Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

The neuro-muscular and musculo-skeletal characterization of children with joint hypermobility

In children, joint hypermobility (typified by structural instability of joints) manifests clinically as neuro-muscular and musculo-skeletal conditions and conditions associated with development and organization of control of posture and gait (Finkelstein, 1916; Jahss, 1919; Sobel, 1926; Larsson, Mudholkar, Baum and Srivastava, 1995; Murray and Woo, 2001; Hakim and Grahame, 2003; Adib, Davies, Grahame, Woo and Murray, 2005:). The process of control of the relative proportions of joint mobility and stability, whilst maintaining equilibrium in standing posture and gait, is dependent upon the complex interrelationship between skeletal, muscular and neurological function (Massion, 1998; Gurfinkel, Ivanenko, Levik and Babakova, 1995; Shumway-Cook and Woollacott, 1995). The efficiency of this relies upon the integrity of neuro-muscular and musculo-skeletal components (ligaments, muscles, nerves), and the Central Nervous System’s capacity to interpret, process and integrate sensory information from visual, vestibular and proprioceptive sources (Crotts, Thompson, Nahom, Ryan and Newton, 1996; Riemann, Guskiewicz and Shields, 1999; Schmitz and Arnold, 1998) and development and incorporation of this into a representational scheme (postural reference frame) of body orientation with respect to internal and external environments (Gurfinkel et al., 1995; Roll and Roll, 1988). Sensory information from the base of support (feet) makes significant contribution to the development of reference frameworks (Kavounoudias, Roll and Roll, 1998). Problems with the structure and/ or function of any one, or combination of these components or systems, may result in partial loss of equilibrium and, therefore ineffectiveness or significant reduction in the capacity to interact with the environment, which may result in disability and/ or injury (Crotts et al., 1996; Rozzi, Lephart, Sterner and Kuligowski, 1999b). Whilst literature focusing upon clinical associations between joint hypermobility and conditions requiring therapeutic intervention has been abundant (Crego and Ford, 1952; Powell and Cantab, 1983; Dockery, in Jay, 1999; Grahame, 1971; Childs, 1986; Barton, Bird, Lindsay, Newton and Wright, 1995a; Rozzi, et al., 1999b; Kerr, Macmillan, Uttley and Luqmani, 2000; Grahame, 2001), there has been a deficit in controlled studies in which the neuro-muscular and musculo-skeletal characteristics of children with joint hypermobility have been quantified and considered within the context of organization of postural control in standing balance and gait. This was the aim of this project, undertaken as three studies. The major study (Study One) compared the fundamental neuro-muscular and musculo-skeletal characteristics of 15 children with joint hypermobility, and 15 age (8 and 9 years), gender, height and weight matched non-hypermobile controls. Significant differences were identified between previously undiagnosed hypermobile (n=15) and non-hypermobile children (n=15) in passive joint ranges of motion of the lower limbs and lumbar spine, muscle tone of the lower leg and foot, barefoot CoP displacement and in parameters of barefoot gait. Clinically relevant differences were also noted in barefoot single leg balance time. There were no differences between groups in isometric muscle strength in ankle dorsiflexion, knee flexion or extension. The second comparative study investigated foot morphology in non-weight bearing and weight bearing load conditions of the same children with and without joint hypermobility using three dimensional images (plaster casts) of their feet. The preliminary phase of this study evaluated the casting technique against direct measures of foot length, forefoot width, RCSP and forefoot to rearfoot angle. Results indicated accurate representation of elementary foot morphology within the plaster images. The comparative study examined the between and within group differences in measures of foot length and width, and in measures above the support surface (heel inclination angle, forefoot to rearfoot angle, normalized arch height, height of the widest point of the heel) in the two load conditions. Results of measures from plaster images identified that hypermobile children have different barefoot weight bearing foot morphology above the support surface than non-hypermobile children, despite no differences in measures of foot length or width. Based upon the differences in components of control of posture and gait in the hypermobile group, identified in Study One and Study Two, the final study (Study Three), using the same subjects, tested the immediate effect of specifically designed custom-made foot orthoses upon balance and gait of hypermobile children. The design of the orthoses was evaluated against the direct measures and the measures from plaster images of the feet. This ascertained the differences in morphology of the modified casts used to mould the orthoses and the original image of the foot. The orthoses were fitted into standardized running shoes. The effect of the shoe alone was tested upon the non-hypermobile children as the non-therapeutic equivalent condition. Immediate improvement in balance was noted in single leg stance and CoP displacement in the hypermobile group together with significant immediate improvement in the percentage of gait phases and in the percentage of the gait cycle at which maximum plantar flexion of the ankle occurred in gait. The neuro-muscular and musculo-skeletal characteristics of children with joint hypermobility are different from those of non-hypermobile children. The Beighton, Solomon and Soskolne (1973) screening criteria successfully classified joint hypermobility in children. As a result of this study joint hypermobility has been identified as a variable which must be controlled in studies of foot morphology and function in children. The outcomes of this study provide a basis upon which to further explore the association between joint hypermobility and neuro-muscular and musculo-skeletal conditions, and, have relevance for the physical education of children with joint hypermobility, for footwear and orthotic design processes, and, in particular, for clinical identification and treatment of children with joint hypermobility.

