Fabrication of Injectable Kartogenin-Conjugated Composite Hydrogel with a Sustained Drug Release for Cartilage Repair.

Cartilage tissue engineering has attracted great attention in defect repair and regeneration. The utilization of bioactive scaffolds to effectively regulate the phenotype and proliferation of chondrocytes has become an elemental means for cartilage tissue regeneration. On account of the simultaneous requirement of mechanical and biological performances for tissue-engineered scaffolds, in this work we prepared a naturally derived hydrogel composed of a bioactive kartogenin (KGN)-linked chitosan (CS-KGN) and an aldehyde-modified oxidized alginate (OSA) via the highly efficient Schiff base reaction and multifarious physical interactions in mild conditions. On the basis of the rigid backbones and excellent biocompatibility of these two natural polysaccharides, the composite hydrogel demonstrated favorable morphology, easy injectability, good mechanical strength and tissue adhesiveness, low swelling ratio, long-term sustainable KGN release, and facilitated bone marrow mesenchymal stem cell activity, which could simultaneously provide the mechanical and biological supports to promote chondrogenic differentiation and repair the articular cartilage defects. Therefore, we believe this work can offer a designable consideration and potential alternative candidate for cartilage and other soft tissue implants.

Pelargonidin ameliorates inflammatory response and cartilage degeneration in osteoarthritis via suppressing the NF-κB pathway.

Pelargonidin (PG), a derivative of anthocyanins, has anti-oxidant and anti-inflammatory properties. Herein, the protective effect and the mechanism of PG in counteract the osteoarthritis (OA) progression were needed to further evaluate. In the current study, C57BL/6 mice was induced by destabilization of medial meniscus (DMM) surgery to establish the OA model. Primary chondrocytes were acquired from the knee cartilage of newborn mice. Then, PG was administrated to OA mice and IL-1ß-stimulated chondrocytes to evaluate its protective effects, respectively. Results uncovered that no conspicuous cytotoxic effects were observed when chondrocytes were treated with PG at a concentration lower than 40 µM for 24-72 h. Thus, 10 µM, 20 µM, and 40 µM PG were chosen for subsequent experiments in vitro. Then, we observed that 10, 20, and 40 µM PG reduced the levels of IL-6, TNF-α, COX-2 and iNOS in chondrocytes. In line, PG inhibited the IL-1ß-induced ECM catabolism in chondrocytes, as evidenced by deepening toluidine blue staining, increased expression of Collagen II, and decreased expressions of ADAMTS5 and MMP13. Moreover, PG also reduced the IL-1ß-stimulated p-p65 overexpression and nuclear translocation of p65 in chondrocytes. In vivo, Safranin O/Fast green and HE staining showed that articular cartilage surface morphology was basically smooth and complete after PG treatment for 8 weeks. Similarly, OARSI scores and MMP13 expression were apparently decreased, whereas Aggrecan expression was elevated in PG-treated mice 8 weeks after DMM surgery. In conclusion, PG can effectively ameliorate inflammatory reactions and cartilage degeneration via suppressing the NF-κB pathway, thereby restraining the OA progression.

Comparison of biological characteristics of human adipose- and umbilical cord- derived mesenchymal stem cells and their effects on delaying the progression of osteoarthritis in a rat model.

