Knee instability caused by altered graft mechanical properties after anterior cruciate ligament reconstruction:the early onset of osteoarthritis?

Anterior cruciate ligament (ACL) rupture is a very common knee joint injury. Torn ACLs are currently reconstructed using tendon autografts. However, half of the patients develop osteoarthritis (OA) within 10 to 14 years postoperatively. Proposedly, this is caused by altered knee kine(ma)tics originating from changes in graft mechanical properties during the in vivo remodeling response. Therefore, the main aim was to use subject-specific finite element knee models and investigate the influence of decreasing graft stiffness and/or increasing graft laxity on knee kine(ma)tics and cartilage loading. In this research, 4 subject-specific knee geometries were used, and the material properties of the ACL were altered to either match currently used grafts or mimic in vivo graft remodeling, i.e., decreasing graft stiffness and/or increasing graft laxity. The results confirm that the in vivo graft remodeling process increases the knee range of motion, up to &gt;300 percent, and relocates the cartilage contact pressures, up to 4.3 mm. The effect of remodeling-induced graft mechanical properties on knee stability exceeded that of graft mechanical properties at the time of surgery. This indicates that altered mechanical properties of ACL grafts, caused by in vivo remodeling, can initiate the early onset of osteoarthritis, as observed in many patients clinically.</p

A decellularized and sterilized human meniscus allograft for off-the-shelf meniscus replacement

PURPOSE: Meniscus tears are one of the most frequent orthopedic knee injuries, which are currently often treated performing meniscectomy. Clinical concerns comprise progressive degeneration of the meniscus tissue, a change in knee biomechanics, and an early onset of osteoarthritis. To overcome these problems, meniscal transplant surgery can be performed. However, adequate meniscal replacements remain to be a great challenge. In this research, we propose the use of a decellularized and sterilized human meniscus allograft as meniscal replacement.METHODS: Human menisci were subjected to a decellularization protocol combined with sterilization using supercritical carbon dioxide (scCO 2). The decellularization efficiency of human meniscus tissue was evaluated via DNA quantification and Hematoxylin &amp; Eosin (H&amp;E) and DAPI staining. The mechanical properties of native, decellularized, and decellularized + sterilized meniscus tissue were evaluated, and its composition was determined via collagen and glycosaminoglycan (GAG) quantification, and a collagen and GAG stain. Additionally, cytocompatibility was determined in vitro. RESULTS: Human menisci were decellularized to DNA levels of ~ 20 ng/mg of tissue dry weight. The mechanical properties and composition of human meniscus were not significantly affected by decellularization and sterilization. Histologically, the decellularized and sterilized meniscus tissue had maintained its collagen and glycosaminoglycan structure and distribution. Besides, the processed tissues were not cytotoxic to seeded human dermal fibroblasts in vitro.CONCLUSIONS: Human meniscus tissue was successfully decellularized, while maintaining biomechanical, structural, and compositional properties, without signs of in vitro cytotoxicity. The ease at which human meniscus tissue can be efficiently decellularized, while maintaining its native properties, paves the way towards clinical use.</p

Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of single fibronectin fibers enhanced the non-persistent migration of both Fnf/f and Fn-/- MEFs, the migration speed was increased for Fn-/- MEFs on plasma fibronectin fibers compared to Fnf/f MEFs. In contrast, the average speed was the same for all cells on collagen-coated Fn fibers. A Fn-FRET sensor revealed that fibronectin on average was more extended on the microtissue surface compared to fibronectin in the core. Gradients of collagen-to-fibronectin ratios and of the fraction of collagen-adsorbed to stretched fibrillar fibronectin conformations might thereby provide critical cell migration cues. This study highlights a dominant role for fibronectin in tissue morphogenesis and the development of tissue heterogeneities

