Electrospun collagen–chitosan nanofiber: A biomimetic extracellular matrix for endothelial cell and smooth muscle cell

Electrospinning of collagen and chitosan blend solutions in a 1,1,1,3,3,3-hexafluoroisopropanol/trifluoroacetic acid (v/v, 90/10) mixture was investigated for the fabrication of a biocompatible and biomimetic nanostructure scaffold in tissue engineering. The morphology of the electrospun collagen–chitosan nanofibers was observed by scanning electron microscopy (SEM) and stabilized by glutaraldehyde (GTA) vapor via crosslinking. Fourier transform infrared spectra analysis showed that the collagen–chitosan nanofibers do not change significantly, except for enhanced stability after crosslinking by GTA vapor. X-ray diffraction analysis implied that both collagen and chitosan molecular chains could not be crystallized in the course of electrospinning and crosslinking, and gave an amorphous structure in the nanofibers. The thermal behavior and mechanical properties of electrospun collagen–chitosan fibers were also studied by differential scanning calorimetry and tensile testing, respectively. To assay the biocompatibility of electrospun fibers, cellular behavior on the nanofibrous scaffolds was also investigated by SEM and methylthiazol tetrazolium testing. The results show that both endothelial cells and smooth muscle cells proliferate well on or within the nanofiber. The results indicate that a collagen–chitosan nanofiber matrix may be a better candidate for tissue engineering in biomedical applications such as scaffolds.     

Long-term effects of knitted silk–collagen sponge scaffold on anterior cruciate ligament reconstruction and osteoarthritis prevention

Anterior cruciate ligament (ACL) is difficult to heal after injury due to the dynamic fluid environment of joint. Previously, we have achieved satisfactory regeneration of subcutaneous tendon/ligament with knitted silk–collagen sponge scaffold due to its specific “internal-space-preservation” property. This study aims to investigate the long-term effects of knitted silk–collagen sponge scaffold on ACL regeneration and osteoarthritis prevention. The knitted silk–collagen sponge scaffold was fabricated and implanted into a rabbit ACL injury model. The knitted silk–collagen sponge scaffold was found to enhance migration and adhesion of spindle-shaped cells into the scaffold at 2 months post-surgery. After 6 months, ACL treated with the knitted silk–collagen sponge scaffold exhibited increased expression of ligament genes and better microstructural morphology. After 18 months, the knitted silk–collagen sponge scaffold-treated group had more mature ligament structure and direct ligament-to-bone healing. Implanted knitted silk–collagen sponge scaffolds degraded much more slowly compared to subcutaneous implantation. Furthermore, the knitted silk–collagen sponge scaffold effectively protected joint surface cartilage and preserved joint space for up to 18 months post-surgery. These findings thus demonstrated that the knitted silk–collagen sponge scaffold can regenerate functional ACL and prevent osteoarthritis in the long-term, suggesting its clinical use as a functional bioscaffold for ACL reconstruction.     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

Adhesion,migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix

Silk fibroin–chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 ± 4.8% at 15 min, increasing to 92.8 ± 1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47 ± 2.7 μm h^?1 and a mean persistence time of 41.4 ± 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC–ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p < 0.0001) for ASCs seeded on SFCS (69.0 ± 9.0 kPa) than on glass (6.1 ± 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher (p < 0.05) for ASC seeded on SFCS (0.42 ± 0.02 fibers μm^?1) than on glass-seeded controls (0.24 ± 0.03 fibers μm^?1). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell–cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.     

Influence of muscle architecture on the torque and power–velocity characteristics of young and elderly men

