Laryngeal Cancer Cells Metabolize 25-Hydroxyvitamin D3 and Respond to 24R,25-dihydroxyvitamin D3 via a Mechanism Dependent on Estrogen Receptor Levels.

Studies have evaluated vitamin D3's therapeutic potential in estrogen-responsive cancers, with conflicting findings. We have shown that the proliferation of breast cancer cells is regulated by 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) depending on estrogen receptor alpha 66 (ERα66) expression, suggesting that this could also be the case for estrogen-sensitive laryngeal cancer cells. Accordingly, we examined levels of ERα isoforms in ERα66-positive UM-SCC-12 and ERα66-negative UM-SCC-11A cells and their response to 24R,25(OH)2D3. 24R,25(OH)2D3 stimulated proliferation, increased the expression of metastatic markers, and inhibited apoptosis in UM-SCC-12 cells while having the opposite effect in UM-SCC-11A cells. To evaluate if vitamin metabolites could act via autocrine/paracrine mechanisms, we assessed the expression, protein levels, and activity of vitamin D3 hydroxylases CYP24A1 and CYP27B1. Both cell types expressed both mRNAs; but the levels of the enzymes and their activities were differentially regulated by estrogen. ERα66-negative UM-SCC-11A cells produced more 24,25(OH)2D3 than UM-SCC-12 cells, but comparable levels of 1,25(OH)2D3 when treated with 25(OH)D3 These results suggest that the regulation of vitamin D3 metabolism in laryngeal cancer cells is modulated by ERα66 expression, and support a role for 24R,25(OH)2D3 as an autocrine/paracrine regulator of laryngeal cancer. The local metabolism of 25(OH)D3 should be considered when determining the potential of vitamin D3 in laryngeal cancer.

Different Methods to Modify the Hydrophilicity of Titanium Implants with Biomimetic Surface Topography to Induce Variable Responses in Bone Marrow Stromal Cells.

The osteoblastic differentiation of bone marrow stromal cells (bMSCs), critical to the osseointegration of titanium implants, is enhanced on titanium surfaces with biomimetic topography, and this is further enhanced when the surfaces are hydrophilic. This is a result of changing the surface free energy to change protein adsorption, improving cell attachment and differentiation, and improving bone-to-implant contact in patients. In this study, we examined different methods of plasma treatment, a well-accepted method of increasing hydrophilicity, and evaluated changes in surface properties as well as the response of bMSCs in vitro. Commercially pure Ti and titanium-aluminum-vanadium (Ti6Al4V) disks were sand-blasted and acid-etched to impart microscale and nanoscale roughness, followed by treatment with various post-processing surface modification methods, including ultraviolet light (UV), dielectric barrier discharge (DBD)-generated plasma, and plasma treatment under an argon or oxygen atmosphere. Surface wettability was based on a sessile water drop measurement of contact angle; the elemental composition was analyzed using XPS, and changes in topography were characterized using scanning electron microscopy (SEM) and confocal imaging. The cell response was evaluated using bMSCs; outcome measures included the production of osteogenic markers, paracrine signaling factors, and immunomodulatory cytokines. All plasma treatments were effective in inducing superhydrophilic surfaces. Small but significant increases in surface roughness were observed following UV, DBD and argon plasma treatment. No other modifications to surface topography were noted. However, the relative composition of Ti, O, and C varied with the treatment method. The cell response to these hydrophilic surfaces depended on the plasma treatment method used. DBD plasma treatment significantly enhanced the osteogenic response of the bMSCs. In contrast, the bMSC response to argon plasma-treated surfaces was varied, with an increase in OPG production but a decrease in OCN production. These results indicate that post-packaging methods that increased hydrophilicity as measured by contact angle did not change the surface free energy in the same way, and accordingly, cells responded differently. Wettability and surface chemistry alone are not enough to declare whether an implant has an improved osteogenic effect and do not fully explain how surface free energy affects cell response.

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.

Regulatory Pathways in Growth Plate Chondrocytes that Are Impacted by Matrix Vesicle microRNA Identified by Targeted RISC Pulldown and Sequencing of the Resulting Transcriptome.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFß) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

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.

Selective BET inhibitor RVX-208 ameliorates periodontal inflammation and bone loss.

