Comparative transcriptomic analysis identifies distinct molecular signatures and regulatory networks of chondroclasts and osteoclasts.

BACKGROUND: Chondroclasts and osteoclasts have been previously identified as the cells capable of resorbing mineralized cartilage and bone matrices, respectively. While both cell types appear morphologically similar, contain comparable ultrastructural features, and express tartrate-resistant acid phosphatase (TRAP), however, no information is available about the genomic similarities and differences between osteoclasts and chondroclasts. METHODS: To address this question, we laser captured homogeneous populations of TRAP-positive cells that interact with bone (osteoclasts) and TRAP-positive cells that interact with mineralized cartilage (chondroclasts) on the same plane from murine femoral fracture callus sections. We then performed a global transcriptome profiling of chondroclasts and osteoclasts by utilizing a mouse genome Agilent GE 4X44K V2 microarray platform. Multiple computational approaches and interaction networks were used to analyze the transcriptomic landscape of osteoclasts and chondroclasts. RESULTS: Our systematic and comprehensive analyses using hierarchical clustering and principal component analysis (PCA) demonstrate that chondroclasts and osteoclasts are transcriptionally distinct cell populations and exhibit discrete transcriptomic signatures as revealed by multivariate analysis involving scatter plot, volcano plot, and heatmap analysis. TaqMan qPCR was used to validate the microarray results. Intriguingly, the functional enrichment and integrated network analyses revealed distinct Gene Ontology terms and molecular pathways specific to chondroclasts and osteoclasts and further suggest that subsets of metabolic genes were specific to chondroclasts. Protein-protein interaction (PPI) network analysis showed an abundance of structured networks of metabolic pathways, ATP synthesis, and proteasome pathways in chondroclasts. The regulatory network analysis using transcription factor-target gene network predicted a pool of genes including ETV6, SIRT1, and ATF1 as chondroclast-specific gene signature. CONCLUSIONS: Our study provides an important genetic resource for further exploration of chondroclast function in vivo. To our knowledge, this is the first demonstration of genetic landscape of osteoclasts from chondroclasts identifying unique molecular signatures, functional clustering, and interaction network.

Serum Cartilage Biomarkers and Shoulder Instability.

Differences in cartilage biomarkers have been noted in patients with anterior cruciate ligament tears, but little is known about any similar relationship with shoulder instability. This study evaluated the relationship between serum cartilage biomarkers and shoulder instability. The authors present a prospective cohort study of young athletes followed from 2006 to 2010. A nested case-control analysis was conducted within this cohort to evaluate the association between preinjury collagen type II cleavage (a marker for type II collagen cleavage) and procollagen II carboxy propeptide (a marker of cartilage synthesis) and the subsequent likelihood of shoulder instability during the 4-year follow-up period. Preinjury collagen type II cleavage and procollagen II carboxy propeptide levels in 51 subjects who had shoulder instability were compared with levels in 210 subjects without documented anterior cruciate ligament or shoulder instability (control group) with commercially available enzyme-linked immunosorbent assay kits. Mean preinjury collagen type II cleavage levels in patients who subsequently had shoulder instability were significantly lower than those in the control group (73.91 vs 79.24 pg/mL, P=.03). No significant difference was found in preinjury procollagen II carboxy propeptide levels compared with the control group (359.94 vs 396.37, P=.24). This study is the first to examine the relationship between baseline collagen biomarkers and subsequent shoulder instability. The finding of lower baseline collagen type II cleavage levels in patients with subsequent shoulder instability may represent a genetic predisposition or a compensatory mechanism by which cartilage degradation is decreased in those who are more likely to have instability. [Orthopedics. 2017; 40(1):34-36.].

Association Between Serum Relaxin and Subsequent Shoulder Instability.

