THREE NEW BENTHIC SPECIES OF PROROCENTRUM (DINOPHYCEAE) FROM CARRIE BOW CAY,BELIZE: P. SABULOSUM SP. NOV., P. SCULPTILE SP. NOV., AND P. ARENARIUM SP. NOV.1

Three new benthic, sand-dwelling dinqflagellate species, Prorocentrum sabulosum, Prorocentrum scuptile, and Prorocentrum arenarium, from coral rubble are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, ornamentation of thecal plates, and architecture of the periflagellar area and intercalary band. Cells of P. sabulosum are oval with a cell size of 48–50 μm long and 41–48 μm wide. The areolae are round to oval and numerous (332–450 per valve) and range from 1 to 1.6 μm in size. The periflagellar area of P. sabulosum bears a wide V-shaped depression with a flat ridge and lacks ornamentation; it accommodates six pores: one large flagellar pore, an adjacent smaller auxiliary pore, and four pores of unknown function. The flagellar and auxiliary pores are surrounded by a narrow apical collar. The intercalary band of P. sabulosum is smooth. Prorocentrum sculptile cells are broadly oval, 32–37 nm long, and 30–32 μm wide in valve view with a deep-sculptured apical area. The valves are smooth and are marked with shallow depressions (856–975 per valve). Some of these depressions have a small round opening (0.13 μm in diameter). The periflagellar area is V-shaped with a deeply indented depression; it accommodates the two flagella and a thin angled apical plate. The intercalary band is smooth. Prorocentrum arenarium cells are nearly round in valve view 30–32 μm in diameter. Thecal surface is smooth with scattered kidney-shaped valve poroids (65–73 per valve) and marginal poroids (50–57 per valve). Length and width of poroids are 0.62 μm and 0.36 μm, respectively. The periflagellar area is an unornamented, broad triangle into which a large flagellar pore and a smaller auxiliary pore are fitted. Both flagella, longitudinal and transverse, protrude from the flagellar pore. The intercalary band is smooth. The presence of a peduncle-like structure (2–3 μm long) in P. arenarium was observed situated in the flagellar pore.     

The development of the hexagonally structured egg envelope of the C-O sole (Pleuronichthys coenosus)

The surface of a mature, pelagic C-O sole egg is composed of polygonal chambers having four to eight sides, most of which are hexagonally shaped. This honeycomb pattern initially appears on primary oocytes as a thin layer of compact, electron-dense material. Discrete thickenings begin to develop on the envelope of perinuclear stage oocytes. The thickenings lengthen and thin to form the hexagonal walls of the envelope in oocytes undergoing yolk vesicle formation. The walls of each hexagonal chamber occur in an area corresponding to the lateral margins of the adjacent follicle cell, suggesting that the hexagonal walls are produced by the follicle cells. The hexagonal layer is nearly complete at the beginning of vitellogenesis, and as vitellogenesis continues, a striated envelope layer composed of fibrillar lamellae develops between the oocyte and the hexagonal layer. The striated layer appears to be secreted by the oocyte. After vitellogenesis is completed, oocytes are ovulated and double in size during a period of maturation. Concurrently, the striated primary envelope stretches and thins into eight to nine horizontal lamellae. On the mature egg surface, the polygonal chambers are about 24–31 μm in diameter. Within each chamber there is a subpattern of polygonal areas; each polygon is 1.5–2.0 μm in diameter, and circumscribes a pore canal opening. This exceptional envelope may furnish the egg with some degree of protection, resiliency, and buoyancy, but its specific functions are not known.     

PROROCENTRUM BELIZEANUM,PROROCENTRUM ELEGANS,AND PROROCENTRUM CARIBBAEUM,THREE NEW BENTHIC SPECIES (DINOPHYCEAE) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE1

