Effects of Nanofiber Scaffolds Coated with Nanoparticulate and Microparticulate Freeze Dried Bone Allograft on the Morphology,Adhesion, and Proliferation of Human Mesenchymal Stem Cells

Background:Freeze dried bone allograft nanoparticles on a nanofiber membrane may serve as an ideal scaffold for bone regeneration. This study aimed to assess the biological behavior of human MSCs in terms of proliferation and adhesion to nanoparticulate and microparticulate FDBA scaffolds on PLLA nanofiber membrane. Methods:In this experimental study, PLLA nanofiber scaffolds were synthesized by the electrospinning method. The FDBA nanoparticles were synthesized mechanically. The FDBA nanoparticles and microparticles were loaded on the surface of PLLA nanofiber membrane. A total of 64 scaffold samples in four groups of n-FDBA/PLLA, FDBA/PLLA, PLLA and control were placed in 24-well polystyrene tissue culture plates; 16 wells were allocated to each group. Data were analyzed using one-way ANOVA and Bonferroni test. Results:The proliferation rate of MSCs was significantly higher in the nanoparticulate group compared to the microparticulate group at five days (p = 0.034). Assessment of cell morphology at 24 hours revealed spindle-shaped cells with a higher number of appendages in the nanoparticulate group compared to other groups. Conclusion:MSCs on n-FDBA/PLLA scaffold were morphologically more active and flatter with a higher number of cellular appendages, as compared to FDBA/PLLA. It seems that the nanoparticulate scaffold is superior to the microparticulate scaffold in terms of proliferation, attachment, and morphology of MSCs in vitro.Key Words: Allografts, Bone regeneration, Mesenchymal stem cells, Nanofibers     

Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration

Background:One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using 3D-printing. Herein, we aimed to determine whether the much tighter control of microstructure of 3DP PLGA/β-TCP scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods:Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results:The 3DP scaffolds had a smoother surface (R_a: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (R_a: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. ALP activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion:The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation. Key Words: Alkaline phosphatase, β-tricalcium phosphate, Poly(lactic-co-glycolic) acid copolymer     

Comparative study of bone repair using porous hydroxyapatite/ β-tricalcium phosphate and xenograft scaffold in rabbits with tibia defect

Background: Bone tissue engineering requires materials that are biocompatible, mechanically suited for bone function, integrated with the host skeleton, and support osteoinduction of the implanted cells for new bone formation. The aim of this study was to compare the osteogenic potential of xenograft with hydroxyapatite/β- tricalcium phosphate (HA/β-TCP) scaffold. Methods: New Zealand rabbits (n = 9) were divided into 3 groups. Osteoblast cells were originally isolated from rabbit iliac crest and cultured in DMEM/F12. After creating a critical-sized defect (2 × 3 cm) in rabbit tibia bone, the defect was filled with an implant of HA/TCP with osteoblasts and xenograft in the hole of left (as control) and right tibia, respectively. The new bone formation and the development of bone union within the defect were evaluated by x-ray images and eosine and hematoxylin staining at 4, 8, and 12 weeks post-operation. Results: The bone partially formed in both groups was filled with osteoblast cultured on porous implants at 4 weeks. Over time, progressive bone regeneration was observed inside the pores. Moreover, a progressive vascular ingrowth and progressive integration with the host bone were obvious in xenograft when compared to HA/β-TCP. A good integration between the xenograft implants and the bone was observed radiographically and confirmed by histological section. Conclusion: The result showed that the bone defect can be repaired using both synthetic and xenograft implants. However, the xenograft showed a better osteointegration as compared to HA/β-TCP scaffold.Key Words: Osteoblasts, Hydroxyapatite/β-Tricalcium phosphate (TCP), Bone tissue     

Sustained Release of Risedronate from PLGA Microparticles Embedded in Alginate Hydrogel for Treatment of Bony Lesions

