Biomaterial Surface Topography Research Articles

Calcium phosphate ceramic-induced osteoimmunomodulation: Submicron-surface-treated macrophage-derived exosomes driving osteogenesis

The development of osteoimmunology has highlighted the crucial role of biomaterial surface topography in modulating macrophage responses, subsequently affecting bone formation. However, the underlying mechanism remains largely unexplored. This study introduced two biphasic calcium phosphate (BCP) ceramics with different surface architecture, finding that compared to micron-scale counterparts (BCP2, ∼3.07 μm), BCP with submicron-scale structure (BCP1, ∼0.66 μm) enhanced ectopic bone formation and facilitated M2 macrophage polarization in vivo. Previous studies have focused predominantly on soluble cytokines secreted by macrophages, the significance of exosomes in intercellular communication has been overlooked. This study isolated exosomes from macrophages treated with either submicron- or micron-scale surfaces, designated as B1-Exos and B2-Exos, respectively. Notably, B1-Exos could significantly improve osteogenic differentiation of mesenchymal stem cells (MSCs), potentially attributable to miRNA cargos within these exosomes. SmallRNA sequencing, western blotting, qRT-PCR, and bioinformatics analyses indicated that submicron-sized BCP1 ceramic markedly elevated miR-142a-5p levels in macrophage-derived exosomes, activating the PTEN/AKT signaling pathway, and consequently guiding the differentiation of MSCs towards osteoblast lineage. These findings not only deepen our understanding of the biomaterial-mediated osteoinduction mechanism, but also inform the design of bone repair materials with specialized surface design for proper immunomodulation to initiate osteogenesis.

Differentiation-inducing effect of osteoclast microgrooves for the purpose of three-dimensional design of regenerated bone.

In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 μm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 μm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 μm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. STATEMENT OF SIGNIFICANCE: Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-μm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment.

The biomimetic surface topography of Rubus fruticosus leaves stimulate the induction of osteogenic differentiation of rBMSCs

The interaction between cells and biomaterials is essential for the success of biomedical applications in which the implantation of biomaterials in the human body is necessary. It has been demonstrated that material’s chemical, mechanical, and structural properties can influence cell behaviour. The surface topography of biomaterials is a physical property that can have a major role in mediating cell–material interactions. This interaction can lead to different cell responses regarding cell motility, proliferation, migration, and even differentiation. The combination of biomaterials with mesenchymal stem cells (MSCs) for bone regeneration is a promising strategy to avoid the need for autologous transplant of bone. Surface topography was also associated with the capacity to control MSCs differentiation. Most of the topographies studied so far involve machine-generated surface topographies. Herein, our strategy differentiates from the above mentioned since we selected natural surface topographies that can modulate cell functions for regenerative medicine strategies. Rubus fruticosus leaf was the selected topography to be replicated in polycaprolactone (PCL) membranes through polydimethylsiloxane moulding and using soft lithography. Afterwards, rat bone marrow stem cells (rBMSCs) were seeded at the surface of the imprinted PCL membranes to characterize the bioactive potential of our biomimetic surface topography to drive rBMSCs differentiation into the osteogenic lineage. The selected surface topography in combination with the osteogenic inductive medium reveals having a synergistic effect promoting osteogenic differentiation.

Regulation of macrophage polarization and functional status by modulating hydroxyapatite ceramic micro/nano-topography

Recent studies on osteoimmunology have revealed the indispensable role of immune cells in osteogenesis. The surface topography of biomaterials plays a critical role in regulating cellular behaviors; however, little attention is paid to its effects on the immune response. Three dense hydroxyapatite (HA) disks with grain size in nano-scale (∼100 nm) to submicron (∼500 nm) range were prepared to investigate their role in macrophage polarization and functional status. It was found that HA ceramics with nano-topography promoted macrophage phenotypic switching from the pro-inflammatory M1 type to the pro-healing M2 type. A reduction in grain size could significantly inhibit the secretion of pro-inflammatory cytokine and increase the proportion of CD206+ M2 population in vitro. Meanwhile, nano-topography could reduce tissue inflammation, up-regulate gene expression of M2 phenotypic marker, and raise the fraction of ARG+ M2 macrophages in vivo. This might be attributed to the differential integrin expression to regulate the activation of intracellular signaling cascades. These findings indicate that the surface topography of HA ceramics regulates macrophage responses, thereby adjusting inflammation and subsequent tissue regeneration. An improved understanding of the relationship between material features and its immunomodulatory potential may help to design bone substitute materials with good biological performance.

