Cell/Tissue Microenvironment Engineering and Monitoring in Tissue Engineering, Regenerative Medicine, and In Vitro Tissue Models

In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells’ behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial importance for the ability to direct and control cell behaviour in 3-dimensional tissueengineering scaffolds spatially and temporally. In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future. Cartilage extracellular matrix (ECM) is composed primarily of the network type II collagen (COLII) and an interlocking mesh of fibrous proteins and proteoglycans (PGs), hyaluronic acid (HA), and chondroitin sulfate (CS). Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO) signal, protein kinase C (PKC), and retinoic acid (RA) signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis. To investigate the safety and clinical efficacy of AA-PRP injections for pattern hair loss. AA-PRP, prepared from a small volume of blood, was injected on half of the selected patients’ scalps with pattern hair loss. The other half was treated with placebo. Three treatments were given for each patient, with intervals of 1 month. The endpoints were hair re-growth, hair dystrophy as measured by dermoscopy, burning or itching sensation, and cell proliferation as measured by Ki-67 evaluation. At the end of the 3 cycles of treatment, the patients presented clinical improvement in the mean number of hairs, with a mean increase of 18.0 hairs in the target area, and a mean increase in total hair density of 27.7 ( number of hairs/cm 2 ) compared with baseline values. Microscopic evaluation showed the increase of epidermis thickness and of the number of hair follicles two weeks after the last AA-PRP treatment compared to baseline value ( 𝑃 < 0.05 ). We also observed an increase of Ki67 + keratinocytes of epidermis and of hair follicular bulge cells and a slight increase of small blood vessels around hair follicles in the treated skin compared to baseline ( 𝑃 < 0.05 ). Compressive stimulation can modulate articular chondrocyte functions. Nevertheless, the relevant studies are not comprehensive. This is primarily due to the lack of cell culture apparatuses capable of conducting the experiments in a high throughput, precise, and cost-effective manner. To address the issue, we demonstrated the use of a perfusion microcell culture system to investigate the stimulating frequency (0.5, 1.0, and 2.0 Hz) effect of compressive loading (20% and 40% strain) on the functions of articular chondrocytes. The system mainly integrates the functions of continuous culture medium perfusion and the generation of pneumatically-driven compressive stimulation in a high-throughput micro cell culture system. Results showed that the compressive stimulations explored did not have a significant impact on chondrocyte viability and proliferation. However, the metabolic activity of chondrocytes was significantly affected by the stimulating frequency at the higher compressive strain of 40% (2Hz, 40% strain). Under the two compressive strains studied, the glycosaminoglycans (GAGs) synthesis was upregulated when the stimulating frequency was set at 1 Hz and 2 Hz. However, the stimulating frequencies explored had no influence on the collagen production. The results of this study provide useful fundamental insights that will be helpful for cartilage tissue engineering and cartilage rehabilitation. The purpose of this study was to develop the pathway of silk fibroin (SF) biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs) and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM) greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.

In any engineered system, the understanding of the properties and interactions between the system components is of utmost importance for a successful outcome. The main components in engineered tissues are the cells, the materials used in construction of scaffolds, soluble or immobilized bioactive agents, and physical and chemical stimuli presented by the environment. As most of the mammalian tissues are constructed by bringing together repeating units of microscale complex tissue structures, understanding and control of all these components would provide the tissue engineers the capability to overcome clinical challenges as well as to develop technologies for high fidelity tissue models suitable for pharmacology, toxicology, and disease modelling applications.
As tissue engineering and regenerative medicine fields mature, the level of information about how cells interact with the surrounding scaffold materials and/or other cells has increased, too. Cell microenvironment, which can be defined as the sum of all the stimuli that stem from the neighborhood of a cell and have direct or indirect effects on a given cell, has become an important consideration in exerting more control over the interactions of the cells with engineered structures.
Another important aspect of tissue engineering and regenerative medicine is their temporal nature. An engineered tissue is actively remodelled over a period of time either in vitro or in vivo and currently our ability to influence this process in order to interfere with the sequence of events to achieve better regeneration is limited. However, developments in noninvasive monitoring methods and biosensor systems have begun to provide the necessary tools for tissue engineers to have real-time information about engineered tissues.
This special issue set out to demonstrate the current developments and future perspectives in the use of cell microenvironment engineering and monitoring in producing W.-Y. Lin et al. (2014) studied another important aspect of cell microenvironment, namely, the effect of dynamic mechanical stress/strain conditions. They have developed a system which can apply cyclic compressive stress to chondrocytes at physiologically relevant levels. They have shown that the application of stress has a direct effect on the chondrocyte metabolic activity and glycosaminoglycan secretion. Another aspect of cartilage tissue engineering, the extracellular matrix/chondrocyte interactions, was reviewed by Gao et al. (2014), where they focused on the signaling pathways that are active during chondrogenesis. In another review paper, Barthes et al. (2014) gave a comprehensive description of cell microenvironment and how each component can be used to direct cellular activity for tissue engineering applications, together with the current developments in the monitoring of artificial tissues.
The advances in tissue engineering not only established it as a field where solutions to serious clinical problems can be developed but also as a growing area where enabling technologies such as organ-on-a-chip systems for pharmacological purposes can be devised. In order to have models accurately mimicking artificial organs, it is important to have a good grasp of the specific microenvironments pertaining to each tissue type. We believe engineering of the cell microenvironment will be an important part of future tissue engineering activities.