Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

SU-8 has become the favourite photoresist for high-aspect-ratio (HAR) and three-dimensional (3D) lithographic patterning due to its excellent coating, planarization and processing properties as well as its mechanical and chemical stability. However, as feature sizes get smaller and pattern complexity increases, particular difficulties and a number of material-related issues arise and need to be carefully considered. This review presents a detailed description of these effects and describes reported strategies and achieved SU-8 HAR and 3D structures up to August 2006.

https://iopscience.iop.org/article/10.1088/0960-1317/17/6/R01/pdf

SU-8: a photoresist for high-aspect-ratio and 3D submicron lithography

The complement system is an evolutionary old and crucial component of innate immunity key to the detection and removal of invading pathogens. It was initially discovered as a liver-derived sentinel system circulating in serum, the lymph and interstitial fluids that mediates the opsonization and lytic killing of bacteria, fungi and viruses and the initiation of the general inflammatory responses. Although work performed specifically in the last five decades identified complement also as a critical instructor of adaptive immunity – indicating that complement’s function is likely broader than initially anticipated - the dominant opinion among researchers and clinicians was that the key complement functions were in principle defined. However, there is now a growing realization that complement activity goes well beyond ‘classic’ immune functions and that this system is also required for normal (neuronal) development and activity and general cell and tissue integrity and homeostasis. Furthermore, the recent discovery that complement activation is not confined to the extracellular space but occurs within cells led to the surprising understanding that complement is involved in the regulation of basic processes of the cell, particularly those of metabolic nature – mostly via novel crosstalks between complement and intracellular sensor, and effector, pathways that had been overlooked because of their spatial separation. These paradigm shifts in the field led to a renaissance in complement research and provide new platforms to now better understand the molecular pathways underlying the wide-reaching effects of complement functions in immunity and beyond. In this review, we will cover the current knowledge about complement’s emerging relationship with the cellular metabolism machinery with a focus on the functional differences between serum-circulating versus intracellularly active complement during normal cell survival and induction of effector functions. We will also discuss how taking a closer look into the evolution of key complement components not only made the functional connection between complement and metabolism rather ‘predictable’ but how it may also give clues for the discovery of additional roles for complement in basic cellular processes.

/pdf/keeping-it-all-going-complement-meets-metabolism-zth049hzu4.pdf

Keeping It All Going—Complement Meets Metabolism

GH and IGF-I are important regulators of bone homeostasis and are central to the achievement of normal longitudinal bone growth and bone mass. Although GH may act directly on skeletal cells, most of its effects are mediated by IGF-I, which is present in the systemic circulation and is synthesized by peripheral tissues. The availability of IGF-I is regulated by IGF binding proteins. IGF-I enhances the differentiated function of the osteoblast and bone formation. Adult GH deficiency causes low bone turnover osteoporosis with high risk of vertebral and nonvertebral fractures, and the low bone mass can be partially reversed by GH replacement. Acromegaly is characterized by high bone turnover, which can lead to bone loss and vertebral fractures, particularly in patients with coexistent hypogonadism. GH and IGF-I secretion are decreased in aging individuals, and abnormalities in the GH/IGF-I axis play a role in the pathogenesis of the osteoporosis of anorexia nervosa and after glucocorticoid exposure.

/pdf/growth-hormone-insulin-like-growth-factors-and-the-skeleton-48qvgpxetz.pdf

Growth Hormone, Insulin-Like Growth Factors, and the Skeleton

The cell microenvironment has emerged as a key determinant of cell behavior and function in development, physiology, and pathophysiology. The extracellular matrix (ECM) within the cell microenvironment serves not only as a structural foundation for cells but also as a source of three-dimensional (3D) biochemical and biophysical cues that trigger and regulate cell behaviors. Increasing evidence suggests that the 3D character of the microenvironment is required for development of many critical cell responses observed in vivo, fueling a surge in the development of functional and biomimetic materials for engineering the 3D cell microenvironment. Progress in the design of such materials has improved control of cell behaviors in 3D and advanced the fields of tissue regeneration, in vitro tissue models, large-scale cell differentiation, immunotherapy, and gene therapy. However, the field is still in its infancy, and discoveries about the nature of cell–microenvironment interactions continue to overturn much earl...

Functional and Biomimetic Materials for Engineering of the Three-Dimensional Cell Microenvironment

Currently, the concept of engineered tissues depends on the ability of cultured cells to fabricate new tissue around a scaffold. Ibis is inherently slow and expensive and has had limited success so far. We report here a new process for the cell-independent, controlled engineering of biomimetic scaffolds by rapid removal of fluid from hyperhydrated collagen gel (or other) constructs, using plastic compression (PC). PC fabrication produces dense, cellular, mechanically strong native collagen structures with controllable nano- and microscale biomimetic structures. The huge-scale shrinkage (> 100-fold) provides the ability to introduce controllable mechanical properties, microlayering, and embossed interface. topography without cell participation, but with high cell viability. Critically, this takes minutes rather than the conventional days and weeks. The rapidity and biomimetic potential of the PC fabrication process at the mesoscale opens a new route for the production of biomaterials and patient-customized tissues. It also represents a new concept in 'engineering' tissues.