Increased Cell Traction-Induced Prestress in Dynamically Cultured Microtissues

Prestress is a phenomenon present in many cardiovascular tissues and has profound implications on their in vivo functionality. For instance, the in vivo mechanical properties are altered by the presence of prestress, and prestress also influences tissue growth and remodeling processes. The development of tissue prestress typically originates from complex growth and remodeling phenomena which yet remain to be elucidated. One particularly interesting mechanism in which prestress develops is by active traction forces generated by cells embedded in the tissue by means of their actin stress fibers. In order to understand how these traction forces influence tissue prestress, many have used microfabricated, high-throughput, micrometer scale setups to culture microtissues which actively generate prestress to specially designed cantilevers. By measuring the displacement of these cantilevers, the prestress response to all kinds of perturbations can be monitored. In the present study, such a microfabricated tissue gauge platform was combined with the commercially available Flexcell system to facilitate dynamic cyclic stretching of microtissues. First, the setup was validated to quantify the dynamic microtissue stretch applied during the experiments. Next, the microtissues were subjected to a dynamic loading regime for 24 h. After this interval, the prestress increased to levels over twice as high compared to static controls. The prestress in these tissues was completely abated when a ROCK-inhibitor was added, showing that the development of this prestress can be completely attributed to the cell-generated traction forces. Finally, after switching the microtissues back to static loading conditions, or when removing the ROCK-inhibitor, prestress magnitudes were restored to original values. These findings show that intrinsic cell-generated prestress is a highly controlled parameter, where the actin stress fibers serve as a mechanostat to regulate this prestress. Since almost all cardiovascular tissues are exposed to a dynamic loading regime, these findings have important implications for the mechanical testing of these tissues, or when designing cardiovascular tissue engineering therapies

How periosteum is involved in long bone growth

Skeletal growth is a tightly controlled phenomenon. In general, it is difficult to correct for skeletal malformations that develop during growth. One of the tissues that are proposed to be of major importance in regulating long bone growth is the periosteum. Previously, it was proposed that periosteum regulates growth via a direct mechanical feedback mechanism where pressure in growing cartilage, balanced by tension in the periosteum, modulates growth processes of chondrocytes. The presence of this tension is attributed to the periosteal collagen. We support the existence of a load-dependent feedback mechanism between cartilage pressure and periosteum tension by showing that the global orientation of collagen in perichondrium and periosteum concurs with the assessed growth directions of cartilage. Nevertheless, the absolute magnitude of periosteum tension determines the extent to which regulation by this mechanical feedback mechanism is present. From measurements described in this thesis, it is concluded that residual periosteum force does not directly dominate modulation of cartilage growth. Hence, a mechano-biological feedback mechanism must prevail. We demonstrate this mechano-biological feedback loop between growing cartilage and tension in the periosteum, whereby the expression of soluble growth-inhibiting factors by periosteum cells is dependent on intracellular tension. Because cartilage growth lengthens the periosteum at a very high rate, a mechanism of fibrous tissue adaptation that preserves low periosteum tension is required. We show that this mechanism is through adaptation towards a state of equilibrium, characterized by a residual tissue strain that corresponds to the strain in between the pliant and stiffer region of the force-strain curve. This process is cell-mediated and involves a structural organization of the collagen network, determining the tissue stiffness, which is not directly coupled to the collagen or HP crosslink density. This thesis takes us closer towards understanding the regulation of bone growth, by not only demonstrating the critical involvement of the periosteum, but also demonstrating mechanisms for the interaction between periosteum and growing cartilage, and for periosteum adaptation

Tendon injury and repair - A perspective on the basic mechanisms of tendon disease and future clinical therapy

Tendon is an intricately organized connective tissue that efficiently transfers muscle force to the bony skeleton. Its structure, function, and physiology reflect the extreme, repetitive mechanical stresses that tendon tissues bear. These mechanical demands also lie beneath high clinical rates of tendon disorders, and present daunting challenges for clinical treatment of these ailments. This article aims to provide perspective on the most urgent frontiers of tendon research and therapeutic development. We start by broadly introducing essential elements of current understanding about tendon structure, function, physiology, damage, and repair. We then introduce and describe a novel paradigm explaining tendon disease progression from initial accumulation of damage in the tendon core to eventual vascular recruitment from the surrounding synovial tissues. We conclude with a perspective on the important role that biomaterials will play in translating research discoveries to the patient. STATEMENT OF SIGNIFICANCE Tendon and ligament problems represent the most frequent musculoskeletal complaints for which patients seek medical attention. Current therapeutic options for addressing tendon disorders are often ineffective, and the need for improved understanding of tendon physiology is urgent. This perspective article summarizes essential elements of our current knowledge on tendon structure, function, physiology, damage, and repair. It also describes a novel framework to understand tendon physiology and pathophysiology that may be useful in pushing the field forward

Tissue alignment enhances remodeling potential of tendon-derived cells - Lessons from a novel microtissue model of tendon scarring

