Poly(amidoamine)-alginate hydrogels: directing the behavior of mesenchymal stem cells with charged hydrogel surfaces.

The surface charge of a biomaterial represents a promising tool to direct cellular behavior, which is crucial for therapeutic approaches in regenerative medicine. To expand the understanding of how the material surface charge affects protein adsorption and mesenchymal stem cell behavior, differently charged surfaces with zeta potentials spanning from -25 mV to +15 mV were fabricated by the conjugation of poly(amidoamine) to alginate-based hydrogels. We showed that the increase of the biomaterials surface charge resulted in enhanced quantities of biologically available, surface-attached proteins. Since different surface charges were equalized after protein adsorption, mesenchymal stem cells interacted rather with diverse protein compositions instead of different surface features. Besides an enhanced cell attachment to increasingly positively charged surfaces, the cell spreading area and the expression of adhesion-related genes integrin α5 and tensin 1 were found to be increased after adhesion. Moreover, first results indicate a potential impact of the surface charge on mesenchymal stem cell differentiation towards bone and fat cells. The improved understanding of surface charge-related cell behavior has significant impact on the design of biomedical devices and artificial organs.

A coating machine for coating filaments with bioactive nanomaterials for extrusion 3D printing.

Extrusion printing based on biocompatible filaments offers a wide variety of targeted medical and dental applications in the area of personalized medicine, if combined with bioactive nanomaterials. However, this requires filament to be coated with bioactive nanomaterial. This study introduces a concept of a machine to coat filament with bioactive nanomaterials and its application. A machine was constructed with modules manufactured using additive manufacturing. A filament spool of polylactide (PLA) or glycol-modified polyethylene terephthalate (PETG) was transported through a copper tube, with the outer surface of the filament heated to the appropriate glass transition temperature to incorporate added nanomaterials such as nano-hydroxyapatite (nHA) or nano-fluorapatite(nFA). Coatings with nHA led to an increase in diameter of around 3 µm, while coatings with nFA increased the diameter by 4 µm. Printing of cubes with a standard extrusion printer platform using PLA or PETG filaments with added nHA or nFA has been successfully carried out. Scanning electron microscope (SEM) images of coated filaments and printed cubes showed an irregular distribution of nHA or nFA, which could be verified by energy dispersive X-ray analysis (EDX). Adding and adjusting bioactive nanomaterials to filament with a coating machine for filament proved to generate printable filaments. With the wide range of possible applications by different nanomaterials it is anticipated that extrusion printing can cover needs for personalized medicine and dentistry.

In vivo biocompatibility of a new hydrophobic coated Al/Al2O3 nanowire surface on stents.

BACKGROUND: Intima proliferation and in-stent restenosis is a challenging situation in interventional treatment of small vessel obstruction. Al/Al2O3 nanowires have been shown to accelerate vascular endothelial cell proliferation and migration in vitro, while suppressing vascular smooth muscle cell growth. Moreover, surface modification of Al/Al2O3 nanowires with poly[bis(2,2,2-trifluoromethoxy)phosphazene (PTFEP) coating enables further advantages such as reduced platelet adhesion. Therefore, the study's goal was to compare the biocompatibility of novel Al/Al2O3 + PTFEP coated nanowire bare-metal stents to uncoated control stents in vivo using optical coherence tomography (OCT), quantitative angiography and histomorphometric assessment. METHODS: 15 Al/Al2O3 + PTFEP coated and 19 control stents were implanted in the cervical arteries of 9 Aachen minipigs. After 90 days, in-stent stenosis, thrombogenicity, and inflammatory response were assessed. Scanning electron microscopy was used to analyse the stent surface. RESULTS: OCT analysis revealed that neointimal proliferation in Al/Al2O3 + PTFEP coated stents was significantly reduced compared to control stents. The neointimal area was 1.16 ± 0.77 mm2 in Al/Al2O3 + PTFEP coated stents vs. 1.98 ± 1.04 mm2 in control stents (p = 0.004), and the neointimal thickness was 0.28 ± 0.20 vs. 0.47 ± 0.10 (p = 0.003). Quantitative angiography showed a tendency to less neointimal growth in coated stents. Histomorphometry showed no significant difference between the two groups and revealed an apparent inflammatory reaction surrounding the stent struts. CONCLUSIONS: At long-term follow-up, Al/Al2O3 + PTFEP coated stents placed in peripheral arteries demonstrated good tolerance with no treatment-associated vascular obstruction and reduced in-stent restenosis in OCT. These preliminary in vivo findings indicate that Al/Al2O3 + PTFEP coated nanowire stents may have translational potential to be used for the prevention of in-stent restenosis.