Protein Adsorption at Nanorough Titanium Oxide Surfaces: The Importance of Surface Statistical Parameters beyond Surface Roughness

The nanoscale surface topography of biomaterials can have strong effects on protein adsorption. While there are numerous surface statistical parameters for the characterization of nanorough surfaces, none of them alone provides a complete description of surface morphology. Herein, a selection of nanorough titanium oxide surfaces has been fabricated with root-mean-square roughness (Sq) values below 2.7 nm but very different surface morphologies. The adsorption of the proteins myoglobin (MGB), bovine serum albumin (BSA), and thyroglobulin (TGL) at these surfaces was investigated in situ by ellipsometry to assess the importance of six of the most common surface statistical parameters. For BSA adsorption, both protein film thickness and time constant of adsorption were found to scale linearly with Sq s. For TGL, however, the same adsorption characteristics depend linearly on the surface skewness (Ssk), which we attribute to the rather extreme size of this protein. Finally, a mixed behavior is observed for MGB adsorption, showing different linear correlations with Sq and Ssk. These results demonstrate the importance of a thorough morphological characterization of the surfaces employed in protein adsorption and possibly also cell adhesion studies.

Systematic Investigation of Polyurethane Biomaterial Surface Roughness on Human Immune Responses in vitro.

It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how different topographies contribute to pro-inflammatory or anti-inflammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with defined roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 μm to 18 μm in average roughness (Ra), which was confirmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inflammatory and anti-inflammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no influence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inflammatory immune response. Therefore, we propose that such modifications do not affect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material.

Collagen and fibronectin surface modification of nanoporous anodic alumina and macroporous silicon for endothelial cell cultures

BackgroundThe ability to direct the cellular response by means of biomaterial surface topography is important for biomedical applications. Substrate surface topography has been shown to be an effective cue for the regulation of cellular response. Here, the response of human aortic endothelial cells to nanoporous anodic alumina and macroporous silicon with collagen and fibronectin functionalization has been studied.MethodsConfocal microscopy and scanning electron microscopy were employed to analyse the effects of the material and the porosity on the adhesion, morphology, and proliferation of the cells. Cell spreading and filopodia formation on macro- and nanoporous material was characterized by atomic force microscopy. We have also studied the influence of the protein on the adhesion.ResultsIt was obtained the best results when the material is functionalized with fibronectin, regarding cells adhesion, morphology, and proliferation.ConclusionThese results permit to obtain chemical modified 3D structures for several biotechnology applications such as tissue engineering, organ-on-chip or regenerative medicine.

Cellular response to nano-structured Zr and ZrO2 alloyed layers on Ti-6Al-4V

The surface topography of biomaterials is known to influence cellular response such as adhesion, spreading and differentiation. In this work, the behavior of osteoblasts and endothelial cells on nano-structured Zr and micro/nano-structured ZrO2 alloyed layers of Ti-6Al-4V (TC4) was investigated. Zr alloyed layer (Zr@TC4) decreases the hydrophilicity whereas ZrO2 alloyed layers (ZrO2@TC4) is more hydrophilic than TC4 and more proteins adsorb on ZrO2@TC4 followed by Zr@TC4. The cells proliferate steadily on the smooth TC4 and nano-structured Zr@TC4 surfaces and the osteoblast activity is more pronounced on Zr@TC4 than TC4. The micro/nano-structured surface on ZrO2@TC4 restricts cellular adhesion and spreading independent of the surface wettability and protein adsorption. The findings provide insights into the design of micro/nano-structured biomaterials and interfaces with controlled tissue response.

Influence of patterned titanium coatings on polarization of macrophage and osteogenic differentiation of bone marrow stem cells

Biomaterial surface topography plays a vital role in the osteointegration of implants by regulating the early cell responses and tissue growth-in. However, most of the previous researches focused on the effects of osteogenic cells, only a little is known about the immune cells which dominate osteogenesis after implanting. In this paper, patterned titanium coatings were fabricated and the effects of surface topography on the macrophage behaviors were investigated. On patterned titanium surface, macrophages preferred to polarize to M2, while macrophages on traditional titanium coatings presented higher M1 polarization. Nearly 70% higher expression of anti-inflammatory genes, including interleukin-4, interleukin-10, interleukin-1ra, and arginase, were detected on the patterned titanium coatings. While the pro-inflammatory genes, such as interleukin-1β, interleukin-6, tumor necrosis factor-α, interferon-γ, and inducible nitric oxide synthase were notably depressed. Up-regulation of the osteoinductive cytokines were also detected on the patterned coatings, which indicated advantageous osteogenic microenvironment provided by macrophages. Immunomodulation effect on osteogenesis was also investigated in this study. Stimulated with RAW cells/patterned coatings conditioned medium, bone marrow stem cells presented nearly 1.5 fold higher expression of osteogenic genes and more mineralization nodules than the traditional sprayed Ti coatings. All these results suggested that modulating materials with a patterned surface might be a valuable strategy to endow the implants with favorable osteoimmunomodulatory properties.