Background:Inflammatory bone resorption in periodontitis can lead to tooth loss. Systemic administration of bisphosphonates such as risedronate for preventing bone resorption can cause adverse effects. ALG and PLGA microparticles have been studied as drug delivery systems for sustained release of drugs. Therefore, the release pattern of risedronate from PLGA microparticles embedded with ALG was studied as a drug delivery system for sustained release of the drug, which can be used in local administrations. Methods:Risedronate-containing PLGA microparticles were fabricated using double emulsion solvent evaporation technique. Ionic cross-linking method was used to fabricate risedronate-loaded ALG. Risedronate-containing PLGA microparticles were then coated with ALG. The calibration curve of risedronate was traced to measure EE and study the release pattern. SEM imaging was carried out, and cell toxicity was examined using MTT assay. Statistical analysis of data was carried out using SPSS ver. 20 software, via one-way ANOVA and Tukey’s tests. Results:SEM imaging showed open porosities on ALGs. The mean EE of PLGA microparticles for risedronate was 57.14 ± 3.70%. Risedronate released completely after 72 h from ALG, and the cumulative release was significantly higher (p = 0.000) compared to PLGA microspheres coated with ALG, which demonstrated sustained released of risedronate until day 28. Risedronate-loaded ALG showed a significant decrease in gingival fibroblasts cell viability (p < 0.05). Conclusion: Alginate-coated PLGA microspheres could release risedronate in a sustained and controlled way and also did not show cell toxicity. Therefore, they seem to be an appropriate system for risedronate delivery in local applications. Key Words: Alginates, Hydrogels, Polylactic acid-polyglycolic acid copolymer, Risedronic acid     

Fabrication of silk fibroin based three dimensional scaffolds for tissue engineering

Herein we report successful synthesis of silk fibroin (SF) three dimensional scaffolds (SF 3D-scaffold) from SF sponge and SF nanofibers. Both the nanofibers and sponge were prepared from Bombyx mori fibroin. The SF 3D-scaffold was prepared by electrospinning the fibroin nanofibers over the sponge. Surface morphology was determined by scanning electron microscopy (SEM), while nanofiber diameter and pore size were measured using imageJ software. Effect of spinning time on the pore size and cell adhesion was determined. Average diameter of the SF nanofibers was measured to be 320 nm and pore size was found to reduce with increasing spinning time, such that, for 1 h spinning time pore size was 231 µm and the same for 3.5 h was 4.1 µm. However, the number of pores increased with spinning time. The results confirmed adhesion of MC3T3-E1 cells on the SF sponge, SF nanofibers and SF three dimensional scaffolds. Higher cell adhesion was found on the three dimensional scaffold in comparison to the nanofibers and sponge, possibly due to highly porous structure with very small and numerous pores in the resultant composite; hence more cell adhesion sites. The cell adhesion result confirmed biocompatibility of the SF 3D-scaffold and hence its suitability for applications in tissue engineering.     

Silk fibroin/hyaluronic acid porous scaffold for dermal wound healing

The use of silk protein as a biomaterial has been studied for decades. In this study, silk fibroin (SF)/hyaluronic acid (HA) blend scaffolds were prepared by freeze-drying technique. The structure and properties of the blend scaffolds were examined and analyzed. The results demonstrated that the secondary structures of the SF/HA scaffolds were mainly amorphous and β-sheet structures. The pore radius and porosity of the scaffolds decreased with a decrease in the freezing temperature decrease and an increase in the HA ratio. The pore radius and porosity were regulated from 32.22 μm to 290.76 μm and from 74.1 % to 91.15 %, respectively. In vitro, the SF/HA scaffolds could support the fibroblast cell adhesion and proliferation and showed good cytocompatibility. In vivo, the SF/HA scaffolds were implanted into the dorsum of Sprague Dawley rats to evaluate their bioactivity for dermal tissue reconstruction. The vascular-like structures appeared more rapidly in SF/HA scaffolds than that in the PVA group, and a new dermal layer was formed, as determined by histological analysis. The SF/HA porous scaffolds have promise as a dermal substitute.     