Three new benthic dinoflagellate species, Prorocentrum belizeanum, Prorocentrum elegans, and Prorocentrum caribbaeum, from mangrove floating detritus are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, ornamentation of thecal plates, and architecture of the periflagellar area and intercalary band. Cells of P. belizeanum are round to slightly oval with a cell size of 55–60 μm long and 50–55 μm wide. Areolae are round and numerous (853–1024 per valve) and range from 0.66 to 0.83 μm in size. The periflagellar area of P. belizeanum is a broad V-shaped depression; it accommodates a flagellar and an auxiliary pore and a flared, curved apical collar. The intercalary band of P. belizeanum is horizontally striated. Prorocentrum elegans is a small species 15–20 μm long and 10–14 μm wide, with an ovate cell shape. The thecal surface is smooth. Two sizes of valve pores were recognized: large, round pores (20–22 per valve) arranged in a distinct pattern and smaller pores situated in an array along the intercalary band. The periflagellar area is V-shaped; it accommodates an uneven sized flagellar pore, an auxiliary pore, and an angled protuberant flagellar plate. The intercalary band is transversely striated. It is a bloom-forming species. Prorocentrum caribbaeum cells are heart-shaped with a rounded anterior end and a pointed posterior end. Cells range from 40 to 45 μm long and 30 to 35 μm wide. Thecal surface has two different-sized pores: large, round pores (145–203 per valve) arranged perpendicularly from the posterior margins, and small, round pores unevenly distributed on the thecal surface. The periflagellar area is ornate. It is V-shaped with a curved apical collar located next to the auxiliary pore; a smaller protuberant apical plate is adjacent to the flagellar pore. The intercalary band is transversely striated and sinuous. Cells are active swimmers.     

Growth of fibroblasts on linear and planar anchorages of limiting dimensions

BHK and 3T3 cells were grown on small glass fibres and platelets (size range 20–250 μm) in agar gel, over a layer of mouse primary feeder cells. Under these conditions the growth of colonies (4–20 cells) from single control cells, suspended freely in the gel, was less than 1 %, while cells attached to the larger platelets yielded more than 95 % colonies. Fibres (average diameter 0.625 μm) induced somewhat fewer colonies (45–70 %). However, there was a significant induction of single divisions by fibres as short as 30 μm, while on 60 μm fibres more than half the cells had gone through at least one division, compared with 4–12% of freely suspended controls. Longer fibres promoted multiple divisions. Colony formation increased sigmoidally with fibre length from 30 to 250 μm, with a d₅₀ around 115 μm. These figures resembled the observed range of lengths of freely growing BHK and 3T3 fibroblasts in tissue culture dishes. In another experiment, finer fibres (average diameter 0.075 μm) performed almost as well as platelets of equal length but of width 20–30 μm.Thus linear extension provides a stimulus for division of anchorage-dependent fibroblasts. It is suggested that this stimulus may arise from a change in conformation or tension in the cell membrane, rather than from increased surface, cell motility or interaction with substrate surface.The present results are discussed in relation to recent findings on the induction of tumours by solid particles.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

OBSERVATIONS ON THE MORPHOLOGY AND SEXUAL REPRODUCTION OF COOLIA MONOTIS (DINOPHYCEAE)1

The surface morphology of the dinoflagellate Coolia monotis Meunier was compared with the surface morphology of Ostreopsis, The apical pore of C. monotis is similar in architecture to that of Ostreopsis but considerably longer (12 μm) than in O. heptagona (8–9 μm) and O. ovata (6–7 μm). A ventral pore in C. monotis is located on the right ventral margin between apical plate l′ and precingular plate 6″ and is similar in appearance and location to the ventral pore of O. ovata. The longitudinal flagellum (20 μm) in C. monotis is longer than in O. ovata (12 μ). Although Coolia and Ostreopsis appear to be distinctly different and should remain as two separate genera, they appear to be related. Cells of C. monotis divided by binary fission. Doubling time was 3–4 days in the logarithmic phase of growth at 23°C, 12:12 h L:D, 30–90 μE-m^?2·s^?1, and a salinity of 36%. Cultures reached cell densities of 2.5 × 10³ cells·L^?1 after 15 days of growth. The sexual process in C. monotis occurred in Erdschreiber's medium when Danish soil extract was substituted with mangrove sediment extract under the culture conditions described above. Gamete fusion produced large biflagellated planozygotes (70–75 μm diam). Planozygote maturation involved cytoplasmic reorganization, loss of motility, development of a spherical shape (80–90 μm diam), and two to three orange accumulation bodies. The cells at this stage appeared to be thin-walled cysts. Further development included reorganization of cyst contents, emergence of non-motile gametes, and development of chloroplasts, sulcus, and girdle. The nucleus of the newly formed cells occupied 50% or more of the total cell volume. Meiosis occurred in the cyst, but nuclear cyclosis was not observed. Four daughter cells were produced within 36–48 h, and motile gametes developed. The gametes exhibited sexuality for 2 months and completed the sexual life cycle by going through a thin-walled cyst stage.     

