Differential Effects of PTH (1-34), PTHrP (1-36), and Abaloparatide on the Murine Osteoblast Transcriptome.

Teriparatide (PTH (1-34)), PTHrP (1-36), and abaloparatide (ABL) have been used for the treatment of osteoporosis, but their efficacy long term is significantly limited. The 3 peptides exert time- and dose-dependent differential responses in osteoblasts, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. Treatment of mouse calvarial osteoblasts with 1 nM of the peptides for 4 hours results in RNA sequencing data with PTH (1-34) regulating 367 genes, including 194 unique genes; PTHrP (1-36) regulating 117 genes, including 15 unique genes; and ABL regulating 179 genes, including 20 unique genes. There were 83 genes shared among all 3 peptides. Gene ontology analyses showed similarities in Wnt signaling, cAMP-mediated signaling, ossification, but differences in morphogenesis of a branching structure in biological processes; receptor ligand activity, transcription factor activity, and cytokine receptor/binding activity in molecular functions. The peptides increased Vdr, Cited1, and Pde10a messenger RNAs (mRNAs) in a pattern similar to Rankl, that is, PTH (1-34) greater than ABL greater than PTHrP (1-36). mRNA abundance of other genes, including Wnt4, Wnt7, Wnt11, Sfrp4, Dkk1, Kcnk10, Hdac4, Epn3, Tcf7, Crem, Fzd5, Ppp2r2a, and Dvl3, showed that some genes were regulated similarly by all 3 peptides; others were not. Finally, small interfering RNA knockdowns of SIK1/2/3 and CRTC1/2/3 in PTH (1-34)-treated cells revealed that Vdr and Wnt4 genes are regulated by salt-inducible kinases (SIKs) and CREB-regulated transcriptional coactivators (CRTCs), while others are not. Although many studies have examined PTH signaling in the osteoblast/osteocyte, ours is the first to compare the global effects of these peptides on the osteoblast transcriptome or to analyze the roles of the SIKs and CRTCs.

Differential effects of PTH (1-34), PTHrP (1-36) and abaloparatide on the murine osteoblast transcriptome.

Teriparatide (PTH(1-34)) and its analogs, PTHrP(1-36) and abaloparatide (ABL) have been used for the treatment of osteoporosis, but their efficacy over long-term use is significantly limited. The 3 peptides exert time- and dose-dependent differential responses in osteoblasts, leading us to hypothesize that they may also differentially modulate the osteoblast transcriptome. We show that treatment of mouse calvarial osteoblasts with 1 nM of the 3 peptides for 4 h results in RNA-Seq data with PTH(1-34) regulating 367 genes, including 194 unique genes; PTHrP(1-36) regulating 117 genes, including 15 unique genes; and ABL regulating 179 genes, including 20 unique genes. There were 83 genes shared among all 3 peptides. Gene ontology analyses showed differences in Wnt signaling, cAMP-mediated signaling, bone mineralization, morphogenesis of a branching structure in biological processes; receptor ligand activity, transcription factor activity, cytokine receptor/binding activity and many other actions in molecular functions. The 3 peptides increased Vdr, Cited1 and Pde10a mRNAs in a pattern similar to Rankl , i.e., PTH(1-34) > ABL > PTHrP(1-36). mRNA abundance of other genes based on gene/pathway analyses, including Wnt4, Wnt7, Wnt11, Sfrp4, Dkk1, Kcnk10, Hdac4, Epha3, Tcf7, Crem, Fzd5, Pp2r2a , and Dvl3 showed that some genes were regulated similarly by all 3 peptides; others were not. Finally, siRNA knockdowns of SIK1/2/3 and CRTC1/2/3 in PTH(1-34)-treated cells revealed that Vdr and Wnt4 genes are regulated by SIKs and CRTCs, while others are not. Although many studies have examined PTH signaling in the osteoblast/osteocyte, ours is the first to examine the global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH(1-34), PTHrP(1-36) or ABL exclusively and which are shared among all 3 will help improve our understanding of the effects these peptides have on the osteoblast and lead to the refinement of PTH-derived treatments for osteoporosis.