Fabrication and Characterization of Nanocomposite Hydrogel Based on Alginate/Nano-Hydroxyapatite Loaded with Linum usitatissimum Extract as a Bone Tissue Engineering Scaffold

In the current paper, we fabricated, characterized, and applied nanocomposite hydrogel based on alginate (Alg) and nano-hydroxyapatite (nHA) loaded with phenolic purified extracts from the aerial part of Linum usitatissimum (LOH) as the bone tissue engineering scaffold. nHA was synthesized based on the wet chemical technique/precipitation reaction and incorporated into Alg hydrogel as the filler via physical cross-linking. The characterizations (SEM, DLS, and Zeta potential) revealed that the synthesized nHA possess a plate-like shape with nanometric dimensions. The fabricated nanocomposite has a porous architecture with interconnected pores. The average pore size was in the range of 100–200 µm and the porosity range of 80–90%. The LOH release measurement showed that about 90% of the loaded drug was released within 12 h followed by a sustained release over 48 h. The in vitro assessments showed that the nanocomposite possesses significant antioxidant activity promoting bone regeneration. The hemolysis induction measurement showed that the nanocomposites were hemocompatible with negligible hemolysis induction. The cell viability/proliferation confirmed the biocompatibility of the nanocomposites, which induced proliferative effects in a dose-dependent manner. This study revealed the fabricated nanocomposites are bioactive and osteoactive applicable for bone tissue engineering applications.     

Growth of primary hippocampal neurons on multichannel silk fibroin scaffold

Particular attention has been given to axonal outgrowth of neurons to understand how topographical surface cues influence attachment and subsequent directional migration and growth. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional freeze-drying processes. The average pore diameter, the porosity, and pore density of the scaffold are 120 µm, 88 %, and 203 mm^?2, respectively. Further, hippocampal neurons were seeded onto the scaffold and the hippocampal neurons morphology was investigated. Cell-cell networks and cell-matrix interactions had been established by newly formed axons and the diversity of neurons was much higher after culturing 7 days. The neurons expressed β-III-tubulin and nerve filament, while glial fibrillary acidic protein immunofluorescence was barely above background. These results indicated that the SF scaffolds with uniaxial multichannels could be guided axons of neurons spread along the channels. SF scaffolds with oriented pores have a potential for nerve tissue regeneration.     

Preparation and Characterization of Antioxidant Nanoparticles Composed of Chitosan and Fucoidan for Antibiotics Delivery

In this study, we developed novel chitosan/fucoidan nanoparticles (CS/F NPs) using a simple polyelectrolyte self-assembly method and evaluated their potential to be antioxidant carriers. As the CS/F weight ratio was 5/1, the CS/F NPs were spherical and exhibited diameters of approximately 230–250 nm, as demonstrated by TEM. These CS/F NPs maintained compactness and stability for 25 day in phosphate-buffered saline (pH 6.0–7.4). The CS/F NPs exhibited highly potent antioxidant effects by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing the concentration of intracellular reactive oxygen species (ROS) and superoxide anion (O₂⁻) in stimulated macrophages. The DPPH scavenging effect of CS/F NPs primarily derives from fucoidan. Furthermore, these CS/F NPs activated no host immune cells into inflammation-mediated cytotoxic conditions induced by IL-6 production and NO generation. The MTT cell viability assay revealed an absence of toxicity in A549 cells after exposure to the formulations containing 0.375 mg NPs/mL to 3 mg NPs/mL. Gentamicin (GM), an antibiotic, was used as a model drug for an in vitro releasing test. The CS/F NPs controlled the release of GM for up to 72 h, with 99% of release. The antioxidant CS/F NPs prepared in this study could thus be effective in delivering antibiotics to the lungs, particularly for airway inflammatory diseases.     

