Estradiol induces bone osteolysis in triple-negative breast cancer via its membrane-associated receptor ERα36.

Triple-negative breast cancer (TNBC) is thought to be an estradiol-independent, hormone therapy-resistant cancer because of lack of estrogen receptor alpha 66 (ERα66). We identified a membrane-bound splice variant, ERα36, in TNBC cells that responds to estrogen (E2) and may contribute to bone osteolysis. We demonstrated that the MDA-MB-231 TNBC cell line, which expresses ERα36 similarly to MCF7 cells, is responsive to E2, forming osteolytic tumors in vivo. MDA-MB-231 cells activate osteoclasts in a paracrine manner. Conditioned media (CM) from MDA-MB-231 cells treated with bovine serum albumin-bound E2 (E2-BSA) increased activation of human osteoclast precursor cells; this was blocked by addition of anti-ERα36 antibody to the MDA-MB-231 cultures. Osteoclast activation and bone resorption genes were elevated in RAW 264.7 murine macrophages following treatment with E2-BSA-stimulated MDA-MB-231 CM. E2 and E2-BSA increased phospholipase C (PLC) and protein kinase C (PKC) activity in MDA-MB-231 cells. To examine the role of ERα36 signaling in bone osteolysis in TNBC, we used our bone-cancer interface mouse model in female athymic homozygous Foxn1nu mice. Mice with MDA-MB-231 tumors and treated with tamoxifen (TAM), E2, or TAM/E2 exhibited increased osteolysis, cortical bone breakdown, pathologic fracture, and tumor volume; the combined E2/TAM group also had reduced bone volume. These results suggest that E2 increased osteolytic lesions in TNBC through a membrane-mediated PLC/PKC pathway involving ERα36, which was enhanced by TAM, demonstrating the role of ERα36 and its membrane-associated signaling pathway in bone tumors. This work suggests that ERα36 may be a potential therapeutic target in patients with TNBC.

Reduced osseointegration in disuse and denervation rat models results from impaired cellular responses to multiscale microstructured titanium surfaces.

Immobilization-induced skeletal unloading results in muscle atrophy and rapid bone loss, thereby increasing the risk of falling and the need for implant therapy in patients with extended bed rest or neuromuscular injuries. Skeletal unloading causes bone loss by altering bone growth and resorption, suggesting that implant performance might be affected. To test this, we focused on early events in implant osseointegration. We used the rat sciatic neurectomy-induced disuse model under two different settings. In Study 1, 16 Sprague Dawley rats (SD) were separated into control, sham operated+cast immobilization, and sciatic neurectomy+casting groups; titanium implants with multiscale microtextured topography and hydrophilic chemistry (modSLA) were inserted in the distal femoral metaphysis. Neurectomy surgeries and casting were performed at the same surgical setting as implant placement; rats were euthanized 4 weeks post-implantation. In Study 2, we established the unloaded condition before implantation. A total of 12 SD rats were divided into control and sciatic+femoral neurectomy groups. A total of 24 days after sciatic and femoral neurectomy surgery, rats received implants. Study 2 rats were euthanized at 4 weeks post-implantation. MicroCT and histomorphometry showed that trabecular bone and osseointegration were reduced when disuse was established before implantation. Osteoblasts isolated from Study 1 sciatic neurectomy tibial bones exhibited impaired differentiation on modSLA culture disks, revealing a possible mechanism responsible for the decreased osseointegration observed in the Study 2 rats. This study addressed the importance of considering the mechanical unloading and muscle function history before implant insertion and suggests that implant performance was reduced due to poor cellular ability to regenerate.

Wnt16 Increases Bone-to-Implant Contact in an Osteopenic Rat Model by Increasing Proliferation and Regulating the Differentiation of Bone Marrow Stromal Cells.