Femtosecond laser microstructured Alumina toughened Zirconia: A new strategy to improve osteogenic differentiation of hMSCs

The use of topographic patterns has been a continuously growing area of research for tissue engineering and it is widely accepted that the surface topography of biomaterials can influence and modulate the initial biological response. Ultrafast lasers are extremely powerful tools to machine and pattern the surface of a wide range of biomaterials, however, only few work has been performed on ceramics with the intent of biomedical applications, and the biological characterization of these structured materials is scarce.In this work, relevance is given to the biological performance of such materials. A femtosecond laser ablation technique was used to modify Alumina toughened Zirconia (ATZ) surface topography, developing surfaces structured at the micro and nanoscale levels (μATZ), in a controlled and reproducible manner. Materials characterization was performed before and after laser treatment, and both materials were compared in terms of osteogenic response of human bone marrow derived mesenchymal stem cells cultured under basal conditions, expecting that the micro/nanofeatures will improve the biological response of cells. Cells metabolic activity and proliferation increased with the culture time and surface microtopography modulated cells alignment and guided proliferation. The modified surface, displayed significantly higher expression of osteogenic transcription factors and genes and, additionally, the formation of a mineralized extracellular matrix, when compared to the control surface, i.e. unmodified ATZ.

Cell patterning via laser micro/nano structured silicon surfaces

The surface topography of biomaterials can have an important impact on cellular adhesion, growth and proliferation. Apart from the overall roughness, the detailed morphological features, at all length scales, significantly affect the cell-biomaterial interactions in a plethora of applications including structural implants, tissue engineering scaffolds and biosensors. In this study, we present a simple, one-step direct laser patterning technique to fabricate nanoripples and dual-rough hierarchical micro/nano structures to control SW10 cell attachment and migration. It is shown that, depending on the laser processing conditions, distinct cell-philic or cell-repellant patterned areas can be attained with a desired motif. We envisage that our technique could enable spatial patterning of cells in a controllable manner, giving rise to advanced capabilities in cell biology research.

Combination of flow and micropattern alignment affecting flow-resistant endothelial cell adhesion

Assuring cell adhesion to an underlying biomaterial surface under blood flow is vital to functional vascular grafts design. In vivo endothelial cells (ECs) are located under the microenvironment of both surface topography of the basement membrane and the mechanical loading resulting from blood flow. Both topographical and mechanical factors should thus be considered when designing vascular grafts to enhance the flow-resistant EC adhesion. This study aims to investigate effects of integrating biomaterial surface topography and flow on EC adhesion, which was a deficit in previous studies. Human umbilical vein endothelial cells (HUVECs) were cultured on different fibronectin (FN) micropatterns parallel or perpendicular to the flow direction and exposed to sustained flow with physiological levels of shear stress (15 dyne/cm2). We demonstrated that micropattern alignment parallel to the flow direction enhanced flow-resistant EC adhesion, while micropattern alignment perpendicular to the flow direction attenuated it. Experimental and numeric modeling analysis underlined that the flow-induced mechanic distribution on the surface of cells that were aligned on the micropatterned surfaces and the subsequent cytoskeleton rearrangement were responsible for the significant difference in EC adhesion. Furthermore, pressure on the surface of cells that were aligned on the micropatterned surfaces induced by flow provided a more critical role in EC adhesion than shear stress. These findings highlight the importance of proper combination of topographical and flow cues in enhancement of EC adhesion and may suggest new strategies for designing functional vascular grafts.

The pathology of the foreign body reaction against biomaterials.

