A dual synergistic effect of titanium and curcumin co-embedded on extracellular matrix hydrogels of decellularized bone: Potential application in osteoblastic differentiation of adipose-derived mesenchymal stem cells.

This research aims to design and fabricate a novel hydrogel-based composite as a functional biomimetic and biocompatible scaffold for amended osteoblastic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). The extracellular matrix (ECM) hydrogel is an ideal scaffold in tissue engineering in terms of its structure mimics natural tissue. In this study, the fresh bovine femur was demineralized and decellularized; next, ECM hydrogel was obtained by digesting these matrices. Then, TiO2 and curcumin-loaded hydrogel (Hy/Ti/Cur) was fabricated besides TiO2-loaded hydrogel (Hy/Ti) and curcumin-loaded hydrogel (Hy/Cur). Comparing the scanning electron microscopy (SEM) images of the pure network hydrogel and the rough morphology of Hy/Ti/Cur revealed that curcumin and titanium dioxide were successfully loaded into the hydrogel. In addition, FTIR spectroscopy and X-ray diffraction (XRD) validated these findings. The findings of the hydrogels' swelling test indicated the favourable impact of curcumin and titanium dioxide in hydrogels, which enhances water absorption capacity. Our results showed that the hydrogels were cytocompatible, and the cell viability on the hydrogels was elevated compared to the control. The synergistic effect of TiO2 and Cur co-embedded on ECM hydrogel (Hy/Ti/Cur) stimulates bone differentiation markers, such as Runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN) in ADMSCs cultured in normal and osteogenic medium. Moreover, Alkaline Phosphatase (ALP) activity and calcium deposition of ADMSCs cultured on engineered hydrogels were increased. These experiments showed that newly fabricated hydrogel has the potential to induce osteogenesis, which is recommended as an attractive scaffold in bone tissue engineering.

Angiogenic effects of cell therapy within a biomaterial scaffold in a rat hind limb ischemia model.

Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1-10% of humans worldwide with peripheral arterial disease. Cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs) have been used for the treatment of CLI. However, little information is available regarding the angiogenic potency of MSCs and mast cells (MC) in angiogenesis. The aim of this study was to evaluate the ability of MCs and MSCs to induce angiogenesis in a rat model of ischemic hind limb injury on a background of a tissue engineered hydrogel scaffold. Thirty rats were randomly divided into six control and experimental groups as follows: (a) Control healthy (b) Ischemic positive control with right femoral artery transection, (c) ischemia with hydrogel scaffold, (d) ischemia with hydrogel plus MSC, (e) ischemia with hydrogel plus MC and (f) ischemia with hydrogel plus MSC and MCs. 106 of each cell type, isolated from bone marrow stroma, was injected into the transected artery used to induce hind limb ischemia. The other hind limb served as a non-ischemic control. After 14 days, capillary density, vascular diameter, histomorphometry and immunohistochemistry at the transected location and in gastrocnemius muscles were evaluated. Capillary density and number of blood vessels in the region of the femoral artery transection in animals receiving MSCs and MCs was increased compared to control groups (P < 0.05). Generally the effect of MCs and MSCs was similar although the combined MC/MSC therapy resulted in a reduced, rather than enhanced, effect. In the gastrocnemius muscle, immunohistochemical and histomorphometric observation showed a great ratio of capillaries to muscle fibers in all the cell-receiving groups (P < 0.05). The data indicates that the combination of hydrogel and cell therapy generates a greater angiogenic potential at the ischemic site than cell therapy or hydrogels alone.

Phenolated alginate-collagen hydrogel induced chondrogenic capacity of human amniotic mesenchymal stem cells.