Osseointegration is a complex biological cascade that regulates bone regeneration after implant placement. Implants possessing complex multiscale surface topographies augment this regenerative process through the regulation of bone marrow stromal cells (MSCs) that are in contact with the implant surface. One pathway regulating osteoblastic differentiation is Wnt signaling, and upregulation of non-canonical Wnts increases differentiation of MSCs on these titanium substrates. Wnt16 is a non-canonical Wnt shown to regulate bone morphology in mouse models. This study evaluated the role of Wnt16 during surface-mediated osteoblastic differentiation of MSCs in vitro and osseointegration in vivo. MSCs were cultured on Ti substrates with different surface properties and non-canonical Wnt expression was determined. Subsequently, MSCs were cultured on Ti substrates +/-Wnt16 (100 ng/mL) and anti-Wnt16 antibodies (2 µg/mL). Wnt16 expression was increased in cells grown on microrough surfaces that were processed to be hydrophilic and have nanoscale roughness. However, treatment MSCs on these surfaces with exogenous rhWnt16b increased total DNA content and osteoprotegerin production, but reduced osteoblastic differentiation and production of local factors necessary for osteogenesis. Addition of anti-Wnt16 antibodies blocked the inhibitor effects of Wnt16. The response to Wnt16 was likely independent of other osteogenic pathways like Wnt11-Wnt5a signaling and semaphorin 3a signaling. We used an established rat model of cortical and trabecular femoral bone impairment following botox injections (2 injections of 8 units/leg each, starting and maintenance doses) to assess Wnt16 effects on whole bone morphology and implant osseointegration. Wnt16 injections did not alter whole bone morphology significantly (BV/TV, cortical thickness, restoration of trabecular bone) but were effective at increasing cortical bone-to-implant contact during impaired osseointegration in the botox model. The mechanical quality of the increased bone was not sufficient to rescue the deleterious effects of botox. Clinically, these results are important to understand the interaction of cortical and trabecular bone during implant integration. They suggest a role for Wnt16 in modulating bone remodeling by reducing osteoclastic activity. Targeted strategies to temporally regulate Wnt16 after implant placement could be used to improve osseointegration by increasing the net pool of osteoprogenitor cells.

Internal surface modification of additively manufactured macroporous TiAl6V4 biomimetic implants via a calciothermic reaction-based process and osteogenic in vivo responses.

Three-dimensional macroporous titanium-aluminum-vanadium (TiAl6V4) implants produced by additive manufacturing (AM) can be grit blasted (GB) to yield microtextured exterior surfaces, with additional micro/nano-scale surface features provided by subsequent acid etching (AE). However, the line-of-sight nature of GB causes the topography of exterior GB + AE-modified surfaces to differ from internal GB-inaccessible surfaces. Previous in vitro studies using dense TiAl6V4 substrates indicated that a nonline-of-sight, calciothermic-reaction (CaR)-based process provided homogeneous osteogenic nanotextures on GB + AE surfaces, suggesting it could be used to achieve a homogeneous nanotopography on external and internal surfaces of macroporous AM constructs. Macroporous TiAl6V4 (3D) constructs were produced by direct laser melting and modified by GB + AE, with the CaR process then applied to 50% of constructs (3DCaR). The CaR process yielded nanoporous/nanorough internal surfaces throughout the macroporous constructs. Skeletally mature, male Sprague-Dawley rats were implanted with these constructs using a cranial on-lay model. Prior to implantation, a Cu++-free click hydrogel was applied to half of the constructs (3D + H, 3DCaR + H) to act as a challenge to osseointegration. Osseointegration was compared between the four implant groups (3D, 3DCaR, 3D + H, 3DCaR + H) at 4w. 3D + H implants exhibited lower bone volume (BV) and percent bone ingrowth (%BI) than the 3D implants. In contrast, osseointegrated 3DCaR + H implants had similar BV and %BI as the 3DCaR implants. Implant pull-off forces correlated with these results. In vitro analyses indicated that human bone marrow stromal cells (MSCs) exhibited enhanced production of osteoblast differentiation markers and factors associated with osteogenesis when grown on CaR-modified 3D substrates relative to control (TCPS) substrates. This work confirms that the CaR process provides osteogenic nanotextures on internal surfaces of macroporous 3D implants, and suggests that CaR-modified surfaces can promote osseointegration in cases where osteogenesis is impaired.

Osseointegration of Titanium Implants in a Botox-Induced Muscle Paralysis Rat Model Is Sensitive to Surface Topography and Semaphorin 3A Treatment.

Reduced skeletal loading associated with many conditions, such as neuromuscular injuries, can lead to bone fragility and may threaten the success of implant therapy. Our group has developed a botulinum toxin A (botox) injection model to imitate disease-reduced skeletal loading and reported that botox dramatically impaired the bone formation and osseointegration of titanium implants. Semaphorin 3A (sema3A) is an osteoprotective factor that increases bone formation and inhibits bone resorption, indicating its potential therapeutic role in improving osseointegration in vivo. We first evaluated the sema3A effect on whole bone morphology following botox injections by delivering sema3A via injection. We then evaluated the sema3A effect on the osseointegration of titanium implants with two different surface topographies by delivering sema3A to cortical bone defect sites prepared for implant insertion and above the implants after insertion using a copper-free click hydrogel that polymerizes rapidly in situ. Implants had hydrophobic smooth surfaces (PT) or multiscale biomimetic micro/nano topography (SLAnano). Sema3A rescued the botox-impaired bone formation. Furthermore, biomimetic Ti implants improved the bone-to-implant contact (BIC) and mechanical properties of the integrated bone in the botox-treated rats, which sema3A enhanced. This study demonstrated the value of biomimetic approaches combining multiscale topography and biologics in improving the clinical outcomes of implant therapy.

Semaphorin 3A delivered by a rapidly polymerizing click hydrogel overcomes impaired implant osseointegration in a rat type 2 diabetes model.

