An insight into morphometric descriptors of cell shape that pertain to regenerative medicine.

Cellular morphology has recently been indicated as a powerful indicator of cellular function. The analysis of cell shape has evolved from rudimentary forms of microscopic visual inspection to more advanced methodologies that utilize high-resolution microscopy coupled with sophisticated computer hardware and software for data analysis. Despite this progress, there is still a lack of standardization in quantification of morphometric parameters. In addition, uncertainty remains as to which methodologies and parameters of cell morphology will yield meaningful data, which methods should be utilized to categorize cell shape, and the extent of reliability of measurements and the interpretation of the resulting analysis. A large range of descriptors has been employed to objectively assess the cellular morphology in two-dimensional and three-dimensional domains. Intuitively, simple and applicable morphometric descriptors are preferable and standardized protocols for cell shape analysis can be achieved with the help of computerized tools. In this review, cellular morphology is discussed as a descriptor of cellular function and the current morphometric parameters that are used quantitatively in two- and three-dimensional environments are described. Furthermore, the current problems associated with these morphometric measurements are addressed. Copyright © 2015 John Wiley & Sons, Ltd.

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.

Regeneration of the limb: opinions on the reality.

Whenever the topic of re-growing human limbs is posed for discussion, it is often argued that 'if a newt can do it, then so can we'. This notion, albeit promising, is somewhat like watching a science-fiction film; the individual components are currently available but we are far from realizing the complete picture. Today's reality is that if we are faced with a limb-severing injury, any regenerative attempt would endeavour to accelerate the pace at which the tissue heals to a clinically relevant/functional state. The science of limb regeneration can be approached from three different angles, developmental biology; regenerative medicine; and tissue engineering. This opinion piece describes how each approach can be used to understand the concepts behind regeneration, how far each approach has advanced and the hurdles faced by each of the approaches.

Simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell sheets and silk scaffolds for annulus fibrosus regeneration.

Most studies on the intervertebral disc (IVD) focus on the regeneration of the nucleus pulposus (NP). However, without a proper strategy to regenerate the damaged annulus fibrosus (AF), the NP replacements are bound to fail. Therefore the objective of this study was to investigate whether the use of bone marrow-derived mesenchymal stem cells (BMSCs) to form cell sheets, and incorporating them onto silk scaffolds, has the potential to regenerate the annulus fibrosus. The BMSC cell sheets and silk scaffolds were wrapped around a silicone NP substitute to form a simulated IVD-like assembly. The simulated IVD-like assembly was cultured for 4 weeks in static conditions and it was shown that the BMSC cell sheets remained viable, with no significant change in cell numbers. Histological analysis showed that the BMSC cell sheets adhered well onto the silk scaffolds and glycosaminoglycans (GAGs) were detected within the extracellular matrix (ECM). The ratio of collagen type I to collagen type II within the ECM of the BMSC cell sheets also decreased significantly over the period of culture. The results suggested that extensive remodelling of the ECM occurred within the simulated IVD-like assembly, and it is suitable for the regeneration of the inner AF.

Effects of radial compression on a novel simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell cell-sheets for annulus fibrosus regeneration.

STUDY DESIGN: The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement. OBJECTIVE: To determine whether a rehabilitative simulation regime on bone marrow­derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. SUMMARY OF BACKGROUND DATA: No previous study has used bone marrow­derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. METHODS: The approach was to use bone marrow­derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. RESULTS: With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. CONCLUSION: The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.

Multilineage potential of bone-marrow-derived mesenchymal stem cell cell sheets: implications for tissue engineering.

Bone-marrow-derived mesenchymal stem cells (BMSCs) are a promising source of cells for tissue engineering due to their multilineage mesenchymal differentiation potential. Their ability to proliferate and differentiate into the osteogenic, chondrogenic, and adipogenic lineage makes them an attractive cell source as compared to the terminally differentiated cells. In tissue engineering, use of cell sheet technology is gaining popularity. It is based on culturing cells until hyperconfluence, and it has resulted in the reduction of the number of cells lost when seeding onto scaffolds. Thus, formation of cell sheets with multipotent cells, such as BMSCs, would be a promising alternative to the conventional method of cell seeding, that is, single-cell suspension. However, the multilineage potential of BMSC cell sheets has yet to be verified. Therefore, the aim of this study was to characterize the formation of a hyperconfluent BMSC cell sheet as well as the effects of the hyperconfluent culture conditions on the multipotentiality of BMSCs. Our results showed that the BMSC cell sheets remained viable. The cell sheets were rich with type I collagen and were shown to have retained their multipotentiality. Hence, the use of BMSC cell sheets for tissue engineering application seems promising.

Technique to accurately quantify collagen content in hyperconfluent cell culture.

Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.