Ligamentous laxity correlates with shoulder instability. Relaxin is a hormone that has been linked to laxity in the knee and has been shown to be a risk factor for anterior cruciate ligament (ACL) injury. This study prospectively evaluated the association between relaxin and acute shoulder instability. A prospective cohort study of 1050 young athletes was performed between 2006 and 2010. The authors conducted a nested case-control analysis within this cohort to evaluate the association between preinjury serum relaxin concentration and the likelihood of subsequent shoulder instability. The study compared 53 patients who had shoulder instability and 53 control subjects who were matched for sex, age, height, and weight. The serum relaxin concentration in preinjury baseline samples was tested with enzyme-linked immunosorbent assay analysis in duplicate. Independent t tests were performed to identify differences in mean serum relaxin concentration between patients with shoulder instability and uninjured control subjects. Logistic regression was used to evaluate whether preinjury baseline serum relaxin concentration was associated with the subsequent likelihood of shoulder instability. Of the 53 patients with instability, 13 (25%) had a detectable serum relaxin concentration compared with 9 (17%) of uninjured control subjects (P=.34). Mean serum relaxin concentration in the injury group was 3.69±1.78 pg/mL and 2.20±0.97 pg/mL in uninjured control subjects (P=.02). Increased serum relaxin concentration was associated with the subsequent likelihood of acute shoulder instability. Subjects were 2.18 times (odds ratio, 2.18; 95% confidence interval, 1.01-4.76) more likely to have acute shoulder instability during the follow-up period for every 1-pg/mL increase in serum relaxin concentration at baseline. The findings suggest that serum relaxin concentration is associated with a risk of subsequent shoulder instability in young athletes. Further research on the role of relaxin in shoulder instability is warranted. [Orthopedics. 2016; 39(4):e724-e728.].

Pregnancy-associated plasma protein-A modulates the anabolic effects of parathyroid hormone in mouse bone.

Intermittent parathyroid hormone (PTH) is a potent anabolic therapy for bone, and several studies have implicated local insulin-like growth factor (IGF) signaling in mediating this effect. The IGF system is complex and includes ligands and receptors, as well as IGF binding proteins (IGFBPs) and IGFBP proteases. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease expressed by osteoblasts in vitro that has been shown to enhance local IGF action through cleavage of inhibitory IGFBP-4. This study was set up to test two specific hypotheses: 1) Intermittent PTH treatment increases the expression of IGF-I, IGFBP-4 and PAPP-A in bone in vivo, thereby increasing local IGF activity. 2) In the absence of PAPP-A, local IGF activity and the anabolic effects of PTH on bone are reduced. Wild-type (WT) and PAPP-A knock-out (KO) mice were treated with 80 µg/kg human PTH 1-34 or vehicle by subcutaneous injection five days per week for six weeks. IGF-I, IGFBP-4 and PAPP-A mRNA expression in bone were significantly increased in response to PTH treatment. PTH treatment of WT mice, but not PAPP-A KO mice, significantly increased expression of an IGF-responsive gene. Bone mineral density (BMD), as measured by DEXA, was significantly decreased in femurs of PAPP-A KO compared to WT mice with PTH treatment. Volumetric BMD, as measured by pQCT, was significantly decreased in femoral midshaft (primarily cortical bone), but not metaphysis (primarily trabecular bone), of PAPP-A KO compared to WT mice with PTH treatment. These data suggest that stimulation of PAPP-A expression by intermittent PTH treatment contributes to PTH bone anabolism in mice.

Detection of relaxin receptor in the dorsoradial ligament, synovium, and articular cartilage of the trapeziometacarpal joint.