AIM: To determine the effects of RVX-208, a selective bromodomain and extra-terminal domain (BET) inhibitor targeting bromodomain 2 (BD2), on periodontal inflammation and bone loss. MATERIALS AND METHODS: Macrophage-like cells (RAW264.7) and human gingival epithelial cells were challenged by Porphyromonas gingivalis (Pg) with or without RVX-208. Inflammatory gene expression and cytokine production were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RAW264.7 cells were induced to osteoclast differentiation. After RVX-208 treatment, osteoclast differentiation was evaluated by histology, tartrate-resistant-acid-phosphatase (TRAP) activity and the expression of osteoclast-specific genes. The effect of RVX-208 on osteoclast transcriptome was studied by RNA sequencing. Periodontitis was induced in rats by ligature and local RVX-208 treatment was administered every other day. Alveolar bone loss was measured by micro-computed tomography. RESULTS: RVX-208 inhibited inflammatory gene expression and cytokine production in Pg-infected cells. Osteoclast differentiation was inhibited by RVX-208, as evidenced by reduced osteoclast number, TRAP activity and osteoclast-specific gene expression. RVX-208 displayed a more selective and less profound suppressive impact on transcriptome compared with pan-BET inhibitor, JQ1. RVX-208 administration prevented the alveolar bone loss in vivo. CONCLUSIONS: RVX-208 regulated both upstream (inflammatory cytokine production) and downstream (osteoclast differentiation) events that lead to periodontal tissue destruction, suggesting that it may be a promising 'epi-drug' for the prevention of periodontitis.

Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation.

Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-ß-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1.

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

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.

1α,25-Dihydroxyvitamin D3 Regulates microRNA Packaging in Extracellular Matrix Vesicles and Their Release in the Matrix.

Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation, including transforming growth factor beta-1 (TGFb1), bone morphogenetic protein 2 (BMP2), insulin-like growth factor-1 (IFG1), parathyroid hormone and parathyroid hormone related peptide (PTH, PTHrP), and Indian hedgehog (IHH). Chondrocytes in the growth plate's growth zone (GC) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 regulates MV enzyme composition genomically and 1α,25(OH)2D3 secreted by the cells acts on the MV membrane nongenomically, destabilizing it and releasing MV enzymes. This study examined the regulatory role 1α,25(OH)2D3 has over production and packaging of microRNA (miRNA) into MVs by GC cells and the release of miRNA by direct action on MVs. Costochondral cartilage GC cells were treated with 1α,25(OH)2D3 and the miRNA in the cells and MVs sequenced. We also treated MVs with 1α,25(OH)2D3 and determined if the miRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)2D3, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)2D3 regulated production and packaging of a unique population of miRNA into MVs compared to the vehicle control population. 1α,25(OH)2D3 treatment of MVs did not release miRNA. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)2D3 treatment. This study adds new regulatory roles for 1α,25(OH)2D3 with respect to packaging and transport of MV miRNAs.

Bone marrow stromal cells generate an osteoinductive microenvironment when cultured on titanium-aluminum-vanadium substrates with biomimetic multiscale surface roughness.

