Epithelial Cdc42 Deletion Induced Enamel Organ Defects and Cystogenesis.

Cdc42, a Rho family small GTPase, regulates cytoskeleton organization, vesicle trafficking, and other cellular processes in development and homeostasis. However, Cdc42's roles in prenatal tooth development remain elusive. Here, we investigated Cdc42 functions in mouse enamel organ. Cdc42 showed highly dynamic temporospatial patterns in the developing enamel organ, with robust expression in the outer enamel epithelium, stellate reticulum (SR), and stratum intermedium layers. Strikingly, epithelium-specific Cdc42 deletion resulted in cystic lesions in the enamel organ. Cystic lesions were first noted at embryonic day 15.5 and progressively enlarged during gestation. At birth, cystic lesions occupied the bulk of the entire enamel organ, with intracystic erythrocyte accumulation. Ameloblast differentiation was retarded upon epithelial Cdc42 deletion. Apoptosis occurred in the Cdc42 mutant enamel organ prior to and synchronously with cystogenesis. Transmission electron microscopy examination showed disrupted actin assemblies, aberrant desmosomes, and significantly fewer cell junctions in the SR cells of Cdc42 mutants than littermate controls. Autophagosomes were present in the SR cells of Cdc42 mutants relative to the virtual absence of autophagosome in the SR cells of littermate controls. Epithelium-specific Cdc42 deletion attenuated Wnt/ß-catenin and Shh signaling in dental epithelium and induced aberrant Sox2 expression in the secondary enamel knot. These findings suggest that excessive cell death and disrupted cell-cell connections may be among multiple factors responsible for the observed cystic lesions in Cdc42 mutant enamel organs. Taken together, Cdc42 exerts multidimensional and pivotal roles in enamel organ development and is particularly required for cell survival and tooth morphogenesis.

Insulin-like peptides regulate vitellogenesis and oviposition in the green lacewing, Chrysopa septempunctata.

Insulin-like peptides (ILPs) act through a conserved insulin signaling pathway and play crucial roles in insect metabolism, growth, reproduction, and aging. Application of bovine insulin is able to increase vitellogenin (Vg) mRNA and protein levels in female insects. Here, we first show that injection of bovine insulin into previtellogenic Chrysopa septempunctata female adults promoted ovarian growth, increased Vg protein abundance, elevated reproductive performance, and enhanced protease activity. These data suggested that ILPs play crucial roles in reproductive regulation of the green lacewing, C. septempunctata.

Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal resorption in the root canal, pulp/dentin regeneration, and root resorption in orthodontic tooth movement.

Soft tissue ossification and condylar cartilage degeneration following TMJ disc perforation in a rabbit pilot study.

OBJECTIVE: There are limited clinical treatments for temporomandibular joint (TMJ) pathologies, including degenerative disease, disc perforation and heterotopic ossification (HO). One barrier hindering the development of new therapies is that animal models recapitulating TMJ diseases are poorly established. The objective of this study was to develop an animal model for TMJ cartilage degeneration and disc pathology, including disc perforation and soft tissue HO. METHODS: New Zealand white rabbits (n = 9 rabbits) underwent unilateral TMJ disc perforation surgery and sham surgery on the contralateral side. A 2.5 mm defect was created using a punch biopsy in rabbit TMJ disc. The TMJ condyles and discs were evaluated macroscopically and histologically after 4, 8 and 12 weeks. Condyles were blindly scored by four independent observers using OARSI recommendations for macroscopic and histopathological scoring of osteoarthritis (OA) in rabbit tissues. RESULTS: Histological evidence of TMJ condylar cartilage degeneration was apparent in experimental condyles following disc perforation relative to sham controls after 4 and 8 weeks, including surface fissures and loss of Safranin O staining. At 12 weeks, OARSI scores indicated experimental condylar cartilage erosion into the subchondral bone. Most strikingly, HO occurred within the TMJ disc upon perforation injury in six rabbits after 8 and 12 weeks. CONCLUSION: We report for the first time a rabbit TMJ injury model that demonstrates condylar cartilage degeneration and disc ossification, which is indispensible for testing the efficacy of potential TMJ therapies.