This study investigated the contribution of muscle architecture to the differences in the torque–velocity and power–velocity relationships between older (OM n = 9, aged 69–82 years) and younger men (YM n = 15, aged 19–35 years). Plantarflexors’ (PF) maximal isometric and concentric torques were recorded at 0.87, 1.75, 2.62, 3.49 and 4.36 rad s⁻¹. Physiological cross-sectional area (PCSA) was calculated as the ratio of muscle volume (determined by magnetic resonance imaging) to muscle fascicle length (L _f, measured by ultrasonography). GM PCSA and L _f of the OM were, respectively, 14.3% (P < 0.05) and 19.3% (P < 0.05) smaller than of the YM. In the OM, GM maximum isometric torque and maximum contraction velocity (V _max), estimated from Hill’s equation were, respectively, 48.5 and 38.2% lower (P < 0.001) than in the YM. At all contraction velocities, the OM produced less torque than the YM (46.3% of YM at 0.87 rad s⁻¹ to 14.7% at 4.36 rad s⁻¹, P < 0.001). Peak power (PP) of the OM was 80% lower than that of the YM and normalisation of PP to muscle volume only reduced this difference by 10%. Normalisation of torque to PCSA reduced, but did not eliminate, differences in torque between YM and OM (9.6%) and differences in torque/PCSA increased with contraction velocity (P < 0.05). After normalisation of velocity to L _f, the difference in V _max between the OM and the YM was reduced to 15.9%. Thus, although muscle architecture contributes significantly to the differences in the torque– and power–velocity properties of OM and YM, other contractile factors, intrinsic to the muscle, seem to play a role. It is noteworthy that the deficit in PP between OM and YM is far greater than that of muscle torque, even after normalisation of PP to muscle volume. This finding likely plays an important role in the loss of mobility in old age.     

Effects of crystalline phase on the biological properties of collagen–hydroxyapatite composites

The objective of this study was to investigate the effects of spatial structure and crystalline phase on the biological performance of collagen–hydroxyapatite (Col–HA) composite prepared by biomineralization crystallization. Two types of Col–HA composites were prepared using mineralization crystallization (MC composites) and pre-crystallization (PC composites), respectively. Structural characteristics were analyzed by scanning electron microscopy and transmission electron microscopy. Surface elemental compositions were measured by electron spectroscopy for chemical analysis (ESCA). These composites were used in in vivo repair of bone defects. The effects of the crystalline phase on the biological performance of Col–HA composites were investigated using radionuclide bone scan, histopathology and morphological observation. It was observed that in MC composites, HA was located on the surface of the collagen fibers and aggregated into crystal balls, whereas HA in PC composites was scattered among the collagen fibers. ESCA showed that phosphorus and calcium were 8.99% and 17.56% on MC composite surface, compared with 4.39% and 5.86% on the PC composite surface. In vivo bone defect repair experiments revealed that radionuclide uptake was significantly higher in the area implanted with the PC composite than in the contralateral area implanted with the MC composite. Throughout the whole repair process, the PC composite proved to be superior to the MC composite with regard to capillary-forming capacity and the amount of newly formed bone tissue. So it could be concluded that HA placement on collagen fibers affected the biological performance of Col–HA composites. Pre-crystallization made HA scattered among collagen fibers, creating a better structure for bone defect repair in comparison with MC Col–HA composites.     

Platelet-derived growth-factor-releasing aligned collagen–nanoparticle fibers promote the proliferation and tenogenic differentiation of adipose-derived stem cells

In order to enhance the healing potential of an injured tendon, we have prepared a novel biomimetic aligned collagen–nanoparticle (NP) composite fiber using an electrochemical process. The aligned collagen–NP composite fiber is designed to affect the cellular activity of adipose-derived stem cells (ADSCs) through two different ways: (i) topographic cues from the alignment of collagen fibril and (ii) controlled release of platelet-derived growth factors (PDGFs) from the NPs. PDGF released from collagen–NP fibers significantly enhanced the proliferation of ADSCs when tested for up to 7 days. Moreover, compared to random collagen fibers with PDGFs, aligned collagen–NP fibers significantly promoted the desirable tenogenic differentiation of ADSCs, as evidenced by an increased level of tendon markers such as tenomodulin and scleraxis. On the other hand, no undesirable osteogenic differentiation, as measured by the unchanged level of alkaline phosphatase and osteocalcin, was observed. Together, these results indicate that the aligned collagen–NP composite fiber induced the tenogenic differentiation of ADSCs through both a topographic cue (aligned collagen fibril) and a chemical cue (PDGF released from NPs). Thus, our novel aligned collagen–NP composite fiber has a significant potential to be used for tendon tissue engineering and regeneration.     

Laboratory analysis of canine packed red blood cells—effects of collection and processing on haemolysis,haemoglobin concentration,haematocrit and blood culture

The aim of this study was to evaluate the relationship between blood collection and processing techniques and the quality of canine packed red blood cell (pRBC) units. This prospective study analysed 235 canine blood donations, followed by processing and evaluation of pRBC units. The need for sedation, number of venepunctures, whole blood volume, time to processing and time to analysis were registered, and five different centrifugation protocols were performed. The final pRBC volume, haematocrit, total haemoglobin concentration, haemolysis and bacterial contamination of pRBC units were evaluated. Obtained results were within the reference range of human blood banks' guidelines. One unit presented a positive bacteriologic analysis. No significant differences in haemolysis were detected when factors related to sedation, blood collection, time to processing or centrifugation protocols were studied. Significantly higher haematocrit values were detected in units centrifuged in a faster and longer programme (3,500×g, 15 min). There was a direct increase of haemolysis with longer time spent between centrifugation and analysis. This study demonstrates that haemolysis is significantly higher when analyses post centrifugations take 48–72 h to be performed than in short periods.     

