL-6 cytokines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial and biocompatible.

Ovariectomy vs. Hypofunction: Their Effects on Rat Mandibular Bone

Previous studies have suggested that the mandible may be more influenced by mechanical loading than by circulating hormone levels. We tested the hypothesis that hypofunction has a greater influence than ovariectomy on mandibular bone. Two-month-old rats were ovariectomized (OVX) or had maxillary molars removed from one side to induce unilateral mandibular hypofunction. Control animals remained untreated. After 5 months, animals were killed, and bones were assessed by micro-tomography (μCT), quantitative back-scattered electron analysis in an SEM (qBSE-SEM), and light microscopy. Mineralization density was reduced in calvarial, maxillary, and mandibular alveolar bone following OVX, yet was increased in lingual mandibular alveolar bone of the hypo-function animals compared with controls. OVX caused a reduction in osteocyte density in alveolar bone, while hypofunction showed an increase compared with controls. Hypofunction led to alveolar bone becoming more highly mineralized and more cellular, while ovariectomy caused a reduction in both mineralization density and osteocyte numbers.