Influence of hydroxyapatite nanoparticles on the formation of calcium fluoride surface layer on enamel and dentine in vitro.

Topical application of different fluoride preparations is considered to be the gold standard of oral prophylaxis measures in preventive dentistry. Hydroxyapatite nanoparticles (nano-HAP) as well, have received considerable attention for dental use in the past few decades. The purpose of this in-vitro study was to analyze the interaction between nano-HAP and different fluoride preparations. In order to investigate the possibility to establish, in the presence of nano-HAP, reproducible calcium fluoride surface layers, specimens were visually examined with regard to the surface coverage's structure, morphology, homogeneity and stability. Test series based on enamel and dentine specimens, that were obtained from extracted bovine teeth, were conducted. Thereby, sodium fluoride, olaflur, elmex Fluid (10.000 ppm) and an aqueous nano-HAP solution (5%) served as test products and sterile water as reference. First, single application of nano- HAP and fluoride was tested. After 5 min of incubation in the test solution, the surface coverage was examined by scanning electron microscopy (SEM). Furthermore, samples were determined by energy dispersive X-ray spectroscopy (EDX) to identify the present elements of the surface layer, particularly fluoride. To test the calcium fluoride layer's persistence and stability, samples were exposed to the spray of a dental multifunctional syringe for 20 s using maximum pressure and maximum water supply. In the second application protocol, fluoride and nano-HAP were applied simultaneously and in the third application protocol they were used sequentially. SEM visualisation showed that the simultaneous or sequential addition of nano-HAP led to a distinct change in the surface layer's structure. Agglomerates of various sizes were formed, with obviously different morphology from the calcium fluoride globules, not covering the surface homogeneously and sprayed off with the multifunctional syringe easily. Application of pure fluoride compounds resulted in a more homogeneous calcium fluoride surface layer with higher persistence in comparison to the combination of fluoride and nano-HAP. Interaction between fluoride and nano-HAP clearly could be proved. On enamel as well as dentine surfaces, the combined application of nano-HAP and fluoride has a negative effect on the stability and persistence of the calcium fluoride surface precipitate.

Influence of a hydroxyapatite suspension on 48-h dental biofilm formation in-situ.

OBJECTIVE: The aim of the present in-situ study was to investigate anti-adherent properties of mouthrinses containing hydroxyapatite (HAP) nanoparticles on oral biofilm formation. DESIGN: Biofilm was formed for 48 h on bovine enamel or dentine specimens that were fixed to maxillary splints and worn intraoral by six volunteers. During biofilm formation, rinsing was performed with sterile water, HAP (5%) or chlorhexidine (0.2%) according to two different rinsing protocols in order to assess substantivity. Scanning electron microscopy was used to investigate biofilm coverage of specimens, biofilm thickness and morphology. In addition, saliva samples were collected and analysed with transmission electron microscopy. RESULTS: Rinsing with sterile water or HAP resulted in 2.1 or 2.3 µm thick biofilms, respectively, covering more than half of specimen' surfaces. Despite single deposits of nanoparticles in saliva and biofilm, HAP did not inhibit biofilm formation. Chlorhexidine on the other hand significantly reduced biofilm thickness and coverage. CONCLUSIONS: Mouthrinses containing HAP nanoparticles showed no anti-adherent effects during 48 h of biofilm formation in-situ.

G-fibres in storage roots of Trifolium pratense (Fabaceae): tensile stress generators for contraction.