BACKGROUND: The prevalence of osteoarthritis (OA) is constantly increasing with age. Adipose-derived (AD-) and umbilical cord-derived (UC-) mesenchymal stem cells (MSCs) are attractive alternatives in OA therapy and regenerative medicine. However, whether there are differences in the efficacy of MSCs derived from different tissues in the cartilage regeneration, and the frequency of administration of MSCs needs to be further studied. EXPERIMENT: UC-MSCs and AD-MSC were isolated from the umbilical cord and subcutaneous fatty tissue of humans respectively and identified by flow cytometry. In vitro, the proliferation ability and chondrogenic potential of AD-MSCs and UC-MSCs were analyzed. In vivo, forty-three Sprague-Dawley rats were used for the OA model induced by ACLT surgery. OA rats were divided into a sham group, an ACLT model group, and two therapy groups (treated with AD-MSCs or UC-MSCs). Therapy groups were treated using a single or repeated twice injection of AD-MSCs and UC-MSCs at a concentration of 1.0 × 106 cells and were followed up for 12 weeks. Serial sections of knees were examined for histological, immunohistochemical and TUNEL analysis. RESULTS: We demonstrated that the proliferation of UC-MSCs was higher than that of AD-MSCs, consistent with the bigger pellets from UC-MSCs in a chondrogenic induction medium. Degeneration of articular cartilage was observed in histological appearance of Safranine O and Toluidine blue staining, and quantitative results of modified Mankin's Score. Importantly, both AD-MSCs and UC-MSCs transplantation significantly attenuated ACLT surgical-induced OA development. In addition, ACLT-induced reduction in cartilage extracellular matrix synthesis (aggrecan) was significantly suppressed by AD-MSCs or UC-MSCs transplantation. TUNEL assay showed that AD-MSCs and UC-MSCs treatments significantly protected chondrocytes against apoptosis compared with the ACLT group. No significant differences were observed between single injections and repeated twice injections. CONCLUSIONS: The current study suggested that, in vitro, AD-MSCs and UC-MSCs showed a comparable chondrogenic potential, although UC-MSCs displayed a superior proliferation capacity. Furthermore, our results confirmed that the injection of AD-MSCs and UC-MSCs, either single or repeated twice, could significantly inhibit the progression of ACLT-induced osteoarthritis with a similar effect, and MSCs transplantation can decrease the apoptosis of articular chondrocytes caused by ACLT.

Overexpression of interleukin-10 in engineered macrophages protects endothelial cells against LPS-induced injury in vitro.

Endothelial dysfunction is a primary pathophysiological change in sepsis. Macrophages are known to interact with vascular endothelial cells during the development of sepsis. Recently, drug delivery based on engineered macrophages was reported as an alternative approach for the management of diseases. Interleukin-10 (IL10) is a well-known anti-inflammatory cytokine, which reduces inflammation and inhibits dysfunction of endothelial cells caused by sepsis. It is currently poorly understood whether genetically modified macrophages with overexpression of IL10 are able to restore endothelial integrity and function at the cellular level. In this study, we used lentiviral vectors to construct RAW264.7 macrophages engineered to overexpress IL10 (IL10-eM) and investigated the effects of the IL10-eM supernatant on LPS-induced endothelial dysfunction using a noncontact coculture system. We found that cotreatment with IL10-eM supernatant significantly attenuates the effects of LPS-induced dysfunction of endothelial cells, including endothelial inflammatory response, endothelial permeability, and apoptosis. In addition, we discovered that LPS-induced downregulation of VE-cadherin and high production of reactive oxygen species were significantly attenuated upon IL10-eM exposure. Furthermore, upregulation of IL6, TNFα, and Bax was decreased after treatment of cells with IL10-eM supernatant. These results demonstrated that supernatant from engineered macrophages genetically modified with IL10 can effectively protect endothelial cells against LPS-induced dysfunction in vitro, suggesting that exosomes from such engineered macrophages may have therapeutic effects against sepsis.

Circular RNA PVT1 inhibits tendon stem/progenitor cell senescence by sponging microRNA-199a-5p.

Tendon stem/progenitor cell (TSPC) senescence can lead to age-dependent tendon maladies and undermines both tendon repair and replacement capacity in humans. The mechanisms underlying TSPC senescence and sensitivity to adverse factors are complicated. In this study, we analyzed involvement of the circular RNA (circRNA) PVT1 (circPVT1) in TSPC senescence. circPVT1 expression was found to be significantly diminished in human TSPCs under prolonged in vitro culture. Accordingly, circPVT1 knockdown promoted senescence progression and suppressed self renewal, migration, and tenogenic differentiation of TSPCs. Furthermore, we found that circPVT1 directly targets microRNA (miR)-199a-5p thereby attenuating its negative regulation of SIRT1 expression. Either miR-199a-5p inhibition or SIRT1 overexpression attenuated the senescence-boosting effect of circPVT1 knockdown, implying that circPVT1 suppresses TSPC senescence in part by upregulating the miR-199a-5p-SIRT1 signaling axis. Our findings conclusively explain the major roles of circPVT1 in TSPC senescence regulation; circPVT1 is a novel potential therapeutic target for reducing tendon senescence.

Intraperitoneal injection of Desferal® alleviated the age-related bone loss and senescence of bone marrow stromal cells in rats.

