Automating three-dimensional osteoarthritis histopathological grading of human osteochondral tissue using machine learning on contrast-enhanced micro-computed tomography.

OBJECTIVE: To develop and validate a machine learning (ML) approach for automatic three-dimensional (3D) histopathological grading of osteochondral samples imaged with contrast-enhanced micro-computed tomography (CEµCT). DESIGN: A total of 79 osteochondral cores from 24 total knee arthroplasty patients and two asymptomatic donors were imaged using CEµCT with phosphotungstic acid -staining. Volumes-of-interest (VOI) in surface (SZ), deep (DZ) and calcified (CZ) zones were extracted depth-wise and subjected to dimensionally reduced Local Binary Pattern -textural feature analysis. Regularized linear and logistic regression (LR) models were trained zone-wise against the manually assessed semi-quantitative histopathological CEµCT grades (diameter = 2 mm samples). Models were validated using nested leave-one-out cross-validation and an independent test set (4 mm samples). The performance was primarily assessed using Mean Squared Error (MSE) and Average Precision (AP, confidence intervals are given in square brackets). RESULTS: Highest performance on cross-validation was observed for SZ, both on linear regression (MSE = 0.49, 0.69 and 0.71 for SZ, DZ and CZ, respectively) and LR (AP = 0.9 [0.77-0.99], 0.46 [0.28-0.67] and 0.65 [0.41-0.85] for SZ, DZ and CZ, respectively). The test set evaluations yielded increased MSE on all zones. For LR, the performance was also best for the SZ (AP = 0.85 [0.73-0.93], 0.82 [0.70-0.92] and 0.8 [0.67-0.9], for SZ, DZ and CZ, respectively). CONCLUSION: We present the first ML-based automatic 3D histopathological osteoarthritis (OA) grading method which also adequately perform on grading unseen data, especially in SZ. After further development, the method could potentially be applied by OA researchers since the grading software and all source codes are publicly available.

Quantifying Subresolution 3D Morphology of Bone with Clinical Computed Tomography.

The aim of this study was to quantify sub-resolution trabecular bone morphometrics, which are also related to osteoarthritis (OA), from clinical resolution cone beam computed tomography (CBCT). Samples (n = 53) were harvested from human tibiae (N = 4) and femora (N = 7). Grey-level co-occurrence matrix (GLCM) texture and histogram-based parameters were calculated from CBCT imaged trabecular bone data, and compared with the morphometric parameters quantified from micro-computed tomography. As a reference for OA severity, histological sections were subjected to OARSI histopathological grading. GLCM and histogram parameters were correlated to bone morphometrics and OARSI individually. Furthermore, a statistical model of combined GLCM/histogram parameters was generated to estimate the bone morphometrics. Several individual histogram and GLCM parameters had strong associations with various bone morphometrics (|r| > 0.7). The most prominent correlation was observed between the histogram mean and bone volume fraction (r = 0.907). The statistical model combining GLCM and histogram-parameters resulted in even better association with bone volume fraction determined from CBCT data (adjusted R2 change = 0.047). Histopathology showed mainly moderate associations with bone morphometrics (|r| > 0.4). In conclusion, we demonstrated that GLCM- and histogram-based parameters from CBCT imaged trabecular bone (ex vivo) are associated with sub-resolution morphometrics. Our results suggest that sub-resolution morphometrics can be estimated from clinical CBCT images, associations becoming even stronger when combining histogram and GLCM-based parameters.

Electromechanical properties of human osteoarthritic and asymptomatic articular cartilage are sensitive and early detectors of degeneration.

