Compressed Prostate Cancer Cells Decrease Osteoclast Activity While Enhancing Osteoblast Activity In Vitro.

Once prostate cancer cells metastasize to bone, they perceive approximately 2 kPa compression. We hypothesize that 2 kPa compression stimulates the epithelial-to-mesenchymal transition (EMT) of prostate cancer cells and alters their production of paracrine signals to affect osteoclast and osteoblast behavior. Human DU145 prostate cancer cells were subjected to 2 kPa compression for 2 days. Compression decreased expression of 2 epithelial genes, 5 out of 13 mesenchymal genes, and increased 2 mesenchymal genes by DU145 cells, as quantified by qPCR. Conditioned medium (CM) of DU145 cells was added to human monocytes that were stimulated to differentiate into osteoclasts for 21 days. CM from compressed DU145 cells decreased osteoclast resorptive activity by 38% but did not affect osteoclast size and number compared to CM from non-compressed cells. CM was also added to human adipose stromal cells, grown in osteogenic medium. CM of compressed DU145 cells increased bone nodule production (Alizarin Red) by osteoblasts from four out of six donors. Compression did not affect IL6 or TNF-α production by PC DU145 cells. Our data suggest that compression affects EMT-related gene expression in DU145 cells, and alters their production of paracrine signals to decrease osteoclast resorptive activity while increasing mineralization by osteoblasts is donor dependent. This observation gives further insight in the altered behavior of PC cells upon mechanical stimuli, which could provide novel leads for therapies, preventing bone metastases.

Shear loaded osteocyte-like-cells affect epithelial and mesenchymal gene expression in DU145 prostate cancer cells, while decreasing their invasion in vitro.

Once prostate cancer (PC) metastasizes towards bone the 5-year survival rates drop with 70%, but it is largely unknown why. Bone is continuously mechanically loaded, which likely modulates the paracrine signaling from osteocytes towards PC cells to affect tumor behavior. We hypothesize that shear loaded osteocytes affect PC cell proliferation, invasion and epithelial and mesenchymal-related gene and protein expression. We cultured human DU145 cells, a commonly used cell line for prostate cancer metastases, in the conditioned medium (CM) from shear loaded or unloaded human osteocyte-like-cells (OCYLCs) for 1 and 3 days and assessed their number by staining nuclei with DAPI, their invasion by performing an invasion assay, and epithelial-to-mesenchymal (EMT)-related gene and protein expression by qPCR and immunocytochemistry. CM of shear loaded OCYLCs did not affect DU145 cell number compared to CM of static cultured OCYLCs, but decreased their invasion 1.34-fold. CM of shear loaded OCYLCs enhanced expression of epithelial genes: SYND1 and CDH1 after day 1, while it also enhanced CDH1 after day 3. CM of shear loaded osteocytes enhanced mesenchymal genes: VMN, Snail and MIP2 after day 1, while it decreased expression of mesenchymal CYR61 after day 3. We conclude that CM of shear loaded OCYLCs does not affect DU145 cell proliferation, but decreases their invasion, and differentially affects their EMT-related gene expression. Identifying paracrine signals from shear loaded osteocytes that decrease PC cell invasion may provide novel leads in developing treatments for bone metastases from PC.

The release of osteoclast-stimulating factors on supraphysiological loading by osteoprogenitors coincides with expression of genes associated with inflammation and cytoskeletal arrangement.