BACKGROUND: Age-related bone loss plays a vital role in the development of osteoporosis and osteoporotic fracture. Bone marrow stromal cell (BMSC) senescence is highly associated with osteoporosis and limits the application of BMSCs in regenerative medicine. Hypoxia is an essential component for maintaining the normal physiology of BMSCs. We have reported that activation of hypoxia-induced factor by deletion of von Hippel-Lindau gene in osteochondral progenitor cells protected mice from aging-induced bone loss. However, whether pharmacologically manipulation of hypoxic niche would attenuate age-related bone loss and dysfunction of BMSCs is not well understood. METHODS: Twelve-month-old Sprague-Dawley rats were used as an aged model and were intraperitoneally injected with Desferal® (20, 60 mg/kg weight or vehicle), three times a week for a continuous 8-week period. Two-month-old young rats were set as a reference. After 8 weeks, micro-CT and HE staining were performed to determine the effect of Desferal® on bone loss. In order to investigate the effects of Desferal® on BMSC senescence, 12-month-old rats were treated with high-dose Desferal® (60 mg/kg weight) daily for 10 days. BMSCs were isolated and evaluated using CCK-8 assay, colony-forming cell assay, cell differentiation assay, laser confocal for reactive oxygen species (ROS) level, senescence-associated ß-galactosidase (SA-ß-gal) staining, and molecular expression test for stemness/senescence-associated genes. RESULTS: Micro-CT and HE staining showed that high-dose Desferal® significantly prevented bone loss in aged rats. Compared with vehicle group, the ex vivo experiments showed that short-term Desferal® administration could promote the potential of BMSC growth (proliferation and colony formation ability) and improve the rebalance of osteogenic and adipogenic differentiation, as well as rejuvenate senescent BMSCs (ROS level and SA-ß-gal staining) and revise the expression of stemness/senescence-associated genes. The potential of BMSCs from 12M-H-Desferal® group at least partly revised to the level close to 2-month-old group. CONCLUSIONS: The current study suggested that Desferal®, an iron-chelating agent, could alleviate age-related bone loss in middle-aged rats. Meanwhile, we found that short-term intraperitoneal injection of Desferal® partly rejuvenate BMSCs from aged rats. Overall, we demonstrated a novel role of Desferal® in rejuvenating aged BMSCs and preventing age-related bone loss.

Blockade of Fgfr1 with PD166866 Protects Cartilage from the Catabolic Effects Induced by Interleukin-1ß: A Genome-Wide Expression Profiles Analysis.

OBJECTIVE: Previously we showed that genetic deletion of Fgfr1 in chondrocytes protected mice from progression of osteoarthritis (OA). The aim of this study is to evaluate the effect of PD166866, a potent selective inhibitor of Fgfr1, on cartilage degeneration induced by interleukin-1ß (IL-1ß) and to clarify underlying global gene expression pattern. DESIGN: Cartilage explants and primary rat chondrocytes were stimulated with IL-1ß to establish an inflammatory OA in vitro model. The effects of PD166866 were determined by measuring the release of glycosaminoglycans (GAG) in cartilage explants and primary rat chondrocytes, and the underlying molecular mechanism was analyzed by microarray and RT-PCR analysis in primary chondrocytes. RESULTS: In cartilage explants, PD166866 significantly counteracts IL-ß stimulated GAG release. In addition, PD166866 impede IL-1ß-stimulated nuclear translocation of p65 in rat chondrocytes. Based on microarray analysis, a total of 67 and 132 genes with more than 1.5-fold changes were identified in IL-1ß-treated versus control and PD166866 cotreatment versus IL-1ß treatment alone, respectively. Only 19 thereof were coregulated by IL-1ß and PD166866 simultaneously. GO and KEGG pathway analysis showed that some pathways, including "cytokine-cytokine receptor interaction," "chemokine signaling pathway," and "complement and coagulation cascades," as well as some key genes like chemokines, complement, and matrix metalloproteinases may relevant for therapeutic application of Fgfr1 blockade in IL-1ß-stimulated chondrocytes. CONCLUSION: Our results clearly demonstrated that blockade of Fgfr1 with PD166866 could effectively suppress the catabolic effects induced by IL-1ß, and elucidated whole genomic targets of Fgfr1 inhibition responsible for the therapeutic effects of Fgfr1 blockade against inflammatory OA.

Delivery of dimethyloxalylglycine in calcined bone calcium scaffold to improve osteogenic differentiation and bone repair.