Semaphorin 3A (sema3A) is an osteoprotective factor that enhances bone formation while inhibiting osteoclast bone resorption. It is produced by rat calvarial osteoblasts cultured on grit-blasted/acid-etched microtextured (SLA) titanium surfaces at higher levels than on tissue culture polystyrene, suggesting that it may improve performance of titanium implants in vivo, particularly in conditions characterized by compromised bone quality. To test this, we established a clinically relevant type 2 diabetes mellitus (T2DM) rat model and used a non-toxic click hydrogel that rapidly polymerizes in situ (GEL) to provide localized controlled delivery of sema3A. In vitro studies confirmed that sema3A released from GEL was biologically active, increasing osteoblast differentiation of a pre-osteoblast cell-line. Whereas increased sema3A production was not observed in T2DM calvarial osteoblasts cultured on SLA, exogenous sema3A enhanced surface-induced osteoblast differentiation, indicating that it would be a viable candidate for in vivo use. Delivery of sema3A either by GEL or by local injection to bone defects enhanced osseointegration of SLA implants in the T2DM rats. Trabecular bone mass and bone-to-implant contact were decreased in T2DM rats compared to normal rats; sema3A delivered locally improved both parameters. These findings suggest that reduced trabecular bone contributes to poor osseointegration in T2DM patients and support GEL as a promising treatment option for sustained release of therapeutic doses of sema3A. Moreover, using this clinically translatable T2DM model and developing a biocompatible, Cu-free click chemistry hydrogel platform for the non-invasive delivery of therapeutics has major implications for regenerative medicine as a whole. STATEMENT OF SIGNIFICANCE: Osseointegration is compromised in patients with poor bone quality due to conditions like type 2 diabetes mellitus (T2DM). Previously, we showed that semaphorin 3A (sema3A) production is increased when human bone marrow stromal cells are cultured on titanium substrates that support osseointegration in vivo, suggesting it may enhance peri-implant osteogenesis in diabetes. Here we established a spontaneously developing T2DM rat model with clinical translatability and used it to assess sema3A effectiveness. Sema3A was delivered to the implant site via a novel copper-free click hydrogel, which has minimal swelling behavior and superior rheological properties. Osseointegration was successfully restored, and enhanced compared to burst release through injections. This study provides scientific evidence for using sema3A to treat impaired osseointegration in T2DM patients.

Differential Effects of Neurectomy and Botox-induced Muscle Paralysis on Bone Phenotype and Titanium Implant Osseointegration.

Metabolic bone is highly innervated by both sensory and sympathetic nerves. In addition to skeletal development, neural regulation participates in local bone remodeling, which is important for successful osseointegration of titanium implants. Neurectomy is a model used to investigate the lack of neural function on bone homeostasis, but the relative impacts of direct denervation to bone or denervation-induced muscle paralysis are less well defined. To investigate this difference, we used two nerve intervention models, sciatic and femoral neurectomy (SFN) v. botox-induced muscle paralysis (BTX) and assessed the resulting femoral bone phenotype and Ti implant osseointegration. Male Sprague Dawley rats (19) were randomly divided into three groups: implant control (n = 5), SFN (n = 7), and BTX (n = 7). Ti implants (microrough/hydrophilic [modSLA], Institut Straumann AG) were placed in the distal metaphysis of each femur on day 24 post-SFN or BTX. Bone and muscle were examined on day 28 after implant insertion. Both nerve intervention models impaired osseointegration. MicroCT and histology indicated that both models had reduced trabecular bone formation. Only BTX reduced cortical bone formation and increased cortical bone porosity. BTX resulted in more bone loss characterized by the least trabecular and cortical bone, as well as osseointegration. Osteoblasts isolated from the tibia exhibited a model-specific phenotype when they were grown on Ti substrates in vitro. Neurectomy caused more severe muscle atrophy than botox injection. These results indicate that neural regulation directly modulates bone formation and osseointegration. Muscle paralysis modulated the effects of loss of neural inputs into bone, supporting the hypothesis that mechanical loading of bone is a factor in achieving successful osseointegration. The different effects of botox and neurectomy on bone phenotype indicated that the sensory and sympathetic nerves had a role in the osseointegration process.

Task-Specific Synthesis of 3D Porous Carbon Nitrides from the Cycloaddition Reaction and Sequential Self-Assembly Strategy toward Photocatalytic Hydrogen Evolution.

