Sarah Calve

A continuum treatment of growth in biological tissue: The coupling of mass transport and mechanics

Growth (and resorption) of biological tissue is formulated in the continuum setting. The treatment is macroscopic, rather than cellular or sub-cellular. Certain assumptions that are central to classical continuum mechanics are revisited, the theory is reformulated, and consequences for balance laws and constitutive relations are deduced. The treatment incorporates multiple species. Sources and fluxes of mass, and terms for momentum and energy transfer between species are introduced to enhance the classical balance laws. The transported species include: (i) a fluid phase, and (ii) the precursors and byproducts of the reactions that create and break down tissue. A notable feature is that the full extent of coupling between mass transport and mechanics emerges from the thermodynamics. Contributions to fluxes from the concentration gradient, chemical potential gradient, stress gradient, body force and inertia have not emerged in a unified fashion from previous formulations of the problem. The present work demonstrates these effects via a physically consistent treatment. The presence of multiple, interacting species requires that the formulation be consistent with mixture theory. This requirement has far-reaching consequences. A preliminary numerical example is included to demonstrate some aspects of the coupled formulation.

Engineering of Functional Tendon

Surgical tendon repair is limited by the availability of viable tissue for transplantation. Because of its relatively avascular nature, tendon is a prime candidate for engineered tissue replacement. To address this problem, cells isolated from rat Achilles tendon were grown to confluence in culture and allowed to self-assemble into a cylinder between two anchor points. The resulting scaffold-free tissue was composed of aligned, small-diameter collagen fibrils, a large number of cells, and an excess of noncollagenous extracellular matrix; all characteristics of embryonic tendon. The stress-strain response of the constructs also resembles the nonlinear behavior of immature tendons, and the ultimate tensile strength is approximately equal to that of embryonic chick tendon, roughly 2 MPa. These physical and mechanical properties indicate that these constructs are the first viable tendons engineered in vitro, without the aid of artificial scaffolding.

A Transitional Extracellular Matrix Instructs Cell Behavior During Muscle Regeneration

Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.

Biochemical and mechanical environment cooperatively regulate skeletal muscle regeneration

During forelimb regeneration in the newt Notophthalmus viridescens, the dynamic expression of a transitional matrix rich in hyaluronic acid, tenascin‐C, and fibronectin controls muscle cell behavior in vivo and in vitro. However, the influence of extracellular matrix (ECM) remodeling on tissue stiffness and the cellular response to mechanical variations during regeneration was unknown. By measuring the transverse stiffness of tissues in situ, we found undifferentiated regenerative blastemas were less stiff than differentiated stump muscle (13.3±1.6 vs. 16.6±1.2 kPa). To directly determine how ECM and stiffness combine to affect skeletal muscle fragmentation, migration, and fusion, we coated silicone‐based substrates ranging from 2 to 100 kPa with matrices representative of transitional (tenascin‐C and fibronectin) and differentiated environments (laminin and Matrigel). Using live‐cell imaging, we found softer tenascin‐C‐coated substrates significantly enhanced migration and fragmentation of primary newt muscle cells. In contrast, stiffer substrates coated with laminin, Matrigel, or fibronectin increased differentiation while suppressing migration and fragmentation. These data support our in vivo observations that a transitional matrix of reduced stiffness regulates muscle plasticity and progenitor cell recruitment into the regenerative blastema. These new findings will enable the determination of how biochemical and mechanical cues from the ECM control genetic pathways that drive regeneration.—Calve, S., Simon, H.‐G. Biochemical and mechanical environment cooperatively regulate skeletal muscle regeneration. FASEB J. 26, 2538‐2545 (2012). www.fasebj.org

Biological remodelling: Stationary energy, configurational change, internal variables and dissipation

Abstract Remodelling is defined as an evolution of microstructure or variations in the configuration of the underlying manifold. The manner in which a biological tissue and its subsystems remodel their structure is treated in a continuum mechanical setting. While some examples of remodelling are conveniently modelled as evolution of the reference configuration—Case I—others are more suited to an internal variable description—Case II. In this paper, we explore the applicability of stationary energy states to remodelled systems. A variational treatment is introduced by assuming that stationary energy states are attained by changes in microstructure via one of the two mechanisms—Cases I and II. The configurational change of a long-chain molecule is presented as an example of Case I, and collagen fibre reorientation in in vitro tissue constructs as an example of Case II. The second example is further studied for its thermodynamic dissipation characteristics. This leads to an important finding on the limitation of purely mechanical treatments of some types of remodelling phenomena.

Hyaluronic acid, HAS1, and HAS2 are significantly upregulated during muscle hypertrophy