As hypoxia plays a vital role in the angiogenic-osteogenic coupling, using proline hydroxylase inhibitors to manipulate hypoxia-inducible factors has become a strategy to improve the osteogenic properties of biomaterials. Dimethyloxallyl glycine (DMOG) is a 2-ketoglutarate analog, a small molecular compound that competes for 2-ketoglutaric acid to inhibit proline hydroxylase. In order to improve the osteogenic ability of calcined bone calcium (CBC), a new hypoxia-mimicking scaffold (DMOG/Collagen/CBC) was prepared by immersing it in the DMOG-Collagen solution, followed by freeze-drying. All coated CBC scaffolds retained the inherent natural porous architecture and showed excellent biocompatibility. A slow release of DMOG by the DMOG-loaded CBC scaffolds for up to one week was observed inin vitroexperiments. Moreover, the DMOG/Collagen/CBC composite scaffold was found to significantly stimulate bone marrow stromal cells to express osteogenic and angiogenic genesin vitro. In addition, the osteogenic properties of three kinds of scaffolds, raw CBC, Collagen/CBC, and DMOG/Collagen/CBC, were evaluated by histology using the rabbit femoral condyle defect model. Histomorphometric analyses showed that the newly formed bone (BV/TV) in the DMOG/Collagen/CBC group was significantly higher than that of the Collagen/CBC group. However, immunostaining of CD31 and Runx2 expression between these two groups showed no significant difference at this time point. Our results indicate that DMOG-coated CBC can promote osteogenic differentiation and bone healing, and show potential for clinical application in bone tissue engineering.

Harnessing the layer-by-layer assembly technique to design biomaterials vaccines for immune modulation in translational applications.

The existence of challenging diseases such as cancers, HIV and Zika requires developing new vaccines that can generate tunable and robust immune responses against the diseases. Biomaterials-based techniques have been broadly explored for designing vaccines that can produce controllable and potent immunity. Among the existing biomaterials-based strategies, the layer-by-layer (LbL) assembly technique is remarkably attractive in vaccine design due to its unique features such as programmed and versatile cargo loading, cargo protection, co-delivery, juxtaposing of immune signals, etc. In this work, we reviewed the existing LbL-based vaccine design techniques for translational applications. Specifically, we discussed nanovaccines constructed by coating polyelectrolyte multilayers (PEMs) on nanoparticles, microcapsule vaccines assembled from PEMs, polyplex/complex vaccines condensed from charged materials and microneedle vaccines deposited with PEMs, highlighting the employment of these techniques to promote immunity against diseases ranging from cancers to infectious and autoimmune diseases (i.e., HIV, influenza, multiple sclerosis, etc.). Additionally, the review specifically emphasized using LbL-based vaccine technologies for tuning the cellular and molecular pathways, demonstrating the unique advantages presented by these vaccination strategies. These studies showed the versatility and potency of using LbL-based techniques for designing the next generation of biomaterials vaccines for translational purposes.

Synergistic Effects of FGF-18 and TGF-ß3 on the Chondrogenesis of Human Adipose-Derived Mesenchymal Stem Cells in the Pellet Culture.

Cell-based therapy serves as an effective way for cartilage repair. Compared with a limited source of autologous chondrocytes, adipose-derived stem cells (ADSCs) are proposed as an attractive cell source for cartilage regeneration. How to drive chondrogenic differentiation of ADSCs efficiently remains to be further investigated. TGF-ß3 has shown a strong chondrogenic action on ADSCs. Recently, fibroblast growth factor 18 (FGF-18) has gained marked attention due to its anabolic effects on cartilage metabolism, but existing data regarding the role of FGF-18 on the chondrogenic potential of mesenchymal stem cells (MSCs) are conflicting. In addition, whether the combined application of FGF-18 and TGF-ß3 would improve the efficiency of the chondrogenic potential of ADSCs has not been thoroughly studied. In the current study, we isolated human ADSCs and characterized the expression of their surface antigens. Also, we evaluated the chondrogenic potential of FGF-18 on ADSCs using an in vitro pellet model by measuring glycosaminoglycan (GAG) content, collagen level, histologic appearance, and expression of cartilage-related genes. We found that FGF-18, similarly to TGF-ß3, had a positive impact on chondrogenic differentiation and matrix deposition when presented throughout the culture period. More importantly, we observed synergistic effects of FGF-18 and TGF-ß3 on the chondrogenic differentiation of ADSCs in the in vitro pellet model. Our results provide critical information on the therapeutic use of ADSCs with the help of FGF-18 and TGF-ß3 for cartilage regeneration.