Ultrastructure and Description of a Fungus-Feeding Amoeba,Trichamoeba mycophaga n. sp. (Amoebidae,Amoebea), from Australia1

ABSTRACT. An amoeba isolated from a wheatfield and a forest soil in Australia has been identified as Trichamoeba mycophaga n. sp. Trophozoites of this amoeba are palmate to elongate and measure 45–136 μm in length and 25–94 μm in width. Amoebae in continuous locomotion may be limax with a villous-bulb uroid. Both the lobose pseudopodia and the advancing margin of a limax trophozoite bear an ectoplasmic crescent. The plasma membrane is coated with an electron-dense amorphous layer ca. 100 nm thick. Endoplasm is granular with elongate to bipyramidal crystals and contains bacterial endosymbionts. Trophozoites have a single, spherical to oval nucleus, 4–10 μm in diameter, which contains a centrally located, spherical to oval nucleolus, 2.8–5.0 μm in diameter. The nucleoplasm contains aggregations of filaments distributed radially within the nuclear membrane. Cysts are 21–60 μm in diameter, with ecto- and endocyst walls separated by an amorphous layer.     

Structure of sensilla trichodea of female Aedes aegypti with comments on innervation of antennal sensilla

On each antenna of female Aedes aegypti four types of sensilla trichodea are distinguishable: long and short, pointed-tipped ones and blunt-tipped types I and II. All types are innervated by two neurones, except the short, pointed-tipped trichodea with which only one neurone is associated. Both pointed-tipped types have unbranched dendrites and relatively thicker hair walls perforated by fewer pores than the blunt-tipped types. The long, pointed-tipped trichodea are 50–60 μm in length and the short ones 20 μm. In both blunt-tipped types the dendrites divide and the hair walls are perforated by numerous pores. Blunt-tipped type I trichodea are 20–40 μm in length and taper somewhat whereas the type II hairs are 11–13 μm in length, do not taper appreciably, and have exceedingly thin walls. All types of sensilla trichodea are olfactory receptors and the blunt-tipped type I are known to respond to repellents.An investigation of the possibility of axon fusion in the flagellar nerve gave negative results.Each flagellar nerve is composed of an estimated 2058 neurones. A majority of these, namely 93%, are involved in the recognition and discrimination of olfactory cues. Approximately 65% of the neurones respond to repellents, 5% to mechanical stimuli, and 2% to heat transferred by convection.     

THREE NEW BENTHIC SPECIES OF PROROCENTRUM (DINOPHYCEAE) FROM BELIZE: P. NORRISIANUM SP. NOV., P. TROPICALIS SP. NOV., and P. RETICULATUM SP. NOV.1

Three new benthic, photosynthetic dinoflagellate species, Prorocentrum norrisianum, Prorocentrum tropicalis, and Prorocentrum reticulatum, from floating detritus and coral rubble of Central America are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, thecal plate ornamentation, and architecture of the periflagellar area and intercalary band. Cells of P. norrisianum are ovate with a cell size of 20–25 μm long and 13–16 μm wide. The theca is delicate, its surface smooth, pores species specific with 95 to 105 pores per valve. Pores are round with a diameter of about 0.1 μm. The periflagellar area is V-shaped, located on the right valve in a shallow depression. It has no ornamentation. The flagellar and auxiliary pores are unequal in size. The intercalary band is smooth. Prorocentrum tropicalis cells are ovoid, 50–55 μm long and 40–45 μm wide in valve view with maximum width behind the middle region, narrow at the anterior end. The periflagellar area, situated in the right valve, is a V-shaped wide triangle with a deeply indented depression; the left valve exhibits a flat ridge. The periflagellar area is unornamented, and the flagellar and auxiliary pores are unequal in size. The valve surface is rugose with evenly distributed valve poroids. Each poroid appears to have a small dome in the center. The intercalary band is rimlike around the cell margin, granulated, and horizontally striated. Prorocentrum reticulatum cells are oblong in valve view; cells are 55–60 μm long and 40–45 μm wide. Thecal surface is reticulated; it is composed of a labyrinth of ridges with alternating depressions that vary in size and shape. Each depression has a narrow, oblong-kidney-shaped opening about 0.6 μm long. The periflagellar area is a deep, V-shaped triangle. The right valve of P. reticulatum is excavated, and contains a large flagellar pore and a smaller auxiliary pore surrounded by a narrow apical collar. The left valve margin exhibits a curved flat ridge. The intercalary band is smooth.     