Effect of Dioxane and N-Methyl-2-pyrrolidone as a Solvent on Biocompatibility and Degradation Performance of PLGA/nHA Scaffolds

Background:Solvent casting/particulate leaching is one of the most conventional methods for fabricating polymer/ceramic composite scaffolds. In this method, the solvent generally affects resulting scaffold properties, including porosity and degradation rate. Methods:Herein, composite scaffolds of PLGA/nHA with different percentages of nHA (25, 35, and 45 wt. %) were prepared by the solvent casting/particle leaching combined with freeze drying. The effects of two different solvents, DIO and NMP, on morphology, porosity, bioactivity, degradation rate, and biocompatibility of the resulting scaffolds were investigated. Results:The results revealed that increasing the nHA percentages had no significant effect on the porosity and interconectivity of scaffolds (p > 0.05), whereas altering the solvent from DIO into NMP decreased the porosity from about 87% into 71%, respectively. Moreover, scaffolds of DIO illustrated the high results of cell proliferation compared to those of NMP; the cell viability of GD25 decreased from 85% to 65% for GN25. The findings also indicated that scaffolds prepared by NMP had a higher rate of losing weight in comparison to DIO. Adding nHA to PLGA had a significant effect on the bioactivity of scaffolds (p < 0.05), composite scaffolds with 45 wt % nHA had at least 30% more weight gain compared to the neat polymer scaffolds. Conclusion:The DIO scaffolds have higher rates of porosity, interconnectivity, bioactivity, and biocompatibility than NMP scaffolds due to its high evaporation rate. Key Words: Freeze drying, Porosity, Solvents     

Mechanism of silk fibroin scaffolds with oriented multichannels and its cytocompatibility

As a biomaterial, besides excellent biocompatibility and biodegradability, suitable macropores and pores structure should be provided to guide cell extension and migration. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional temperature field freezing technique. The average pore diameter, pore density and porosity of the scaffold with oriented channels are ～128.7 µm, ～158 mm^?2 and ～91.4 %, respectively. By controlling of the temperature gradient direction, the oriented multichannels of the scaffolds were formed in longitudinal easily. In process of the scaffolds fabrication, the directional growth of ice crystal could shear and draft to the silk fibroin molecule segments, which resulted in the new crystal nucleus formation in new zone and increase of β-sheet components in the scaffolds. In vitro, L929 cells were seeded on the scaffolds with oriented channels to evaluate the effect on cell behavior. Cell viability, adhesion and morphology were determined by methyl thiazolyl tetrazolium, confocal microscope and scanning electron microscope. The results showed that the cells anchored on the oriented channels, spread along the direction of the channels and hold a higher viability on the scaffolds with oriented channels. These new oriented multichannel scaffold could guide the adhesion and proliferation of L929 cells, which hold a potential in tissue engineering.     

Mesenchymal Stem Cells as an Alternative for Schwann Cells in Rat Spinal Cord Injury

Background: Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Methods: Mesenchymal stem cells were cultured from adult rats’ bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. Results: In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. Conclusion: This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment. Key Words: Rats, Spinal cord injuries (SCI), Bone marrow, Schwann cells, Cell transdifferentiation     

Evaluation of the Prognostic Value of CD56 (140 kDa Isoform) Expression in Breast Cancer Tissues: an Eight-Year Retrospective Study

Background:Identification of specific antigens is highly beneficial for early detection, diagnosis, staging, and outcome prediction of cancer. This study aimed to evaluate the expression and prognostic value of CD56 (140 kDa isoform) in IDC. Methods:Sixty-five patients with IDC who underwent radical surgery or mastectomy as the primary treatment were included. Proper formalin-fixed and paraffin embedded tissue blocks of the patients were prepared and stained by IHC for CD56 (140 kDa isoform) molecule. Chi-square and fisher exact tests were used to compare the results against the clinicopathologic data of patients. Kaplan-Meier and log-rank test were employed to study the prognostic value of the target antigen. Results:The expression pattern of CD56 was granular and cytoplasmic. There were significant associations between the intensity of CD56 expression in invasive cells and carcinoma in situ (p = 0.005) and normal ducts (p = 0.010). Among all clinicipathologic parameters, there was only a significant association between the expression of ER and CD56 (p = 0.023). Neither OS (p = 0.356) nor DFS (p = 0.976) had significant correlation with CD56 expression. Conclusion:Our data indicated that the CD56 marker offers no prognostic value in terms of predicting the OS or DFS for up to eight years after primary surgery. Furthermore, the intensity of its expression is similar between normal, non-invasive, and invasive cells. Considering the generally better outcome of ER+ BC patients than their ER-counterparts, the CD56 marker may be indirectly associated with a more favorable prognosis among IDC patients.Key Words: Breast cancer, Neural cell adhesion molecule, Prognosis     