Fibroblast Growth Factor Receptor 3 Deficiency Does Not Impair the Osteoanabolic Action of Parathyroid Hormone on Mice.

UNLABELLED: PTH stimulates bone formation in Fgfr3 knockout mice through promotion of proliferation and differentiation in osteoblasts. INTRODUCTION: Previous studies showed that endogenous fibroblast growth factor 2 (FGF-2) is required for parathyroid hormone (PTH)-stimulated bone anabolic effects, however, the exact mechanisms by which PTH stimulate bone formation and the function of FGF receptors in mediating these actions are not fully defined. FGF receptor 3 (FGFR3) has been characterized as an important regulator of bone metabolism and is confirmed to cross-talk with PTH/PTHrP signal in cartilage and bone development. METHODS: Fgfr3 knockout and wild-type mice at 2-month-old and 4-month-old were intraperitoneally injected with PTH intermittently for 4 weeks and then the skeletal responses to PTH were assessed by dual energy X-ray absorptiometry (DEXA), micro-computed tomography (µCT) and bone histomorphometry. RESULTS: Intermittent PTH treatment improved bone mineral density (BMD) and femoral mechanical properties in both Fgfr3 (-/-) and wild-type mice. Histomorphometric analysis showed that bone formation and bone resorption were increased in both genotypes following PTH treatment. PTH treatment increased trabecular bone volume (BV/TV) in WT and Fgfr3-deficient mice. The anabolic response in Fgfr3-deficient and wild-type bone is characterized by an increase of both bone formation and resorption-related genes following PTH treatment. In addition, we found that Fgfr3 null osteoblasts (compared to wild-type controls) maintained normal abilities to response to PTH-stimulated increase of proliferation, differentiation, expression of osteoblastic marker genes (Cbfa1, Osteopontin and Osteocalcin), and phosphorylation of Erk1/2. CONCLUSIONS: Bone anabolic effects of PTH were not impaired by the absence of FGFR3, suggesting that the FGFR3 signaling may not be required for osteoanabolic effects of PTH activities.

[A gain-of-function mutation in FGFR2 influences mandibular condylar development on mice].

The development of the skeleton is regulated by numerous signaling molecules expressed in epiphyseal cartilage controlling both chondrogenesis and osteogenesis such as fibroblast growth factor receptors (FGFRs). In order to explore the important effect of fibroblast growth factor receptor 2 (FGFR2) in the process of mandibular condylar growth, we introduced gain-of-function Fgfr2(+/S252W) mice, and investigated mandibular condylar morphology by means of safranin-o/fast green staining at the stage of 1 week, 3 weeks and 6 weeks. The mutant mice displayed narrower width of the mandibular condylar growth plate, stronger stainings of trabecular bone at the stage of 1 week, 3 weeks and 6 weeks and faster degradation of the calcified cartilage cell layer at the stage of 6 weeks. We also assessed the expression of type X collagen (Col X) in mandibular condyle at the stage of 3 weeks by immunohistochemical staining and real-time PCR. The results showed that Col X was increased in the mutant mice. In conclusion, the gain-of-function mutation in FGFR2 resulted in histopathological abnormalities and development deformity of mandibular condyle cartilage in mice, which inhibited endochondral bone formation.

Inhibited Wnt signaling causes age-dependent abnormalities in the bone matrix mineralization in the Apert syndrome FGFR2(S252W/+) mice.

Apert syndrome (AS) is a type of autosomal dominant disease characterized by premature fusion of the cranial sutures, severe syndactyly, and other abnormalities in internal organs. Approximately 70% of AS cases are caused by a single mutation, S252W, in fibroblast growth factor receptor 2 (FGFR2). Two groups have generated FGFR2 knock-in mice Fgfr2S252W/+ that exhibit features of AS. During the present study of AS using the Fgfr2S252W/+ mouse model, an age-related phenotype of bone homeostasis was discovered. The long bone mass was lower in 2 month old mutant mice than in age-matched controls but higher in 5 month old mutant mice. This unusual phenotype suggested that bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to maintain bone homeostasis, might be involved. BMSCs were isolated from Fgfr2S252W/+ mice and found that S252W mutation could impair osteogenic differentiation BMSCs but enhance mineralization of more mature osteoblasts. A microarray analysis revealed that Wnt pathway inhibitors SRFP1/2/4 were up-regulated in mutant BMSCs. This work provides evidence to show that the Wnt/ß-catenin pathway is inhibited in both mutant BMSCs and osteoblasts, and differentiation defects of these cells can be ameliorated by Wnt3a treatment. The present study suggested that the bone abnormalities caused by deregulation of Wnt pathway may underlie the symptoms of AS.

