Stem cell-derived cell-sheets for connective tissue engineering.

Cell-sheet technology involves the recovery of cells with its secreted ECM and cell-cell junctions intact, and thereby harvesting them in a single contiguous layer. Temperature changes coupled with a thermoresponsive polymer grafted culture plate surface are typically used to induce detachment of this cell-matrix layer by controlling the hydrophobicity and hydrophilicity properties of the culture surface. This review article details the genesis and development of this technique as a critical tissue-engineering tool, with a comprehensive discussion on connective tissue applications. This includes applications in the myocardial, vascular, cartilage, bone, tendon/ligament, and periodontal areas among others discussed. In particular, further focus will be given to the use of stem cells-derived cell-sheets, such as those involving bone marrow-derived and adipose tissue-derived mesenchymal stem cells. In addition, some of the associated challenges faced by approaches using stem cells-derived cell-sheets will also be discussed. Finally, recent advances pertaining to technologies forming, detaching, and manipulating cell-sheets will be covered in view of the potential impact they will have on shaping the way cell-sheet technology will be utilized in the future as a tissue-engineering technique.

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets.

Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.

Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals.

Characterization and mechanical performance study of silk/PVA cryogels: towards nucleus pulposus tissue engineering.

Poly (vinyl) alcohol (PVA) cryogels are reported in the literature for application in nucleus pulposus (NP) replacement strategies. However, these studies are mainly limited to acellular approaches-in part due to the high hydrophilicity of PVA gels that renders cellular adhesion difficult. Silk is a versatile biomaterial with excellent biocompatibility. We hypothesize that the incorporation of silk with PVA will (i) improve the cell-hosting abilities of PVA cryogels and (ii) allow better tailoring of physical properties of the composite cryogels for an NP tissue engineering purpose. 5% (wt/vol) PVA is blended with 5% silk fibroin (wt/vol) to investigate the effect of silk : PVA ratios on the cryogels' physical properties. Results show that the addition of silk results in composite cryogels that are able to swell to more than 10 times its original dry weight and rehydrate to at least 70% of its original wet weight. Adding at least 20% silk significantly improves surface hydrophobicity and is correlated with an improvement in cell-hosting abilities. Cell-seeded cryogels also display an increment in compressive modulus and hoop stress values. In all, adding silk to PVA creates cryogels that can be potentially used as NP replacements.

Dynamic imaging with dual-source gated Computed Tomography (CT): implications of motion parameters on image quality for wrist imaging.

OBJECTIVE: Dynamic Computed Tomography (CT) promises insights into the pathophysiology of carpal instability by recording images of the carpus while it is in motion. The purpose of this study was to investigate the effect of motion velocity on image quality for dynamic carpal imaging applications using a clinical dual-source CT (DSCT) scanner. METHODS: A phantom with targets in the axial, coronal and sagittal planes was attached to a motion simulator and imaged using a 64-slice DSCT scanner. Data was acquired when the phantom was stationary and during periodic linear motion. Spatial resolution, motion artifacts and banding artifacts were assessed. RESULTS: Mean spatial resolution was 0.82 mm at 36 mm/s and 0.79 mm at 18 mm/s. Banding artifacts were mild at 36 mm/s and minimal at 18 mm/s. Motion artifacts were minimal at motion velocity of up to 36 mm/s in both the coronal and sagittal planes. Axial plane motion artifacts were moderate at 36 mm/s and mild at 18 mm/s. DISCUSSION: Sub-millimeter resolution is achievable with commercially available DSCT scanners with mild to moderate amounts of motion artifacts at velocities of 18 mm/s and 36 mm/s respectively.

A biomechanical comparison of 3 loop suture materials in a 6-strand flexor tendon repair technique.

PURPOSE: The braided polyblend (FiberWire) suture is recognized for its superiority in tensile strength in flexor tendon repair. The purpose of this study was to compare the biomechanical performance of 3 loop-suture materials used in a locking 6-strand flexor tendon repair configuration: braided polyblend (FiberLoop 4-0), cable nylon (Supramid Extra II 4-0), and braided polyester (Tendo-Loop 4-0). We hypothesized that, using this technique, the braided polyblend suture would give superior tensile strength compared with the other 2 suture materials. METHODS: We divided 30 fresh porcine flexor tendons transversely and repaired each with 1 of the 3 suture materials using a modified Lim-Tsai 6-strand suture technique. We loaded the repaired tendons to failure using a materials testing machine and collected data on the mechanism of failure, ultimate tensile strength, gap strength, and stiffness. RESULTS: Failure mechanisms for the repaired specimens were as follows: the braided polyblend had 50% suture breakage and 50% suture pullout; the cable nylon had 100% suture breakage; and the braided polyester had 80% suture breakage and 20% suture pullout. Specimens repaired with the braided polyblend suture had the highest mean ultimate tensile strength (97 N; standard deviation, 22) and the highest mean gap force (35 N; standard deviation, 7). CONCLUSIONS: This study supports the findings of previous studies showing superior strength of the braided polyblend suture. CLINICAL RELEVANCE: We were able to achieve up to 124 N in ultimate tensile strength and 48 N of gap force with this suture in porcine tendons. This gives greater confidence in starting immediate controlled passive or active rehabilitation after repair of flexor tendon injuries.