Recombinant bone matrix maintains the graft space, induces vascularized bone regeneration and preserves canine tooth extraction socket structure.

AIM: Recombinant bone matrix (RBM) is a newly conceived and engineered porous bone graft granule of average size 600 µm composed of purified recombinant collagen peptide. We sought to examine the behaviour with time of RBM that was grafted in the canine tooth extraction socket. MATERIALS AND METHODS: The canine tooth extraction socket of the hemisectioned mandibular third premolar distal root was grafted with RBM granules, whereas the opposite side extraction socket served as non-grafted control. The mandibular samples were harvested at 1, 3 and 6 months of healing and subjected to micro-CT imaging and decalcified paraffin-embedded histology. Separately, the effect of RBM was compared with that of deproteinized cancellous bovine bone (DCBB) and bovine atelocollagen plug (BACP) in the canine tooth extraction model at 3 months of healing. RESULTS: RBM maintained the grafted space in the socket and the gingival connective tissue until new bone was formed within its porous space. The regenerated bone was highly vascularized and continued to mature, while RBM was completely bioresorbed by 6 months. The buccal and lingual alveolar ridge heights of the RBM-grafted extraction socket was better preserved than those of non-grafted control sockets. The degree of socket preservation by RBM was equivalent to that by DCBB, although their healing mechanisms were different. CONCLUSIONS: This study demonstrated that RBM induced controlled active bone regeneration and preserved the extraction socket structure in a canine model. Bioresorbable RBM engineered without animal or human source materials presents a novel bone graft category with robust bone regenerative property.

Zoledronic acid mediated differential activation of NK cells in different organs of WT and Rag2-/- mice; stark differences between the bone marrow and gingivae.

We have previously shown that natural killer (NK) cells expand, and increase their function after interaction with cells that exhibit a number of different knock-down genes. We hypothesized that deletion or knockdown of a variety of key genes such as RAG may cause de-differentiation of the cells which could lead to increased NK expansion and function since we have shown previously that NK cells are activated and expanded by less differentiated cells. When comparing the function of NK cells from bone marrow (BM), spleen, pancreas, adipose tissue, and gingiva from WT mice to those from Rag2-/- mice, we observed a significant increase in IFN-γ secretion in all tissues of Rag2-/- mice versus in WT mice, with the exception of the gingivae in which similar levels were observed. After injecting WT mice with zoledronic acid (ZOL) and tooth extraction, immune cells from BM, spleen, and purified NK cells from spleen exhibited very high induction of IFN-γ and NK cell-mediated cytotoxicity with the exception of gingiva in which immune cells exhibited the opposite. In Rag2-/- mice, ZOL injection and tooth extraction stimulated IFN-γ secretion from BM immune cells but inhibited IFN-γ secretion from both spleen and gingivae. In both WT and Rag2-/- mice, immune cells from gingivae exhibited decreased IFN-γ secretion when activated, indicating significant regulation of immune cell function in the gingival microenvironment. However, even though significantly lower induction of IFN-γ was observed in both WT and Rag2-/- gingival cells after ZOL injection, ZOL mediated secretion of IFN-γ was still higher in the gingivae of WT mice when compared to those of Rag2-/- gingival cells. These results suggest an important role for IFN-γ in the pathogenesis of osteonecrosis lesions observed in post-tooth extraction jawbone.

Raman Spectroscopy of Oral Candida Species: Molecular-Scale Analyses, Chemometrics, and Barcode Identification.

Oral candidiasis, a common opportunistic infection of the oral cavity, is mainly caused by the following four Candida species (in decreasing incidence rate): Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei. This study offers in-depth Raman spectroscopy analyses of these species and proposes procedures for an accurate and rapid identification of oral yeast species. We first obtained average spectra for different Candida species and systematically analyzed them in order to decode structural differences among species at the molecular scale. Then, we searched for a statistical validation through a chemometric method based on principal component analysis (PCA). This method was found only partially capable to mechanistically distinguish among Candida species. We thus proposed a new Raman barcoding approach based on an algorithm that converts spectrally deconvoluted Raman sub-bands into barcodes. Barcode-assisted Raman analyses could enable on-site identification in nearly real-time, thus implementing preventive oral control, enabling prompt selection of the most effective drug, and increasing the probability to interrupt disease transmission.

