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.

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.

Growth factors produced by bone marrow stromal cells on nanoroughened titanium-aluminum-vanadium surfaces program distal MSCs into osteoblasts via BMP2 signaling.

Statement of Clinical Significance: There remains the need to develop materials and surfaces that can increase the rate of implant osseointegration. Though osteoanabolic agents, like bone morphogenetic protein (BMP), can provide signaling for osteogenesis, the appropriate design of implants can also produce an innate cellular response that may reduce or eliminate the need to use additional agents to stimulate bone formation. Studies show that titanium implant surfaces that mimic the physical properties of osteoclast resorption pits regulate cellular responses of bone marrow stromal cells (MSCs) by altering cell morphology, transcriptomes, and local factor production to increase their differentiation into osteoblasts without osteogenic media supplements required for differentiation of MSCs on tissue culture polystyrene (TCPS). The goal of this study was to determine how cells in contact with biomimetic implant surfaces regulate the microenvironment around these surfaces in vitro. Two different approaches were used. First, unidirectional signaling was assessed by treating human MSCs grown on TCPS with conditioned media from MSC cultures grown on Ti6Al4V biomimetic surfaces. In the second set of studies, bidirectional signaling was assessed by coculturing MSCs grown on mesh inserts that were placed into culture wells in which MSCs were grown on the biomimetic Ti6Al4V substrates. The results show that biomimetic Ti6Al4V surface properties induce MSCs to produce factors within 7 days of culture that stimulate MSCs not in contact with the surface to exhibit an osteoblast phenotype via endogenous BMP2 acting in a paracrine signaling manner.

The Relative Expression of ERα Isoforms ERα66 and ERα36 Controls the Cellular Response to 24R,25-Dihydroxyvitamin D3 in Breast Cancer.

Vitamin D3 and its metabolites have antitumorigenic properties in vitro and in vivo; however, clinical trials and retrospective studies on the effectiveness of vitamin D3 oral supplementation against cancer have been inconclusive. One reason for this may be that clinical trials ignore the complex vitamin D metabolome and the many active vitamin D3 metabolites present in the body. Recent work by our lab showed that 24R,25(OH)2D3, a vitamin D3 metabolite that is active in chondrocyte proliferation and differentiation, has antitumorigenic properties in estrogen receptor alpha-66 (ERα66)-positive (ER+) breast cancer, but not in ERα66-negative (ER-) breast cancer. Here we show that 24R,25(OH)2D3 is protumorigenic in an in vivo mouse model (NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice) of ER- breast cancer, causing greater tumor growth than in mice treated with vehicle alone. In vitro results indicate that the effect of 24R,25(OH)2D3 is via a membrane-associated mechanism involving ERs and phospholipase D. 24R,25(OH)2D3 increased proliferation and reduced apoptosis in ERα66-negative HCC38 breast cancer cells, and stimulated expression of metastatic markers. Overexpressing ESRI, which encodes ERα66, ERα46, and ERα36, reduced the proapoptotic response of ERα66- cells to 24R,25(OH)2D3, possibly by upregulating ERα66. Silencing ESR1 in ERα66+ cells increased apoptosis. This suggests 24R,25(OH)2D3 is differentially tumorigenic in cancers with different ERα isoform profiles. Antiapoptotic actions of 24R,25(OH)2D3 require ERα36 and proapoptotic actions require ERα66. IMPLICATIONS: These results suggest that 24R,25(OH)2D3, which is a major circulating metabolite of vitamin D, is functionally active in breast cancer and that the regulatory properties of 24R,25(OH)2D3 are dependent upon the relative expression of ERα66 and ERα36.

Hot isostatic pressure treatment of 3D printed Ti6Al4V alters surface modifications and cellular response.

Additive manufacturing can be used to create personalized orthopedic and dental implants with varying geometries and porosities meant to mimic morphological properties of bone. These qualities can alleviate stress shielding and increase osseointegration through bone ingrowth, but at the expense of reduced fatigue properties compared to machined implants, and potential for loose build particle erosion. Hot isostatic pressure (HIP) treatment is used to increase fatigue resistance; implant surface treatments like grit-blasting and acid-etching create microroughness and reduce the presence of loose particles. However, it is not known how HIP treatment affects surface treatments and osseointegration of the implant to bone. We manufactured two titanium-aluminum-vanadium constructs, one with simple through-and-through porosity and one possessing complex trabecular bone-like porosity. We observed HIP treatment varied in effect and was dependent on architecture. Micro/meso/nano surface properties generated by grit-blasting and acid-etching were altered on biomimetic HIP-treated constructs. Human mesenchymal stem cells (MSCs) were cultured on constructs fabricated +/- HIP and subsequently surface-treated. MSCs were sensitive to 3D-architecture, exhibiting greater osteogenic differentiation on constructs with complex trabecular bone-like porosity. HIP-treatment did not alter the osteogenic response of MSCs to these constructs. Thus, HIP may provide mechanical and biological advantages during implant osseointegration and function.