Root contraction has been described for many species within the plant kingdom for over a century, and many suggestions have been made for mechanisms behind these contractions. To move the foliage buds deeper into the soil, the proximal part of the storage root of Trifolium pratense contracts by up to 30%. Anatomical studies have shown undeformed fibres next to strongly deformed tissues. Raman imaging revealed that these fibres are chemically and structurally very similar to poplar (Populus) tension wood fibres, which are known to generate high tensile stresses and bend leaning stems or branches upright. Analogously, an almost pure cellulosic layer is laid down in the lumen of certain root fibres, on a thin lignified secondary cell wall layer. To reveal its stress generation capacities, the thick cellulosic layer, reminiscent of a gelatinous layer (G-layer) in tension wood, was selectively removed by enzymatic treatment. A substantial change in the dimensions of the isolated wood fibre bundles was observed. This high stress relaxation indicates the presence of high tensile stress for root contraction. These findings indicate a mechanism of root contraction in T. pratense (red clover) actuated via tension wood fibres, which follows the same principle known for poplar tension wood.

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.

BACKGROUND AIMS: The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. METHODS: We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. RESULTS: The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. CONCLUSIONS: Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.

Hydroxyapatite-Based Solution as Adjunct Treatment for Biofilm Management: An In Situ Study.

Synthetic hydroxyapatite-based solution is a bioinspired material that may present anti-adhesive properties, restraining the dental biofilm formation without causing adverse effects. This in situ study aims to evaluate the effects of three different hydroxyapatite (HAP) watery solutions as a mouthwash against biofilm adhesion on different dental material surfaces under oral conditions. Hence, four volunteers carried maxillary splints containing enamel, titanium, ceramics, and polymethyl-methacrylate resin (PMMA) samples. Three HAP watery solutions (5%) were prepared with HAP particles presenting different shapes and sizes (HAP I, HAP II, HAP III). During 24 h, the volunteers rinsed two times with one of the following selected tested solution: HAP I, HAP II, HAP III, water, or chlorhexidine 0.2% (CHX). The first rinse was performed 3 min after pellicle formation; the second rinse occurred after a 12 h interval. The surface analysis was performed by scanning electron microscopy (SEM), fluorescence microscopy (FM), and transmission electron microscopy (TEM). Statistical and microscopic analysis showed that most samples treated with any HAP solution revealed reduced biofilm coverage presenting comparable results to CHX treated samples, however without altering the microorganisms' viability. In conclusion, the results of this investigation showed that a pure hydroxyapatite-based mouthrinse could be a promising bioinspired adjunct solution for biofilm management.

Cleavage plane after liquid-bubble preparation of Descemet's membrane.

PURPOSE: To investigate the ultrastructure of the cleavage plane of human cornea after liquid-bubble-prepared tissue for Descemet's membrane endothelial keratoplasty (DMEK). METHODS: Experimental study with scanning electron microscopy (SEM) and block-face SEM of 18 corneal specimens. Fresh human research donor corneoscleral discs (n = 12) were prepared with liquid-bubble technique or examined as untreated controls (n = 3). In addition, Descemet's membrane samples, n = 3, were obtained in DMEK surgery. RESULTS: The cleavage plane after liquid-bubble Descemet's membrane (DM) preparation was consistently located between interfacial matrix and posterior stromal collagen lamellae, providing a largely smooth surface exposing the amorphous interfacial zone without any significant amounts of adherent stromal remnants. No demarcation of a distinct pre-DM layer could be detected. CONCLUSION: The DMEK graft preparation performed by liquid-bubble technique showed a smooth cleavage plane and could not reveal any demarcation of a distinct pre-DM layer.

Toward Alginate-Based Membrane Technology for High Performance Recovery of Heavy Metals in Cells.

