Photoimmobilization of biomolecules within a 3-dimensional hydrogel matrix

It has been recognized that a three-dimensional cell invasive scaffold that provides both topographical and chemical cues is desirable in regenerative tissue engineering to encourage cell attachment, migration, regrowth and ultimately tissue repair. Carbohydrate hydrogels are attractive for such applications because they are generally biocompatible and able to match the mechanical properties of most soft tissues. Although carbohydrate hydrogels have been previously modified with cell adhesive peptides and proteins, complicated hydrogel matrix activation was required prior to biomolecule coupling and, perhaps more importantly, the overall immobilization yield was low at approximately 1%. In this study, we report the photo-immobilization of a model biomolecule, ovalbumin (OVA), to agarose gel. We describe two methods of modification where the photoactive moiety is coupled to either the protein (i.e. OVA) or the matrix (i.e. agarose) prior to immobilization. We found that the photo-immobilization yield depends on the location of the photoactive moiety. Using photoactive OVA, 1.8% of the OVA initially incorporated into the agarose gel is immobilized; using photoactive agarose, 9.3% of the OVA initially mixed with the agarose is immobilized. The latter is a significant improvement over previous yields and may be useful in attaining our goal of immobilizing a biomolecule gradient for guided tissue regeneration.     

A Calcium-Cross-Linked Hydrogel Based on Alginate-Modified Atelocollagen Functions as a Scaffold Material

We have developed a scaffold material consisting of a covalently-bonded structure of alginate and atelocollagen (AtCol). Addition of calcium ions caused the material to form a hydrogel (alginate-modified AtCol gel). The condition of the alginate-modified AtCol gel could be controlled by the feed ratio of alginate and the activating reagent. Measurement of temporal stability in culture medium suggested that covalent bonding between alginate and AtCol might contribute to the structural stability of the alginate-modified AtCol gel. Culture with endothelial cells indicated that cell adhesiveness on the alginate-modified AtCol gel was similar to that on native collagen. Collagenase digestion revealed that the alginate-modified AtCol gel had considerable ability to retain basic fibroblast growth factor. Additionally, active cell migration into alginate-modified AtCol was detected by in vitro assay using endothelial cells. These findings indicate that this gel material can be expected to function as a scaffold for inducing vascular in-growth.     

Degradation Behavior of 3D Porous Polydioxanone-b-Polycaprolactone Scaffolds Fabricated Using the Melt-Molding Particulate-Leaching Method

Recently, polydioxanone (PDO) and polycaprolactone (PCL) have been applied in applications for tissue engineering owing to their flexibility, as well as biocompatibility and biodegradability, even though their degradation rates are usually either too fast or too slow for many applications. In this study, we synthesized poly(dioxanone-b-caprolactone) co-polymers (PDOCLs) with different DO/CL ratio (0:10–10:0) by ring-opening polymerization. The synthesized co-polymers were characterized by ¹H-NMR, the measurement of inherent viscosity (IV), GPC and DSC. PDOCL scaffolds with different DO/CL ratio were fabricated by a melt-molding particulate-leaching method without using any organic solvents during the scaffold fabrication process. The degradation behavior (in vitro) of the PDOCL scaffolds was evaluated in PBS at 37°C for up to 56 days by the changes in molecular weight, mechanical strength, gross weight and pH. It was observed that the degradation rate of PDOCL scaffolds could be controlled by adjusting the DO/CL ratio of the co-polymers (increasing CL composition leads to slower degradation rate). The PDOCL scaffolds did not lead to a significant drop in pH during the degradation, not even for the PDO-dominant PDOCL scaffolds showing a fast degradation rate, indicating the formation of a small amount of acidic by-products compared to the PLGA scaffolds. From the results, it was expected that the PDOCLs can be a new flexible scaffolding material with different degradation rate for various tissue-engineering applications.     