Trends in the theory that inflammation plays a causal role in tendinopathy: a systematic review and quantitative analysis of published reviews.

BACKGROUND/AIMS: The contribution of inflammation to tendinopathy has been debated in the scientific literature. Several factors may contribute to this lack of clarity, including inconsistent definitions of inflammation. We hypothesised that the adoption and/or rejection of a causal link between inflammation and tendinopathy varied as a function of the 'inflammatory component' (eg, immune cell and molecular mediators included in published reviews). METHODS: Twenty data items were collected from each review to determine conclusions about the role of inflammation in tendinopathy, specific definitions of the 'inflammatory component,' quality of the review and other potential correlates. Associations between correlates and a review's conclusion about the role of inflammation in tendinopathy were tested using binomial logistic regression. The database searches retrieved 2261 unique publications: 137 fulfilled inclusion criteria after full text screenings. RESULTS: There has been little support for an inflammatory component to tendinopathy until recently (2012-2015). Prior to 2012, the majority of published reviews did not discuss monocytes, macrophages or lymphocytes in tendinopathy; rather they focused on the lack of neutrophils, often referred to as 'the inflammatory infiltrate', or immune cells were not discussed. Reviews including monocytes and lymphocytes in their discussions were 5.23 times more likely to conclude inflammation was important than reviews that did not, p<0.001. CONCLUSIONS: Data collected show growing support for an inflammatory component to tendinopathy, particularly among high-quality reviews and those that used more robust definitions of inflammation. This finding may have implications for explaining dissonance in the literature regarding a causal role for inflammation in the pathogenesis of tendinopathy.

Biomechanics and Microstructural Analysis of the Mouse Knee and Ligaments.

The purpose of this study is to determine the feasibility of using murine models for translational study of knee ligament injury, repair, and reconstruction. To achieve this aim, we provide objective, quantitative data detailing the gross anatomy, biomechanical characteristics, and microscopic structure of knee ligaments of 44 male mice (C57BL6, 12 weeks of age). Biomechanical testing determined the load-to-failure force, stiffness, and the site of ligament failure for the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), and the medial and lateral collateral ligaments (MCL and LCL). These data are complemented by histological characterization of each of the knee ligaments. In addition, the osseous morphology of the mouse knee was examined using high-resolution nanofocus computed tomography (CT), while standard micro-CT was employed to measure bone morphometrics of the distal femur and proximal tibia. Collectively, our findings suggest that the gross anatomy of the mouse knee is similar to the human knee despite some minor differences and features unique to the murine knee. The ACL had the highest load to failure (5.60 ± 0.75 N), the MCL (3.33 ± 1.45 N), and the PCL (3.45 ± 0.84 N) were similar, and the LCL (1.44 ± 0.37 N) had the lowest load to failure and stiffness. Murine models provide a unique opportunity to focus on biological processes that impact ligament pathology and healing due to the availability of transgenic strains. Our data support their use as a translational platform for the in vivo study of ligament injury, repair, and reconstruction.

Differential expression of alarmins-S100A9, IL-33, HMGB1 and HIF-1α in supraspinatus tendinopathy before and after treatment.