One of the major environmental problems is a global metal contamination. Heavy metals are nonbiodegradable and tend to accumulate in living organisms. Therefore, searching for biocompatible materials with enhanced sorption capabilities for selective removal of toxic elements from complex environments, low cost, ease of operation, and large available quantities that meet all requirements of the Green Chemistry concept is a current engineering and analytical task. We present a comprehensive study toward construction of an advanced biomembrane-based technology for recovery of several heavy metals and ruthenium by microdimensional alginate scaffolds. The chosen design of alginate scaffolds and their operational conditions were monitored during removal of Cd(II), Co(II), Pb(II), As(III), and Ru(III) in modeled aqueous solutions, cell culture medium, and in the presence of A549 lung cells by a tandem of biological (live/dead cell test), physical nanoanalytical (TEM/EDX, SEM/EDX), and chemical (FT-IR, HR-ICP-MS) assays. More precisely, the impact of certain experimental conditions, viz., medium acidity and matrix effects on sorption capacity of the above-mentioned elements, was investigated in detail. Remarkably, a different attachment behavior during adsorption of chosen elements by alginate scaffolds was observed. In addition, we revealed an essential concentration dependent effect of loaded heavy metals and ruthenium on cultivated cells. The obtained data allow us to gain a deeper insight into the interactions occurring in the studied biomaterial-inorganic system. Moreover, the obtained dependencies can be widely used for the development of alginate-based membrane technology employed for the protection of environmental and biological samples from the toxic pollutants.

Adhesion of Hydroxyapatite Nanoparticles to Dental Materials under Oral Conditions.

Hydroxyapatite nanoparticles (nano-HAP) are receiving considerable attention for dental applications, and their adhesion to enamel is well established. However, there are no reports concerning the effects of HAP on other dental materials, and most of the studies in this field are based on in vitro designs, neglecting the salivary pellicle-apatite interactions. Thus, this in situ pilot study aims to evaluate the effects of three hydroxyapatite-based solutions and their interactions with different dental material surfaces under oral conditions. Hence, two volunteers carried intraoral splints with mounted samples from enamel and from three dental materials: titanium, ceramics, and polymethyl-methacrylate (PMMA). Three HAP watery solutions (5%) were prepared with different shapes and sizes of nano-HAP (HAP I, HAP II, HAP III). After 3 min of pellicle formation, 10 ml rinse was performed during 30 sec. Rinsing with water served as control. Samples were accessed immediately after rinsing, 30 min and 2 h after rinsing. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the particles, and SEM evaluated the pellicle-HAP interactions. SEM and TEM results showed a high variation in the size range of the particles applied. A heterogeneous HAP layer was present after 2 h on enamel, titanium, ceramics, and PMMA surfaces under oral conditions. Bridge-like structures were visible between the nano-HAP and the pellicle formed on enamel, titanium, and PMMA surfaces. In conclusion, nano-HAP can adhere not only to enamel but also to artificial dental surfaces under oral conditions. The experiment showed that the acquired pellicle act as a bridge between the nano-HAP and the materials' surface.

Modification of in situ Biofilm Formation on Titanium by a Hydroxyapatite Nanoparticle-Based Solution.

Oral biofilms play an essential role on peri-implant disease development. Synthetic hydroxyapatite nanoparticles (nHAP) are a bioinspired material that has structural and functional similarities to dental enamel apatite and may provide preventive properties against biofilm formation. This study aimed to investigate the effects of an experimental nHAP solution on biofilm formation on polished and non-polished titanium under oral conditions. Five volunteers carried maxillary splints with non-polished and polished titanium and followed a 48 h rinsing protocol with the proposed nHAP solution, and with chlorhexidine 0.2% (CHX) and water, as controls. Samples were analyzed by fluorescence microscopy (FM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). FM showed a significant reduction of biofilms on polished samples treated with nHAP (p = 0.0485) compared with water, without differences between nHAP and CHX (p > 0.9999). Analyzing biofilm viability, polished samples rinsed with nHAP showed significantly fewer dead bacteria than CHX (p = 0.0079), but there was no significant difference in viability between polished samples rinsed with water and nHAP (p = 0.9268). A significantly higher biofilm coverage was observed on the non-polished surfaces compared to the polished surfaces when nHAP was applied (p = 0.0317). This difference between polished and non-polished surfaces was not significant when water (p = 0.1587) or CHX (p = 0.3413) rinsing were applied. SEM and TEM analysis supported the FM findings, that polished samples rinsed with nHAP presented fewer biofilm coverage compared to samples rinsed with water. In conclusion, the nHAP solution reduced the biofilm formation on polished Ti surfaces without altering bacterial viability, providing a novel approach for the management of biofilm formation on biomaterials.

Author Correction: Influence of pure fluorides and stannous ions on the initial bacterial colonization in situ.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Influence of pure fluorides and stannous ions on the initial bacterial colonization in situ.