Supraphysiological loading induced by unstable orthopedic implants initiates osteoclast formation, which results in bone degradation. We aimed to investigate which mechanosensitive cells in the peri-implant environment produce osteoclast-stimulating factors and how the production of these factors is stimulated by supraphysiological loading. The release of osteoclast-stimulating factors by different types of isolated bone marrow-derived hematopoietic and mesenchymal stem cells from six osteoarthritic patients was analyzed after one hour of supraphysiological loading (3.0 ± 0.2 Pa, 1 Hz) by adding their conditioned medium to osteoclast precursors. Monocytes produced factors that enhanced osteoclastogenesis by 1.6 ± 0.07-fold and mesenchymal stem cells by 1.4 ± 0.07-fold. Medium from osteoprogenitors and pre-osteoblasts enhanced osteoclastogenesis by 1.3 ± 0.09-fold and 1.4 ± 0.03-fold, respectively, where medium from four patients elicited a response and two did not. Next generation sequencing analysis of osteoprogenitors revealed that genes encoding for inflammation-related pathways and cytoskeletal rearrangements were regulated differently between responders and non-responders. Our data suggest that released osteoclast-stimulating soluble factors by progenitor cells in the bone marrow after supraphysiological loading may be related to cytoskeletal arrangement in an inflammatory environment. This connection could be relevant to better understand the aseptic loosening process of orthopedic implants.

Temporomandibular joint synovial chondromatosis: An analysis of 7 cases and literature review.

OBJECTIVE: To investigate the diagnosis and treatment procedure of synovial chondromatosis (SC) of the temporomandibular joint (TMJ). METHODS: Clinical features, imaging features, surgical methods, and prognosis of 7 patients with SC of the TMJ were analyzed. We also reviewed and analyzed surgery-relevant literature included in the Pubmed database in the past decade using the search terms "synovial chondromatosis" and "temporomandibular joint", and found 181 cases. RESULTS: There was no specific difference in the symptoms of SC in the TMJ in different Milgram's stages in our cases and the cases mentioned in the literature. The main symptoms of SC in the TMJ were pain (100%, 7/7; 64.64%, 117/181), limited mouth opening (57.14%, 4/7; 53.59%, 97/181), swelling (14.29%, 1/7; 28.18%, 51/181), crepitus (28.57%, 2/7; 19.34%, 35/181), and clicking (14.29%, 1/7; 9.94%, 18/181) in our cases and cases from literature separately. The imaging features of SC were occupying lesions (including loose bodies or masses) (71.42%, 5/7; 37.57%, 68/181), bone change in condyle or glenoid fossa (1/7, 14.29%; 34.81%, 63/181), effusion (42.86%, 3/7; 20.99%, 38/181), joint space changes (42.86%, 3/7; 11.05%, 20/181) in our cases and cases from literature separately. The surgical procedures seem to depend mainly on the involved structures and the extension of the lesion rather than the Milgram's stage. CONCLUSIONS: The clinical features of SC in the TMJ are nonspecific and easy to be misdiagnosed. MRI is helpful in the diagnosis of SC in the TMJ. The surgical procedures mainly depend on the involved structures and the extension of the lesion.

Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells.

Osteoblasts derived from mouse skulls have increased osteoclastogenic potential compared to long bone osteoblasts when stimulated with 1,25(OH)2 vitamin D3 (vitD3). This indicates that bone cells from specific sites can react differently to biochemical signals, e.g., during inflammation or as emitted by bioactive bone tissue-engineering constructs. Given the high turn-over of alveolar bone, we hypothesized that human alveolar bone-derived osteoblasts have an increased osteogenic and osteoclastogenic potential compared to the osteoblasts derived from long bone. The osteogenic and osteoclastogenic capacity of alveolar bone cells and long bone cells were assessed in the presence and absence of osteotropic agent vitD3. Both cell types were studied in osteogenesis experiments, using an osteogenic medium, and in osteoclastogenesis experiments by co-culturing osteoblasts with peripheral blood mononuclear cells (PBMCs). Both osteogenic and osteoclastic markers were measured. At day 0, long bones seem to have a more late-osteoblastic/preosteocyte-like phenotype compared to the alveolar bone cells as shown by slower proliferation, the higher expression of the matrix molecule Osteopontin (OPN) and the osteocyte-enriched cytoskeletal component Actin alpha 1 (ACTA1). This phenotype was maintained during the osteogenesis assays, where long bone-derived cells still expressed more OPN and ACTA1. Under co-culture conditions with PBMCs, long bone cells also had a higher Tumor necrose factor-alfa (TNF-α) expression and induced the formation of osteoclasts more than alveolar bone cells. Correspondingly, the expression of osteoclast genes dendritic cell specific transmembrane protein (DC-STAMP) and Receptor activator of nuclear factor kappa-Β ligand (RankL) was higher in long bone co-cultures. Together, our results indicate that long bone-derived osteoblasts are more active in bone-remodeling processes, especially in osteoclastogenesis, than alveolar bone-derived cells. This indicates that tissue-engineering solutions need to be specifically designed for the site of application, such as defects in long bones vs. the regeneration of alveolar bone after severe periodontitis.