Hyaluronic acid (HA) is a component of the extracellular matrix (ECM) in most vertebrate tissues and is thought to play a significant role during development, wound healing, and regeneration. In vitro studies have shown that HA enhances muscle progenitor cell recruitment and inhibits premature myotube fusion, implicating a role for this glycosaminoglycan in functional repair. However, the spatiotemporal distribution of HA during muscle growth and repair was unknown. We hypothesized that inducing hypertrophy via synergist ablation would increase the expression of HA and the HA synthases (HAS1-HAS3). We found that HA and HAS1-HAS3 were significantly upregulated within the plantaris muscle in response to Achilles tenectomy. HA concentration significantly increased 2.8-fold after 2 days but decreased towards levels comparable to age-matched controls by 14 days. Using immunohistochemistry, we found the colocalization of HAS1-HAS3 with macrophages, blood vessel epithelia, and fibroblasts varied in response to time and/or tenectomy. At the level of gene expression, only HAS1 and HAS2 significantly increased with respect to both time and tenectomy. The profiles of additional genes that influence ECM composition during muscle repair, tenascin-C, type I collagen, the HA-degrading hyaluronidases (Hyal) and matrix metalloproteinases (MMP) were also investigated. Hyal1 and Hyal2 were highly expressed in skeletal muscle but did not change after tenectomy; however, indicators of hypertrophy, MMP-2 and MMP-14, were significantly upregulated from 2 to 14 days. These results indicate that HA levels dynamically change in response to a hypertrophic stimulus and various cells may participate in this mechanism of skeletal muscle adaptation.

The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments.

Growth factors play a central role in the development and remodelling of musculoskeletal tissues. To determine which growth factors optimized in vitro ligament formation and mechanics, a Box-Behnken designed array of varying concentrations of growth factors and ascorbic acid were applied to engineered ligaments and the collagen content and mechanics of the grafts were determined. Increasing the amount of transforming growth factor (TGF) β1 and insulin-like growth factor (IGF)-1 led to an additive effect on ligament collagen and maximal tensile load (MTL). In contrast, epidermal growth factor (EGF) had a negative effect on both collagen content and MTL. The predicted optimal growth media (50 μg/ml TGFβ, IGF-1, and GDF-7 and 200 μM ascorbic acid) was then validated in two separate trials: showing a 5.7-fold greater MTL and 5.2-fold more collagen than a minimal media. Notably, the effect of the maximized growth media was scalable such that larger constructs developed the same material properties, but larger MTL. These results show that optimizing the interactions between growth factors and engineered ligament volume results in an engineered ligament of clinically relevant function.

Reduced muscle fiber force production and disrupted myofibril architecture in patients with chronic rotator cuff tears

BACKGROUND A persistent atrophy of muscle fibers and an accumulation of fat, collectively referred to as fatty degeneration, commonly occur in patients with chronic rotator cuff tears. The etiology of fatty degeneration and function of the residual rotator cuff musculature have not been well characterized in humans. We hypothesized that muscles from patients with chronic rotator cuff tears have reduced muscle fiber force production, disordered myofibrils, and an accumulation of fat vacuoles. METHODS The contractility of muscle fibers from biopsy specimens of supraspinatus muscles of 13 patients with chronic full-thickness posterosuperior rotator cuff tears was measured and compared with data from healthy vastus lateralis muscle fibers. Correlations between muscle fiber contractility, American Shoulder and Elbow Surgeons (ASES) scores, and tear size were analyzed. Histology and electron microscopy were also performed. RESULTS Torn supraspinatus muscles had a 30% reduction in maximum isometric force production and a 29% reduction in normalized force compared with controls. Normalized supraspinatus fiber force positively correlated with ASES score and negatively correlated with tear size. Disordered sarcomeres were noted, along with an accumulation of lipid-laden macrophages in the extracellular matrix surrounding supraspinatus muscle fibers. CONCLUSIONS Patients with chronic supraspinatus tears have significant reductions in muscle fiber force production. Force production also correlates with ASES scores and tear size. The structural and functional muscle dysfunction of the residual muscle fibers is independent of the additional area taken up by fibrotic tissue. This work may help establish future therapies to restore muscle function after the repair of chronically torn rotator cuff muscles.

Optical Clearing in Dense Connective Tissues to Visualize Cellular Connectivity In Situ

Visualizing the three-dimensional morphology and spatial patterning of cells embedded deep within dense connective tissues of the musculoskeletal system has been possible only by utilizing destructive techniques. Here we utilize fructose-based clearing solutions to image cell connectivity and deep tissue-scale patterning in situ by standard confocal microscopy. Optical clearing takes advantage of refractive index matching of tissue and the embedding medium to visualize light transmission through a broad range of bovine and whole mount murine tissues, including cartilage, bone, and ligament, of the head and hindlimb. Using non-destructive methods, we show for the first time intercellular chondrocyte connections throughout the bulk of cartilage, and we reveal in situ patterns of osteocyte processes and the lacunar-canalicular system deep within mineralized cortical bone. Optical clearing of connective tissues is expected to find broad application for the study of cell responses in normal physiology and disease pathology.

Incorporation of non-canonical amino acids into the developing murine proteome

Analysis of the developing proteome has been complicated by a lack of tools that can be easily employed to label and identify newly synthesized proteins within complex biological mixtures. Here, we demonstrate that the methionine analogs azidohomoalanine and homopropargylglycine can be globally incorporated into the proteome of mice through facile intraperitoneal injections. These analogs contain bio-orthogonal chemical handles to which fluorescent tags can be conjugated to identify newly synthesized proteins. We show these non-canonical amino acids are incorporated into various tissues in juvenile mice and in a concentration dependent manner. Furthermore, administration of these methionine analogs to pregnant dams during a critical stage of murine development, E10.5–12.5 when many tissues are assembling, does not overtly disrupt development as assessed by proteomic analysis and normal parturition and growth of pups. This successful demonstration that non-canonical amino acids can be directly administered in vivo will enable future studies that seek to characterize the murine proteome during growth, disease and repair.