The healing process after implantation of biomaterials involves the interaction of many contributing factors. Besides their in vivo functionality, biomaterials also require characteristics that allow their integration into the designated tissue without eliciting an overshooting foreign body reaction (FBR). The targeted design of biomaterials with these features, thus, needs understanding of the molecular mechanisms of the FBR. Much effort has been put into research on the interaction of engineered materials and the host tissue. This elucidated many aspects of the five FBR phases, that is protein adsorption, acute inflammation, chronic inflammation, foreign body giant cell formation, and fibrous capsule formation. However, in practice, it is still difficult to predict the response against a newly designed biomaterial purely based on the knowledge of its physical-chemical surface features. This insufficient knowledge leads to a high number of factors potentially influencing the FBR, which have to be analyzed in complex animal experiments including appropriate data-based sample sizes. This review is focused on the current knowledge on the general mechanisms of the FBR against biomaterials and the influence of biomaterial surface topography and chemical and physical features on the quality and quantity of the reaction. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 927-940, 2017.

Poly(L-lactide) crystallization topography directs MC3T3-E1 cells response

Biomaterial surface topography significantly influences cellular form and function. Using poly(L-lactic acid) films with normal spherulites, banded spherulites, and amorphous surfaces as model substrates, we conducted a systematic assessment of the role for polymer crystallization induced surface morphologies on cell growth and contact guidance. Microscopy and image analysis showed that the MC3T3-E1 cells spread out in a random fashion on the amorphous substrate. At 24 h post-seeding, MC3T3-E1 cells on both types of spherulite surfaces were elongated and aligned along the spherulite radius direction. For the banded spherulite surface with radial stripes and coupling annular grooves, the cell orientation and cell nuclear localization were related to the grooves structure. With increasing time, this orientation preference was weaker. These results demonstrate that the patterning of polymer crystallization structure provide important signals for guiding cells to exhibit characteristic orientation and morphology especially in the early stages of regeneration.

Accelerated Osteogenic Differentiation and Bone Formation on Zirconia with Surface Grooves Created with Fiber Laser Irradiation.

Modification of the surface topography of biomaterials is a critical factor for the proliferation and differentiation of osteoblasts. Elucidating the biological response to surface roughening is necessary for clinical application of zirconia implants. To investigate the effects of fiber laser-induced zirconia surface roughening on cultured osteoblast-like cell morphology, proliferation, differentiation, and calcification, and on in vivo bone formation. Sixty-six machine-surfaced yttria-tetragonal zirconia polycrystal plates (S-Zr) and 16 machine-surfaced implants were used as controls. We prepared 66 rough plates (R-Zr) and 16 rough implants by surface treatment using a fiber laser. MC3T3-E1 cells spread well in all directions on S-Zr, whereas elongated cells with poorly organized actin stress fibers were observed on R-Zr. Cell proliferation was significantly greater on R-Zr than on S-Zr. The Runx2 mRNA level increased time dependently in osteogenic culture condition. Alkaline phosphatase activity and osteocalcin mRNA levels were higher on R-Zr compared with S-Zr. Alizarin red S staining revealed greater calcification on R-Zr than on S-Zr. Laser treatment of zirconia implant bodies placed in rat tibiae increased the bone-implant contact ratio and removal torque considerably. Our results suggest that fiber laser irradiation produces adequate surface roughening of zirconia ceramics to support osseointegration.

Evaluation of Surface Microtopography Engineered by Direct Laser Interference for Bacterial Anti-Biofouling

Modification of the biomaterial surface topography is a promising strategy to prevent bacterial adhesion and biofilm formation. In this study, we use direct laser interference patterning (DLIP) to modify polystyrene surface topography at sub-micrometer scale. The results revealed that three-dimensional micrometer structures have a profound impact on bacterial adhesion. Thus, line- and pillar-like patterns enhanced S. aureus adhesion, whereas complex lamella microtopography reduced S. aureus adhesion in static and continuous flow culture conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus adhesion both when the surface is coated with human serum proteins and when the material is implanted subcutaneously in a foreign-body associated infection model.

Impact of surface chemistry and topography on the function of antigen presenting cells.

Antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs) play a crucial role in orchestrating immune responses against foreign materials. The activation status of APCs can determine the outcome of an immune response following implantation of synthetic materials, towards either healing or inflammation. A large range of biomaterials are used in the fabrication of implantable devices and drug delivery systems. These materials will be in close contact with APCs and characteristics such as surface chemistry and topography may have a critical role in initiating pro- or anti-inflammatory immune responses. Controlling biomaterial surface attributes provides a powerful tool for modulating the phenotype and function of immune cells with the aim of reducing detrimental pro-inflammatory responses and promoting beneficial healing responses. In this article, we review recent literature on how biomaterial surface topography and chemistry can modulate APC populations towards distinct pro- or anti-inflammatory phenotypes with specific examples of how these properties can be used to control host response in vivo. Topographical and/or chemical design of biomaterial surfaces with respect to the APC responses can pave the way for a new generation of 'cell instructive' materials with immunomodulatory properties with a wide range of clinical applications.