Both genes and lncRNAs can be used as biomarkers of prostate cancer by using high throughput sequencing data.

OBJECTIVE: To investigate prostate cancer-related genes and lncRNAs by using a high throughput sequencing dataset. MATERIALS AND METHODS: RNA-seq data were obtained from the sequencing read archive database, including both benign and malignant tumor samples. After aligning the RNA-seq reads to human genome reference, gene expression profile as well as lncRNA expression profile was obtained. Next, student's t-test was used to screen both the differentially expressed genes (DEGs) and lncRNAs (DELs) between benign and malignant samples. Finally, goseq was used to conduct the functional annotation of DEGs. RESULTS: A total of 7112 DEGs were screened, such as ZNF512B, UCKL1, STMN3, GMEB2, and PTK6. The top 10 enriched functions of DEGs were mainly related to organism development, including multi-cellular development, system development and anatomical structure development. Also, we discovered 26 differentially expressed lncRNAs. CONCLUSIONS: The analysis used in this study is reliable in screening prostate cancer markers including both genes and lncRNAs by using RNA-seq data, which provides new insight into the understanding of molecular mechanism of prostate cancer.

Physiologic load-bearing characteristics of autografts, allografts, and polymer-based scaffolds in a critical sized segmental defect of long bone: an experimental study.

BACKGROUND: To address the challenge of treating critical sized intercalary defects, we hypothesized that under physiologic cyclic loading, autografts, allografts, and scaffolds loaded with and without human mesenchymal stem cells (hMSCs) would have different biomechanical characteristics. METHODS: Using a rat femoral defect model, 46 rats were assigned to four groups, ie, autograft (n = 12), allograft (n = 10), scaffold (n = 13), and scaffold with hMSCs (n = 11). The scaffold groups used a 5 mm segment of scaffold composed of 80% poly-ε-caprolactone and 20% hydroxyapatite. Rats were sacrificed 4 months postoperatively, and the repairs were assessed radiographically and biomechanically. RESULTS: Autograft and allograft groups exhibited the most bridging callus, while the scaffold/hMSCs group had more callus than the scaffold repairs. Although signs of radiographic healing did not accurately reflect restoration of mechanical properties, addition of hMSCs on the scaffold enhanced bone formation. The scaffold alone group had significantly lower elastic and viscous stiffness and higher phase angles than other repairs and the contralateral controls. Addition of hMSCs increased the elastic and viscous stiffness of the repair, while decreasing the phase angle. CONCLUSION: Further comparative analysis is needed to optimize clinical use of scaffolds and hMSCs for critical sized defect repairs. However, our results suggest that addition of hMSCs to scaffolds enhances mechanical simulation of native host bone.

Sustained inflammation induces degeneration of the temporomandibular joint.

The temporomandibular joint (TMJ) undergoes degenerative changes among patients who suffer from arthritis, and yet the pathogenesis of TMJ osteoarthritis and rheumatoid arthritis is poorly understood. We hypothesized that sustained inflammation in the TMJ induces structural abnormalities, and accordingly characterized the disc and synovium in a novel model with double injections of complete Freund's adjuvant (CFA), using behavioral, morphological, cellular, and molecular assessments. Thirty-five days following double CFA injections in seven-week-old female Sprague-Dawley rats, the disc in the CFA-induced inflammation group demonstrated multiple degenerative changes, including marked thickening, opacity, and deformation. The discs in the CFA group further showed significantly greater wet and net weights, and elevated collagen, aggrecan, and total glycosaminoglycan contents. The synovium in the CFA-induced inflammation group showed marked infiltration of mononucleated cells and accumulated sub-synovial adipose tissue. Both the disc and synovium had significantly higher iNOS and IL-1ß mRNA expression than controls (saline injections). These findings are consistent with our hypothesis that sustained TMJ inflammation, even within the presently observed 35 days, may be a predisposing factor for structural abnormalities. Insight into TMJ inflammation and degeneration is anticipated to improve our understanding of the pathogenesis of TMJ arthritis and help design clinically relevant strategies for tissue engineering.