Microstructural characterization of electron beam evaporated tungsten oxide films for gas sensing applications

Tungsten trioxide is one of the potential semiconducting materials used for sensing NH3, CO, CH4 and acetaldehyde gases. The current research aims at development, microstructural characterization and gas sensing properties of thin films of Tungsten trioxide (WO3). In this paper, we intend to present the microstructural characterization of these films as a function of post annealing heat treatment. Microstructural and elemental analysis of electron beam evaporated WO3 thin films and iron doped WO3 films (WO3:Fe) have been carried out using analytical techniques such as Transmission electron microscopy, Rutherford Backscattered Spectroscopy and XPS analysis. TEM analysis revealed that annealing at 300oC for 1 hour improves cyrstallinity of WO3 film. Both WO3 and WO3:Fe films had uniform thickness and the values corresponded to those measured during deposition. RBS results show a fairly high concentration of oxygen at the film surface as well as in the bulk for both films, which might be due to adsorption of oxygen from atmosphere or lattice oxygen vacancy inherent in WO3 structure. XPS results indicate that tungsten exists in 4d electronic state on the surface but at a depth of 10 nm, both 4d and 4f electronic states were observed. Atomic force microscopy reveals nanosize particles and porous structure of the film. This study shows e-beam evaporation technique produces nanoaparticles and porous WO3 films suitable for gas sensing applications and doping with iron decreases the porosity and particle size which can help improve the gas selectivity.

Thin film deposition and characterization of pure and iron-doped electron-beam evaporated tungsten oxide for gas sensors

Pure Tungsten Oxide (WO3) and Iron-doped (10 at%) Tungsten Oxide (WO3:Fe) nanostructured thin films were prepared using a dual crucible Electron Beam Evaporation techniques. The films were deposited at room temperature in high vacuum condition on glass substrate and post-heat treated at 300 oC for 1 hour. From the study of X-ray diffraction and Raman the characteristics of the as-deposited WO3 and WO3:Fe films indicated non-crystalline nature. The surface roughness of all the films showed in the order of 2.5 nm as observed using Atomic Force Microscopy (AFM). X-Ray Photoelectron Spectroscopy (XPS) analysis revealed tungsten oxide films with stoichiometry close to WO3. The addition of Fe to WO3 produced a smaller particle size and lower porosity as observed using Transmission Electron Microscopy (TEM). A slight difference in optical band gap energies of 3.22 eV and 3.12 eV were found between the as-deposited WO3 and WO3:Fe films, respectively. However, the difference in the band gap energies of the annealed films were significantly higher having values of 3.12 eV and 2.61 eV for the WO3 and WO3:Fe films, respectively. The heat treated samples were investigated for gas sensing applications using noise spectroscopy and doping of Fe to WO3 reduced the sensitivity to certain gasses. Detailed study of the WO3 and WO3:Fe films gas sensing properties is the subject of another paper.

Towards a model of understanding strategic decision-making in micro-firms : exploring the Australian information technology sector

This study explores strategic decision-making (SDM) in micro-firms, an economically significant business subsector. As extant large- and small-firm literature currently proffers an incomplete characterization of SDM in very small enterprises, a multiple-case methodology was used to investigate how these firms make strategic decisions. Eleven Australian Information Technology service micro-firms participated in the study. Using an information-processing lens, the study uncovered patterns of SDM in micro-firms and derived a theoretical micro-firm SDM model. This research also identifies several implications for micro-firm management and directions for future research, contributing to the understanding of micro-firm SDM in both theory and practice.