High shear stress amplitude in combination with prolonged stimulus duration determine induction of osteoclast formation by hematopoietic progenitor cells.

To date, it is unclear how fluid dynamics stimulate mechanosensory cells to induce an osteoprotective or osteodestructive response. We investigated how murine hematopoietic progenitor cells respond to 2 minutes of dynamic fluid flow stimulation with a precisely controlled sequence of fluid shear stresses. The response was quantified by measuring extracellular adenosine triphosphate (ATP), immunocytochemistry of Piezo1, and sarcoplasmic/endoplasmic Ca2+ reticulum ATPase 2 (SERCA2), and by the ability of soluble factors produced by mechanically stimulated cells to modulate osteoclast differentiation. We rejected our initial hypothesis that peak wall shear stress rate determines the response of hematopoietic progenitor cells to dynamic fluid shear stress, as it had only a minor correlation with the abovementioned parameters. Low stimulus amplitudes corresponded to activation of Piezo1, SERCA2, low concentrations of extracellular ATP, and inhibition of osteoclastogenesis and resorption area, while high amplitudes generally corresponded to osteodestructive responses. At a given amplitude (3 Pa) and waveform (square), the duration of individual stimuli (duty cycle) showed a strong correlation with the release of ATP and osteoclast number and resorption area. Collectively, our data suggest that hematopoietic progenitor cells respond in a viscoelastic manner to loading, since a combination of high shear stress amplitude and prolonged duty cycle is needed to trigger an osteodestructive response. PLAIN LANGUAGE SUMMARY: In case of painful joints or missing teeth, the current intervention is to replace them with an implant to keep a high-quality lifestyle. When exercising or chewing, the cells in the bone around the implant experience mechanical loading. This loading generally supports bone formation to strengthen the bone and prevent breaking, but can also stimulate bone loss when the mechanical loading becomes too high around orthopedic and dental implants. We still do not fully understand how cells in the bone can distinguish between mechanical loading that strengthens or weakens the bone. We cultured cells derived from the bone marrow in the laboratory to test whether the bone loss response depends on (i) how fast a mechanical load is applied (rate), (ii) how intense the mechanical load is (amplitude), or (iii) how long each individual loading stimulus is applied (duration). We mimicked mechanical loading as it occurs in the body, by applying very precisely controlled flow of fluid over the cells. We found that a mechanosensitive receptor Piezo1 was activated by a low amplitude stimulus, which usually strengthens the bone. The potential inhibitor of Piezo1, namely SERCA2, was only activated by a low amplitude stimulus. This happened regardless of the rate of application. At a constant high amplitude, a longer duration of the stimulus enhanced the bone-weakening response. Based on these results we deduce that a high loading amplitude tends to be bone weakening, and the longer this high amplitude persists, the worse it is for the bone.

Polymethyl methacrylate does not adversely affect the osteogenic potential of human adipose stem cells or primary osteoblasts.