OBJECTIVE: To evaluate cross-correlations of ex vivo electromechanical properties with cartilage and subchondral bone plate thickness, as well as their sensitivity and specificity regarding early cartilage degeneration in human tibial plateau. METHOD: Six pairs of tibial plateaus were assessed ex vivo using an electromechanical probe (Arthro-BST) which measures a quantitative parameter (QP) reflecting articular cartilage compression-induced streaming potentials. Cartilage thickness was then measured with an automated thickness mapping technique using Mach-1 multiaxial mechanical tester. Subsequently, a visual assessment was performed by an experienced orthopedic surgeon using the International Cartilage Repair Society (ICRS) grading system. Each tibial plateau was finally evaluated with µCT scanner to determine the subchondral-bone plate thickness over the entire surface. RESULTS: Cross-correlations between assessments decreased with increasing degeneration level. Moreover, electromechanical QP and subchondral-bone plate thickness increased strongly with ICRS grade (ρ = 0.86 and ρ = 0.54 respectively), while cartilage thickness slightly increased (ρ = 0.27). Sensitivity and specificity analysis revealed that the electromechanical QP is the most performant to distinguish between different early degeneration stages, followed by subchondral-bone plate thickness and then cartilage thickness. Lastly, effect sizes of cartilage and subchondral-bone properties were established to evaluate whether cartilage or bone showed the most noticeable changes between normal (ICRS 0) and each early degenerative stage. Thus, the effect sizes of cartilage electromechanical QP were almost twice those of the subchondral-bone plate thickness, indicating greater sensitivity of electromechanical measurements to detect early osteoarthritis. CONCLUSION: The potential of electromechanical properties for the diagnosis of early human cartilage degeneration was highlighted and supported by cartilage thickness and µCT assessments.

Magnetic resonance imaging (MRI)-defined cartilage degeneration and joint pain are associated with poor physical function in knee osteoarthritis - the Oulu Knee Osteoarthritis study.

OBJECTIVE: The main aim was to investigate the associations between Magnetic Resonance Imaging (MRI)-defined structural pathologies of the knee and physical function. DESIGN: A cohort study with frequency matching on age and sex with eighty symptomatic subjects with knee pain and suspicion or diagnosis of knee osteoarthritis (OA) and 57 asymptomatic subjects was conducted. The subjects underwent knee MRI, and the severity of structural changes was graded by MRI Osteoarthritis Knee Score (MOAKS) in separate knee locations. WOMAC function subscores were recorded and physical function tests (20-m and 5-min walk, stair ascending and descending, timed up & go and repeated sit-to-stand tests) performed. The association between MRI-defined structural pathologies and physical function tests and WOMAC function subscores were evaluated by linear regression analysis with adjustment for demographic factors, other MRI-features and pain with using effect size (ES) as a measure of the magnitude of an association. RESULTS: Cartilage degeneration showed significant association with poor physical performance in TUG-, stair ascending and descending-, 20-m- and 5-min walk-tests (ESs in the subjects with cartilage degeneration anywhere between 0.134 [95%CI 0.037-0.238] and 0.224 [0.013-0.335]) and with increased WOMAC function subscore (ES in the subjects with cartilage degeneration anywhere 0.088 [0.012-0.103]). Also, lateral meniscus maceration and extrusion were associated with poor performance in stair ascending test (ESs 0.067 [0.008-0.163] and 0.077 [0.012-0.177]). CONCLUSIONS: After adjustments cartilage degeneration was associated with both decreased self-reported physical function and poor performance in the physical function tests. Furthermore, subjects with lateral meniscus maceration and extrusions showed significantly worse performance in stair ascending tests.

Perfluoroalkyl substances in human bone: concentrations in bones and effects on bone cell differentiation.

Perfluoroalkyl substances (PFAS), including two most commonly studied compounds perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are widely distributed environmental pollutants, used extensively earlier. Due to their toxicological effects the use of PFAS is now regulated. Based on earlier studies on PFOA's distribution in bone and bone marrow in mice, we investigated PFAS levels and their possible link to bone microarchitecture of human femoral bone samples (n = 18). Soft tissue and bone biopsies were also taken from a 49-year old female cadaver for PFAS analyses. We also studied how PFOA exposure affects differentiation of human osteoblasts and osteoclasts. PFAS were detectable from all dry bone and bone marrow samples, PFOS and PFOA being the most prominent. In cadaver biopsies, lungs and liver contained the highest concentrations of PFAS, whereas PFAS were absent in bone marrow. Perfluorononanoic acid (PFNA) was present in the bones, PFOA and PFOS were absent. In vitro results showed no disturbance in osteogenic differentiation after PFOA exposure, but in osteoclasts, lower concentrations led to increased resorption, which eventually dropped to zero after increase in PFOA concentration. In conclusion, PFAS are present in bone and have the potential to affect human bone cells partly at environmentally relevant concentrations.

3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography.