Horseradish peroxidase (HRP)-catalyzed hydrogels are considered to be an important platform for tissue engineering applications. In this study, we investigated the chondrogenic capacity of phenolated (1.2%) alginate-(0.5%) collagen hydrogel on human amniotic mesenchymal stem cells after 21 days. Using NMR, FTIR analyses, and SEM imaging, we studied the phenolation and structure of alginate-collagen hydrogel. For physicochemical evaluations, gelation time, mechanical properties, swelling, and degradation rate were assessed. The survival rate was monitored using the MTT assay and DAPI staining. Western blotting was performed to measure the chondrogenic differentiation of cells. NMR showed successful phenolation of the alginate-collagen hydrogel. FTIR exhibited the interaction between the functional groups of collagen with phenolated alginate. SEM showed the existence of collagen microfibrils in the alginate-collagen hydrogel. Compared to phenolated alginate, the addition of collagen increased hydrogel elasticity by 10%. Both swelling rate and biodegradability were reduced in the presence of collagen. We noted an increased survival rate in phenolated alginate-collagen compared to the control cells (p < 0.05). Western blotting revealed the increase of chondrocyte-associated proteins such as SOX9 and COL2A1 in phenolated-alginate-collagen hydrogels after 21 days. These data showed that phenolated alginate-collagen hydrogel is an appropriate 3 D substrate to induce chondrogenic capacity of human mesenchymal stem cells.

Preparation and characterization of polycaprolactone/chitosan-g-polycaprolactone/hydroxyapatite electrospun nanocomposite scaffolds for bone tissue engineering.

Chitosan (CS) and poly (ε-caprolactone) (PCL) are two most usable polymers in biomedical applications. In this study, chitosan has been modified and incorporated with poly (ε-caprolactone) to fabricate bone tissue engineering scaffold. Moreover, hydroxyapatite nanoparticles were added to enhance bioactivity and mechanical properties of scaffold. Bulk and fibrous comparative results showed significant effect of fiber diameter and distribution on mechanical properties. Moreover, the incorporation of chitosan-g-poly (ε-caprolactone) (CS-g-PCL) significantly decreases fiber diameter of pure PCL scaffold. Furthermore, both CS-g-PCL and nHA enhance mineralization and degradation of the scaffold soaked in simulated body fluid (SBF) and phosphate buffered saline (PBS), respectively. In vitro cytocompatibility assays also confirmed high cell viability and proliferation on the samples. Taken together, the results suggest that the microfabricated nanocomposite scaffolds could be used in bone tissue engineering.

Culture of rabbit bone marrow mesenchymal stem cells on polyurethane/pyrrole surface promoted differentiation into endothelial lineage.

Due to the electrical conductivity, pyrrole-based scaffolds are one of the attractive biomaterials in the regeneration of electrically active tissues like the heart and brain. Here, we investigated the impact of polyurethane/pyrrole scaffold on the angiogenesis differentiation of rabbit mesenchymal stem cells toward endothelial lineage in vitro. Nanoelectrospun polyurethane/pyrrole fibers were synthesized and characterized using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrum analysis, scanning electron microscope (SEM) imaging. Mechanical properties, electroconductivity, and hydrophobicity were also measured. The viability of cells was monitored 72 hours after being plated on the polyurethane/pyrrole surface. The endothelial differentiation of stem cells was explored using western blotting. ATR-FTIR revealed that the pyrrole was successfully polymerized to polypyrrole and blend with polyurethane fibers. The addition of pyrrole to polyurethane increased the tensile strength compared to the polyurethane group. These features coincided with the reduction of the hydrophilic properties of polyurethane. Based on our data, the electro-conductivity of polyurethane/pyrrole was superior compared to the polyurethane group. SEM imaging showed an appropriate cell attachment to the surface of polyurethane/pyrrole and polyurethane groups synthesized membranes. MTT assay revealed a significantly increased survival rate in the polyurethane/pyrrole group compared to the polyurethane group (P < .05). We noted a statistically significant increase of endothelial-associated proteins, CD31, von Willebrand factor, and CD34, in cells expanded on polyurethane/pyrrole compared to the polyurethane group (P < .05). As a more general note, it could be hypothesized that the polyurethane/pyrrole blend could improve the angiogenesis potency of rabbit bone marrow mesenchymal stem cells for regenerative purposes.

Histomorphometrical evaluation of extensor digitorum longus muscle in sciatic nerve regeneration using tissue engineering in rats.