BACKGROUND: Alarmins, endogenous molecules released on tissue damage have been shown to play an important role in inflammatory musculoskeletal conditions including fracture repair andrheumatoid arthritis. However, the contribution of alarmins to the pathogenesis of tendon disease is not fully understood. METHODS: We investigated expression of alarmin proteins (S100A9, high-mobility group box 1 (HMGB1) and interleukin-33 (IL-33) and hypoxia-inducible factor 1α (HIF-1α), a subunit of an oxygen sensitive transcription factor, in three cohorts of human supraspinatus tissues: healthy (n=6), painful diseased (n=13) and post-treatment pain-free tendon samples (n=5). Tissue samples were collected during shoulder stabilisation surgery (healthy) or by biopsy needle (diseased/treated). Immunohistochemistry was used to investigate the protein expression of these factors in these healthy, diseased and treated tendons. Kruskal-Wallis with pairwise post hoc Mann-Whitney U tests were used to test for differences in immunopositive staining between these tissue cohorts. Additionally, costaining was performed to identify the cell types expressing HIF-1α, S100A9, IL-33 and HMGB1 in diseased tendons. RESULTS: Immunostaining showed HIF-1α and S100A9 were increased in diseased compared with healthy and post-treatment pain-free tendons (p<0.05). IL-33 was reduced in diseased compared with healthy tendons (p=0.0006). HMGB1 was increased in post-treatment pain-free compared with healthy and diseased tendons (p<0.01). Costaining of diseased tendon samples revealed that HIF-1α, S100A9 and IL-33 were expressed by CD68+ and CD68- cells, whereas HMGB1 was predominantly expressed by CD68- cells. CONCLUSIONS: This study provides insight into the pathways contributing to the progressionand resolution of tendon disease. We found potential pro-inflammatory and pathogenic roles for HIF-1α and S100A9, a protective role fornuclear IL-33 and a potentially reparative function for HMGB1 in diseased supraspinatus tendons.

Involvement of Indian hedgehog signaling in mesenchymal stem cell-augmented rotator cuff tendon repair in an athymic rat model.

BACKGROUND: Bone marrow aspirate has been used in recent years to augment tendon-to-bone healing, including in rotator cuff repair. However, the healing mechanism in cell-based therapy has not been elucidated in detail. METHODS: Sixteen athymic nude rats were randomly allocated to 2 groups: experimental (human mesenchymal stem cells in fibrin glue carrier) and control (fibrin glue only). Animals were sacrificed at 2 and 4 weeks. Immunohistochemical staining was performed to evaluate Indian hedgehog (Ihh) signaling and SOX9 signaling in the healing enthesis. Macrophages were identified using CD68 and CD163 staining, and proliferating cells were identified using proliferating cell nuclear antigen staining. RESULTS: More organized and stronger staining for collagen II and a higher abundance of SOX9+ cells were observed at the enthesis in the experimental group at 2 weeks. There was significantly higher Gli1 and Patched1 expression in the experimental group at the enthesis at 2 weeks and higher numbers of Ihh+ cells in the enthesis of the experimental group vs control at both 2 weeks and 4 weeks postoperatively. There were more CD68+ cells localized to the tendon midsubstance at 2 weeks compared with 4 weeks, and there was a higher level of CD163 staining in the tendon midsubstance in the experimental group than in the control group at 4 weeks. CONCLUSION: Stem cell application had a positive effect on fibrocartilage formation at the healing rotator cuff repair site. Both SOX9 and Ihh signaling appear to play an important role in the healing process.

Platelet-rich plasma for muscle injuries: game over or time out?

Muscle injuries are common and may be associated with impaired functional capacity, especially among athletes. The results of healing with conventional therapy including rest, ice, compression, and elevation (RICE) are often inadequate, generating substantial interest in the potential for emerging technologies such as platelet-rich plasma (PRP) to enhance the process of soft-tissue healing and to decrease time to recovery. In vitro studies and animal research have suggested that PRP may have benefits associated with the increased release of cytokines and growth factors resulting from supraphysiological concentrations of platelets that facilitate muscle repair, regeneration, and remodeling. Despite the promise of basic science, there is a paucity of clinical data to support the theoretical benefits of PRP. The only double-blind controlled clinical trial was recently reported and showed no benefit of PRP in the time to resume sports activity among athletes with hamstring muscle injury. This review examines the current evidence and the theoretical framework for PRP and muscle healing. Scientific gaps and technological barriers are discussed that must be addressed if the potential promise of PRP as a therapeutic modality for muscle injury is to be realized.