OBJECTIVE: Histopathological grading of osteochondral (OC) tissue is widely used in osteoarthritis (OA) research, and it is relatively common in post-surgery in vitro diagnostics. However, relying on thin tissue section, this approach includes a number of limitations, such as: (1) destructiveness, (2) sample processing artefacts, (3) 2D section does not represent spatial 3D structure and composition of the tissue, and (4) the final outcome is subjective. To overcome these limitations, we recently developed a contrast-enhanced µCT (CEµCT) imaging technique to visualize the collagenous extracellular matrix (ECM) of articular cartilage (AC). In the present study, we demonstrate that histopathological scoring of OC tissue from CEµCT is feasible. Moreover, we establish a new, semi-quantitative OA µCT grading system for OC tissue. RESULTS: Pathological features were clearly visualized in AC and subchondral bone (SB) with µCT and verified with histology, as demonstrated with image atlases. Comparison of histopathological grades (OARSI or severity (0-3)) across the characterization approaches, CEµCT and histology, excellent (0.92, 95% CI = [0.84, 0.96], n = 30) or fair (0.50, 95% CI = [0.16, 0.74], n = 27) intra-class correlations (ICC), respectively. A new µCT grading system was successfully established which achieved an excellent cross-method (µCT vs histology) reader-to-reader intra-class correlation (0.78, 95% CI = [0.58, 0.89], n = 27). CONCLUSIONS: We demonstrated that histopathological information relevant to OA can reliably be obtained from CEµCT images. This new grading system could be used as a reference for 3D imaging and analysis techniques intended for volumetric evaluation of OA pathology in research and clinical applications.

Imaging of Osteoarthritic Human Articular Cartilage using Fourier Transform Infrared Microspectroscopy Combined with Multivariate and Univariate Analysis.

The changes in chemical composition of human articular cartilage (AC) caused by osteoarthritis (OA) were investigated using Fourier transform infrared microspectroscopy (FTIR-MS). We demonstrate the sensitivity of FTIR-MS for monitoring compositional changes that occur with OA progression. Twenty-eight AC samples from tibial plateaus were imaged with FTIR-MS. Hyperspectral images of all samples were combined for K-means clustering. Partial least squares regression (PLSR) analysis was used to compare the spectra with the OARSI grade (histopathological grading of OA). Furthermore, the amide I and the carbohydrate regions were used to estimate collagen and proteoglycan contents, respectively. Spectral peak at 1338 cm(-1) was used to estimate the integrity of the collagen network. The layered structure of AC was revealed using the carbohydrate region for clustering. Statistically significant correlation was observed between the OARSI grade and the collagen integrity in the superficial (r = -0.55) and the deep (r = -0.41) zones. Furthermore, PLSR models predicted the OARSI grade from the superficial (r = 0.94) and the deep (r = 0.77) regions of the AC with high accuracy. Obtained results suggest that quantitative and qualitative changes occur in the AC composition during OA progression, and these can be monitored by the use of FTIR-MS.

Associations between MRI-defined structural pathology and generalized and localized knee pain - the Oulu Knee Osteoarthritis study.

OBJECTIVE: To determine the associations between multi-feature structural pathology assessed using magnetic resonance imaging (MRI) and the presence of knee pain, and to determine the associations between the locations of structural changes and different knee pain patterns. METHOD: Eighty symptomatic subjects with knee pain and suspicion or diagnosis of knee OA and 63 asymptomatic subjects underwent knee MRI. Severity of structural changes was graded by MRI Osteoarthritis Knee Score (MOAKS) in separate knee locations. The associations between cartilage damage, bone marrow lesions (BMLs), osteophytes, Hoffa's synovitis, effusion-synovitis, meniscal damage and structural pathologies in ligaments, tendons and bursas and both the presence of pain and the knee pain patterns were assessed. RESULTS: The presence of Hoffa's synovitis (adjusted RR 1.6, 95% CI 1.2-1.3) and osteophytes in any region (2.07, 1.19-3.60) was significantly associated with the presence of pain. Any Hoffa's synovitis was associated with patellar pain (adjusted RR 4.70, 95% CI 1.19-3.60) and moderate-to-severe Hoffa's synovitis with diffuse pain (2.25, 1.13-4.50). Medial knee pain was associated with cartilage loss in the medial tibia (adjusted RR 2.66, 95% CI 1.22-5.80), osteophytes in the medial tibia (2.66, 1.17-6.07) and medial femur (2.55, 1.07-6.09), medial meniscal maceration (2.20, 1.01-4.79) and anterior meniscal extrusions (2.78, 1.14-6.75). CONCLUSIONS: Hoffa's synovitis and osteophytes were strongly associated with the presence of knee pain. Medial pain was associated most often with medially located structural pathologies.

Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways.