Three-Dimensional Culture of Cartilage Tissue on Nanogel-Cross-Linked Porous Freeze-Dried Gel Scaffold for Regenerative Cartilage Therapy: A Vibrational Spectroscopy Evaluation.

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.

Design and Synthesis of Cathepsin-K-Activated Osteoadsorptive Fluorogenic Sentinel (OFS) Probes for Detecting Early Osteoclastic Bone Resorption in a Multiple Myeloma Mouse Model.

We describe the design and synthesis of OFS-1, an Osteoadsorptive Fluorogenic Sentinel imaging probe that is adsorbed by hydroxyapatite (HAp) and bone mineral surfaces, where it generates an external fluorescent signal in response to osteoclast-secreted cathepsin K (Ctsk). The probe consists of a bone-anchoring bisphosphonate moiety connected to a Förster resonance energy transfer (FRET) internally quenched fluorescent (IQF) dye pair, linked by a Ctsk peptide substrate, GHPGGPQG. Key structural features contributing to the effectiveness of OFS-1 were defined by structure-activity relationship (SAR) and modeling studies comparing OFS-1 with two cognates, OFS-2 and OFS-3. In solution or when preadsorbed on HAp, OFS-1 exhibited strong fluorescence when exposed to Ctsk (2.5-20 nM). Time-lapse photomicrographs obtained after seeding human osteoclasts onto HAp-coated well plates containing preadsorbed OFS-1 revealed bright fluorescence at the periphery of resorbing cells. OFS-1 administered systemically detected early osteolysis colocalized with orthotopic engraftment of RPMI-8226-Luc human multiple myeloma cells at a metastatic skeletal site in a humanized mouse model. OFS-1 is thus a promising new imaging tool for detecting abnormal bone resorption.

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice.

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

Plasticity of Myeloid Cells during Oral Barrier Wound Healing and the Development of Bisphosphonate-related Osteonecrosis of the Jaw.

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Osteonecrosis of the Jaw Developed in Mice: DISEASE VARIANTS REGULATED BY γδ T CELLS IN ORAL MUCOSAL BARRIER IMMUNITY.

Osteonecrosis of the jaw (ONJ), an uncommon co-morbidity in patients treated with bisphosphonates (BP), occurs in the segment of jawbone interfacing oral mucosa. This study aimed to investigate a role of oral mucosal barrier γδ T cells in the pathogenesis of ONJ. Female C57Bl/6J (B6) mice received a bolus zoledronate intravenous injection (ZOL, 540 µg/kg), and their maxillary left first molars were extracted 1 week later. ZOL-treated mice (WT ZOL) delayed oral wound healing with patent open wounds 4 weeks after tooth extraction with characteristic oral epithelial hyperplasia. γδ T cells appeared within the tooth extraction site and hyperplastic epithelium in WT ZOL mice. In ZOL-treated γδ T cell null (Tcrd(-/-) ZOL) mice, the tooth extraction open wound progressively closed; however, histological ONJ-like lesions were identified in 75 and 60% of WT ZOL and Tcrd(-/-) ZOL mice, respectively. Although the bone exposure phenotype of ONJ was predominantly observed in WT ZOL mice, Tcrd(-/-) ZOL mice developed the pustule/fistula disease phenotype. We further addressed the role of γδ T cells from human peripheral blood (h-γδ T cells). When co-cultured with ZOL-pretreated human osteoclasts in vitro, h-γδ T cells exhibited rapid expansion and robust IFN-γ secretion. When h-γδ T cells were injected into ZOL-treated immunodeficient (Rag2(-/-) ZOL) mice, the oral epithelial hyperplasia developed. However, Rag2(-/-) ZOL mice did not develop osteonecrosis. The results indicate that γδ T cells are unlikely to influence the core osteonecrosis mechanism; however, they may serve as a critical modifier contributing to the different oral mucosal disease variations of ONJ.

Novel bisphosphonate-based cathepsin K-triggered compound targets the enthesis without impairing soft tissue-to-bone healing.