Sol-Gel Synthesis,in vitro Behavior,and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S

Background:Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods:The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion:The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering. Key Words: Bioactive glass 58S, Gene expression, Mesenchymal stem cells     

Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering

Over the last few years, significant research has been conducted in the construction of artificial bone scaffolds. In the present study, different types of polymer scaffolds, such as chitosan-alginate (Chi-Alg) and chitosan-alginate with fucoidan (Chi-Alg-fucoidan), were developed by a freeze-drying method, and each was characterized as a bone graft substitute. The porosity, water uptake and retention ability of the prepared scaffolds showed similar efficacy. The pore size of the Chi-Alg and Chi-Alg-fucoidan scaffolds were measured from scanning electron microscopy and found to be 62–490 and 56–437 µm, respectively. In vitro studies using the MG-63 cell line revealed profound cytocompatibility, increased cell proliferation and enhanced alkaline phosphatase secretion in the Chi-Alg-fucoidan scaffold compared to the Chi-Alg scaffold. Further, protein adsorption and mineralization were about two times greater in the Chi-Alg-fucoidan scaffold than the Chi-Alg scaffold. Hence, we suggest that Chi-Alg-fucoidan will be a promising biomaterial for bone tissue regeneration.     

Effects of including bioactive legumes in grass silage on digestion parameters,nitrogen balance and methane emissions in sheep

The aim of this work was to investigate the effects of feeding sheep with silage mixtures containing bioactive legumes on intake and digestive parameters. The bioactive legumes used were sainfoin (SF, Onobrychis viciifolia) and red clover (RC, Trifolium pratense), which contain condensed tannins (CT) and polyphenol oxidase respectively. Five treatments were assigned to two groups of sheep according to a replicated 5 × 5 Latin square design. The five types of silages tested were, on a dry matter (DM) basis: pure timothy grass silage (Phleum pratense, control, T), three binary mixtures of T‐SF, T‐RC and RC‐SF (500 g/kg each) and a ternary mixture of T‐RC‐SF (500, 250 and 250 g/kg respectively). The daily voluntary DM intake of silage mixtures containing both SF and RC was greater than for pure T silage, while the presence of SF resulted in lower organic matter digestibility compared to pure T. The rumen disappearance rate measured in situ increased linearly with the presence of SF and RC in silage. The nitrogen (N) digestibility was greater for pure T and T‐RC than for T‐SF, and the amount of N retained daily by the animals was greater for RC‐containing silages than for T and T‐SF. The methane (CH₄) yield was greater for pure T than for the silage mixtures containing SF. We conclude that the presence of RC in silage could boost performances through intake and N retention, while SF‐based mixtures appear to have reduced negative environmental impacts through the reduction of CH₄ emissions.     

Mesenchymal Stem/Stromal-Like Cells from Diploid and Triploid Human Embryonic Stem Cells Display Different Gene Expression Profiles

Background:hESCs-MSCs open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods:Herein, hESCs-MSCs were characterized by IF technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using RT-PCR technique. FACS was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results:MSCs were derived from diploid and triploid hESC lines using sequential 3D and 2D cultures and characterized with the specific markers. IF showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. The hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Conclusion:Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications. Key Words: Human embryonic stem cells, Mesenchymal stem/stromal cells, Regenerative medicine     

Preparation, structure, and in vitro degradation behavior of the electrospun poly(lactide-co-glycolide) ultrafine fibrous vascular scaffold