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography.

OBJECTIVE: Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. METHOD: We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland-Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. RESULTS: Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4). CONCLUSIONS: PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

In vivo comparison of delayed gadolinium-enhanced MRI of cartilage and delayed quantitative CT arthrography in imaging of articular cartilage.

OBJECTIVE: To compare delayed gadolinium-enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) and delayed quantitative computed tomography (CT) arthrography (dQCTA) to each other, and their association to arthroscopy. Additionally, the relationship between dGEMRIC with intravenous (dGEMRIC(IV)) and intra-articular contrast agent administration (dGEMRIC(IA)) was determined. DESIGN: Eleven patients with knee pain were scanned at 3 T MRI and 64-slice CT before arthroscopy. dQCTA was performed at 5 and 45 min after intra-articular injection of ioxaglate. Both dGEMRIC(IV) and dGEMRIC(IA) were performed at 90 min after gadopentetate injection. dGEMRIC indices and change in relaxation rates (ΔR(1)) were separately calculated for dGEMRIC(IV) and dGEMRIC(IA). dGEMRIC and dQCTA parameters were calculated for predetermined sites at the knee joint that were International Cartilage Repair Society (ICRS) graded in arthroscopy. RESULTS: dQCTA normalized with the contrast agent concentration in synovial fluid (SF) and dGEMRIC(IV) correlated significantly, whereas dGEMRIC(IA) correlated with the normalized dQCTA only when dGEMRIC(IA) was also normalized with the contrast agent concentration in SF. Correlation was strongest between normalized dQCTA at 45 min and ΔR(1,IV) (r(s) = 0.72 [95% CI 0.56-0.83], n = 49, P < 0.01) and ΔR(1,IA) normalized with ΔR(1) in SF (r(s) = 0.70 [0.53-0.82], n = 52, P < 0.01). Neither dGEMRIC nor dQCTA correlated with arthroscopic grading. dGEMRIC(IV) and non-normalized dGEMRIC(IA) were not related while ΔR(1,IV) correlated with normalized ΔR(1,IA) (r(s) = 0.52 [0.28-0.70], n = 50, P < 0.01). CONCLUSIONS: This study suggests that dQCTA is in best agreement with dGEMRIC(IV) at 45 min after CT contrast agent injection. dQCTA and dGEMRIC were not related to arthroscopy, probably because the remaining cartilage is analysed in dGEMRIC and dQCTA, whereas in arthroscopy the absence of cartilage defines the grading. The findings indicate the importance to take into account the contrast agent concentration in SF in dQCTA and dGEMRIC(IA).

Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13.

Human mesenchymal stem cells (hMSCs) are multipotent cells that are found in the bone marrow. Inflammation and tissue damage mobilize MSCs and induce their migration towards the damaged site through mechanisms that are not well defined. Toll-like receptor-9 (TLR9) is a cellular receptor for microbial and vertebrate DNA. Stimulation of TLR9 induces inflammatory and invasive responses in TLR9-expressing cells. We studied here the expression of TLR9 in human MSCs and the effects of synthetic TLR9-agonists on their invasion. Constitutive expression of TLR9 was detected in human MSCs but the expression was suppressed when MSCs were induced to differentiate into osteoblasts. Using standard invasion assays and a novel organotypic culture model based on human myoma tissue, we discovered that stimulation with the TLR9 agonistic, CpG oligonucleotides increased the invasion capacity of undifferentiated MSCs. Simultaneously, an increase in MMP-13 synthesis and activity was detected in the CpG-activated MSCs. Addition of anti-MMP-13 antibody significantly diminished the CpG-induced hMSC invasion. We conclude that treatment with TLR9-ligands increases MSC invasiveness, and this process is at least partially MMP-13-mediated.

Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor-avidin fusion protein.

The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force microscopy studies revealed the expression of the fusion protein on cell membranes, and demonstrated specific and high-affinity binding of biotin to the low-density lipoprotein receptor (LDLR)-avidin fusion protein in vitro. Additionally, systemically administered biotinylated ligand targeted with high specificity the intracerebral tumors of rats that were expressing fusion protein after the virus-mediated gene transfer. These results suggest that local gene transfer of the fusion protein to target tissues may offer a novel tool for the delivery of biotinylated molecules in vitro and in vivo for therapeutic and imaging purposes.

Alendronate disturbs vesicular trafficking in osteoclasts.

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.

Behaviour of nitinol in osteoblast-like ROS-17 cell cultures.