Effect of surface topography and bioactive properties on early adhesion and growth behavior of mouse preosteoblast MC3T3-E1 cells.

The effects of bioactive properties and surface topography of biomaterials on the adhesion and spreading properties of mouse preosteoblast MC3T3-E1 cells was investigated by preparation of different surfaces. Poly lactic-co-glycolic acid (PLGA) electrospun fibers (ES) were produced as a porous rough surface. In our study, coverslips were used as a substrate for the immobilization of 3,4-dihydroxyphenylalanine (DOPA) and collagen type I (COL I) in the preparation of bioactive surfaces. In addition, COL I was immobilized onto porous electrospun fibers surfaces (E-COL) to investigate the combined effects of bioactive molecules and topography. Untreated coverslips were used as controls. Early adhesion and growth behavior of MC3T3-E1 cells cultured on the different surfaces were studied at 6, 12, and 24 h. Evaluation of cell adhesion and morphological changes showed that the all the surfaces were favorable for promoting the adhesion and spreading of cells. CCK-8 assays and flow cytometry revealed that both topography and bioactive properties were favorable for cell growth. Analysis of β1, α1, α2, α5, α10 and α11 integrin expression levels by immunofluorescence, real-time RT-PCR, and Western blot and indicated that surface topography plays an important role in the early stage of cell adhesion. However, the influence of topography and bioactive properties of surfaces on integrins is variable. Compared with any of the topographic or bioactive properties in isolation, the combined effect of both types of properties provided an advantage for the growth and spreading of MC3T3-E1 cells. This study provides a new insight into the functions and effects of topographic and bioactive modifications of surfaces at the interface between cells and biomaterials for tissue engineering.

Involvement of ILK/ERK1/2 and ILK/p38 pathways in mediating the enhanced osteoblast differentiation by micro/nanotopography

The hierarchical micro/nanotextured topography (MNT) on titanium (Ti) implant surface significantly enhances osteoblast differentiation. We have demonstrated that integrin-linked kinase (ILK) is a key underlying signal molecule and β-catenin is one of its downstream mediators in MNT-regulated osteoblast behavior. Here we propose that mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun NH2-terminal kinase (JNK), are other mediators downstream of ILK, and this study aims to confirm this. Firstly, the levels of ILK and MAPK activity in MG63 cells on MNT are examined by Western blot analysis. The ILK, ERK1/2 and p38 signals are significantly up-regulated by MNT, whereas the JNK activity is undetectable by Western blot. The MG63 cell morphology, proliferation and differentiation are studied in the absence and presence of the MAPK subgroup inhibitors to confirm their roles in cell functions on the Ti surface. The MAPK subgroup inhibitors obviously change the cell shape and depress cell proliferation. Blocking the ERK1/2 or p38 signaling, but not the JNK signaling, significantly down-regulates the cell osteogenesis-related gene expression, ALP production, collagen secretion and matrix mineralization. Afterwards, the ILK expression is down-regulated using ILK-specific siRNA (ILKsi) and then the MAPK activity is determined. ILKsi significantly attenuates the phosphorylated ERK1/2 and p38 levels on MNT, explicitly demonstrating that the ERK1/2 and p38 signalings are downstream effectors of ILK. In conclusion, these data demonstrate that both ILK/ERK1/2 and ILK/p38 pathways are involved in the mechanisms mediating the enhanced osteoblast differentiation by biomaterial surface topography, hopefully directing the biomaterial modification and biofunctionalization.

Three-dimensional reconstruction of surface nanoarchitecture from two-dimensional datasets

The design of biomaterial surfaces relies heavily on the ability to accurately measure and visualize the three-dimensional surface nanoarchitecture of substrata. Here, we present a technique for producing three-dimensional surface models using displacement maps that are based on the data obtained from two-dimensional analyses. This technique is particularly useful when applied to scanning electron micrographs that have been calibrated using atomic force microscopy (AFM) roughness data. The evaluation of four different surface types, including thin titanium films, silicon wafers, polystyrene cell culture dishes and dragonfly wings confirmed that this technique is particularly effective for the visualization of conductive surfaces such as metallic titanium. The technique is particularly useful for visualizing surfaces that cannot be easily analyzed using AFM. The speed and ease with which electron micrographs can be recorded, combined with a relatively simple process for generating displacement maps, make this technique useful for the assessment of the surface topography of biomaterials.