Skeletal muscle atrophy induced by denervation is one of the common disorders in traumatic nerve injuries. The aim of this study was the evaluation of histomorphometrical changes of extensor digitorum longus muscle after denervation and its regeneration by tissue engineering. Ninety adult male Wistar rats were randomly divided into six main groups (n = 15) in three time periods (2, 4 and 8 weeks; n = 5). Control group was treated without surgery, in transection (Tr) group left sciatic nerve was transected, in scaffold (S) group only collagen gel scaffold was used, in mast cell (MC) group mast cells were used, mesenchymal stem cell (MSC) group was treated with mesenchymal stem cells and in MC+MSC group, mast cells along with mesenchymal stem cells were used. In the cellular groups, the scaffold and cells were mixed and placed in the transected nerve gap. The average diameter of muscle fibers, ratio of the muscle fibers nuclei to the fibrocytes nuclei (mn/fn), ratio of the muscle fibers nuclei number to the muscle fibers number (mn/mf), the average ratio of blood vessels to muscle fibers number (v/mf) and muscles weight in Tr group were the lowest compared to the other groups; but, in cellular and S groups, amelioration was observed according to the time period. However, in MC+MSC group, there were the highest ameliorative results. This study revealed that simultaneous use of MCs and MSCs mixed with collagen gel scaffold can be considered as a suitable approach to improve denervated skeletal muscle atrophy associated with sciatic nerve injury.

Enzymatically gellable gelatin improves nano-hydroxyapatite-alginate microcapsule characteristics for modular bone tissue formation.

To maintain gelatin (Gel) as adhesive motifs inside alginate microcapsule as building blocks of modular approach, phenol moiety (Ph) was introduced into gelatin (Gel Ph). Addition of Gel Ph to alginate (Alg-Gel Ph) dramatically altered the physical properties of alginate-based hydrogels as compared to unmodified gelatin (Alg-Gel) addition. Alg-Gel Ph hydrogels revealed a dramatically lower swelling ratios (63%) as compared to Alg-Gel hydrogels (150%). Moreover, Gel Ph decreased 40% degradation rate of alginate-based hydrogels after 72 hr, while increasing compressive modulus 3.5-fold as compared to Alg-Gel hydrogels. Introducing nano-hydroxyapatite (nHA) to Alg-Gel Ph hydrogel (Alg-Gel Ph-nHA) could reduce degradation rate to 41.5% and improve compressive modulus of hydrogels significantly, reaching to 294 ± 2.5 kPa. The microencapsulated osteoblast-like cells proliferated considerably and showed more metabolic activities (two times) in Alg-Gel Ph-nHA microcapsules during a 21-day culture period, resulting in more calcium deposition and alkaline phosphatase (ALP) activities. The subcutaneous microcapsules could also be identified readily without complete absorption and signs of toxicity or any untoward reactions and viable osteoblast-like cells were seen as red colored areas in the central regions of cell-laden microcapsules after 1 month. The study demonstrated Alg-Gel Ph-nHA microcapsule as a promising 3D microenvironment for modular bone tissue formation.

Histomorphometric and immunohistochemical evaluation of angiogenesis in ischemia by tissue engineering in rats: Role of mast cells.

The aim of this study was to find a proper method for improvement of ischemic condition in the rat hind limb and also to observe the efficacy of cell engraftment with alginate/gelatin three-dimensional scaffolds. Eighteen male Wistar rats weighing 200 to 250 g were randomly divided into three groups (n = 6) including a) ischemia group; in which femoral artery was removed after ligation at the distance of 5 mm, b) scaffold group; in which hydrogel scaffold was added to the site of transected femoral artery and c) test group; in which in addition to hydrogel scaffold, mast cells (MCs) were also added (1 × 106 cells). Analysis of capillary density, artery diameter, histomorphometric parameters and immunohistochemistry in transected location were done on day 14 after femoral artery transection. The average number of blood capillary was significantly higher in the test group than other groups. Also, the average number of medium and large blood vessels was significantly higher in the test group compared to ischemia and scaffold groups. Application of MCs through the use of hydrogel scaffolds (alginate/gelatin) can be considered as a new approach in the application of stem cells for therapeutic angiogenesis under ischemic conditions which can improve the angiogenesis process in patients with peripheral artery diseases.