Identification of WA-type three-line hybrid rice with real-time polymerase chain reaction (PCR) method.

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2₋630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R2₋630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3₋14) were designed based on the R2₋630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg²âº concentration is 3.5 mmol/L.

Induced migration of dental pulp stem cells for in vivo pulp regeneration.

Dental pulp has intrinsic capacity for self-repair. However, it is not clear whether dental pulp cells can be recruited endogenously for regenerating pulp tissues, including mineralizing into dentin. This work is based on a hypothesis that dental pulp stem/progenitor cells can be induced to migrate by chemotactic cytokines and act as endogenous cell sources for regeneration and mineralization. Dental stem cells (DSCs) were isolated from adult human tooth pulp and seeded on the surfaces of 3D collagen gel cylinders that were incubated in chemically defined media with stromal-derived factor-1α (SDF1), basic fibroblast growth factor (bFGF), or bone morphogenetic protein-7 (BMP7). Significantly more cells were recruited into collagen gel by SDF1 or bFGF than without cytokines in 7 days, whereas BMP7 had little effect on cell recruitment. BMP7, however, was highly effective, equally to dexamethasone, in orchestrating mineralization of cultured DSCs. Cell membrane receptors for SDF1, bFGF, and BMP7 were up-regulated in treated DSCs. Upon in vivo delivery, bFGF induced re-cellularization and re-vascularization in endodontically treated human teeth implanted into the dorsum of rats. Thus, endogenous dental pulp cells, including stem/progenitor cells, may be recruited and subsequently differentiated by chemotaxis of selective cytokines in the regeneration of dental pulp.

Stroke after treatment with bortezomib and dexamethasone in a Chinese patient with extramedullary relapse of multiple myeloma.

Thromboembolic complications commonly occur in patients with multiple myeloma (MM). The risk of such complications may be elevated by the use of immunomodulatory agents such as thalidomide and lenalidomide as initial therapy for MM. However, arterial thrombosis after treatment with bortezomib is rare. Herein we report a case of a 70-year-old Chinese male patient with extramedullary relapse of MM. After treatment with bortezomib and dexamethasone he developed a nonfatal thrombotic stroke. Administration of bortezomib and dexamethasone was then discontinued and he obtained partial remission.

Anatomically shaped tooth and periodontal regeneration by cell homing.

Tooth regeneration by cell delivery encounters translational hurdles. We hypothesized that anatomically correct teeth can regenerate in scaffolds without cell transplantation. Novel, anatomically shaped human molar scaffolds and rat incisor scaffolds were fabricated by 3D bioprinting from a hybrid of poly-epsilon-caprolactone and hydroxyapatite with 200-microm-diameter interconnecting microchannels. In each of 22 rats, an incisor scaffold was implanted orthotopically following mandibular incisor extraction, whereas a human molar scaffold was implanted ectopically into the dorsum. Stromal-derived factor-1 (SDF1) and bone morphogenetic protein-7 (BMP7) were delivered in scaffold microchannels. After 9 weeks, a putative periodontal ligament and new bone regenerated at the interface of rat incisor scaffold with native alveolar bone. SDF1 and BMP7 delivery not only recruited significantly more endogenous cells, but also elaborated greater angiogenesis than growth-factor-free control scaffolds. Regeneration of tooth-like structures and periodontal integration by cell homing provide an alternative to cell delivery, and may accelerate clinical applications.

Craniofacial tissue engineering by stem cells.

Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

Tissue-engineered rabbit cranial suture from autologous fibroblasts and BMP2.