Basilar thumb osteoarthritis (OA) is postulated to occur due to ligament attenuation of the trapeziometacarpal (TM) joint. Relaxin is a peptide hormone, which loosens ligaments before childbirth, through remodeling of the extracellular matrix via upregulation of matrix metalloproteases (MMPs). We postulated that relaxin family peptide receptor 1 (RXFP-1), the receptor for circulating relaxin, was present in tissues of the TM joint. Ligaments and synovium were sampled from 15 patients during surgery for TM arthritis. We obtained trapezial cartilage from two autopsy donors and four patients. Tissues were fixed, paraffin embedded, and sectioned at 5 µm, then were immunostained for RXFP-1, as well as MMP-1, and MMP-13, using rabbit anti-human polyclonal antibodies. Eight DRL samples showed positive immunostaining for relaxin receptor, with 14/15 positively stained in synovium. Greater staining was seen in specimens obtained from women with more severe TM arthritis. Trapezial cartilage demonstrated receptor staining within chondrocytes in the middle and deep zones. Immunostaining for MMPs co-localized with relaxin receptor staining. Relaxin receptors are present at the ligament, cartilage, and synovium of the TM joint, indicating that it is a potential target for relaxin. This suggests that circulating relaxin may impact joint stability. The role of relaxin in cartilage and synovium may be related to its role in collagen regulation as a possible tissue response to OA.

Effects of Wnt5a Haploinsufficiency on Bone Repair.

OBJECTIVES: Wnt5a expression is upregulated during fracture repair and has previously been implicated as a potential regulator of skeletal development and bone mass accrual and maintenance. Our objective was to evaluate the function of Wnt5a in fracture healing. METHODS: Femoral fracture experiments on Wnt5a and Wnt5a mice were carried out. To better understand the effect of the Wnt5a on bone repair, we evaluated radiographs using a previously validated qualitative scoring system and performed microcomputed tomography analyses. Histomorphometric analyses determined the temporal distribution of stroma, cartilage matrix, and woven bone in the fracture callus. Finally, we performed tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining to visualize and quantify bone resorbing cells. RESULTS: Radiographic evaluations at day 21 demonstrated significantly higher cortical remodeling and bridging parameters for the Wnt5a group compared with the Wnt5a group. The bone volume fraction by microcomputed tomography was also significantly increased in Wnt5a mice. Histological and histomorphometric analyses showed that although Wnt5a mice exhibit decreased cartilage matrix production at day 7 postfracture, they displayed increased residual cartilaginous callus at days 14 and 21 compared with the Wnt5a group. In addition, the total number of multinucleated tartrate-resistant acid phosphatase-positive cells was significantly lower in the Wnt5a group than in the Wnt5a group. CONCLUSIONS: The data indicate that decreased Wnt5a signaling impaired proper fracture healing, possibly through decreased cartilaginous callus formation, and delayed cartilage matrix and mineralized tissue remodeling within the fracture callus.

Relationship of serum relaxin to generalized and trapezial-metacarpal joint laxity.

PURPOSE: The reproductive hormone relaxin acts to loosen pelvic ligaments in preparation for childbirth and is thought to be a mediator of joint laxity. The purpose of this study was to evaluate the correlation of serum relaxin with radiographic laxity at the trapezial-metacarpal joint and with generalized joint laxity. METHODS: We enrolled 289 healthy subjects prospectively. Participants completed a demographic questionnaire and were examined for generalized joint hypermobility using the Beighton-Horan scale. Stress radiographs of the trapezial-metacarpal joint were obtained in 163 subjects (56%). Blood samples were collected, and serum relaxin was measured for 287 subjects using enzyme-linked immunosorbent assay for human relaxin-2. RESULTS: The mean serum relaxin level among all subjects was 1.84 pg/mL (range, 0-45.25 pg/mL). Relaxin was not detectable in 166 of 287 samples, whereas the mean serum relaxin level among the 121 subjects with a detectable relaxin level (of 287 total relaxin samples) was 4.37 pg/mL (range, 0.46-45.25 pg/mL). Mean trapezial-metacarpal subluxation ratio scores were higher among those with a detectable relaxin level compared to those without a detectable relaxin level (0.34 vs 0.30 pg/mL). The average Beighton-Horan laxity score was 1.8 (range, 0-9). There was no correlation between generalized joint laxity measures and serum relaxin levels. CONCLUSIONS: In a large volunteer population, we demonstrated a relationship between circulating relaxin and trapezial-metacarpal joint laxity. However, we were unable to show a direct link between serum relaxin and generalized joint laxity. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic II.