Background: Osteoadsorptive fluorogenic sentinel 3 (OFS-3) is a recently described compound that contains a bone-targeting bisphosphonate (BP) and cathepsin K (Ctsk)-triggered fluorescence signal. A prior study in a murine Achilles repair model demonstrated its effectiveness at targeting the site of tendon-to-bone repair, but the intrinsic effect of this novel bisphosphonate chaperone on tendon-to-bone healing has not been previously explored. We hypothesized that application of this bisphosphonate-fluorophore cargo conjugate would not affect the biomechanical properties or histologic appearance of tendon-bone repairs. Materials and Methods: Right hindlimb Achilles tendon-to-bone repair was performed on 12-week old male mice. Animals were divided into 2 groups of 18 each: 1) Achilles repair with OFS-3 applied directly to the repair site prior to closure, and 2) Achilles repair with saline applied prior to closure. Repaired hindlimbs from 12 animals per group were harvested at 6 weeks for biomechanical analysis with a custom 3D-printed jig. At 4 and 6 weeks, repaired hindlimbs from the remaining animals were assessed histologically using H&E, immunohistochemistry (IHC) staining for the presence of Ctsk, and second harmonic generation (SHG) imaging to evaluate collagen fibers. Results: At 6 weeks, there was no significant difference in failure load, stiffness, toughness, or displacement to failure between repaired hindlimbs that received OFS-3 versus saline. There was no difference in tissue healing on H&E or Ctsk staining on immunohistochemistry between animals that received OFS-3 versus saline. Finally, second harmonic generation imaging demonstrated no difference in collagen fiber parameters between the two groups. Conclusion: OFS-3 did not significantly affect the biomechanical properties or histologic appearance of murine Achilles tendon-to-bone repairs. This study demonstrates that OFS-3 can target the site of tendon-to-bone repair without causing intrinsic negative effects on healing. Further development of this drug delivery platform to target growth factors to the site of tendon-bone repair is warranted.

Mathematical filtering minimizes metallic halation of titanium implants in MicroCT images.

Microcomputed tomography (MicroCT) images containing titanium implant suffer from x-rays scattering, artifact and the implant surface is critically affected by metallic halation. To improve the metallic halation artifact, a nonlinear Total Variation denoising algorithm such as Split Bregman algorithm was applied to the digital data set of MicroCT images. This study demonstrated that the use of a mathematical filter could successfully reduce metallic halation, facilitating the osseointegration evaluation at the bone implant interface in the reconstructed images.

Current perspectives of residual ridge resorption: Pathological activation of oral barrier osteoclasts.

PURPOSE: Tooth extraction is a last resort treatment for resolving pathological complications of dentition induced by infection and injury. Although the extraction wound generally heals uneventfully, resulting in the formation of an edentulous residual ridge, some patients experience long-term and severe residual ridge reduction. The objective of this review was to provide a contemporary understanding of the molecular and cellular mechanisms that may potentially cause edentulous jawbone resorption. STUDY SELECTION: Clinical, in vivo, and in vitro studies related to the characterization of and cellular and molecular mechanisms leading to residual ridge resorption. RESULTS: The alveolar processes of the maxillary and mandibular bones uniquely juxtapose the gingival tissue. The gingival oral mucosa is an active barrier tissue that maintains homeostasis of the internal organs through its unique barrier immunity. Tooth extraction not only generates a bony socket but also injures oral barrier tissue. In response to wounding, the alveolar bone socket initiates regeneration and remodeling through coupled bone formation and osteoclastic resorption. Osteoclasts are also found on the external surface of the alveolar bone, interfacing the oral barrier tissue. Osteoclasts in the oral barrier region are not coupled with osteoblastic bone formation and often remain active long after the completion of wound healing, leading to a net decrease in the alveolar bone structure. CONCLUSIONS: The novel concept of oral barrier osteoclasts may provide important clues for future clinical strategies to maintain residual ridges for successful prosthodontic and restorative therapies.

Mouse gingival single-cell transcriptomic atlas identified a novel fibroblast subpopulation activated to guide oral barrier immunity in periodontitis.

Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulation activated to guide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete topical application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils, together with AG fibroblasts, appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis of Rag2-/- and Rag2-/-Il2rg-/- mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose the AG fibroblast-neutrophil-ILC3 axis as a previously unrecognized mechanism which could be involved in the complex interplay between oral barrier immune cells contributing to pathological inflammation in periodontitis.

Mouse gingival single-cell transcriptomic atlas: An activated fibroblast subpopulation guides oral barrier immunity in periodontitis.

Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulation activated to guide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils together with AG fibroblasts appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis of Rag2-/- and Rag2γc-/- mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose that AG fibroblasts function as a previously unrecognized surveillant to initiate gingival inflammation leading to periodontitis through the AG fibroblast-neutrophil-ILC3 axis.

Cyclic pressure-induced cytokines from gingival fibroblasts stimulate osteoclast activity: Clinical implications for alveolar bone loss in denture wearers.

Purpose The involvement of oral mucosa cells in mechanical stress-induced bone resorption is unclear. The aim of this study was to investigate the effects of cyclic pressure-induced cytokines from oral mucosal cells (human gingival fibroblasts: hGFs) on osteoclast activity in vitro.Methods Cyclic pressure at 50 kPa, which represents high physiologic occlusal force of dentures on the molar area, was applied to hGFs. NFAT-reporter stable RAW264.7 preosteoclasts (NFAT/Luc-RAW cells) were cultured in conditioned medium collected from hGF cultures under cyclic pressure or static conditions. NFAT activity and osteoclast formation were determined by luciferase reporter assay and TRAP staining, respectively. Cyclic pressure-induced cytokines in hGF culture were detected by ELISA, real-time RT-PCR, and cytokine array analyses.Results Conditioned media from hGFs treated with 48 hours of cyclic pressure significantly induced NFAT activity and increased multinucleated osteoclast formation. Furthermore, the cyclic pressure significantly increased the bone resorption activity of RAW264.7 cells. Cyclic pressure significantly increased the expression of major inflammatory cytokines including IL-1ß/IL-1ß, IL-6/IL-6, IL-8/IL-8 and MCP-1/CCL2 in hGFs compared to hGFs cultured under static conditions, and it suppressed osteoprotegerin (OPG/OPG) expression. A cytokine array detected 12 cyclic pressure-induced candidates. Among them, IL-8, decorin, MCP-1 and ferritin increased, whereas IL-28A and PDGF-BB decreased, NFAT activation of NFAT/Luc-RAW cells.Conclusions These results suggest that cyclic pressure-induced cytokines from hGFs promote osteoclastogenesis, possibly including up-regulation of IL-1ß, IL-6, IL-8 and MCP-1, and down-regulation of OPG. These findings introduce the possible involvement of GFs in mechanical stress-induced alveolar ridge resorption, such as in denture wearers.

Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ.

Introduction: The potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals. Methods: We used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice. Results: Percentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly. Discussion: ZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw.

Biologically-coupled bisphosphonate chaperones effectively deliver molecules to the site of soft tissue-bone healing.

Tendon injuries are common and often treated surgically, however, current tendon repair healing results in poorly organized fibrotic tissue. While certain growth factors have been reported to improve both the strength and organization of the repaired enthesis, their clinical applicability is severely limited due to a lack of appropriate delivery strategies. In this study, we evaluated a recently developed fluorescent probe, Osteoadsorptive Fluorogenic Sentinel-3 that is composed of a bone-targeting bisphosphonate (BP) moiety linked to fluorochrome and quencher molecules joined via a cathepsin K-sensitive peptide sequence. Using a murine Achilles tendon-to-bone repair model, BP-based and/or Ctsk-coupled imaging probes were applied either locally or systemically. Fluorescence imaging was used to quantify the resultant signal in vivo. After tendon-bone repair, animals that received either local or systemic administration of imaging probes demonstrated significantly higher fluorescence signal at the repair site compared to the sham surgery group at all time points (p < 0.001), with signal peaking at 7-10 days after surgery. Our findings demonstrate the feasibility of using a novel BP-based targeting and Ctsk-activated delivery of molecules to the site of tendon-to-bone repair and creates a foundation for further development of this platform as an effective strategy to deliver bioactive molecules to sites of musculoskeletal injury.