The present clinical-experimental study aims to examine the effect of pure experimental fluoride solutions and stannous chloride on the initial oral bioadhesion under in situ conditions. After 1 min of pellicle formation on bovine enamel slabs, 12 subjects rinsed with 8 ml of the fluoride test solutions (NaF, Na2PO3F, AmF, SnF2,) with 500 ppm fluoride concentration each for 1 min. Additionally, rinsing without a solution (control) and rinsing with 1563 ppm SnCl2 solution took place for 1 min. Afterwards, fluorescence microscopy took place to visualize bacterial adhesion and glucan formation (8 h oral exposition) with DAPI and ConA and the BacLight method. TEM was performed to visualize the pellicle ultrastructure together with EDX to detect stannous ions. The rinsing solutions with pure SnF2 and SnCl2 reduced significantly the initial bacterial colonization (DAPI). While, NaF and Na2PO3F showed no significant effect compared to the control. There was no significant difference between AmF, SnF2 and SnCl2. All tested experimental solutions showed no reducing effect on the glucan formation. Considerable alterations of the pellicle ultrastructure resulted from rinsing with the Sn-containing solutions. SnF2 appears to be the most effective type of fluoride to reduce initial bacterial colonization in situ. The observed effects primarily have to be attributed to the stannous ions' content.

Evaluation of novel organosilane modifications of paper spray mass spectrometry substrates for analyzing polar compounds.

Paper spray mass spectrometry (PSMS) is currently used in different analytical fields, but less effort has been made so far to use PSMS for highly polar compounds. Such analytes usually show poor performance in PSMS due to their high affinity for common paper substrates in addition to high matrix effects. In this study, strategies for hydrophobic modifications of commercially available paper substrates using ten different organosilanes were developed. The modified substrates were generated, characterized, and applied for PSMS analysis of polar toxins. By using the modified paper, PSMS performance of some of the toxins could be considerably increased, especially for orellanine, showing a more than 80-fold signal enhancement when substrates modified with chlorotrimethylsilane were used. For other toxins like ricinine, only small beneficial effects could be shown on PSMS performance when using modified substrates. Statistical equivalence tests showed sufficient ruggedness of the developed procedures also compared to conventional substrates. Thus, further systematic development of paper substrates modified with organosilane derivatives based on the presented study for application in PSMS should be encouraged.

Controlling fibroblast adhesion and proliferation by 1D Al2O3 nanostructures.

The fibrotic encapsulation, which is mainly accompanied by an excessive proliferation of fibroblasts, is an undesired phenomenon after the implantation of various medical devices. Beside the surface chemistry, the topography plays also a major role in the fibroblast-surface interaction. In the present study, one-dimensional aluminium oxide (1D Al2O3) nanostructures with different distribution densities were prepared to reveal the response of human fibroblasts to the surface topography. The cell size, the cell number and the ability to form well-defined actin fibres and focal adhesions were significantly impaired with increasing distribution density of the 1D Al2O3 nanostructures on the substratum.

Reduced myofibroblast differentiation on femtosecond laser treated 316LS stainless steel.

In-stent restenosis is a common complication after stent surgery which leads to a dangerous wall narrowing of a blood vessel. Laser assisted patterning is one of the effective methods to modify the stent surface to control cell-surface interactions which play a major role in the restenosis. In this current study, 316 LS stainless steel substrates are structured by focusing a femtosecond laser beam down to a spot size of 50 µm. By altering the laser induced spot density three distinct surfaces (low density (LD), medium density (MD) and high density (HD)) were prepared. While such surfaces are composed of primary microstructures, due to fast melting and re-solidification by ultra-short laser pulses, nanofeatures are also observed as secondary structures. Following a detailed surface characterization (chemical and physical properties of the surface), we used a well-established co-culture assay of human microvascular endothelial cells and human fibroblasts to check the cell compatibility of the prepared surfaces. The surfaces were analyzed in terms of cell adherence, proliferation, cell morphology and the differentiation of the fibroblast into the myofibroblast, which is a process indicating a general fibrotic shift within a certain tissue. It is observed that myofibroblast proliferation decreases significantly on laser treated samples in comparison to non-treated ones. On the other hand endothelial cell proliferation is not affected by the surface topography which is composed of micro- and nanostructures. Such surfaces may be used to modify stent surfaces for prevention or at least reduction of restenosis.