Serum relaxin levels in young athletic men are comparable with those in women.

Relaxin was originally described as a reproductive hormone that mediated joint laxity in pregnant women and has been minimally studied in men. The purpose of this descriptive laboratory and clinical study was to evaluate serum relaxin in a young, primarily male population and compare levels between the sexes. In addition, the authors evaluated the relationship between relaxin and generalized laxity.

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen.

Consequences of age on the effects of hyperbaric oxygen (HBO) on bone marrow (BM) derived stem cells and progenitors (SCPs) are largely unknown. We treated 2- and 18-month old C57BL/6 female mice by HBO. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-ß and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold). Our data demonstrate the impact of aging on the response of BM SCPs to HBO and indicate the potentially different age-related benefit of HBO in wound healing and tissue remodeling.

Pregnancy-associated plasma protein-A2 (PAPP-A2): tissue expression and biological consequences of gene knockout in mice.

Pregnancy-associated plasma protein-A2 (PAPP-A2) is a novel homolog of PAPP-A in the metzincin superfamily. However, compared with the accumulating data on PAPP-A, very little is known about PAPP-A2. In this study, we determined the tissue expression pattern of PAPP-A2 mRNA in wild-type (WT) mice and characterized the phenotype of mice with global PAPP-A2 deficiency. Tissues expressing PAPP-A2 in WT mice were more limited than those expressing PAPP-A. The highest PAPP-A2 mRNA expression was found in the placenta, with abundant expression in fetal, skeletal, and reproductive tissues. Heterozygous breeding produced the expected Mendelian distribution for the pappa2 gene and viable homozygous PAPP-A2 knockout (KO) mice that were normal size at birth. The most striking phenotype of the PAPP-A2 KO mouse was postnatal growth retardation. Male and female PAPP-A2 KO mice had 10 and 25-30% lower body weight, respectively, than WT littermates. Adult femur and body length were also reduced in PAPP-A2 KO mice, but without significant effects on bone mineral density. PAPP-A2 KO mice were fertile, but with compromised fecundity. PAPP-A expression was not altered to compensate for the loss of PAPP-A2 expression, and proteolysis of PAPP-A2's primary substrate, IGF-binding protein-5, was not altered in fibroblasts from PAPP-A2 KO embryos. In conclusion, tissue expression patterns and biological consequences of gene KO indicate distinct physiological roles for PAPP-A2 and PAPP-A in mice.

How tough is bone? Application of elastic-plastic fracture mechanics to bone.

Bone, with a hierarchical structure that spans from the nano-scale to the macro-scale and a composite design composed of nano-sized mineral crystals embedded in an organic matrix, has been shown to have several toughening mechanisms that increases its toughness. These mechanisms can stop, slow, or deflect crack propagation and cause bone to have a moderate amount of apparent plastic deformation before fracture. In addition, bone contains a high volumetric percentage of organics and water that makes it behave nonlinearly before fracture. Many researchers used strength or critical stress intensity factor (fracture toughness) to characterize the mechanical property of bone. However, these parameters do not account for the energy spent in plastic deformation before bone fracture. To accurately describe the mechanical characteristics of bone, we applied elastic-plastic fracture mechanics to study bone's fracture toughness. The J integral, a parameter that estimates both the energies consumed in the elastic and plastic deformations, was used to quantify the total energy spent before bone fracture. Twenty cortical bone specimens were cut from the mid-diaphysis of bovine femurs. Ten of them were prepared to undergo transverse fracture and the other 10 were prepared to undergo longitudinal fracture. The specimens were prepared following the apparatus suggested in ASTM E1820 and tested in distilled water at 37 degrees C. The average J integral of the transverse-fractured specimens was found to be 6.6 kPa m, which is 187% greater than that of longitudinal-fractured specimens (2.3 kPa m). The energy spent in the plastic deformation of the longitudinal-fractured and transverse-fractured bovine specimens was found to be 3.6-4.1 times the energy spent in the elastic deformation. This study shows that the toughness of bone estimated using the J integral is much greater than the toughness measured using the critical stress intensity factor. We suggest that the J integral method is a better technique in estimating the toughness of bone.