Crystallographic characterization of the first reported crystalline form of the potent hallucinogen (R)-2-amino-1-(8-bromobenzo[1,2-b;5,4-b']difuran-4-yl)propane or `bromodragonfly': the 1:1 anhydrous proton-transfer compound with 3,5-dinitrosalicylic acid

The 1:1 proton-transfer compound of the potent substituted amphetamine hallucinogen (R)-1-(8-bromobenzo[1,2-b; 4,5-b']difuran-4-yl)-2-aminopropane (common trivial name 'bromodragonfly') with 3,5-dinitrosalicylic acid, 1-(8-bromobenzo[1,2-b;4,5-b']difuran-4-yl)-2-mmoniopropane 2-carboxy-4,6-dinitrophenolate, C13H13BrNO2+ C7H3N2O7- forms hydrogen-bonded cation-anion chain substructures comprising undulating head-to-tail anion chains formed through C(8) carboxyl O-H...O(nitro) associations and incorporating the aminium groups of the cations. The intra-chain cation-anion hydrogen-bonding associations feature proximal cyclic R33(8) interactions involving both a N+-H...O(phenolate) and the carboxyl O--H...O(nitro)associations. Also present are aromatic pi-pi ring interactions [minimum ring centroid separation, 3.566(2)A; inter-plane dihedral angle, 5.13(1)deg]. A lateral hydrogen-bonding interaction between the third aminium proton and a carboxyl O acceptor link the chain substructures giving a two-dimensional sheet structure. This determination represents the first of any form of this compound and confirms that it has the (R) absolute configuration. The atypical crystal stability is attributed both to the hydrogen-bonded chain substructures provided by the anions, which accommodate the aminium proton-donor groups of the cations and give cross-linking, and to the presence of cation--anion aromatic ring pi-pi interactions.

Use of micro-ATR/FTIR imaging to study heterogeneous polymer oxidation by direct solvent casting onto the ATR IRE

A technique is described whereby micro-ATR/FTIR imaging can be used to follow polymer degradation reactions in situ in real time. The internal reflection element (IRE) assembly is removed from the ATR objective and polymer is solvent cast directly onto the IRE surface. The polymer is then subjected to degradation conditions and molecular structural changes monitored by periodically replacing the IRE assembly back in the ATR objective and collecting spectra which can be used to construct images. This approach has the benefit that the same part of the sample is always studied, and that contact by pressure which might damage the polymer surface is not required. The technique is demonstrated using the polymer Topas which was degraded by exposure to UVC light in air.

Fabrication using a rapid prototyping system and in vitro characterization of PEG-PCL-PLA scaffolds for tissue engineering

n the field of tissue engineering new polymers are needed to fabricate scaffolds with specific properties depending on the targeted tissue. This work aimed at designing and developing a 3D scaffold with variable mechanical strength, fully interconnected porous network, controllable hydrophilicity and degradability. For this, a desktop-robot-based melt-extrusion rapid prototyping technique was applied to a novel tri-block co-polymer, namely poly(ethylene glycol)-block-poly(epsi-caprolactone)-block-poly(DL-lactide), PEG-PCL-P(DL)LA. This co-polymer was melted by electrical heating and directly extruded out using computer-controlled rapid prototyping by means of compressed purified air to build porous scaffolds. Various lay-down patterns (0/30/60/90/120/150°, 0/45/90/135°, 0/60/120° and 0/90°) were produced by using appropriate positioning of the robotic control system. Scanning electron microscopy and micro-computed tomography were used to show that 3D scaffold architectures were honeycomb-like with completely interconnected and controlled channel characteristics. Compression tests were performed and the data obtained agreed well with the typical behavior of a porous material undergoing deformation. Preliminary cell response to the as-fabricated scaffolds has been studied with primary human fibroblasts. The results demonstrated the suitability of the process and the cell biocompatibility of the polymer, two important properties among the many required for effective clinical use and efficient tissue-engineering scaffolding.