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.     

Drug carrier systems based on collagen–alginate composite structures for improving the performance of GDNF-secreting HEK293 cells

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor. Development of drug delivery technologies facilitating controlled release of GDNF is critical to applying GDNF in treating neurodegenerative diseases. We previously developed 3D collagen microspheres and demonstrated enhanced GDNF secretion after encapsulation of HEK293 cells, which were transduced to overexpress GDNF in these microspheres. However, the entrapped HEK293 cells were able to migrate out of the collagen microspheres, making it undesirable for clinical applications. In this report, we investigate two new carrier designs, namely collagen–alginate composite gel and collagen microspheres embedded in alginate gel in preventing cell leakage, maintaining cell growth and controlling GDNF secretion in the HEK293 cells. We demonstrated that inclusion of alginate gel in both designs is efficient in preventing cell leakage to the surrounding yet permitting the GDNF secretion, although the cellular growth rate is reduced in an alginate concentration dependent manner. Differential patterns of GDNF secretion in the two designs were demonstrated. The collagen–alginate composite gel maintains a more or less constant GDNF secretion over time while the collagen microspheres embedded in alginate gel continue to increase the secretion level of GDNF over time. This study contributes towards the development of cell-based GDNF delivery devices for the future therapeutics of neurodegenerative diseases.     

Varying effects of different β-glucans on the maturation of porcine monocyte-derived dendritic cells

β-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, β-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of β-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 μg/ml) of five commercial β-glucan preparations, differing in structure and source, on porcine monocyte-derived dendritic cells (MoDCs). Although all β-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another β-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlan-stimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1β (IL-1β), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when β-glucans are used as dietary supplement.     

Substrate stiffness and contractile behaviour modulate the functional maturation of osteoblasts on a collagen–GAG scaffold

Anchorage-dependent cells respond to the mechanical and physical properties of biomaterials. One such cue is the mechanical stiffness of a material. We compared the osteogenic potential of collagen–glycosaminoglycan (CG) scaffolds with varying stiffness for up to 6 weeks in culture. The mechanical stiffness of CG scaffolds were varied by cross-linking by physical (dehydrothermal (DHT)) and chemical (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC) and glutaraldehyde (GLUT)) methods. The results showed that all CG substrates allowed cellular attachment, infiltration and osteogenic differentiation. CG scaffolds treated with EDAC and GLUT were mechanically stiffer, retained their original scaffold structure and resisted cellular contraction. Consequently, they facilitated a 2-fold greater cell number, probably due to the pore architecture being maintained, allowing improved diffusion of nutrients. On the other hand, the less stiff substrates cross-linked with DHT allowed increased cell-mediated scaffold contraction, contracting by 70% following 6 weeks (P < 0.01) of culture. This reduction in scaffold area resulted in cells reaching the centre of the scaffold quicker up to 4 weeks; however, at 6 weeks all scaffolds showed similar levels of cellular infiltration, with higher cell numbers found on the stiffer EDAC- and GLUT-treated scaffolds. Analysis of osteogenesis showed that scaffolds cross-linked with DHT expressed higher levels of the late stage bone formation markers osteopontin and osteocalcin (P < 0.01) and increased levels of mineralisation. In conclusion, the more compliant CG scaffolds allowed cell-mediated contraction and supported a greater level of osteogenic maturation of MC3T3 cells, while the stiffer, non-contractible scaffolds resulted in lower levels of cell maturation, but higher cell numbers on the scaffold. Therefore, we found scaffold stiffness had different effects on differentiation and cell number whereby the increased cell-mediated contraction facilitated by the less stiff scaffolds positively modulated osteoblast differentiation while reducing cell numbers.     