Osseointegration of titanium-based implants possessing complex macroscale/microscale/mesoscale/nanoscale (multiscale) topographies support a direct and functional connection with native bone tissue by promoting recruitment, attachment and osteoblastic differentiation of bone marrow stromal cells (MSCs). Recent studies show that the MSCs on these surfaces produce factors, including bone morphogenetic protein 2 (BMP2) that can cause MSCs not on the surface to undergo osteoblast differentiation, suggesting they may produce an osteogenic environmentin vivo. This study examined if soluble factors produced by MSCs in contact with titanium-aluminum-vanadium (Ti6Al4V) implants possessing a complex multiscale biomimetic topography are able to induce osteogenesis ectopically. Ti6Al4V disks were grit-blasted and acid-etched to create surfaces possessing macroscale and microscale roughness (MM), micro/meso/nanoscale topography (MN), and macro/micro/meso/nanoscale topography (MMNTM). Polyether-ether-ketone (PEEK) disks were also fabricated by machining to medical-grade specifications. Surface properties were assessed by scanning electron microscopy, contact angle, optical profilometry, and x-ray photoelectron spectroscopy. MSCs were cultured in growth media (GM). Proteins and local factors in their conditioned media (CM) were measured on days 4, 8, 10 and 14: osteocalcin, osteopontin, osteoprotegerin, BMP2, BMP4, and cytokines interleukins 6, 4 and 10 (IL6, IL4, and IL10). CM was collected from D14 MSCs on MMNTMand tissue culture polystyrene (TCPS) and lyophilized. Gel capsules containing active demineralized bone matrix (DBM), heat-inactivated DBM (iDBM), and iDBM + MMN-GM were implanted bilaterally in the gastrocnemius of athymic nude mice (N= 8 capsules/group). Controls included iDBM + GM; iDBM + TCPS-CM from D5 to D10 MSCs; iDBM + MMN-CM from D5 to D10; and iDBM + rhBMP2 (R&D Systems) at a concentration similar to D5-D10 production of MSCs on MMNTMsurfaces. Legs were harvested at 35D. Bone formation was assessed by micro computed tomography and histomorphometry (hematoxylin and eosin staining) with the histology scored according to ASTM 2529-13. DNA was greatest on PEEK at all time points; DNA was lowest on MN at early time points, but increased with time. Cells on PEEK exhibited small changes in differentiation with reduced production of BMP2. Osteoblast differentiation was greatest on the MN and MMNTM, reflecting increased production of BMP2 and BMP4. Pro-regenerative cytokines IL4 and IL10 were increased on Ti-based surfaces; IL6 was reduced compared to PEEK. None of the media from TCPS cultures was osteoinductive. However, MMN-CM exhibited increased bone formation compared to iDBM and iDBM + rhBMP2. Furthermore, exogenous rhBMP2 alone, at the concentration found in MMN-CM collected from D5 to D10 cultures, failed to induce new bone, indicating that other factors in the CM play a critical role in that osteoinductive microenvironment. MSCs cultured on MMNTMTi6Al4V surfaces differentiate and produce an increase in local factors, including BMP2, and the CM from these cultures can induce ectopic bone formation compared to control groups, indicating that the increased bone formation arises from the local response by MSCs to a biomimetic, multiscale surface topography.

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.

Bone marrow stromal cells are sensitive to discrete surface alterations in build and post-build modifications of bioinspired Ti6Al4V 3D-printed in vitro testing constructs.

Current standards in bone-facing implant fabrication by metal 3D (M3D) printing require post-manufacturing modifications to create distinct surface properties and create implant microenvironments that promote osseointegration. However, the biological consequences of build parameters and surface modifications are not well understood. This study evaluated the relative contributions of build parameters and post-manufacturing modification techniques to cell responses that impact osseointegration in vivo. Biomimetic testing constructs were created by using a M3D printer with standard titanium-aluminum-vanadium (Ti6Al4V) print parameters. These constructs were treated by either grit-blasting and acid-etching (GB + AE) or GB + AE followed by hot isostatic pressure (HIP) (GB + AE, HIP). Next, nine constructs were created by using a M3D printer with three build parameters: (1) standard, (2) increased hatch spacing, and (3) no infill, and additional contour trace. Each build type was further processed by either GB + AE, or HIP, or a combination of HIP treatment followed by GB + AE (GB + AE, HIP). Resulting constructs were assessed by SEM, micro-CT, optical profilometry, XPS, and mechanical compression. Cellular response was determined by culturing human bone marrow stromal cells (MSCs) for 7 days. Surface topography differed depending on processing method; HIP created micro-/nano-ridge like structures and GB + AE created micro-pits and nano-scale texture. Micro-CT showed decreases in closed pore number and closed porosity after HIP treatment in the third build parameter constructs. Compressive moduli were similar for all constructs. All constructs exhibited ability to differentiate MSCs into osteoblasts. MSCs responded best to micro-/nano-structures created by final post-processing by GB + AE, increasing OCN, OPG, VEGFA, latent TGFß1, IL4, and IL10. Collectively these data demonstrate that M3D-printed constructs can be readily manufactured with distinct architectures based on the print parameters and post-build modifications. MSCs are sensitive to discrete surface topographical differences that may not show up in qualitative assessments of surface properties and respond by altering local factor production. These factors are vital for osseointegration after implant insertion, especially in patients with compromised bone qualities.

Tailoring of TiAl6V4 Surface Nanostructure for Enhanced In Vitro Osteoblast Response via Gas/Solid (Non-Line-of-Sight) Oxidation/Reduction Reactions.