Bioactive SrO–SiO2 glass with well-ordered mesopores : characterization, physiochemistry and biological properties

For a biomaterial to be considered suitable for bone repair it should ideally be both bioactive and have a capacity for controllable drug delivery; as such, mesoporous SiO2 glass has been proposed as a new class of bone regeneration material by virtue of its high drug-loading ability and generally good biocompatibility. It does, however, have less than optimum bioactivity and controllable drug delivery properties. In this study, we incorporated strontium (Sr) into mesoporous SiO2 in an effort to develop a bioactive mesoporous SrO–SiO2 (Sr–Si) glass with the capacity to deliver Sr2+ ions, as well as a drug, at a controlled rate, thereby producing a material better suited for bone repair. The effects of Sr2+ on the structure, physiochemistry, drug delivery and biological properties of mesoporous Sr–Si glass were investigated. The prepared mesoporous Sr–Si glass was found to have an excellent release profile of bioactive Sr2+ ions and dexamethasone, and the incorporation of Sr2+ improved structural properties, such as mesopore size, pore volume and specific surface area, as well as rate of dissolution and protein adsorption. The mesoporous Sr–Si glass had no cytotoxic effects and its release of Sr2+ and SiO44− ions enhanced alkaline phosphatase activity – a marker of osteogenic cell differentiation – in human bone mesenchymal stem cells. Mesoporous Sr–Si glasses can be prepared to porous scaffolds which show a more sustained drug release. This study suggests that incorporating Sr2+ into mesoporous SiO2 glass produces a material with a more optimal drug delivery profile coupled with improved bioactivity, making it an excellent material for bone repair applications. Keywords: Mesoporous Sr–Si glass; Drug delivery; Bioactivity; Bone repair; Scaffolds

Deformation behaviours of coarse-grained and nanocrystalline Mg-5wt% Al alloys

Magnesium alloys have been of growing interest to various engineering applications, such as the automobile, aerospace, communication and computer industries due to their low density, high specific strength, good machineability and availability as compared with other structural materials. However, most Mg alloys suffer from poor plasticity due to their Hexagonal Close Packed structure. Grain refinement has been proved to be an effective method to enhance the strength and alter the ductility of the materials. Several methods have been proposed to produce materials with nanocrystalline grain structures. So far, most of the research work on nanocrystalline materials has been carried out on Face-Centered Cubic and Body-Centered Cubic metals. However, there has been little investigation of nanocrystalline Mg alloys. In this study, bulk coarse-grained and nanocrystalline Mg alloys were fabricated by a mechanical alloying method. The mixed powder of Mg chips and Al powder was mechanically milled under argon atmosphere for different durations of 0 hours (MA0), 10 hours (MA10), 20 hours (MA20), 30 hours (MA30) and 40 hours (MA40), followed by compaction and sintering. Then the sintered billets were hot-extruded into metallic rods with a 7 mm diameter. The obtained Mg alloys have a nominal composition of Mg–5wt% Al, with grain sizes ranging from 13 μm down to 50 nm, depending on the milling durations. The microstructure characterization and evolution after deformation were carried out by means of Optical microscopy, X-Ray Diffraction, Scanning Electron Microscopy, Transmission Electron Microscopy, Scanning Probe Microscopy and Neutron Diffraction techniques. Nanoindentaion, compression and micro-compression tests on micro-pillars were used to study the size effects on the mechanical behaviour of the Mg alloys. Two kinds of size effects on the mechanical behaviours and deformation mechanisms were investigated: grain size effect and sample size effect. The nanoindentation tests were composed of constant strain rate, constant loading rate and indentation creep tests. The normally reported indentation size effect in single crystal and coarse-grained crystals was observed in both the coarse-grained and nanocrystalline Mg alloys. Since the indentation size effect is correlated to the Geometrically Necessary Dislocations under the indenter to accommodate the plastic deformation, the good agreement between the experimental results and the Indentation Size Effect model indicated that, in the current nanocrystalline MA20 and MA30, the dislocation plasticity was still the dominant deformation mechanism. Significant hardness enhancement with decreasing grain size, down to 58 nm, was found in the nanocrystalline Mg alloys. Further reduction of grain size would lead to a drop in the hardness values. The failure of grain refinement strengthening with the relatively high strain rate sensitivity of nanocrystalline Mg alloys suggested a change in the deformation mechanism. Indentation creep tests showed that the stress exponent was dependent on the loading rate during the loading section of the indentation, which was related to the dislocation structures before the creep starts. The influence of grain size on the mechanical behaviour and strength of extruded coarse-grained and nanocrystalline Mg alloys were investigated using uniaxial compression tests. The macroscopic response of the Mg alloys transited from strain hardening to strain softening behaviour, with grain size reduced from 13 ìm to 50 nm. The strain hardening was related to the twinning induced hardening and dislocation hardening effect, while the strain softening was attributed to the localized deformation in the nanocrystalline grains. The tension–compression yield asymmetry was noticed in the nanocrystalline region, demonstrating the twinning effect in the ultra-fine-grained and nanocrystalline region. The relationship k tensions < k compression failed in the nanocrystalline Mg alloys; this was attributed to the twofold effect of grain size on twinning. The nanocrystalline Mg alloys were found to exhibit increased strain rate sensitivity with decreasing grain size, with strain rate ranging from 0.0001/s to 0.01/s. Strain rate sensitivity of coarse-grained MA0 was increased by more than 10 times in MA40. The Hall-Petch relationship broke down at a critical grain size in the nanocrystalline region. The breakdown of the Hall-Petch relationship and the increased strain rate sensitivity were due to the localized dislocation activities (generalization and annihilation at grain boundaries) and the more significant contribution from grain boundary mediated mechanisms. In the micro-compression tests, the sample size effects on the mechanical behaviours were studied on MA0, MA20 and MA40 micro-pillars. In contrast to the bulk samples under compression, the stress-strain curves of MA0 and MA20 micro-pillars were characterized with a number of discrete strain burst events separated by nearly elastic strain segments. Unlike MA0 and MA20, the stress-strain curves of MA40 micro-pillars were smooth, without obvious strain bursts. The deformation mechanisms of the MA0 and MA20 micro-pillars under micro-compression tests were considered to be initially dominated by deformation twinning, followed by dislocation mechanisms. For MA40 pillars, the deformation mechanisms were believed to be localized dislocation activities and grain boundary related mechanisms. The strain hardening behaviours of the micro-pillars suggested that the grain boundaries in the nanocrystalline micro-pillars would reduce the source (nucleation sources for twins/dislocations) starvation hardening effect. The power law relationship of the yield strength on pillar dimensions in MA0, MA20 supported the fact that the twinning mechanism was correlated to the pre-existing defects, which can promote the nucleation of the twins. Then, we provided a latitudinal comparison of the results and conclusions derived from the different techniques used for testing the coarse-grained and nanocrystalline Mg alloy; this helps to better understand the deformation mechanisms of the Mg alloys as a whole. At the end, we summarized the thesis and highlighted the conclusions, contributions, innovations and outcomes of the research. Finally, it outlined recommendations for future work.