Relationship of axonal voltage-gated sodium channel 1.8 (NaV1.8) mRNA accumulation to sciatic nerve injury-induced painful neuropathy in rats.

Painful peripheral neuropathy is a significant clinical problem; however, its pathological mechanism and effective treatments remain elusive. Increased peripheral expression of tetrodotoxin-resistant voltage-gated sodium channel 1.8 (NaV1.8) has been shown to associate with chronic pain symptoms in humans and experimental animals. Sciatic nerve entrapment (SNE) injury was used to develop neuropathic pain symptoms in rats, resulting in increased NaV1.8 mRNA in the injured nerve but not in dorsal root ganglia (DRG). To study the role of NaV1.8 mRNA in the pathogenesis of SNE-induced painful neuropathy, NaV1.8 shRNA vector was delivered by subcutaneous injection of cationized gelatin/plasmid DNA polyplex into the rat hindpaw and its subsequent retrograde transport via sciatic nerve to DRG. This in vivo NaV1.8 shRNA treatment reversibly and repeatedly attenuated the SNE-induced pain symptoms, an effect that became apparent following a distinct lag period of 3-4 days and lasted for 4-6 days before returning to pretreatment levels. Surprisingly, apparent knockdown of NaV1.8 mRNA occurred only in the injured nerve, not in the DRG, during the pain alleviation period. Levels of heteronuclear NaV1.8 RNA were unaffected by SNE or shRNA treatments, suggesting that transcription of the Scn10a gene encoding NaV1.8 was unchanged. Based on these data, we postulate that increased axonal mRNA transport results in accumulation of functional NaV1.8 protein in the injured nerve and the development of painful neuropathy symptoms. Thus, targeted delivery of agents that interfere with axonal NaV1.8 mRNA may represent effective neuropathic pain treatments.

RB1CC1 protein suppresses type II collagen synthesis in chondrocytes and causes dwarfism.

RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations.

High throughput screening of biologically functional small molecules for modulating the expression of FGFR1OP2/wit3.0 in fibroblasts.

Oral wounds heal rapidly without scarring through yet unknown molecular mechanisms. A small cytoskeleton molecule identified in oral wound fibroblasts, FGFR1OP2/wit3.0, has been shown to accelerate wound closure in vitro and in vivo. The objective of this study was to elucidate the transcriptional mechanism of FGFR1OP2/ wit3.0 in fibroblasts using a high throughput drug-screening platform. This pilot study identified chemical compounds that could effectively modulate the FGFR1OP2/wit3.0 expression for future studies on effective wound management.

Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment.

Chronic pain arising from peripheral nerve injuries represents a significant clinical challenge because even the most efficacious anticonvulsant drug treatments are limited by their side effects profile. We investigated pain behavior, changes in axonal signal conduction and excitability of trigeminal neurons, and expression of voltage-gated sodium channels (NaVs) in the infraorbital nerve and trigeminal ganglion (TG) after infraorbital nerve entrapment (IoNE). Compared to Sham, IoNE rats had increased A- and C-fiber compound action potentials (CAPs) and Aδ component of A-CAP area from fibers innervating the vibrissal pad. After IoNE, A- and C-fiber CAPs were more sensitive to blockade by tetrodotoxin (TTX), and those fibers that were TTX-resistant were more sensitive to blockade by the NaV1.8 selective blocker, A-803467. Although NaV1.7 blocker, ICA-121431 alone, did not affect Aδ-fiber signal propagation, cumulative application with A-803467 and 4,9-anhydro-TTX significantly reduced the Aδ-fiber CAP in IoNE rats. In patch clamp recordings from small- and medium-sized TG neurons, IoNE resulted in reduced action potential (AP) depolarizing current threshold, hyperpolarized AP voltage threshold, increased AP duration, and a more depolarized membrane potential. While the transcripts of most NaVs were reduced in the ipsilateral TG after IoNE, NaV1.3, NaV1.7, and NaV1.8 mRNAs, and NaV1.8 protein, were significantly increased in the nerve. Altogether, our data suggest that axonal redistribution of NaV1.8, and to a lesser extent NaV1.3, and NaV1.7 contributes to enhanced nociceptive signal propagation in peripheral nerve after IoNE.