A collagen–phosphophoryn sponge as a scaffold for bone tissue engineering

Non-collagenous phosphoproteins that interact with a type-I collagen are thought to nucleate bone mineral into collagen networks of mineralized tissues. Previously, phosphophoryn cross-linked to type-I collagen was reported to be an effective nucleator of appatite. However, free phosphophoryn molecules inhibit the formation of apatite in vitro. On the basis of the above study, we expected a collagen-phosphophoryn sponge to be a good scaffold for bone-tissue engineering and examined the formation of bone in orthotopically transplanted composites of the sponge and bone marrow osteoblasts in vivo in Fischer rats. Osteoblastic primary cells were obtained from the bone shaft of femorae of Fisher rats, according to the method of Maniatopoulous et al. A suspension of marrow cells was distributed through a flask with standard culture medium and incubated at 37°C. When cultures were nearly confluent after 10 days, they were concentrated by centrifugation to 10⁶ cells/ml and subcultured onto the synthesized collagen-phosphophoryn sponge and a collagen sponge (control). After 14 days, the composites of collagen-phosphophoryn and osteoblastic cells as well as control composites were transplanted into bone-defect sites of Fisher rats (holes 2 mm in diameter) and then the wounds were sutured. The composites were harvested at 1-8 weeks after implantation, and stained with hematoxylin and eosin. It was found that more bone was formed in the composites of collagen-phosphophoryn sponge and osteoblasts than control composites from 1 week to 8 weeks, suggesting that the collagen-phosphophoryn sponge is a good candidate as a scaffold for bone-tissue engineering.     

Development of Porous Alginate-Based Scaffolds Covalently Cross-Linked through a Peroxidase-Catalyzed Reaction

Porous scaffolds are important in tissue engineering. We developed porous scaffolds from the hydrogels of an alginate derivative bearing phenolic hydroxyl groups. The hydrogels were prepared using horseradish peroxidase (HRP) to catalyze the cross-linking between the phenolic hydroxyl groups. A porous structure with a pore size of approx. 200 μm was developed through simultaneous water-extraction and ionic cross-linking by calcium ions by soaking frozen hydrogels in the mixture of ethanol and CaCl₂ solution at –20°C. Due to the existence of the covalent cross-links developed through the enzymatic reaction, the porous form had a higher stability from a loss of cross-linked calcium ions than that obtained from non-modified sodium alginate (Na-Alg). The porous specimen developed from the hydrogel obtained with 10 U/ml HRP and 10 mM H₂O₂ showed about 1.5-times greater repulsion forces than those detected for the porous specimen obtained from Na-Alg toward compressions. No harmful effects of the enzymatically cross-linked specimens were detected on the growth and morphology of the entrapped cells: cells in the enzymatically cross-linked specimens showed almost the same growth profile and morphology with those in the porous specimen obtained from Na-Alg.     

In Situ Fabrication of Nano-hydroxyapatite in a Macroporous Chitosan Scaffold for Tissue Engineering

A chitosan (CS)/hydroxyapatite (HAP) nanohybrid scaffold with high porosity and homogeneous nanostructure was fabricated through a bionic treatment combined with thermally-induced phase separation. The nano-HAP particles were formed in situ in the scaffold at room temperature instead of mechanically mixing the powders with the polymer component. The scaffold was macroporous with a pore size of about 100–136 μm. The nano-sized HAP particles with diameters of 90–200 nm were scattered homogeneously in the interactively connective pores. Both the improvement of the compressive modulus and yield strength of the scaffolds showed that the in situ nano-HAP particles reinforced the microstructure of the scaffold. The in vitro bioactivity study carried out in simulated body fluid (SBF) indicated good mineralization activity. The crystallization phenomenon suggested that the nano-HAP particles have positive impacts on directing apatite crystallization in the scaffold and led to the good bioactivity of the nanohybrid scaffold.     

A novel construct as a cell carrier for tissue engineering

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.     

Morphological and Histological Evaluations of 3D-Layered Blood Vessel Constructs Prepared by Hierarchical Cell Manipulation

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.     

Anisotropic Mechanical Properties of Collagen Hydrogels Induced by Uniaxial-Flow for Ocular Applications

Engineering of well-organized tissue constructs is active in the field of material science for biomedical applications. Here, we propose a method for orienting collagen in transparent high-density collagen hydrogels using a simple rolling method. Structural organization and mechanical function were adjusted by regulating the thickness of the construct and the cross-linking reagent. Directionality of collagen alignment on the microscopic scale was achieved parallel to the extensional flow. The preferential alignment of collagen significantly affected the mechanical properties of the construct, with strong tensile strength in the direction parallel to the collagen, and high elastic strain in the perpendicular direction. The tensile strength in the parallel direction was effectively increased by 67% by increasing the cross-linking reagents by 33%, without affecting transparency which remained at 70–80% to visible light. The constructs exhibit good biocompatibility for as a substrate for the expansion of corneal epithelial cells isolated from human donor cornea, indicating the potential for tissue engineering and biomedical applications, particularly for ocular treatments.     