Custom-made polymethyl methacrylate (PMMA) bone cement is used to treat cranial bone defects but whether it is cytotoxic is still unsure. Possible PMMA-induced adverse effects in vivo affect mesenchymal stem cells and osteoblasts at the implant site. We aimed to investigate whether PMMA affects osteogenic and osteoclast activation potential of human mesenchymal stem cells and/or osteoblasts. Immediately after polymerization, PMMA was added to cultured human adipose stem cells (hASCs) or human osteoblasts (hOBs). Medium lactate dehydrogenase was measured (day 1), metabolic activity, proliferation, osteogenic and osteoclast-activation marker expression (day 1 and 7), and mineralization (day 14). PMMA did not affect lactate dehydrogenase, KI67 gene expression, or metabolic activity in hASCs and hOBs. PMMA transiently decreased DNA content in hOBs only. PMMA increased COL1 gene expression in hASCs, but decreased RUNX2 in hOBs. PMMA did not affect osteocalcin or alkaline phosphatase (ALP) expression, ALP activity, or mineralization. Only in hOBs, PMMA decreased RANKL/OPG ratio. In conclusion, PMMA is not cytotoxic and does not adversely affect the osteogenic potential of hASCs or hOBs. Moreover, PMMA does not enhance production of osteoclast factors by hASCs and hOBs in vitro. Therefore, PMMA bone cement seems highly suitable to treat patients with cranial bone defects.

IL-6 counteracts the inhibitory effect of IL-4 on osteogenic differentiation of human adipose stem cells.

Fracture repair is characterized by cytokine production and hypoxia. To better predict cytokine modulation of mesenchymal stem cell (MSC)-aided bone healing, we investigated whether interleukin 4 (IL-4), IL-6, and their combination, affect osteogenic differentiation, vascular endothelial growth factor (VEGF) production, and/or mammalian target of rapamycin complex 1 (mTORC1) activation by MSCs under normoxia or hypoxia. Human adipose stem cells (hASCs) were cultured with IL-4, IL-6, or their combination for 3 days under normoxia (20% O 2 ) or hypoxia (1% O 2 ), followed by 11 days without cytokines under normoxia or hypoxia. Hypoxia did not alter IL-4 or IL-6-modulated gene or protein expression by hASCs. IL-4 alone decreased runt-related transcription factor 2 (RUNX2) and collagen type 1 (COL1) gene expression, alkaline phosphatase (ALP) activity, and VEGF protein production by hASCs under normoxia and hypoxia, and decreased mineralization of hASCs under hypoxia. In contrast, IL-6 increased mineralization of hASCs under normoxia, and enhanced RUNX2 gene expression under normoxia and hypoxia. Neither IL-4 nor IL-6 affected phosphorylation of the mTORC1 effector protein P70S6K. IL-4 combined with IL-6 diminished the inhibitory effect of IL-4 on ALP activity, bone nodule formation, and VEGF production, and decreased RUNX2 and COL1 expression, similar to IL-4 alone, under normoxia and hypoxia. In conclusion, IL-4 alone, but not in combination with IL-6, inhibits osteogenic differentiation and angiogenic stimulation potential of hASCs under normoxia and hypoxia, likely through pathways other than mTORC1. These results indicate that cytokines may differentially affect bone healing and regeneration when applied in isolation or in combination.

Mechanical loading releases osteoclastogenesis-modulating factors through stimulation of the P2X7 receptor in hematopoietic progenitor cells.

Mechanical instability of bone implants stimulate osteoclast differentiation and peri-implant bone loss, leading to prosthetic loosening. It is unclear which cells at the periprosthetic interface transduce mechanical signals into a biochemical response, and subsequently facilitate bone loss. We hypothesized that mechanical overloading of hematopoietic bone marrow progenitor cells, which are located near to the inserted bone implants, stimulates the release of osteoclast-inducing soluble factors. Using a novel in vitro model to apply mechanical overloading, we found that hematopoietic progenitor cells released adenosine triphosphate (ATP) after only 2 min of mechanical loading. The released ATP interacts with its specific receptor P2X7 to stimulate the release of unknown soluble factors that inhibit (physiological loading) or promote (supraphysiological loading) the differentiation of multinucleated osteoclasts derived from bone marrow cultures. Inhibition of ATP-receptor P2X7 by Brilliant Blue G completely abolished the overloading-induced stimulation of osteoclast formation. Likewise, stimulation of P2X7 receptor on hematopoietic cells by BzATP enhanced the release of osteoclastogenesis-stimulating signaling molecules to a similar extent as supraphysiological loading. Supraphysiological loading affected neither gene expression of inflammatory markers involved in aseptic implant loosening (e.g., interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α, and PTGES2) nor expression of the osteoclast modulators receptor activator of nuclear factor κ-Β ligand and osteoprotegerin. Our findings suggest that murine hematopoietic progenitor cells are a potential key player in local mechanical loading-induced bone implant loosening via the ATP/P2X7-axis. Our approach identifies potential therapeutic targets to prevent prosthetic loosening.