Ultrarapid Engineering of Biomimetic Materials and Tissues: Fabrication of Nano‐ and Microstructures by Plastic Compression

3D in vitro models have been used in cancer research as a compromise between 2-dimensional cultures of isolated cancer cells and the manufactured complexity of xenografts of human cancers in immunocompromised animal hosts. 3D models can be tailored to be biomimetic and accurately recapitulate the native in vivo scenario in which they are found. These 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Approaches to create more biomimetic 3D models of cancer include, but are not limited to, (i) providing the appropriate matrix components in a 3D configuration found in vivo, (ii) co-culturing cancer cells, endothelial cells and other associated cells in a spatially relevant manner, (iii) monitoring and controlling hypoxia- to mimic levels found in native tumours and (iv) monitoring the release of angiogenic factors by cancer cells in response to hypoxia. This article aims to overview current 3D in vitro models of cancer and review strategies employed by researchers to tackle these aspects with special reference to recent promising developments, as well as the current limitations of 2D cultures and in vivo models. 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Here we review current strategies in the field of modelling cancer, with special reference to advances in complex 3D in vitro models.

3D tumour models: novel in vitro approaches to cancer studies

Collagen gel is a poroelastic/biphasic system consisting of a fibrillar loose lattice structure filled with >99% fluid. Its mechanical behaviour is governed by the inherent viscoelasticity of the fibrils, and their interaction with the fluid. This study investigated the underlying mechanisms of plastic compression (PC), a recently introduced technique for the production of dense collagen matrices for tissue engineering. Unconfined compressive loading results in the rapid expulsion of the fluid phase to produce scaffolds with improved mechanical properties potentially suitable for direct implantation and suturing. The controllability of the PC, as a single or multi-stage process was investigated in terms of fluid loss, remaining protein concentration, and morphological characteristics. Time dependent analysis, and quasi-static mechanical (compressive and tensile) properties of hyper-hydrated and PC collagen, produced by single (SC) and double (DC) compression, were also investigated on the non-covalently cross-linked gels. Under unconfined compressive creep, the behaviour of hyper hydrated gel was dictated by the fluid movement relative to the solid (i.e. poroelasticity) with negligible recovery upon load removal. Similar behaviour was achieved in multiple compressed gels; however, these progressively dense matrices displayed an instantaneous recovery that was in line with the increase in fibrillar collagen concentration. Under tension, where the mechanical response of the gels is dominated by the fibrils, there was significant increase in both break strength and modulus with increasing fibril concentration due to multiple compression as DC provided greater opportunity for physical interaction between the nano-sized fibrils.

Use of multiple unconfined compression for control of collagen gel scaffold density and mechanical properties.

It has been shown that the insulin-like growth factor (IGF-I) gene is spliced in response to mechanical signals producing forms of IGF-I which have different actions. In order to study how mechanical signals influence this gene splicing in developing muscle, C2C12 cells were grown in three-dimensional (3D) culture and subjected to different regimens of mechanical strain. IGF-IEa which initiates the fusion of myoblasts to form myotubes was found to be constitutively expressed in myoblasts and myotubes (held under endogenous tension) and its expression upregulated by a single ramp stretch of 1-h duration but reduced by repeated cyclical stretch. In contrast, mechano growth factor (MGF), which is involved in the proliferation of mononucleated myoblasts that are required for secondary myotube formation and to establish the muscle satellite (stem) cell pool, showed no significant constitutive expression in static cultures, but was upregulated by a single ramp stretch and by cycling loading. The latter types of force simulate those generated in myoblasts by the first contractions of myotubes. These data indicate the importance of seeking to understand the physiological signals that determine the ratios of splice variants of some growth factor/tissue factor genes in the early stages of development of skeletal muscle.

Mechanical signals and IGF-I gene splicing in vitro in relation to development of skeletal muscle.

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse c2c12 myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 Ea and MGF expression on day 3 of the developing construct. This peak of IGF-1 Ea and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 Ea stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 Ea on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues. Cell Motil. Cytoskeleton 54:226-236, 2003. (C) 2003 Wiley-Liss, Inc.

Umber Cheema

Papers

Ultrarapid Engineering of Biomimetic Materials and Tissues: Fabrication of Nano‐ and Microstructures by Plastic Compression

3D tumour models: novel in vitro approaches to cancer studies

Use of multiple unconfined compression for control of collagen gel scaffold density and mechanical properties.

Mechanical signals and IGF-I gene splicing in vitro in relation to development of skeletal muscle.

3-D in vitro model of early skeletal muscle development.