Physical Properties and Biocompatibility of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Blended with Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was blended with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) to improve physical properties and biocompatibility of PHBHHx for a wide range of biomedical applications. PHBHHx was completely miscible with P3HB4HB in their blends. All the PHBHHx/P3HB4HB blends showed improved physical properties compared with PHBHHx, including higher thermal stability, flexibility and mechanical strength. All the blends had more hydrophilic surface, higher polar component and rougher surface than PHBHHx. The PHBHHx/P3HB4HB blend in 4:2 weight ratio showed the roughest surface and also had the highest chondrocyte viability among all the blends and the polymers tested, which was 59% higher than that on PHBHHx and 32% higher than that on P3HB4HB. The blend with 4:2 weight ratio also had the maximum cartilage-specific collagen II mRNA expression among all the blends and the polymers tested, which was 9-times higher than that on PHBHHx and 8-times higher than that on P3HB4HB. These results demonstrated that PHBHHx had improved physical properties and biocompatibility after blending with P3HB4HB. The blends could be used for cartilage tissue engineering.     

Improvement of Stem Cell Viability in Hyaluronic Acid Hydrogels Using Dextran Microspheres

Although hyaluronic acid (HA) has been widely used in clinics as an injectable biomaterial, it may not be appropriate as an injectable stem cell carrier because highly hydrophilic HA hydrogels provide an unfavorable environment in which the encapsulated stem cells are likely to be constrained to a round shape, thereby losing their native morphology. Herein, we hypothesized that dextran microspheres (DMs) can improve stem cell viability in HA hydrogels because they can act as substrates for stem cell adhesion, spreading and proliferation. DMs with a mean diameter of 80 μm were mixed with HA hydrogels. Human adipose-derived stem cells (hASCs) were isolated from human adipose tissue and seeded into the DM-incorporated HA hydrogels. When compared with the hydrogels alone, the number of viable cells was significantly increased in the presence of the DMs. Initially, hASCs appeared to be round in the HA hydrogels. At 12 h after seeding, the hASCs apparently attached onto the DMs and became slightly flattened. One day after seeding, the hASCs seemed to spread onto the surface of the DMs. Fluorescence micrography of live and dead cells confirmed that the cell viability was significantly improved by use of the DMs in HA hydrogels. Overall results demonstrated that the microsphere/hydrogel composite supported stem cell survival and spreading. These characteristics show the potential for use of the composite in cell-delivery and tissue-engineering applications.     

Characterization of neural stem cells on electrospun poly(L-lactic acid) nanofibrous scaffold

Nanofibrous poly(L-lactic acid) (PLLA) scaffolds were fabricated by an electrospinning technique and characterized by scanning electron microscopy, mercury porosimeter, atomic force microscopy and contact-angle test. The produced PLLA fibers with diameters ranging from 150 to 350 nm were randomly orientated with interconnected pores varying from several μm to about 140 μm in-between to form a three-dimensional architecture, which resembles the natural extracellular matrix structure in human body. The in vitro cell culture study was performed and the results indicate that the nanofibrous scaffold not only supports neural stem cell (NSC) differentiation and neurites out-growth, but also promotes NSC adhesion. The favorable interaction between the NSCs and the nanofibrous scaffold may be due to the greatly improved surface roughness of the electrospun nanofibrous scaffold. As evidenced by this study, the electrospun nanofibrous scaffold is expected to play a significant role in neural tissue engineering.     

三维数字规划和二维胶片模板测量辅助全髋关节置换的效果比较

目的 比较运用三维数字规划技术进行三维规划和传统二维胶片模板测量进行初次全髋关节置换术前规划的效果.方法 2018年12月至2019年10月在哈尔滨医科大学附属第一医院行初次单侧全髋关节置换(THA)的102例患者根据其采用的术前规划的类型分为未规划组(37例)、二维规划组(27例)、三维规划组(38例),分别对各组手...     