Anti-fibrotic effect of iguratimod on pulmonary fibrosis by inhibiting the fibroblast–to-myofibroblast transition

PurposePulmonary fibrosis (PF) is a severe lung disease causing significant morbidity and mortality. PF pathogenesis is attributed to the fibroblast-to-myofibroblast transition (FMT) driven by the most potent pro-fibrogenic factor TGF-β1 activating the Smad3-dependent TGF-β1 canonical pathway. Iguratimod (IGU) is a novel anti-rheumatic drug that suppresses the secretion of inflammatory factors, but is also able to modulate the differentiation of multiple cells. Therefore, the aim of this work was to investigate the effect of IGU on FMT.Materials/methodsPF mouse model was induced in C57BL/6 male mice by bleomycin. The effect of IGU was assessed through the evaluation of lung morphology by H&E and through the collagen accumulation in the lung by Masson staining. Primary human lung fibroblasts (pHLFs) were also used to evaluate the effect of IGU in vitro on TGF-β1-stimulated cells, and proliferation, migration and invasion were measured, together with genes and proteins involved in FMT.ResultsIGU attenuated bleomycin-induced PF in mice and improved the pathological changes in their lungs. In addition, IGU significantly inhibited proliferation, migration and invasion in TGF-β1-stimulated pHLFs without causing apoptosis. Moreover, IGU significantly reduced TGF-β1-induced increase of collagen I and III mRNA expression, thus reducing lung function impairment, and α-SMA, Smad2 and Smad3 phosphorylation, fibronectin expression and F-actin microfilament formation, thus attenuating FMT through the inhibition of the Smad3 pathway. Conclusions: Our results collectively revealed the beneficial effect of IGU on the inhibition of FMT, thus suggesting that it might act as an effective anti-fibrotic agent in preventing the progression of PF.     

In vitro effects of bone- and platelet-derived transforming growth factor-β on the growth of Walker 256 carcinosarcoma cells

Conditioned media from fetal rat calvarial cultures has previously been shown to stimulate the growth of the bone-metastasizing Walker 256 carcinosarcoma cell line. In the current investigation we looked at the possibility that transforming growth factor- (TGF-), present in conditioned media, and positively correlated with resorption in vitro, may be responsible for the enhanced proliferation of Walker cells cultured in these conditioned media. Purified platelet-derived TGF- produced a dose-dependent growth response in Walker cells with an ED₅₀ equal to 0·05 ng/ml. Bone-derived TGF- activity in conditioned media, measured by NRK fibroblast colony formation, correlated well with percentage resorption in bone cultures, and growth activity in Walker cell culture. In addition to this, the growth response normally seen with conditioned media cultures of Walker cells was significantly inhibited by the addition of anti-TGF-1 neutralizing antibody. We conclude that TGF- is an important growth stimulatory component from fetal rat calvaria.     

Edaravone effects on the expression levels of collagen type i and iv after spinal cord injury in rats北大核心

BACKGROUND: Edaravone, an effective free radical scavenger, has been reported to significantly improve the rehabilitation of limb locomotion after spinal cord injury (SCI), but the underlying mechanism remains unclear. OBJECTIVE: To explore the mechanism underlying edaravone promoting the recovery of limb locomotion in rats with SCI by observing the Basso, Beattie, Bresnahan scores and expression levels of collagen type I and IV. METHODS: Thirty-six rats were randomly allocated into three groups (n=12 per group): sham group (laminectomy plus intraperitoneal injection of normal saline), model group (SCI model by NYU impactor plus intraperitoneal injection of normal saline), and edaravone group (SCI model by NYU impactor plus intraperitoneal injection of edaravone). All rats were given the administration at the 1st day post-SCI for consecutive 7 days. The Basso, Beattie, Bresnahan scores were tested at 1, 3, 5 and 7 days post treatment. On day 7, all rats were sacrificed to remove the spinal cord, and the morphology of neurons in the spinal cord were observed by Nissl staining; the expression levels of collagen type I and IV were detected by immunohistochemistry and western blot assays. RESULTS AND CONCLUSION: Compared with the model group, the Basso, Beattie, Bresnahan scores in the edaravone group were significantly increased at day 5 post treatment (P < 0.05). Nissl staining showed a clear boundary between grey matter and white matter, and a large nucleolus in the neurocytoplasm in the sham group; there was a complete structure of neurons, slight cellular swelling and small hematoma area in the edaravone group; many and large cavitations and swollen nucleus were found in the neurons, even without nucleolus. Immunohistochemistry and western blot assay results showed that the expression levels of collagen type I and IV in the edaravone group were significantly higher than those in the model group (P < 0.05). These results indicate that edaravone can promote the recovery of limb locomotion of rats with SCI, probably via up-regulating the expression levels of collagen type I and IV. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.