Long-Distance and Trans-Generational Stomatal Patterning by CO2 Across Arabidopsis Organs.

Stomata control water loss and carbon dioxide uptake by both altering pore aperture and developmental patterning. Stomatal patterning is regulated by environmental factors including atmospheric carbon dioxide (p[CO2]), which is increasing globally at an unprecedented rate. Mature leaves are known to convey developmental cues to immature leaves in response to p[CO2], but the developmental mechanisms are unknown. To characterize changes in stomatal patterning resulting from signals moving from mature to developing leaves, we constructed a dual-chamber growth system in which rosette and cauline leaves of Arabidopsis thaliana were subjected to differing p[CO2]. Young rosette tissue was found to adjust stomatal index (SI, the proportion of stomata to total cell number) in response to both the current environment and the environment experienced by mature rosette tissue, whereas cauline leaves appear to be insensitive to p[CO2] treatment. It is likely that cauline leaves and cotyledons deploy mechanisms for controlling stomatal development that share common but also deploy distinctive mechanisms to that operating in rosette leaves. The effect of p[CO2] on stomatal development is retained in cotyledons of the next generation, however, this effect does not occur in pre-germination stomatal lineage cells but only after germination. Finally, these data suggest that p[CO2] affects regulation of stomatal development specifically through the development of satellite stomata (stomata induced by signals from a neighboring stomate) during spacing divisions and not the basal pathway. To our knowledge, this is the first report identifying developmental steps responsible for altered stomatal patterning to p[CO2] and its trans-generational inheritance.

Application of Optical Topometry to Analysis of the Plant Epidermis.

The plant epidermis regulates key physiological functions contributing to photosynthetic rate, plant productivity, and ecosystem stability. Yet, quantitative characterization of this interface between a plant and its aerial environment is laborious and destructive with current techniques, making large-scale characterization of epidermal cell parameters impractical. Here, we present our exploration of optical topometry (OT) for the analysis of plant organ surfaces. OT is a mature, confocal microscopy-based implementation of surface metrology that generates nanometer-scale digital characterizations of any surface. We report epidermal analyses in Arabidopsis (Arabidopsis thaliana) and other species as well as dried herbarium specimens and fossilized plants. We evaluate the technology's analytical potential for identifying an array of epidermal characters, including cell type distributions, variation in cell morphology and stomatal depth, differentiation of herbarium specimens, and real-time deformations in living tissue following detachment. As applied to plant material, OT is very fast and nondestructive, yielding richly mineable data sets describing living tissues and rendering a variety of their characteristics accessible for statistical, quantitative genetic, and structural analysis.

Heritable variation in the inflorescence replacement program of Arabidopsis thaliana.

Owing to their sessile habits and trophic position within global ecosystems, higher plants display a sundry assortment of adaptations to the threat of predation. Unlike animals, nearly all higher plants can replace reproductive structures lost to predators by activating reserved growing points called axillary meristems. As the first step in a program aimed at defining the genetic architecture of the inflorescence replacement program (IRP) of Arabidopsis thaliana, we describe the results of a quantitative germplasm survey of developmental responses to loss of the primary reproductive axis. Eighty-five diverse accessions were grown in a replicated common garden and assessed for six life history traits and four IRP traits, including the number and lengths of axillary inflorescences present on the day that the first among them re-flowered after basal clipping of the primary inflorescence. Significant natural variation and high heritabilities were observed for all measured characters. Pairwise correlations among the 10 focal traits revealed a multi-dimensional phenotypic space sculpted by ontogenic and plastic allometries as well as apparent constraints and outliers of genetic interest. Cluster analysis of the IRP traits sorted the 85 accessions into 5 associations, a topology that establishes the boundaries within which the evolving Arabidopsis genome extends and restricts the species' IRP repertoire to that observable worldwide.