Enantiomeric resolution by HPLC of axial chiral amides using amylose tris[(S)-1-phenylethylcarbamate]

The enantiomeric resolution of a series of N-arylamides was examined on amylose tris[(S)-1-phenylethylcarbamate] coated onto aminopropylated 7 μm silica with 500 Å diameter pores and on naked silica 5 μm particle size with 500 Å diameter pores. The enantiomeric resolution obtained for this series was excellent on both columns. The enantioselectivity of cellulose and amylose tris (3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (120 Å pore size, 5 μm particle size) was also investigated for this series of compounds. Chirality 9:109–112, 1997. © 1997 Wiley-Liss, Inc.     

Plant Regeneration from Supersweet Maize Protoplasts Derived from Gametophytic Cell Suspensions

Plant regeneration from protoplasts isolated from haploid cell suspensions of commercial supersweet maize (SS 7700) was achieved and the plants were survival after transfer into soil in pots. Protoplast plating efficiency obtained from feeder layer system was 130 folds higher as compared with conventional liquid culture method, the composition of protoplast culture medium, the pore size of supportive membrane filter and the relationship between protoplasts and feeder cells were critical for callus formation. An enriched medium containing vitamins, organic acids, amino-acids and other organic substances such as coconut water could extremely improve callus formation. Filters with pore size within the range of 0.22–8.0 μm in diameter was useful. Filters with smaller pore size of 0.04 μm or larger 11 μm appeared to decrease the frequency of protocolony formation. The feeder cells which belong to the same species (Zea mays) as protoplasts greatly increased protoplast plating efficiencies as compared to those of feeder cells belonging to other species such as Avena nuda and Nicotiana tabacum. Among 11 protoplast-regenerated plants examined, 10 plants were haploid and one plant was diploid.     

Further Observations on Zosterophllum yunnanicum Hsü

Zosterophyllum yunnanicum, an important and widely distributedspecies in the Lower Devonian of southern China, was first described by Hsü in 1966.Later then, Li and Cai (1977), Cai and Schweitzer (1983) described the details of itsmorpholegy, and compered it with other members of the genus Zosterophyllum. In thispaper, the materials of its tracheids, epidermal ceils, stomata and its spores have beenstudied. The tracheids of Z. Yunnanicum are annular, and 9–30 gm across. Epidermal cellsof the axes are elongate, polygonaI, ranging from 70–280 μm by 12–60 μm. The stomata are mainly fusiform, 65–80 μm in length and 40–70 μm in width, probably consisting of a pair of guard-cells enclosing a pore, 8–20μm long and about 1.5μm wide. The density of stomata evidently varied in different parts of the plant. The portion of the sporangium near the rim is composed of rectangle and elongate cells. Theremainder wall of the valve consists of elongate cells, folding often. Spores from the sporangia are 20–55 gm (average 37.1μm) in diameter, smooth with thickened in area of trilete mark, which are 1/4–2/3 of the radius of spore. Some spores look similar to the dispersed spore Retusotriles (Naum.) Streel 1964. Apparently this Plant is homosporous.     

Detection of complement activity by using a polysaccharide-protected membrane

The complement system of mammalian blood is a nonspecific part of the immune system involved in a number of disease conditions. We report the observation of pore creation caused by its activation in blood applied to the front gel layer of a bilayer membrane formed from dioleoylphosphatidyl choline and protected by a polysaccharide gel. The pores were detected by measuring the DC conductivity between nonblocking Ag/AgCl electrodes. The thickness of the protective gel was approximately 100 μm, and the complement response was seen within 3 min after application of activator. The lifetime of such membranes is limited only by hydrolysis of the phospholipid constituting the membrane. This easily prepared system is suitable for examining the kinetics of complement component interactions with inhibitors.     

