Effect of HIP/ribosomal protein L29 deficiency on mineral properties of murine bones and teeth

Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues

CK2.1, a bone morphogenetic protein receptor type Ia mimetic peptide, repairs cartilage in mice with destabilized medial meniscus

Abstract Background Osteoarthritis (OA) of the knee involves degeneration of articular cartilage of the diarthrodial joints. Current treatment options temporarily relieve the joint pain but do not restore the lost cartilage. We recently designed a novel bone morphogenetic protein receptor type I (BMPRI) mimetic peptide, CK2.1, that activates BMPRIa signaling in the absence of bone morphogenetic protein (BMP). Our previous research demonstrated that CK2.1 induced chondrogenesis in vitro and in vivo; however, it is unknown if CK2.1 restores damaged articular cartilage in vivo. In this study, we demonstrate that CK2.1 induced articular cartilage (AC) repair in an OA mouse model. Methods We designed hyaluronic acid (HA)-based hydrogel particles (HGPs) that slowly release CK2.1. HGP-CK2.1 particles were tested for chondrogenic potency on pluripotent mesenchymal stem cells (C3H10T1/2 cells) and locally injected into the intra-articular capsule in mice with cartilage defects. C57BL/6J mice were operated on to destabilize the medial meniscus and these mice were kept for 6 weeks after surgery to sustain OA-like damage. Mice were then injected via the intra-articular capsule with HGP-CK2.1; 4 weeks after injection the mice were sacrificed and their femurs were analyzed for cartilage defects. Results Immunohistochemical analysis of the cartilage demonstrated complete repair of the AC compared to sham-operated mice. Immunofluorescence analysis revealed collagen type IX production along with collagen type II in the AC of mice injected with HGP-CK2.1. Mice injected with phosphate-buffered saline (PBS) and HGP alone had greater collagen type X and osteocalcin production, in sharp contrast to those injected with HGP-CK2.1, indicating increased chondrocyte hypertrophy. Conclusions Our results demonstrate that the slow release HGP-CK2.1 drives cartilage repair without the induction of chondrocyte hypertrophy. The peptide CK2.1 could be a powerful tool in understanding the signaling pathways contributing to the repair process, and also may be used as a potential therapeutic for treating degenerative cartilage diseases such as OA

Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes

Publisher's PDF.Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3- E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degenerationUniversity of Delaware. Department of Biological Sciences.University of Delaware. Biomedical Engineering Program.University of Delaware. Department of Mechanical Engineering

Decrease of OA damage in T-VSCC KO vs. WT controls following <i>in vivo</i> knee loading.

<p>A: boxes and whiskers graph showing the median (central line), 25–75% (box) and the entire range (whiskers) of average histological OA scores obtained three weeks after final loading in the four compartments (MT-Medial Tibia, MF- Medial Femur, LT-Lateral Tibia, LF- Lateral Femur) of wild type control and T-VSCC KO mouse knees. B and C are coronal knee sections stained with Safranin O and Fast green from either a WT or a T-VSCC KO loaded knee, respectively. Arrow in B indicates a focal lesion caused by loading in the lateral knee compartment of a WT mouse knee. * p = 0.012, ** p = 0.027 and *** p = 0.001; n = 10 for wild type and n = 7 for T-VSCC KO mice. An average of 15 slides representative of the entire knee were blinded and scored by two independent observers using scoring system modified from Glasson <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127290#pone.0127290.ref024" target="_blank">24</a>].</p

The fluid shear stress (FSS)-induced expression of the early shear response marker cyclooxygenase 2 and the late shear response shear response gene osteopontin are inhibited in MC3T3-E1 cells with the addition of T-VSCC-specific inhibitor, NNC55-0396 (NNC).

<p><b>A</b>: quantitative PCR analysis shows that the marked increase in Cox2 (Ptgs2) mRNAs observed 2hrs following FSS relative to the static untreated (Static UT) control condition is significantly inhibited in the presence of NNC. The conditions are FSS untreated (FSS UT), FSS treated with NNC (FSS NNC), static treated with NNC (static NNC). Error bars represent standard error of mean of biological duplicates and * indicates p = 0.026 between FSS UT and FSS NNC, ** p = 0.006 between FSS UT and static NNC. <b>B</b>: western blot analysis performed under the same conditions described in A indicates that the FSS-induced increase of COX2 protein is decreased to control levels in the presence of NNC. Vinculin was used as a loading control. <b>C</b>: Quantitative PCR analysis shows that the increase in osteopontin (Spp1) mRNAs observed 20hrs following FSS relative to the static untreated (Static UT) control condition is significantly inhibited in the presence of NNC. The conditions are FSS untreated (FSS UT), FSS treated with NNC 55–0396 (FSS NNC), static treated with NNC (static NNC). Error bars represent standard error of mean of biological duplicates and *** p<0.0001 between FSS UT and FSS NNC or FSS UT and static NNC.</p

<i>In-vivo</i> loading system used to experimentally induce knee OA and compare progression of disease severity between T-VSCC and WT control knees.

<p>(A) Radiograph of mouse knee joint during loading using the Bose ElectroForce loading apparatus, (B) loading cycle waveform including a 0.025sec of rise time, 0.05sec peak load time, 0.025sec fall time and 4.9sec holding/resting time, (C) loading regimen: Five loading episodes were performed over a period of eight days followed by a 26-day period of non loading prior to sacrifice (week 5) and histological processing.</p

FSS triggers a response in chondrocytes that is reversed in the presence of NNC55-0396 (NNC) without altering the expression of transcripts encoding for markers of cartilage ECM.

<p>Real time PCR analysis showing the relative fold changes in the mRNA levels of cyclooxygenase 2 (ptgs2), collagen X (Col10a1), matrix metalloproteinase 13 (Mmp13), aggrecan (Acan), collagen II (Col2a1), and alkaline phosphatase (Alpl) in primary mouse chondrocytes grown in monolayer and collected 20hrs after FSS alone (FSS UT), FSS with NNC (FSS NNC) or maintained under static conditions with NNC (Static NNC) compared with the untreated static control condition (Static UT). The error bars represent standard error of mean of biological duplicates.</p