Effect of temperature on the fracture toughness of compact bone.

Bone is a composite composed mainly of organics, minerals, and water. Many researchers have studied effects such as crack velocity, density, orientation, storage media, porosity, and age on the fracture toughness (K(C), also called critical stress intensity factor) of compact bone. Most of these studies were conducted at room temperature. Considering that the body temperature of animals is greater than room temperature, and that bone has a large volumetric percentage of organics and water (generally, 55-65%), it is hypothesized that temperature has a significant effect on the fracture toughness of compact bone. Single-edge V-notched (SEVN) specimens were prepared to measure the fracture toughness of bovine femur and manatee rib in water at 0, 10, 23, 37, and 50 degrees C in four-point flexure. The fracture toughness values of bovine femur and manatee rib were found to decrease from 7.0 to 4.3MPam(1/2) and from 5.5 to 4.0MPam(1/2), respectively, as temperature increased over a temperature range of 50 degrees C. The results support the hypothesis that temperature has a significant effect on the fracture toughness of compact bone. Therefore, we suggest that study on fracture toughness of bone should be done at physiologically relevant temperatures.

Application of fracture mechanics to failure in manatee rib bone.

BACKGROUND: The Florida manatee (Trichechus manatus latirostris) is listed as endangered by the U.S. Department of the Interior. Manatee ribs have different microstructure from the compact bone of other mammals. Biomechanical properties of the manatee ribs need to be better understood. Fracture toughness (K(C)) has been shown to be a good index to assess the mechanical performance of bone. Quantitative fractography can be used in concert with fracture mechanics equations to identify fracture initiating defects/cracks and to calculate the fracture toughness of bone materials. METHOD OF APPROACH: Fractography is a standard technique for analyzing fracture behavior of brittle and quasi-brittle materials. Manatee ribs are highly mineralized and fracture in a manner similar to quasi-brittle materials. Therefore, quantitative fractography was applied to determine the fracture toughness of manatee ribs. RESULTS: Average fracture toughness values of small flexure specimens from six different sizes of manatees ranged from 1.3 to 2.6 MPa(m)(12). Scanning electron microscope (SEM) images show most of the fracture origins were at openings for blood vessels and interlayer spaces. CONCLUSIONS: Quantitative fractography and fracture mechanics can be combined to estimate the fracture toughness of the material in manatee rib bone. Fracture toughness of subadult and calf manatees appears to increase as the size of the manatee increases. Average fracture toughness of the manatee rib bone materials is less than the transverse fracture toughness of human and bovine tibia and femur.

Fracture toughness of manatee rib and bovine femur using a chevron-notched beam test.

Bone is an anisotropic material with a hierarchical structure consisting of organic matrix, minerals and water. Fracture toughness (K(C)) has been shown to be a good index to assess the mechanical performance of bone. A chevron-notched (CN) beam test, a standard fracture mechanics test successfully applied to many other materials, was used to determine the transverse-direction fracture toughness in manatee rib and bovine femur cortical bone. Although human and bovine bone has been well studied, there is virtually no information on the toughness of manatee rib bone. As a biological material, manatee rib is interesting for study in that it is a highly mineralized bone. Three major advantages of the CN specimen test are: (1) it is easier to reach plane strain condition; (2) there is no fatigue-precracking needed; and (3) it is relatively easy to produce stable crack propagation before catastrophic fracture. The fracture toughness values of manatee rib and bovine femur were measured to be 4.5 +/- 0.5 MPa m(1/2) and 5.8 +/- 0.5 MPa m(1/2), respectively. Based on the microstructures shown in SEM images, two features that contributed to the greater fracture toughness of bovine femur were identified as greater osteon density and lesser porosity.