An aging global population is accelerating the need for better, longer-lasting orthopaedic and dental implants. Additive manufacturing can provide patient-specific, titanium-alloy-based implants with tailored, three-dimensional, bone-like architecture. Studies using two-dimensional substrates have demonstrated that osteoblastic differentiation of bone marrow stromal cells (MSCs) is enhanced on surfaces possessing hierarchical macro/micro/nano-scale roughness that mimics the topography of osteoclast resorption pits on the bone surface. Conventional machined implants with these surfaces exhibit successful osseointegration, but the complex architectures produced by 3D printing make consistent nanoscale surface texturing difficult to achieve, and current line-of-sight methods used to roughen titanium alloy surfaces cannot reach all internal surfaces. Here, we demonstrate a new, non-line-of-sight, gas/solid-reaction-based process capable of generating well-controlled nanotopographies on all open (gas-exposed) surfaces of titanium alloy implants. Dense 3D-printed titanium-aluminum-vanadium (TiAl6V4) substrates were used to evaluate the evolution of surface nanostructure for development of this process. Substrates were either polished to be smooth (for easier evaluation of surface nanostructure evolution) or grit-blasted and acid-etched to present a microrough biomimetic topography. An ultrathin (90 ± 16 nm) conformal, titania-based surface layer was first formed by thermal oxidation (600 °C, 6 h, air). A calciothermic reduction (CaR) reaction (700 °C, 1 h) was then used to convert the surface titania (TiO2) into thin layers of calcia (CaO, 77 ± 16 nm) and titanium (Ti, 51 ± 20 nm). Selective dissolution of the CaO layer (3 M acetic acid, 40 min) then yielded a thin nanoporous/nanorough Ti-based surface layer. The changes in surface nanostructure/chemistry after each step were confirmed by scanning and transmission electron microscopies with energy-dispersive X-ray analysis, X-ray diffraction, selected area electron diffraction, atomic force microscopy, and mass change analyses. In vitro studies indicated that human MSCs on CaR-modified microrough surfaces exhibited increased protein expression associated with osteoblast differentiation and promoted osteogenesis compared to unmodified microrough surfaces (increases of 387% in osteopontin, 210% in osteocalcin, 282% in bone morphogenic protein 2, 150% in bone morphogenic protein 4, 265% in osteoprotegerin, and 191% in vascular endothelial growth factor). This work suggests that this CaR-based technique can provide biomimetic topography on all biologically facing surfaces of complex, porous, additively manufactured TiAl6V4 implants.

Prophylactic administration of miR-451 inhibitor decreases osteoarthritis severity in rats.

Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the growth plate, upregulates production of enzymes association with extracellular matrix degradation. miR-451 is also present in articular cartilage and exacerbates IL-1ß effects in articular chondrocytes. Moreover, when osteoarthritis (OA) was induced in Sprague Dawley rats via bilateral anterior cruciate ligament transection (ACLT), miR-451 expression was increased in OA cartilage compared to control, suggesting its inhibition might be used to prevent or treat OA. To examine the prophylactic and therapeutic potential of inhibiting miR-451, we evaluated treatment with miR-451 power inhibitor (451-PI) at the onset of joint trauma and treatment after OA had developed. The prophylactic animal cohort received twice-weekly intra-articular injections of either 451-PI or a negative control (NC-PI) beginning on post-surgical day 3. OA was allowed to develop for 24 days in the therapeutic cohort before beginning injections. All rats were killed on day 45. Micro-CT, histomorphometrics, OARSI scoring, and muscle force testing were performed on samples. 451-PI mitigated OA progression compared to NC-PI limbs in the prophylactic cohort based on histomorphometric analysis and OARSI scoring, but no differences were detected by micro-CT. 451-PI treatment beginning 24 days post-surgery was not able to reduce OA severity. Prophylactic administration of 451-PI mitigates OA progression in a post-trauma ACLT rat model supporting its potential to prevent OA development following an ACLT injury clinically.

Osseointegration and Remodeling of Mineralized Bone Graft Are Negatively Impacted by Prior Treatment with Bisphosphonates.