Craniosynostosis is a congenital disorder of premature ossification of cranial sutures, occurring in one of approximately every 2500 live human births. This work addressed a hypothesis that a cranial suture can be tissue-engineered from autologous cells. Dermal fibroblasts were isolated subcutaneously from growing rabbits, culture-expanded, and seeded in a gelatin scaffold. We fabricated a composite tissue construct by sandwiching the fibroblast-seeded gelatin scaffold between two collagen sponges loaded with recombinant human BMP2. Surgically created, full-thickness parietal defects were filled with the composite tissue construct in the same rabbits from which dermal fibroblasts had been obtained. After four-week in vivo implantation, there was de novo formation of tissue-engineered cranial suture, microscopically reminiscent of the adjacent natural cranial suture. The tissue-engineered cranial suture showed radiolucency on radiographic images, in contrast to radio-opacity of microscopically ossified calvarial defects filled with fibroblast-free, BMP2-loaded constructs. This approach may be refined for tissue engineering of cranial sutures for craniosynostosis patients.

Nanomechanical properties of facial sutures and sutural mineralization front.

The mechanical properties of craniofacial sutures have rarely been investigated. Three facial sutures-the pre-maxillomaxillary (PMS), the nasofrontal (NFS), and the zygomaticotemporal (ZTS)-and their corresponding sutural mineralization fronts in 8 young New Zealand White rabbits were subjected to nano-indentation with atomic force microscopy as a test of the hypothesis that they have different mechanical properties. The average elastic modulus of the PMS was 1.46 +/- 0.24 MPa (mean +/- SD), significantly higher than both the ZTS (1.20 +/- 0.20) and NFS (1.16 +/- 0.18). The average elastic moduli of sutural mineralization fronts 30 micro m away were significantly higher than their corresponding sutures and had the same distribution pattern: the PMS (2.07 +/- 0.24 MPa) significantly higher than both the ZTS (1.56 +/- 0.29) and NFS (1.71 +/- 0.22). Analysis of these data suggests that facial sutures and their immediately adjacent sutural mineralization fronts have different capacities for mechanical deformation. The elastic properties of sutures and sutural mineralization fronts are potentially useful for improving our understanding of their roles in development.

Tissue-engineered neogenesis of human-shaped mandibular condyle from rat mesenchymal stem cells.

The temporomandibular joint is susceptible to diseases and trauma that may ultimately lead to structural degeneration. Current approaches for replacing degenerated mandibular condyles suffer from deficiencies such as donor site morbidity, immunorejection, implant wear and tear, and pathogen transmission. The hypothesis of this study was that a human-shaped mandibular condyle can be tissue-engineered from rat mesenchymal stem cells (MSCs) encapsulated in a biocompatible polymer. Rat bone marrow MSCs were isolated and induced to differentiate into chondrogenic and osteogenic cells in vitro, and encapsulated in poly(ethylene glycol)-based hydrogel in two stratified layers molded into the shape of a cadaver human mandibular condyle. Eight weeks following in vivo implantation of the bilayered osteochondral constructs in the dorsum of immunodeficient mice, mandibular condyles formed de novo. Microscopic evaluation of the tissue-engineered mandibular condyle demonstrated two stratified layers of histogenesis of cartilaginous and osseous phenotypes. The current approach is being refined for ultimate therapeutic applications.

Mechanobiology of craniofacial sutures.

Craniofacial sutures are soft connective-tissue joints between mineralized skull bones. Suture mechanobiology refers to the understanding of how mechanical stimuli modulate sutural growth. This review's hypothesis is that novel mechanical stimuli can effectively modulate sutural growth. Exogenous forces with static, sinusoidal, and square waveforms induce corresponding waveforms of sutural strain. Sutural growth is accelerated upon small doses of oscillatory strain, as few as 600 cycles delivered 10 min/day over 12 days. Interestingly, both oscillatory tensile and compressive strains induce anabolic sutural responses beyond natural growth. Mechanistically, oscillatory strain likely turns on genes and transcription factors that activate cellular machinery via mechanotransduction pathways. Thus, sutural growth is determined by hereditary and mechanical signals via the common pathway of genes. It is concluded that small doses of oscillatory mechanical stimuli have the potential to modulate sutural growth effectively: either accelerating it or initiating net sutural bone resorption for various therapeutic objectives.