Supraphysiological loading induces osteocyte-mediated osteoclastogenesis in a novel in vitro model for bone implant loosening.

We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability-induced osteoclast differentiation and peri-implant bone loss. MLO-Y4-osteocytes were mechanically stimulated for 1 h by fluid shear stress using regimes simulating: (i) supraphysiological loading in the peri-prosthetic interface (2.9 ± 2.9 Pa, 1 Hz, square wave); (ii) physiologic loading in the cortical bone (0.7 ± 0.7 Pa, 5 Hz, sinusoidal wave); and (iii) stress shielding. Cellular morphological parameters, membrane-bound RANKL expression, gene expression influencing osteoclast differentiation, nitric oxide release and caspase 3/7-activity were determined. Either Mouse bone marrow cells were cultured on top of loaded osteocytes or osteocyte-conditioned medium was added to bone marrow cells. Osteoclast differentiation was assessed after 6 days. We found that osteocytes subjected to supraphysiological loading showed similar morphology and caspase 3/7-activity compared to simulated physiological loading or stress shielding. Supraphysiological stimulation of osteocytes enhanced osteoclast differentiation by 1.9-fold compared to physiological loading when cell-to-cell contact was permitted. In addition, it enhanced the number of osteoclasts using conditioned medium by 1.7-fold, membrane-bound RANKL by 3.3-fold, and nitric oxide production by 3.2-fold. The stimulatory effect of supraphysiological loading on membrane-bound RANKL and nitric oxide production was higher than that achieved by stress shielding. In conclusion, the in vitro model developed recapitulated the catabolic biological situation in the peri-prosthetic interface during instability that is associated with osteoclast differentiation and enhanced RANKL expression. The model thus provides a platform for pre-clinical testing of pharmacological interventions with potential to stop instability-induced bone implant loosening. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1425-1434, 2018.

Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells.

Renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. Shear stress is altered in renal diseases caused by tubular dilation, obstruction, and hyperfiltration, which occur to compensate for lost nephrons. Fundamental in regulation of shear stress are primary cilia and other mechano-sensors, and defects in cilia formation and function have profound effects on development and physiology of kidneys and other organs. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in renal epithelial cells. Functional enrichment-analysis revealed TGF-ß, MAPK, and Wnt signaling as core signaling pathways up-regulated by shear. Inhibitors of TGF-ß and MAPK/ERK signaling modulate a wide range of mechanosensitive genes, identifying these pathways as master regulators of shear-induced gene expression. However, the main down-regulated pathway, that is, JAK/STAT, is independent of TGF-ß and MAPK/ERK. Other up-regulated cytokine pathways include FGF, HB-EGF, PDGF, and CXC. Cellular responses to shear are modified at several levels, indicated by altered expression of genes involved in cell-matrix, cytoskeleton, and glycocalyx remodeling, as well as glycolysis and cholesterol metabolism. Cilia ablation abolished shear induced expression of a subset of genes, but genes involved in TGF-ß, MAPK, and Wnt signaling were hardly affected, suggesting that other mechano-sensors play a prominent role in the shear stress response of renal epithelial cells. Modulations in signaling due to variations in fluid shear stress are relevant for renal physiology and pathology, as suggested by elevated gene expression at pathological levels of shear stress compared to physiological shear.