The effect of alginate-gelatin encapsulation on the maturation of human myelomonocytic cell line U937.

Today, many attempts have been collected in the field of tissue engineering for reconstitution of injured bone marrow capacity by transplantation of functional cell source. By having a three-dimensional condition, microcapsules are appropriate candidates for cells transplantation to target sites. Here, we examined the effect of alginate-gelatin microcapsules on functional maturation of human myelomonocytic cell line U937 after 7 days in vitro. U937 cells were encapsulated by the mixture of alginate-gelatin and cultured for 7 days. Trypan blue staining was used to show cell survival rate. Morphological changes were determined by haematoxylin and eosin staining. The expression of monocyte (CD14) and leukocyte (CD33) factors were measured by flow cytometry. The functional maturation of encapsulated cells was shown by immunocytochemistry targeting myeloperoxidase (MPO) activity and level of CD68. Transcription level of adhesion molecules CD68L, CD18, CD11b, and CD49d/VLA was detected by real-time polymerase chain reaction. In vivo constitutive capacity of encapsulated U937 was investigated in rabbits via administration to bone marrow. We showed enhanced U937 viability and monocyte and band cell-like appearance 7 days after encapsulation. These changes coincided with increasing CD33 and CD14 levels and a decrease of CD15, confirming cell maturation (p < 0.05). High level of MPO and CD68-positive cells showed the functional maturation of U937 cells into neutrophils and macrophages. Compared with that of nonencapsulated cells, the level of adhesion factor was up-regulated. We found labelled cells in the peripheral blood after cell transplantation to bone marrow. These data suggest that alginate-gelatin encapsulation of U937 cells promotes functional leukopoiesis and monocytopoiesis.

Preparation, characterization and in vitro anticancer activity of paclitaxel conjugated magnetic nanoparticles.

In this study, magnetic nanoparticles (MNPs) coated with L-aspartic acid (F-Asp NPs) were synthesized through a co-precipitation method and conjugated with paclitaxel (PTX) (F-Asp-PTX NPs) by esterification reaction between the carboxylic acid end groups on MNPs surface and the hydroxyl groups of the PTX and studied its cytotoxic effect in vitro. The successful conjugating of PTX onto the nanoparticles (NPs) was confirmed by X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM) and transmission electron microscopy (TEM) techniques. The results showed that the average size was 46.11 ± 7.8 (mean ± SD (n = 25)) nm. The cytotoxicity of void of PTX and F-Asp-PTX NPs were compared to each other by MTT assay of the treated MCF-7 cell line. The F-Asp-PTX NPs showed pH-dependent drug release behavior. These studies specify that F-Asp-PTX NPs have a very remarkable anticancer effect, for breast cancer cell line.

Barium-cross-linked alginate-gelatine microcapsule as a potential platform for stem cell production and modular tissue formation.

Influence of gelatine concentration and cross-linker ions of Ca2+ and Ba2+ was evaluated on characteristics of alginate hydrogels and proliferation behaviours of model adherent and suspendable stem cells of fibroblast and U937 embedded in alginate microcapsules. Increasing gelatine concentration to 2.5% increased extent of swelling to 15% and 25% for barium- and calcium-cross-linked hydrogels, respectively. Mechanical properties also decreased with increasing swelling of hydrogels. Both by increasing gelatine concentration and using barium ions increased considerably the proliferation of encapsulated model stem cells. Barium-cross-linked alginate-gelatine microcapsule tested for bone building block showed a 13.5 ± 1.5-fold expansion for osteoblast cells after 21 days with deposition of bone matrix. The haematopoietic stem cells cultured in the microcapsule after 7 days also showed up to 2-fold increase without adding any growth factor. The study demonstrates that barium-cross-linked alginate-gelatine microcapsule has potential for use as a simple and efficient 3D platform for stem cell production and modular tissue formation.