Determining the Cellular Structure of Two Cereal Food Foams by X-ray Micro-Tomography

The cellular structure of two products, an extruded breakfast cereal and a short dough biscuit, was characterized by two different X-ray micro computed tomographic systems. Acquisitions were made by a compact desktop system Skyscan 1174 (Bruker μCT, Belgium) and at the European Synchrotron Radiation Facility (ESRF, beamline ID19, France) at different resolutions (voxel size of 6.5 μm, 7.5 μm, 16.2 μm and 25.8 μm). 3D images were processed for the density, the connectivity index and the granulometry of cells and cell walls. These experiments underlined the importance of the resolution for determination of quantitative measurements such as densities and thicknesses. The median width calculated for the cell walls distribution in the biscuit dropped from 141 to 50 μm when the voxel size changed from 25.8 to 7.5 μm. Images well showed that even though the food products had close values of porosity 0.6 and 0.7 for biscuit and extruded breakfast cereal respectively, their cellular structures were very different. The biscuit had small cells (median value of the distribution varied from 125 to 152 μm, according to resolution) and larger cell walls (50–141 μm) than the extrudate (32–109 μm) which, on the contrary, exhibited very large cells (307–400 μm). Beyond methodological issues, these differences could be clearly attributed to the differences of compositions and processes.     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied. Silica gels were used directly and characterized by mercuric porosimetry. Agitation rates (100–740 rpm) and particles size (35–75 to 250–500 μm) of silica gels did not affect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limiting factor of the trypsin adsorption onto the mesopores structure of silica gels. The adsorption of trypsin was determined as a function of their initial concentration and multilayer formed at high trypsin concentration.     

Tuberculina-rusts: a unique basidiomycetous interfungal cellular interaction with horizontal nuclear transfer

Cellular interaction of the basidiomycete Tuberculina persicina with the haploid stages of two rusts Puccinia silvatica and Tranzschelia pruni-spinosae was analyzed by serial-section electron microscopy of chemically fixed samples and samples subjected to high-pressure freezing and freeze substitution. Tuberculina persicina is a contact parasite, forming neither haustoria nor other intracellular structures. However, at contact areas between T. persicina and its hosts, distinct interfungal interactions are present. At the beginning, a hyphal cell of T. persicina invades the host cell wall with a protuberance and the cell walls of both protuberance and host cell dissolve at the point of contact. Thus, the plasma membranes of the two organisms contact and fuse to form a pore that enlarges to a final diameter of approximately 1 μm. The membrane of the fusion pore is continuous with the plasma membranes of both cells, and Tuberculina nuclei and other organelles are transferred to the rust cells. Phylogenetic and functional aspects of this curious basidiomycetous interfungal interaction are discussed, and a hypothesis of the evolutionary derivation of the Tuberculina mycoparasitism from a sexual interaction is presented.     

An electron microscope study of gametangia in the green seaweed Halimeda tuna

Abstract

The gametangia of the green seaweed Halimeda tuna are spherical bodies of diameter up to 250–300 μm. They are clustered in groups of 8–10 on hundreds of threads sprouting from all pale white segments of the fertile individuals. In addition to gametes, starch-containing chloroplasts, naked starch grains and two types of spherical bodies different in size and ultrastructure are the main corpuscular components. A layer of amorphous material of irregular thickness underlies the walls which are finely and evenly rough, structureless, electron translucent and 1–1.5 μm thick. Gametangia with superficial wall warts were found also.     

Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds

Mean pore size is an essential aspect of scaffolds for tissue-engineering. If pores are too small cells cannot migrate in towards the center of the construct limiting the diffusion of nutrients and removal of waste products. Conversely, if pores are too large there is a decrease in specific surface area available limiting cell attachment. However the relationship between scaffold pore size and cell activity is poorly understood and as a result there are conflicting reports within the literature on the optimal pore size required for successful tissue-engineering. Previous studies in bone tissue-engineering have indicated a range of mean pore sizes (96–150 µm) to facilitate optimal attachment. Other studies have shown a need for large pores (300–800 µm) for successful bone growth in scaffolds. These conflicting results indicate that a balance must be established between obtaining optimal cell attachment and facilitating bone growth. In this commentary we discuss our recent investigations into the effect of mean pore size in collagen-glycosaminoglycan (CG) scaffolds with pore sizes ranging from 85–325 μm and how it has provided an insight into the divergence within the literature.