Diet and Exercise: a Match Made in Bone.

PURPOSE OF REVIEW: Multiple dietary components have the potential to positively affect bone mineral density in early life and reduce loss of bone mass with aging. In addition, regular weight-bearing physical activity has a strong positive effect on bone through activation of osteocyte signaling. We will explore possible synergistic effects of dietary components and mechanical stimuli for bone health by identifying dietary components that have the potential to alter the response of osteocytes to mechanical loading. RECENT FINDINGS: Several (sub)cellular aspects of osteocytes determine their signaling towards osteoblasts and osteoclasts in response to mechanical stimuli, such as the osteocyte cytoskeleton, estrogen receptor α, the vitamin D receptor, and the architecture of the lacunocanalicular system. Potential modulators of these features include 1,25-dihydroxy vitamin D3, several forms of vitamin K, and the phytoestrogen genistein. Multiple dietary components potentially affect osteocyte function and therefore may have a synergistic effect on bone health when combined with a regime of physical activity.

Histological and micro Computed Tomography analysis of a femoral stress fracture associated with prolonged bisphosphonate use.

BACKGROUND: The origin of atypical femoral fractures (AFF) associated with bisphosphonate therapy remains to be elucidated. In this study, a biopsy of an AFF site is analyzed to determine whether microdamage and/or morphological changes are present in the area of the AFF. MATERIAL AND METHODS: Cortical bone from an AFF region was obtained during a preventive stabilization in a patient with a symptomatic AFF. This bone was scanned using microCT (resolution=0.01 mm), stained with basic fuchsin and analyzed histologically. RESULTS: The diameter of the Haversian canals was higher in the vicinity of the AFF compared to the bone further away from the AFF. The bone mineral density within the cortex ranged from 1020 to 1080 mg HA/cm3. We observed penetration of basic fuchsin into the matrix, which is a tell-tale sign of diffuse damage. DISCUSSION: The higher diameter of haversian canals is likely to result in higher local stresses and consequently increased microdamage. The diffuse microdamage in the biopsy may furthermore be directly related to bisphosphonate use, preventing repair of microdamage, and consequently the development of the AFF. CONCLUSION: Increased porosity of the cortex and accumulation of microdamage might have lead to a stress fracture and ultimately a complete AFF.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia.

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation.

Cyclic Tensile Strain Reduces TNF-α Induced Expression of MMP-13 by Condylar Temporomandibular Joint Cells.

To investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ-derived cells to tumor necrosis factor-α (TNF-α), and whether mechanical loading affects this response. The effect of TNF-α (0.1-10 ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT-qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12 h with TNF-α (10 ng/ml), followed by 8 h of 6% cyclic tensile strain, and gene expression of MMPs was quantified. TNF-α (10 ng/ml) reduced the expression of the matrix proteins collagen types I and II after 6 h of incubation. Aggrecan gene expression was increased in the presence of 0.1 ng/ml TNF-α. The fossa and disc cells responded to TNF-α with an increased expression of the aggrecanase ADAMTS4. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect. Cells isolated from the different cartilaginous structures reacted differently to TNF-α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF-α induced MMP-13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF-α. J. Cell. Physiol. 232: 1287-1294, 2017. © 2016 Wiley Periodicals, Inc.

Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and CXCL8) was observed followed by complete reepithelialization. Seven days after wounding, tissue integrity, metabolic activity, and cytokine levels had returned to the prewounded state. In conclusion, immortalized human gingiva KC and fibroblasts can be used to make physiologically relevant GE, which resemble either the healthy gingiva or a neoplastic disease model. These organotypic models will provide valuable tools to investigate oral mucosa biology and can also be used as an animal alternative for drug targeting, vaccination studies, microbial biofilm studies, and testing new therapeutics.

Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

Biocompatibility of Polypyrrole with Human Primary Osteoblasts and the Effect of Dopants.

Polypyrrole (PPy) is a conducting polymer that enables controlled drug release upon electrical stimulation. We characterized the biocompatibility of PPy with human primary osteoblasts, and the effect of dopants. We investigated the biocompatibility of PPy comprising various dopants, i.e. p-toluene sulfonate (PPy-pTS), chondroitin sulfate (PPy-CS), or dodecylbenzenesulfonate (PPy-DBS), with human primary osteoblasts. PPy-DBS showed the roughest appearance of all surfaces tested, and its wettability was similar to the gold-coated control. The average number of attached cells was 45% higher on PPy-DBS than on PPy-CS or PPy-pTS, although gene expression of the proliferation marker Ki-67 was similar in osteoblasts on all surfaces tested. Osteoblasts seeded on PPy-DBS or gold showed similar vinculin attachment points, vinculin area per cell area, actin filament structure, and Feret's diameter, while cells seeded on PPY-CS or PPY-pTS showed disturbed focal adhesions and were enlarged with disorganized actin filaments. Osteoblasts grown on PPy-DBS or gold showed enhanced alkaline phosphatase activity and osteocalcin gene expression, but reduced osteopontin gene expression compared to cells grown on PPy-pTS and PPy-CS. In conclusion, PPy doped with DBS showed excellent biocompatibility, which resulted in maintaining focal adhesions, cell morphology, cell number, alkaline phosphatase activity, and osteocalcin gene expression. Taken together, conducting polymers doped with DBS are well tolerated by osteoblasts. Our results could provide a basis for the development of novel orthopedic or dental implants with controlled release of antibiotics and pharmaceutics that fight infections or focally enhance bone formation in a tightly controlled manner.

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts.

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2-200 pg/ml) or CCL20 (5-500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5-2.7-fold), ALP (1.6-1.7-fold), and IL-6 protein production (1.3-1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3-5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3-5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.

Mechanical loading reduces inflammation-induced human osteocyte-to-osteoclast communication.

Multiple factors contribute to bone loss in inflammatory diseases such as rheumatoid arthritis (RA), but circulating inflammatory factors and immobilization play a crucial role. Mechanical loading prevents bone loss in the general population, but the effects of mechanical loading in patients with RA are less clear. Therefore, we aimed to investigate whether mechanical stimuli reverse the stimulatory effect of RA serum on osteocyte-to-osteoclast communication. Human primary osteocytes were pretreated with 10 % RA serum or healthy control serum for 7 days, followed by 1 h ± mechanical loading by pulsating fluid flow (PFF). Nitric oxide (NO) and prostaglandin E2 were measured in the medium. Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), interleukin-6 (IL-6), cyclooxygenase-2 (COX2), matrix-extracellular phosphoglycoprotein (MEPE), cysteine-rich protein 61 (CYR61), and SOST gene expression was quantified by qPCR. Osteoclast precursors were cultured with PFF-conditioned medium (PFF-CM) or static-conditioned medium (stat-CM), and osteoclast formation was assessed. RA serum alone did not affect IL-6, CYR61, COX2, MEPE, or SOST gene expression in osteocytes. However, RA serum enhanced the RANKL/OPG expression ratio by 3.4-fold, while PFF nullified this effect. PFF enhanced NO production to the same extent in control serum (2.6-3.5-fold) and RA serum-pretreated (2.7-3.6-fold) osteocytes. Stat-CM from RA serum-pretreated osteocytes enhanced osteoclastogenesis compared with stat-CM from control serum-pretreated osteocytes, while PFF nullified this effect. In conclusion, RA serum, containing inflammatory factors, did not alter the intrinsic capacity of osteocytes to sense mechanical stimuli, but upregulated osteocyte-to-osteoclast communication. Mechanical loading nullified this upregulation, suggesting that mechanical stimuli could contribute to the prevention of osteoporosis in inflammatory disease.