A Ser252Trp mutation in fibroblast growth factor receptor 2 (FGFR2) mimicking human Apert syndrome reveals an essential role for FGF signaling in the regulation of endochondral bone formation.

A S252W mutation of fibroblast growth factor receptor 2 (FGFR2), which is responsible for nearly two-thirds of Apert syndrome (AS) cases, causes retarded development of the skeleton and skull malformation resulting from premature fusion of the craniofacial sutures. We utilized a Fgfr2(+/S252W) mouse (a knock-in mouse model mimicking human AS) to demonstrate decreased bone mass due to reduced trabecular bone volume, reduced bone mineral density, and shortened growth plates in the long bones. In vitro bone mesenchymal stem cells (BMSCs) culture studies revealed that the mutant mice showed reduced BMSC proliferation, a reduction in chondrogenic differentiation, and reduced mineralization. Our results suggest that these phenomena are caused by up-regulation of p38 and Erk1/2 phosphorylation. Treatment of cultured mutant bone rudiments with SB203580 or PD98059 resulted in partial rescue of the bone growth retardation. The p38 signaling pathway especially was found to be responsible for the retarded long bone development. Our data indicate that the S252W mutation in FGFR2 directly affects endochondral ossification, resulting in growth retardation of the long bone. We also show that the p38 and Erk1/2 signaling pathways partially mediate the effects of the S252W mutation of FGFR2 on long bone development.

Inactivation of Vhl in osteochondral progenitor cells causes high bone mass phenotype and protects against age-related bone loss in adult mice.

Previous studies have shown that disruption of von Hippel-Lindau gene (Vhl) coincides with activation of hypoxia-inducible factor α (HIFα) signaling in bone cells and plays an important role in bone development, homeostasis, and regeneration. It is known that activation of HIF1α signaling in mature osteoblasts is central to the coupling between angiogenesis and bone formation. However, the precise mechanisms responsible for the coupling between skeletal angiogenesis and osteogenesis during bone remodeling are only partially elucidated. To evaluate the role of Vhl in bone homeostasis and the coupling between vascular physiology and bone, we generated mice lacking Vhl in osteochondral progenitor cells (referred to as Vhl cKO mice) at postnatal and adult stages in a tamoxifen-inducible manner and changes in skeletal morphology were assessed by micro-computed tomography (µCT), histology, and bone histomorphometry. We found that mice with inactivation of Vhl in osteochondral progenitor cells at the postnatal stage largely phenocopied that of mice lacking Vhl in mature osteoblasts, developing striking and progressive accumulation of cancellous bone with increased microvascular density and bone formation. These were accompanied with a significant increase in osteoblast proliferation, upregulation of differentiation marker Runx2 and osteocalcin, and elevated expression of vascular endothelial growth factor (VEGF) and phosphorylation of Smad1/5/8. In addition, we found that Vhl deletion in osteochondral progenitor cells in adult bone protects mice from aging-induced bone loss. Our data suggest that the VHL-mediated signaling in osteochondral progenitor cells plays a critical role in bone remodeling at postnatal/adult stages through coupling osteogenesis and angiogenesis. © 2014 American Society for Bone and Mineral Research.

Efficient multi-site-directed mutagenesis directly from genomic template.

In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for combination, deletion or insertion of DNA fragments.

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice.

OBJECTIVE: Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR-1) in the development of osteoarthritis (OA) and its underlying mechanisms. METHODS: FGFR-1 was deleted from the articular chondrocytes of adult mice in a cartilage-specific and tamoxifen-inducible manner. Two OA models (aging-associated spontaneous OA, and destabilization-induced OA), as well as an antigen-induced arthritis (AIA) model, were established and tested in Fgfr1-deficient and wild-type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR-1 inhibitor on human articular chondrocytes was examined. RESULTS: The gross morphologic features of Fgfr1-deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12-month-old Fgfr1-deficient mice displayed greater aggrecan staining compared to 12-month-old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR-1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP-13) and up-regulation of FGFR-3 in mouse and human articular chondrocytes. CONCLUSION: Disruption of FGFR-1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down-regulation of MMP-13 expression and up-regulation of FGFR-3 levels may contribute to the phenotypic changes observed in Fgfr1-deficient mice.

