Enhanced osteogenic potential of human mesenchymal stem cells on electrospun nanofibrous scaffolds prepared from eri-tasar silk fibroin.

This study evaluated the mechanical properties and osteogenic potential of a silk fibroin scaffold prepared from a 70:30 blend of Eri (Philosamia ricini) and Tasar (Antheraea mylitta) silk, respectively (ET scaffolds). An electrospinning process was used to prepare uniformly blended, fibrous scaffolds of nanoscale dimensions, as confirmed by scanning and transmission electron microscopy (fiber diameter < 300 nm). Similarly prepared scaffolds derived from gelatin and Bombyx mori (BM) silk fibroin were used as controls. Mechanical testing and atomic force microscopy showed that the ET scaffolds had significantly higher tensile strength (1.83 ± 0.13 MPa) and surface roughness (0.44 µm) compared with BM (1.47 ± 0.10 MPa; 0.37 µm) and gelatin scaffolds (0.6 ± 0.07 MPa; 0.28 µm). All scaffolds were exposed to mesenchymal stem cells isolated to human chord blood (hMSCs) for up to 28 days in vitro. Alamar blue and alkaline phosphatase assay showed greater attachment and proliferation for both ET and BM scaffolds compared with gelatin. The ET scaffolds also promoted greater differentiation of the attached hMSCs as evidenced by higher expression of RunX2, osteocalcin, and CD29/CD44 expression. ET scaffolds also showed significantly higher mineralization, as evidenced by glycosaminoglycan assay, alizarin red staining, and elemental analysis of crystalline composites isolated from the scaffolds.

Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs.

Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me2SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofibrous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40mM), ectoin (40mM), catalase (100µg) as antioxidant and Me2SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered. The performance of the freezing medium towards cryopreservation of TEC was superior than the performance achieved using conventional Me2SO and similar to the non cryopreserved TEC. Overall we have formulated an efficient freezing medium that may pave the way of long term preservation of TECs with maintaining its integrity, MSCs viability and differentiation potentiality. It was observed that the performance of freezing medium for cryopreservation of TECs was better than the Me2SO.

Extraction and characterization of biocompatible hydroxyapatite from fresh water fish scales for tissue engineering scaffold.

In bone tissue engineering, porous hydroxyapatite (HAp) is used as filling material for bone defects, augmentation, artificial bone graft and scaffold material. The present paper compares the preparation and characterization of HAp from fish scale (FS) and synthetic body fluid (SBF) solution. Thermo gravimetric analysis, differential thermal analysis, Fourier transform infrared spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and particle size analysis of the samples have been performed. The analysis indicates that synthesized HAp consists of sub-micron HAp particle with Ca/P ratio corresponding to FS and SBF 1.62 and 1.71, respectively. MTT assay and quantitative DNA analysis show growth and proliferation of cells over the HA scaffold with the increase in time. The shape and size (morphology) of mesenchymal stem cells after 3 days show a transition from rounded shape to elongated and flattened shape expressing its spreading behavior. These results confirm that HAp bio-materials from fish scale are physico-chemically and biologically equivalent to the chemically synthesized HAp from SBF. Biological HAp, thus, possesses a great potential for conversion of industrial by-product into highly valuable compounds using simple effective and novel processes.

Development and evaluation of cross-linked collagen-hydroxyapatite scaffolds for tissue engineering.

This study examines the tissue engineering potential of type I collagen cross-linked in the presence of hydroxyapatite (HAp). Scaffolds were prepared by controlled freezing followed by lyophilization of composite mixtures of collagen and HAp in acetic acid, followed by cross-linking with 0.3% glutaraldehyde. Scaffolds of three ratios were prepared, corresponding to collagen/HAp ratios of 1:2, 1:4, and 1:6. The scaffolds were evaluated for their microstructure, chemical and physical properties, swelling behavior, mechanical strength, biodegradability hemocompatability, cytocompatibility, and histopathology following subcutaneous implantation in Sprague Dawley rats. The collagen/HAp matrices showed a smaller pore size of 10-40 µm compared to 50-100 µm for pure collagen scaffolds. Pure collagen showed a mechanical strength of 0.25 MPa, and the value almost doubled for cross-linked composites with collagen/HAp ratio 1:6. The improvement in mechanical strength corresponded to a decrease in swelling and enzymatic degradation (measured by resistance to collagenases). FTIR spectra results in conjunction with scanning electron micrographs showed that cross-linking in the presence of HAp did not significantly alter the structure of collagen. MTT assay and calcein AM staining revealed prominent and healthy growth of mesenchymal stem cells in both the pure collagen as well as collagen:HAp composites of ratio 1:2. In vivo implantation in Sprague Dawley rats showed an initial acute inflammatory response during days 3 and 7, followed by a chronic, macrophage-mediated inflammatory response on days 14 and 28. Overall, a cross-linked collagen/HAp composite scaffold of ratio 1:2 was identified as having potential for further development in tissue engineering.