The PLGA ultrafine fibrous scaffold was successfully fabricated by electrospinning. The morphology and properties of the PLGA vascular scaffolds were examined. In particular, the in vitro degradation behavior of the electrospun PLGA vascular scaffolds was investigated by means of morphology, microstructure, mass loss, M_w, and breaking strength characterization. The results showed that electrospun scaffold possessed ultrafine fibrous and porous structure, and had adequate mechanical properties to be developed as a substitute for native blood vessels. In vitro degradation study showed that the PLGA ultrafine fibrous scaffold could biodegrade in the PBS solution, and the mass loss, M_w, and breaking strength studies indicated that degradation rate of the electrospun PLGA nanofibers was greater in the first 2 weeks. After the degradation of 2 weeks, the degradation slowed down. Furthermore, with the extension of the degradation time, the thermal decomposition temperature of the PLGA scaffold decreased gradually. The results indicated that the electrospun PLGA vascular scaffold could be considered as an ideal candidate for tissue-engineered blood vessel.     

Silages containing bioactive forage legumes: a promising protein‐rich feed source for growing lambs

Forage legumes that contain secondary compounds are considered to be less susceptible to proteolysis than other legumes, with improved silage quality and possibly improved animal performance. This was investigated feeding five groups of growing lambs for 10 weeks, five silages composed of pure timothy (T; Phleum pratense), mixtures of T with red clover (Trifolium pratense; T‐RC, 50/50 on DM basis), sainfoin (Onobrychis viciifolia; T‐SF), or both (T‐RC‐SF, 50/25/25), or a mixture of the two legumes (RC‐SF). Including SF and/or RC in silages improved silage fermentation as shown by higher lactic acid and lower soluble N and NH₃ contents than in T silage. Voluntary intakes were higher with all the RC‐containing silages than with T and T‐SF. The T‐SF silage had the lowest, and RC‐containing silages the highest average values for daily gain, feed conversion efficiency, live weight and carcass weight. The differing effects of these silages cannot be totally explained by differences in their nutritive value and so may be due to their bioactive compounds impacting differently on feeding motivation and digestive efficiency. These results suggest that including RC in silages is a promising strategy to combine improved animal performance with reduced environmental pressure.     

The Effect of Melanocyte Stimulating Hormone and Hydroxyapatite on Osteogenesis in Pulp Stem Cells of Human Teeth Transferred into Polyester Scaffolds

Presently, tissue engineering is employed in the restoration and repair of tissue defects. Degradable scaffolds, stem cells and stimulating factors are employed in this method. In this study, the effect of melanocyte-stimulating hormone (MSH) and/or hydroxyapatite (HA) on proliferation, osteoblast differentiation, and mineralization of human dental pulp stem cells (hDPSCs) seeded on PLLA-PCL nanofibrous scaffolds was evaluated. For this aim, PLLA-PCL-HA nanofibrous scaffolds were fabricated using electrospinning method. FE-SEM images exhibited that all nanofibers had bead-free morphologies with average diameters ranging from 150–205 nm. Human DPSCs seeded into PLLA-PCL nanofibers were treated with MSH. Cell viability, proliferation, morphology, osteogenic potential, and the expression of tissue-specific genes were assessed by means of MTT assay, FE-SEM, alizarin red S staining, and RT-PCR analysis. hDPSCs exhibited improved adhesion and proliferation capacity on the PLLA-PCL-HA nanofibers treated with MSH compared to other groups (p<0.05). Additionally, PLLA-PCL-HA nanofibers treated with MSH exhibited significantly higher mineralization and alkaline phosphatase activity than other groups. RT-PCR results confirmed that PLLA-PCL-HA nanofibers enriched with MSH could significantly unregulated the gene expression of BMP2, osteocalcin, RUNX2 and DSPP that correlated to osteogenic differentiation (p<0.05). Based on results, incorporation of HA nanoparticles in PLLA-PCL nanofibers and addition of MSH in media exhibited synergistic effects on the adhesion, proliferation, and osteogenesis differentiation of hDPSCs, and therefore assumed to be a favorable scaffold for bone tissue engineering applications.