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones : a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeo-stasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, im-munohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the os-teocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogene-sis.

3D-printing of highly uniform CaSiO3 ceramic scaffolds : preparation, characterization and in vivo osteogenesis

Calcium silicate (CaSiO3, CS) ceramics have received significant attention for application in bone regeneration due to their excellent in vitro apatite-mineralization ability; however, how to prepare porous CS scaffolds with a controllable pore structure for bone tissue engineering still remains a challenge. Conventional methods could not efficiently control the pore structure and mechanical strength of CS scaffolds, resulting in unstable in vivo osteogenesis. The aim of this study is to set out to solve these problems by applying a modified 3D-printing method to prepare highly uniform CS scaffolds with controllable pore structure and improved mechanical strength. The in vivo osteogenesis of the prepared 3D-printed CS scaffolds was further investigated by implanting them in the femur defects of rats. The results show that the CS scaffolds prepared by the modified 3D-printing method have uniform scaffold morphology. The pore size and pore structure of CS scaffolds can be efficiently adjusted. The compressive strength of 3D-printed CS scaffolds is around 120 times that of conventional polyurethane templated CS scaffolds. 3D-Printed CS scaffolds possess excellent apatite-mineralization ability in simulated body fluids. Micro-CT analysis has shown that 3D-printed CS scaffolds play an important role in assisting the regeneration of bone defects in vivo. The healing level of bone defects implanted by 3D-printed CS scaffolds is obviously higher than that of 3D-printed b-tricalcium phosphate (b-TCP) scaffolds at both 4 and 8 weeks. Hematoxylin and eosin (H&E) staining shows that 3D-printed CS scaffolds induce higher quality of the newly formed bone than 3D-printed b-TCP scaffolds. Immunohistochemical analyses have further shown that stronger expression of human type I collagen (COL1) and alkaline phosphate (ALP) in the bone matrix occurs in the 3D-printed CS scaffolds than in the 3D-printed b-TCP scaffolds. Considering these important advantages, such as controllable structure architecture, significant improvement in mechanical strength, excellent in vivo osteogenesis and since there is no need for second-time sintering, it is indicated that the prepared 3D-printed CS scaffolds are a promising material for application in bone regeneration.