The surface topography of the choroid plexus. Environmental, low and high vacuum scanning electron microscopy.

Environmental scanning electron microscopy (ESEM) allows the examination of hydrated and dried specimens without a conductive metal coating which could be advantageous in the imaging of biological and medical objects. The aim of this study was to assess the performance and benefits of wet-mode and low vacuum ESEM in comparison to high vacuum scanning electron microscopy (SEM) using the choroid plexus of chicken embryos as a model, an organ of the brain involved in the formation of cerebrospinal fluid in vertebrates. Specimens were fixed with or without heavy metals and examined directly or after critical point drying with or without metal coating. For wet mode ESEM freshly excised specimens without any pre-treatment were also examined. Conventional high vacuum SEM revealed the characteristic morphology of the choroid plexus cells at a high resolution and served as reference. With low vacuum ESEM of dried but uncoated samples the structure appeared well preserved but charging was a problem. It could be reduced by a short beam dwell time and averaging of images or by using the backscattered electron detector instead of the gaseous secondary electron detector. However, resolution was lower than with conventional SEM. Wet mode imaging was only possible with tissue that had been stabilized by fixation. Not all surface details (e.g. microvilli) could be visualized and other structures, like the cilia, were deformed. In summary, ESEM is an additional option for the imaging of bio-medical samples but it is problematic with regard to resolution and sample stability during imaging.

Seed germination and seedling morphology of Smilax polyantha (Smilacaceae) / Germinação e morfologia de plântulas de Smilax polyantha (Smilacaceae)

Brazilians have been using the underground organs of Smilax species in alternative medicine since the 19th century because of their anti-rheumatic qualities. However, even nowadays, these species are explored only by extractivism. Studies on seed germination and development of these organs could be useful to preserve these plants. After germination, seedling development of Smilax polyantha was analyzed to understand underground stem formation. Furthermore, to analyze the ontogenesis of the underground system, seedlings aged from one to twelve months were sectioned. One of the most striking features of this species is the presence of two stem branching systems. The plumule gives rise to the first stem branching system with negative geotropism. Its first underground axillary bud sprouted into the other caulinar axis with positive geotropism. The horizontal growth and the subsequent thickening of this underground organ depended on the development of axillary buds from basal nodes of the previous branches. The cotyledonary bud did not play a role in the underground formation, as previously described in the literature for this genus, but the buds of the basal cataphylls built the second stem branching system. In this study we discuss the terminology and suggest calling this second stem branching system a rhizophore.

As espécies de Smilax L. são utilizadas na medicina popular brasileira desde o século 19 devido às propriedades anti-reumáticas atribuídas aos órgãos subterrâneos de todas as espécies. No entanto, ainda hoje, essas espécies são exploradas apenas por extrativismo. Estudos sobre a germinação e o desenvolvimento dos órgãos subterrâneos podem ser úteis para preservar essas plantas. Após a germinação, o desenvolvimento de plântulas de Smilax polyantha foi analisado para compreender a formação do sistema subterrâneo. Para a análise da ontogênese do sistema subterrâneo foram seccionadas plantas em diferentes estágios de desenvolvimento entre um e doze meses. Uma das características mais marcantes desta espécie é a presença de dois sistemas de ramificação caulinar. A plúmula dá origem ao primeiro sistema caulinar de ramificações com geotropismo negativo. As gemas axilares subterrâneas desse primeiro eixo caulinar originam o segundo eixo caulinar com geotropismo positivo. O crescimento horizontal e o espessamento do órgão subterrâneo dependem do desenvolvimento de gemas axilares de nós basais dos ramos anteriores. A gema cotiledonar não participa da formação do caule subterrâneo, como descrito anteriormente na literatura para este gênero, mas as gemas axilares basais dos ramos caulinares aéreos originam o segundo eixo de ramificação caulinar subterrâneo. Neste estudo, após ser discutida a terminologia mais adequada, sugere-se chamar o caule subterrâneo dessas espécies de rizóforo.