BACKGROUND: Bisphosphonates limit resorption by inhibiting osteoclast formation and activation. They are removed during preparation of demineralized bone matrix (DBM) particles, but it is not known if osteogenesis and incorporation of mineralized bone allografts from patients treated with oral bisphosphonates are affected in vivo. METHODS: Human block allografts from 3 bisphosphonate-treated donors and 3 age and sex-matched control donors who had not received bisphosphonates were obtained (Musculoskeletal Transplant Foundation); one-half from each donor was demineralized. In the first study, 3 × 2-mm mineralized and demineralized cylindrical grafts were implanted bilaterally in the femoral metaphysis of 56 rats. In the second study, samples from each group were pooled, prepared as particles, and implanted bilaterally in the femoral marrow canal of 24 rats. Osseointegration, defined as native bone in contact with allograft, was assessed at 10 weeks by micro-computed tomography (CT) and histomorphometry. RESULTS: Micro-CT showed greater bone volume in sites treated with demineralized samples compared with the control mineralized and bisphosphonate-exposed mineralized samples. More new bone was generated along the cortical-endosteal interface compared with mineralized samples. Histology showed significantly less new bone in contact with the mineralized bisphosphonate-exposed allograft (10.4%) compared with mineralized samples that did not receive bisphosphonates (22.8%) and demineralized samples (31.7% and 42.8%). A gap was observed between native bone and allograft in the bisphosphonate-exposed mineralized samples (0.50 mm 2 ). The gap area was significantly greater compared with mineralized samples that did not receive bisphosphonates (0.16 mm 2 ) and demineralized samples (0.10 and 0.03 mm 2 ). CONCLUSIONS: Mineralized allografts were osseointegrated, but not remodeled or replaced by living bone, preventing full regeneration of the bone defect. Prior treatment of the donor with bisphosphonates affected osteogenesis, preventing osteointegration and remodeling of the allograft into the regenerating bone. CLINICAL RELEVANCE: Clinical use of mineralized allografts from patients who had received bisphosphonate therapy needs to be evaluated; in this animal model, such grafts were not integrated into the host bone or remodeled, and full regeneration of the bone defects was prevented.

The Role of Matrix-Bound Extracellular Vesicles in the Regulation of Endochondral Bone Formation.

Matrix vesicles are key players in the development of the growth plate during endochondral bone formation. They are involved in the turnover of the extracellular matrix and its mineralization, as well as being a vehicle for chondrocyte communication and regulation. These extracellular organelles are released by the cells and are anchored to the matrix via integrin binding to collagen. The exact function and makeup of the vesicles are dependent on the zone of the growth plate in which they are produced. Early studies defined their role as sites of initial calcium phosphate deposition based on the presence of crystals on the inner leaflet of the membrane and subsequent identification of enzymes, ion transporters, and phospholipid complexes involved in mineral formation. More recent studies have shown that they contain small RNAs, including microRNAs, that are distinct from the parent cell, raising the hypothesis that they are a distinct subset of exosomes. Matrix vesicles are produced under complex regulatory pathways, which include the action of steroid hormones. Once in the matrix, their maturation is mediated by the action of secreted hormones. How they convey information to cells, either through autocrine or paracrine actions, is now being elucidated.

A Review of Biomimetic Topographies and Their Role in Promoting Bone Formation and Osseointegration: Implications for Clinical Use.

The use of metallic and polymeric materials for implants has been increasing over the past decade. This trend can be attributed to a variety of factors including a significant increase in basic science research focused on implant material characteristics and how various surface modifications may stimulate osseointegration and, ultimately, fusion. There are many interbody fusion devices and dental implants commercially available; however, detailed information about their surface properties, and the effects that various materials and surface modifications may have on osteogenesis, is lacking in the literature. While the concept of bone-implant osseointegration is a relatively recent addition to the spine fusion literature, there is a comparatively large body of literature related to dental implants. The purpose of this article is to summarize the science of surface modified bone-facing implants, focusing on biomimetic material chemistry and topography of titanium implants, to promote a better understanding of how these characteristics may impact bone formation and osseointegration. This manuscript has the following aspects: highlights the role of titanium and its alloys as potent osteoconductive bioactive materials; explores the importance of biomimetic surface topography at the macro-, micro- and nano-scale; summarizes how material surface design can influence osteogenesis and immune responses in vitro; focuses on the kinds of surface modifications that play a role in the process. Biomimetic surface modifications can be varied across many clinically available biomaterials, and the literature supports the hypothesis that those biomaterial surfaces that exhibit physical properties of bone resorption pits, such as roughness and complex hierarchical structures at the submicron and nanoscale, are more effective in supporting osteoblast differentiation in vitro and osteogenesis in vivo.