Carbon nitride has drawn widespread attention as a low-cost alternative to metal-based materials in the field of photocatalysis. However, the traditionally synthesized carbon nitrides always suffer a bulky architecture, which limits their intrinsic activities. Here, a cycloaddition reaction is proposed to synthesize a triazine-based precursor with implanted sodium and cyano groups, which are mostly retained in the resulting carbon nitride after the following polymerization. Incorporated sodium and cyano defects can not only tune the band structure of the carbon nitride but also provide more additive active sites. The optimized properties enable it an adorable photocatalytic hydrogen evolution rate of 1070 µmol h-1 g-1, varying by almost an order of magnitude from the pristine carbon nitride (79 µmol h-1 g-1). Moreover, a sequential self-assembly strategy has been adopted to further improve its architecture. As a consequence, a three-dimensional (3D) porous carbon nitride microtube cluster is constructed, indicating abundant exposed active sites and the faster separation of charge carriers. The corresponding photocatalytic hydrogen evolution rate is 1681 µmol h-1 g-1, which is very competitive compared with the reported pure carbon nitride photocatalysts. Briefly, this new approach may offer opportunities to fabricate task-specific carbon- and nitrogen-based materials from the molecular level.

Introgression of bread wheat chromatin into tall wheatgrass via somatic hybridization.

Regenerates were obtained following somatic hybridization between tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum cv. Jinan177) protoplasts. Two lines (CU and XI) were self-fertile in the first (R0) and subsequent (R1 and R2) generations. The phenotype of each R1 population was uniform. All CU progeny were phenotypically similar to the tall wheatgrass parent, while XI progeny had thinner, smoother and softer leaves. Cytological analysis showed that more wheat chromatin was present in the hybrid callus than in the R1 and R2 plants, and that some intercalary translocations of wheat chromosome segments were retained in the R2 generation. AFLP profiling confirmed the presence of wheat DNA in the introgression lines. Analysis of the high molecular weight glutenin subunit content of derived seed identified three novel subunits, not present in either the wheat or the tall wheatgrass parent. Microsatellite-based profiling of the chloroplast genome of the introgression lines suggested that only chloroplast sequences from the tall wheatgrass parent were present. The specifically inherited phenomena and possible application of these hybrids are discussed.

Analysis of remote asymmetric somatic hybrids between common wheat and Arabidopsis thaliana.

Callus-derived protoplasts of common wheat (Triticum aestivum L. cv. Hesheng 3) irradiated with ultraviolet light were fused by using the PEG method with cell suspension-derived protoplasts of Arabidopsis thaliana. Regenerated calli and green plants resembling that of wheat were obtained. The hybrid nature of putative calli and plants were confirmed by isozyme, random amplified polymorphic DNA and genomic in situ hybridization (GISH) analyses. GISH results indicated that 1 approximately 3 small chromosome fragments of A. thaliana were found introgression into the terminals of wheat chromosomes, forming highly asymmetric hybrids. Cytoplasmic genome tests did not show any cytoplasmic genetic materials from A. thaliana. However, variations from the normal wheat cytoplasmic genome were found, indicating recombination or rearrangement occurred during the process of somatic hybridization. The chromosome elimination in the asymmetric somatic hybridization of remote phylogenetic relationship was discussed. A miniature inverted-repeat transposable element related sequence was found by chance in the hybrids which might accompany and impact the process of somatic hybridization.

Phylogenetic relationships of 12 penaeoidea shrimp species deduced from mitochondrial DNA sequences.

DNA sequences of an 847 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) gene and a 514 bp fragment of 16s rRNA gene were determined to examine the phylogenetic relationships of 12 Penaeoidea shrimp species (Penaeus chinensis, Penaeus japonicus, Penaeus penicillatus, Penaeus vannamei, Penaeus canaliculatus, Trachypenaeus curvirostris, Metapenaeus affinis, Metapenaeus ensis, Metapenaeopsis barbata, Parapenaeus fissuroides, Parapenaeopsis hardiwickii, Solenocera crassicomis). Both fragments of the swimming crab Portunus trituberculaus chosen as the outgroup were also sequenced. Intraspecific sequence divergence of 0.24-1.2% in the COI gene was found in 5 species, while no intraspecific variation was observed in the 16s rRNA gene. Three phylogenetic trees based on the 1361 bp combined sequences of COI and 16s rRNA were concordant in indicating the following suggestions: (1) phylogenetic relationship of the 11 Penaeidae species based on our result support the opinion of Burkenroad (Burkenroad, M.D. (1983). Crustacean Issues 3:279-290) on the basis of morphological features; (2) it seems more reasonable to class Solenocera crassicorni in the family Penaeidae; (3) the fragment of the COI gene chosen here appears to be a good marker for speciation studies and population analysis in Crustaceans, while the 16s rRNA gene fragment here seems suitable for examining phylogenetic relationships at the species or genus levels in Crustaceans. Our time estimates suggest that Penaeus and Metapenaeus might have separated about 6.38 x 10(6)-7.98 x 10(6) years BP in the post-Miocene, and the species separation within Metapenaeus and Penaeus might occur 0.08 x 10(6)-0.4 x 10(6) years BP in the late Pleistocene.