Nickel titanium shape memory metal alloy Nitinol (NiTi) has been used in dental wares and in gastrointestinal surgery. Nitinol is a promising implant material in orthopedics, but its biocompatibility, especially in long-term implantation is not confirmed yet. We studied Nitinol's effect on a cell culture model. Comparisons to stainless steel, pure titanium and pure nickel were performed. The effects of Nitinol on cell death rate, the apoptosis rate and the formation of local contacts were studied on rat osteosarcoma cell line ROS-17 in 48-h cultures. The cell death rate was assessed with combined calcein-ethidium-homodimer labelling. The amount of dead cells 1000 cells were as follows: four in the NiTi, 21 in the Stst, 4.8 in the Ti and 51 in the Ni group. In the NiTi and Ti groups, the number of dead cells was significantly lower (p < or = 0.01) than in Ni group. The rate of apoptosis was detected with TUNEL-assay. The assay results were: 1.93 apoptotic cells 1000 cells in the NiTi, 1.1 in the Stst, 2.98 in the Ti and 0.62 in the Ni group. A comparison of these two results shows that 48% of the dead cells were apoptotic in the NiTi, 56.6 in the Stst, 62% in the Ti and only 1.8% in the Ni group. The focal contacts were stained with a paxillin antibody and counted. There were marked differences in the number of focal contacts per unit area compared to NiTi (774 focal contacts): 335 in Stst (p < or = 0.01), 462 in Ti (p < or = 0.01) and 261 in Ni (p < or = 0.005). Our results show that NiTi is well tolerated by the osteoblastic type ROS-17 cells.

Estrogen reduces the depth of resorption pits by disturbing the organic bone matrix degradation activity of mature osteoclasts.

Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.

Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions.

In this study, atomic force microscopy (AFM) was used to mechanically stimulate primary osteoblasts. In response to mechanical force applied by the AFM, the indented cell increased its intracellular calcium concentration. The material properties of the cell could be estimated and the membrane strains calculated. We proceeded to validate this technique experimentally and a 20% error was found between the predicted and the measured diameter of indentation. We also determined the strain distributions within the cell that result from AFM indentation using a simple finite element model. This enabled us to formulate hypotheses as to the mechanism through which cells may sense the applied mechanical strains. Finally, we report the effect of the Poisson ratio and the cell thickness on the strain distributions. Varying the Poisson ratio did not change the order of magnitude of the strains; whereas the cellular thickness dramatically changed the order of magnitude of the cellular strains. We conclude that AFM can be used for controlled mechanical stimulation of osteoblasts and that cellular strain distributions can be computed with a good accuracy when the cell is indented in its highest part.

Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptor subtypes in mouse calvarial osteoblasts: presence of VIP-2 receptors and differentiation-induced expression of VIP-1 receptors.

Three distinct complementary DNAs for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been cloned and designated VIP-1 receptor (VIP-1R), VIP-2 receptor (VIP-2R), and PACAP receptor (PACAP-R). In the present study, we have characterized the binding sites on primary mouse calvarial osteoblasts for VIP and related peptides. By analyzing the cAMP response, the rank order of response observed was PACAP 38 > PACAP 27 > helodermin > VIP > helospectin > glucagon > PHI >>> secretin. The VIP-2R/PACAP-R antagonist, PACAP 6-38, inhibited both VIP- and PACAP-stimulated cAMP formation. Binding studies using an atomic force microscopy (AFM) technique showed high affinity binding for VIP and PACAP 38, but not for secretin. Radioligand binding studies using (125)I-VIP and (125)I-PACAP 38 demonstrated a more specific and higher affinity binding for PACAP 38 than for VIP. Secretin failed to inhibit both (125)I-VIP and (125)I-PACAP 38 binding. RT-PCR demonstrated that undifferentiated mouse calvarial osteoblasts express messenger RNA for VIP-2R, but not for VIP-1R or PACAP-R. When the osteoblasts were cultured for 20 days to induce bone noduli formation, VIP-1R, in addition to VIP-2R, were expressed when the nodules started to mineralize at 12 days. Taken together, these data demonstrate that mouse calvarial osteoblasts express functional VIP-2R with higher affinity binding for PACAP than for VIP and that the VIP-1R expression is induced during osteoblastic differentiation.

Vasoactive intestinal peptide regulates osteoclast activity via specific binding sites on both osteoclasts and osteoblasts.

Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.