Dynamic morphological changes in the skulls of mice mimicking human Apert syndrome resulting from gain-of-function mutation of FGFR2 (P253R).

Apert syndrome is caused mainly by gain-of-function mutations of fibroblast growth factor receptor 2. We have generated a mouse model (Fgfr2(+/P253R)) mimicking human Apert syndrome resulting from fibroblast growth factor receptor 2 Pro253Arg mutation using the knock-in approach. This mouse model in general has the characteristic skull morphology similar to that in humans with Apert syndrome. To characterize the detailed changes of form in the overall skull and its major anatomic structures, euclidean distance matrix analysis was used to quantitatively compare the form and growth difference between the skulls of mutants and their wild-type controls. There were substantial morphological differences between the skulls of mutants and their controls at 4 and 8 weeks of age (P < 0.01). The mutants showed shortened skull dimensions along the rostrocaudal axis, especially in their face. The width of the frontal bone and the distance between the two orbits were broadened mediolaterally. The neurocrania were significantly increased along the dorsoventral axis and slightly increased along the mediolateral axis, and also had anteriorly displayed opisthion along the rostrocaudal axis. Compared with wild-type, the mutant mandible had an anteriorly displaced coronoid process and mandibular condyle along the rostrocaudal axis. We further found that there was catch-up growth in the nasal bone, maxilla, zygomatic bone and some regions of the mandible of the mutant skulls during the 4-8-week interval. The above-mentioned findings further validate the Fgfr2(+/P253R) mouse strain as a good model for human Apert syndrome. The changes in form characterized in this study will help to elucidate the mechanisms through which the Pro253Arg mutation in fibroblast growth factor receptor 2 affects craniofacial development and causes Apert syndrome.

Osteoblastic molecular scaffold Gab1 is required for maintaining bone homeostasis.

The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, plays a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. However, its biological role in osteoblast activity and postnatal bone metabolism remains unclear. To elucidate the in vivo function of Gab1 in postnatal bone remodeling, we generated osteoblast-specific Gab1 knockout mice. Disruption of Gab1 expression in osteoblasts led to decreased trabecular bone mass with a reduced bone formation rate and a decreased bone resorption. Bones from Gab1 mutants also exhibited inferior mechanical properties. Moreover, primary osteoblasts from Gab1 mutant mice demonstrated markedly suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased expression of receptor activator of NF-kappaB ligand (RANKL). Activation of serine-threonine Akt kinase and extracellular signal-regulated kinase in response to insulin and insulin-like growth factor 1 was attenuated in Gab1 mutant osteoblasts. Our results show that Gab1-mediated signals in osteoblasts are crucial for normal postnatal bone homeostasis.

Disruption of Smad4 in odontoblasts causes multiple keratocystic odontogenic tumors and tooth malformation in mice.

Keratocystic odontogenic tumors (KCOTs) are cystic epithelial neoplasias with a high recurrence rate. However, the molecular mechanisms underlying the initiation and progression of KCOTs are still largely unknown. Here, we show that specific ablation of Smad4 in odontoblasts unexpectedly resulted in spontaneous KCOTs in mice. The mutant mice exhibited malformed teeth characterized by fractured incisors and truncated molar roots. These abnormalities were mainly caused by disrupted odontoblast differentiation that led to irregular dentin formation. The cystic tumors arising from the reactivation of epithelial rests of Malassez (ERM), in which Smad4 remained intact, proliferated and formed stratified and differentiated squamous epithelia that exhibited a dramatic upregulation of Hedgehog signaling. Odontoblasts, which are responsive to transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) signals, may produce signal molecules to inhibit the activation of ERM. Indeed, we observed a downregulation of BMP signals from Smad4 mutant odontoblasts to the adjacent Hertwig's epithelial root sheath (HERS). Intriguingly, KCOTs frequently emerged from Smad4-deficient ERM in keratinocyte-specific Smad4 knockout mice, suggesting a novel mechanism in which reciprocal TGF-beta/BMP signaling between odontoblasts and HERS was required for tooth root development and suppression of KCOT formation. These findings provide insight into the genetic basis underlying KCOTs and have important implications for new directions in KCOT treatment.