A Micro-Scale Surface-Structured PCL Scaffold Fabricated by a 3D Plotter and a Chemical Blowing Agent

To study cell responses, polymeric scaffolds with a controllable pore size and porosity have been fabricated using rapid-prototyping methods. However, the scaffolds fabricated by rapid prototyping have very smooth surfaces, which tend to discourage initial cell attachment. Initial cell attachment, migration, differentiation and proliferation are strongly dependent on the chemical and physical characteristics of the scaffold surface. In this study, we propose a three-dimensional (3D) plotting method supplemented with a chemical blowing agent to produce a surface-modified 3D scaffold in which the surface is inscribed with nano- and micro-sized pores. The chemically-blown 3D polymeric scaffold exhibited positive qualities, including the compressive modulus, hydrophilicity and initial cell adhesion. Cell cultures on the scaffolds demonstrated that chondrocytes interacted better with the surface-modified scaffold than with a normal 3D scaffold.     

The role of endothelial cells in the retinal stem and progenitor cell niche within a 3D engineered hydrogel matrix

Cell-cell interactions are critical to understanding functional tissues. A number of stem cell populations have been shown to receive key regulatory information from endothelial cells (ECs); however, the role of ECs in the retinal stem and progenitor cell (RSPC) niche has been largely unexplored. To gain greater insight into the role of ECs on RSPC fate, a three-dimensional (3D) co-culture model, incorporating cell-cell interactions, was designed by covalently-modifying agarose hydrogels with growth factors and cell-adhesive peptides in defined volumes. Therein ECs adopted tubular-like morphologies similar to those observed in vivo, but not observed in two-dimensional (2D) cultures. Unexpectedly, ECs inhibited proliferation and differentiation of RSPCs, revealing, for the first time, the possible role of ECs on RSPC fate. This 3D hydrogel scaffold provides a simple, reproducible and versatile method with which to answer biological questions related to the cellular microenvironment.     

3D position of pericentromeric heterochromatin within the nucleus of a patient with ICF syndrome

ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome is a rare autosomal recessive disorder characterised by severe immunodeficiency, craniofacial anomalies and chromosome instability. Chromosome analyses from blood samples show a high frequency of decondensation of pericentromeric heterochromatin (PH) and rearrangements involving chromosomes 1 and 16. It is the first and, as far as we know, the only disease associated with a mutation in a DNA methyltransferase gene, DNMT3B, with significant hypomethylation of the classical satellite DNA, the major component of the juxtacentromeric heterochromatin. To better understand the complex links between the hypomethylation of the satellite DNA, the cytogenetic anomalies and the clinical features of ICF syndrome, we performed three-dimensional (3D) FISH on preserved cells from a patient with a suspected ICF phenotype. Analysis of DNMT3B did not reveal any mutation in our patient, making this case an ICF type 2. The results of 3D-FISH showed a statistically significant change in the intranuclear position of PH of chromosome 1 in cells of the patient as compared to normal cells. It is difficult to understand how a defect in the methylation pathway can be responsible for the various symptoms of this condition. From our observations we suggest a mechanistic link between the reorganisation of the nuclear architecture and the altered gene expression.     

The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel

The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.     

Novel biodegradable films and scaffolds of chitosan blended with poly(3-hydroxybutyrate)

In order to develop a novel biomaterial, films of chitosan blended with poly(3-hydroxybutyrate) (PHB) were prepared by an emulsion blending technique and their properties were characterized. Scanning electron microscopy (SEM) showed that PHB microspheres were formed and were entrapped in chitosan matrices, which made the film surface rough. With increasing PHB content, the roughness of the film surface increased, while the swelling capability of the films decreased. In a wet state, the blended films exhibited a lower elastic modulus, a higher elongation-at-break and a higher tensile strength compared with chitosan films. Cell-culture experiments revealed that the blended films had better cytocompatibility than chitosan films. To explore the potential application of the blended material in tissue engineering, the porous blended scaffolds were fabricated and their pore morphology was observed by SEM. The results revealed that not only pore structure but also pore wall morphology of the blended scaffolds could be controlled by selecting the parameters of the fabrication process. These advantageous properties indicate that the blended chitosan/PHB material is promising for tissue engineering applications.