A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200-950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.
We developed tricalcium phosphate-chitosan-fucoidan biocomposite scaffold (TCP-Ch-Fu) by using the freeze-drying technique. The fabricated biocomposite scaffolds were analyzed by spectroscopy and porosity measurement. The biomechanical properties of scaffolds were assessed by compression test and the results suggested that the incorporation of Fucoidan into the biocomposite improves the compression strength of scaffolds. Biomineralization of scaffolds was evaluated by soaking them in simulated body fluid and the results revealed that the addition of Fucoidan into the scaffolds enhanced the formation of apatite layer on the surface of biocomposite after 7 days of immersion. Alamar Blue assay confirmed that the cell viability of human-derived bone marrow stromal cell was superior in the TCP-Ch-Fuscaffold. The addition of Fucoidan to TCP-Ch increased the release of osteocalcin, confirming that it can support osteogenic differentiation of human mesenchymal stromal cells in in vitro culture. Thus, TCP-Ch-Fu could be a potential candidate for bone-tissue engineering applications.
Tissue engineering aims to generate or facilitate regrowth or healing of damaged tissues by applying a combination of biomaterials, cells, and bioactive signaling molecules. In this regard, growth factors clearly play important roles in regulating cellular fate. However, uncontrolled release of growth factors has been demonstrated to produce severe side effects on the surrounding tissues. In this study, poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) incorporated three-dimensional (3D) CORAGRAF scaffolds were engineered to achieve controlled release of platelet-derived growth factor-BB (PDGF-BB) for the differentiation of stem cells within the 3D polymer network. Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, scanning electron microscopy, and microtomography were applied to characterize the fabricated scaffolds. In vitro study revealed that the CORAGRAF-PLGA-PDGF-BB scaffold system enhanced the release of PDGF-BB for the regulation of cell behavior. Stromal cell attachment, viability, release of osteogenic differentiation markers such as osteocalcin, and upregulation of osteogenic gene expression exhibited positive response. Overall, the developed scaffold system was noted to support rapid cell expansion and differentiation of stromal cells into osteogenic cells in vitro for bone tissue engineering applications.
Gellan-chitosan (GC) incorporated with CS: 0% (GC-0 CS), 10% (GC-10 CS), 20% (GC-20 CS) or 40% (GC-40 CS) w/w was prepared using freeze-drying method to investigate its physicochemical, biocompatible, and osteoinductive properties in human bone-marrow mesenchymal stromal cells (hBMSCs). The composition of different groups was reflected in physicochemical analyses performed using BET, FTIR, and XRD. The SEM micrographs revealed excellent hBMSCs attachment in GC-40 CS. The Alamar Blue assay indicated an increased proliferation and viability of seeded hBMSCs in all groups on day 21 as compared with day 0. The hBMSCs seeded in GC-40 CS indicated osteogenic differentiation based on an amplified alkaline-phosphatase release on day 7 and 14 as compared with day 0. These cells supported bone mineralization on GC-40 CS based on Alizarin-Red assay on day 21 as compared with day 7 and increased their osteogenic gene expression (RUNX2, ALP, BGLAP, BMP, and Osteonectin) on day 21. The GC-40 CS-seeded hBMSCs initiated their osteogenic differentiation on day 7 as compared with counterparts based on an increased expression of type-1 collagen and BMP2 in immunocytochemistry analysis. In conclusion, the incorporation of 40% (w/w) calcium silicate in gellan-chitosan showed osteoinduction potential in hBMSCs, making it a potential biomaterial to treat critical bone defects.
Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering.
In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology.
In this study, effect of degree of deacetylation on property and adsorption capacity of chitosan/polyvinyl Alcohol electrospun membrane has been investigated. Resulting nanofibers were characterized by FESEM, FTIR, XRD, TGA, tensile testing, weight loss test and adsorption test. FESEM result shows, finer nanofiber was fabricated from 42h hydrolyzed chitosan and PVA blend solution. FTIR and XRD result showed a strong interaction between chitosan and polyvinyl alcohol. Higher tensile strength was observed for the nanofiber having 42h hydrolyzed chitosan. Blend solution of chitosan/PVA having low DD chitosan had higher viscosity. The nanofibrous membrane was stable in distilled water, acidic and basic medium. The isotherm study shows that the adsorption capacity (qm) of nanofiber containing higher DD chitosan was higher for Cr(VI). In contrary, the membrane containing chitosan with lower DD showed the higher adsorption capacity for Fe(III) and methyl orange. Moreover, the effect of DD on removal percentage of adsorbate was dependent on the initial concentration of the adsorbate.
Given their highly porous nature and excellent water retention, hydrogel-based biomaterials can mimic critical properties of the native cellular environment. However, their potential to emulate the electromechanical milieu of native tissues or conform well with the curved topology of human organs needs to be further explored to address a broad range of physiological demands of the body. In this regard, the incorporation of nanomaterials within hydrogels has shown great promise, as a simple one-step approach, to generate multifunctional scaffolds with previously unattainable biological, mechanical, and electrical properties. Here, recent advances in the fabrication and application of nanocomposite hydrogels in tissue engineering applications are described, with specific attention toward skeletal and electroactive tissues, such as cardiac, nerve, bone, cartilage, and skeletal muscle. Additionally, some potential uses of nanoreinforced hydrogels within the emerging disciplines of cyborganics, bionics, and soft biorobotics are highlighted.