Chondrocyte proliferation of the cranial base cartilage upon in vivo mechanical stresses.

Whereas the growth of the cranial base cartilage is thought to be regulated solely by genes, epiphyseal growth plates are known to respond to mechanical stresses. This disparity has led to our hypothesis that chondrocyte proliferation is accelerated by mechanical stimuli above natural growth. Two-Newton tensile forces with static and cyclic waveforms were delivered in vivo to the premaxillae of actively growing rabbits for 20 min/day over 12 consecutive days. The average number of BrdU-labeled chondrocytes in the proliferating zone treated with cyclic forces was significantly higher than both static forces of matching peak magnitude and sham controls representing natural chondral growth. Cyclic forces also evoked greater area of the proliferating zone than both static forces and sham controls. Thus, chondrocyte proliferation is enhanced by mechanical stresses in vivo, especially those with oscillatory waveform. Analysis of these data suggests that genetically coded chondral growth is up-regulated by mechanical signals.

Bone strain patterns of the zygomatic complex in response to simulated orthopedic forces.

Craniofacial bone strain upon orthopedic loading has rarely been characterized, despite its fundamental importance in our understanding of the anabolic and catabolic effects of orthopedic forces. The present study tested the hypothesis that zygomatic bone strain is modulated upon loading by headgear, a device widely used in craniofacial orthopedics. Ramp forces from 0 to 50 Newtons were applied via headgear attached to the permanent maxillary first molars in four juvenile and five adult human skulls. The average peak bone strain of the juvenile temporal articular eminence was significantly higher than the adult articular eminence (p < 0.05). Contrasting bone strain patterns were identified in the zygomatic arch: tensile in its lateral surface but compressive in its medial surface. The peak bone strain of the temporal articular eminence and the zygomatic arch both depend upon loading direction. Thus, headgear-generated orthopedic forces evoke bending of the zygomatic arch and stresses of the temporal articular eminence in vitro, suggesting the need to verify whether bone strain induces in vivo bone modeling and remodeling.

The relationship between alphaB-crystallin and neurofibrillary tangles in Alzheimer's disease.

AlphaB-crystallin is known as a small heat shock protein with a cytoprotective function. This study was undertaken to investigate the relationship between alphaB-crystallin and changes seen in Alzheimer's disease. The distribution and immunohistochemical characteristics of alphaB-crystallin positive neurones in the cerebral cortices of 4 patients with Alzheimer's disease were examined. AlphaB-crystallin positive neurones were mainly distributed in the limbic and paralimbic regions, namely parahippocampal gyrus, fusiform gyrus, cingulate cortex, middle and superior frontal gyrus, and insular cortex, which corresponded to commonly affected regions in Alzheimer's disease. Moreover, such neurones were present predominantly in the III or V layer of the cerebral cortex. The number of alphaB-crystallin positive neurones increased in parallel with the neuronal loss. Logistic regression analysis revealed a significant relation between the density of alphaB-crystallin positive neurones and that of extracellular neurofibrillary tangles (NFTs), with a correlation coefficient (r) of 0.57 and P < 0.0001 in 14 regions of the cerebral cortex. In contrast, the relation was not statistically significant between the density of alphaB-crystallin positive neurones and that of classical senile plaques, diffuse plaques or intracellular NFTs. Modified Gallyas-Braak (GB) staining on alphaB-crystallin positive neurone demonstrated several patterns of the structures: faint GB positive structures in the swollen perikaryon with straight neurites, fine granules compressed and contorted into fuzzy bundles, intensely GB positive filamentous structures together with fine granules and very intensely GB positive ring-like NFTs in a swollen perikaryon with curved neurites. In positive neurones, the density of ring-like NFTs correlated with that of atrophic perikaryon, or bent neurites and a decrease in the immunoreactivity of alphaB-crystallin. These data suggest that a close relationship exists between the appearance of alphaB-crystallin in neurones, extracellular NFTs, and neurofibrillary formation in alphaB-crystallin positive neurones in Alzheimer brain.