Tendinopathy is a widespread and unresolved clinical challenge, in which associated pain and hampered mobility present a major cause for work-related disability. Tendinopathy associates with a change from a healthy tissue with aligned extracellular matrix (ECM) and highly polarized cells that are connected head-to-tail, towards a diseased tissue with a disorganized ECM and randomly distributed cells, scar-like features that are commonly attributed to poor innate regenerative capacity of the tissue. A fundamental clinical dilemma with this scarring process is whether treatment strategies should focus on healing the affected (disorganized) tissue or strengthen the remaining healthy (anisotropic) tissue. The question was thus asked whether the intrinsic remodeling capacity of tendon-derived cells depends on the organization of the 3D extracellular matrix (isotropic vs anisotropic). Progress in this field is hampered by the lack of suitable in vitro tissue platforms. We aimed at filling this critical gap by creating and exploiting a next generation tissue platform that mimics aspects of the tendon scarring process; cellular response to a gradient in tissue organization from isotropic (scarred/non-aligned) to highly anisotropic (unscarred/aligned) was studied, as was a transient change from isotropic towards highly anisotropic. Strikingly, cells residing in an 'unscarred' anisotropic tissue indicated superior remodeling capacity (increased gene expression levels of collagen, matrix metalloproteinases MMPs, tissue inhibitors of MMPs), when compared to their 'scarred' isotropic counterparts. A numerical model then supported the hypothesis that cellular remodeling capacity may correlate to cellular alignment strength. This in turn may have improved cellular communication, and could thus relate to the more pronounced connexin43 gap junctions observed in anisotropic tissues. In conclusion, increased tissue anisotropy was observed to enhance the cellular potential for functional remodeling of the matrix. This may explain the poor regenerative capacity of tenocytes in chronic tendinopathy, where the pathological process has resulted in ECM disorganization. Additionally, it lends support to treatment strategies that focus on strengthening the remaining healthy tissue, rather than regenerating scarred tissue

Shaping tissues by balancing active forces and geometric constraints

The self-organization of cells into complex tissues during growth and regeneration is a combination of physical–mechanical events and biochemical signal processing. Cells actively generate forces at all stages in this process, and according to the laws of mechanics, these forces result in stress fields defined by the geometric boundary conditions of the cell and tissue. The unique ability of cells to translate such force patterns into biochemical information and vice versa sets biological tissues apart from any other material. In this topical review, we summarize the current knowledge and open questions of how forces and geometry act together on scales from the single cell to tissues and organisms, and how their interaction determines biological shape and structure. Starting with a planar surface as the simplest type of geometric constraint, we review literature on how forces during cell spreading and adhesion together with geometric constraints impact cell shape, stress patterns, and the resulting biological response. We then move on to include cell–cell interactions and the role of forces in monolayers and in collective cell migration, and introduce curvature at the transition from flat cell sheets to three-dimensional (3D) tissues. Fibrous 3D environments, as cells experience them in the body, introduce new mechanical boundary conditions and change cell behaviour compared to flat surfaces. Starting from early work on force transmission and collagen remodelling, we discuss recent discoveries on the interaction with geometric constraints and the resulting structure formation and network organization in 3D. Recent literature on two physiological scenarios—embryonic development and bone—is reviewed to demonstrate the role of the force-geometry balance in living organisms. Furthermore, the role of mechanics in pathological scenarios such as cancer is discussed. We conclude by highlighting common physical principles guiding cell mechanics, tissue patterning and matrix organization under geometric constraints across multiple length and time scales

Serum deprivation limits loss and promotes recovery of tenogenic phenotype in tendon cell culture systems

Current knowledge gaps on tendon tissue healing can partly be ascribed to the limited availability of physiologically relevant culture models. An unnatural extracellular matrix, high serum levels and random cell morphology in vitro mimic strong vascularization and lost cell elongation in pathology, and discord with a healthy, in vivo cell microenvironment. The thereby induced phenotypic drift in tendon-derived cells (TDCs) compromises the validity of the research model. Therefore, this research quantified the extracellular matrix (ECM)-, serum-, and cell morphology-guided phenotypic changes in tendon cells of whole tendon fascicle explants with intact ECM and TDCs cultured in a controlled microenvironmental niche. Explanted murine tail tendon fascicles were cultured in serum-rich or serum-free medium and phenotype was assessed using transcriptome analysis. Next, phenotypic marker gene expression was measured in in vitro expanded murine tail TDCs upon culture in serum-rich or serum-free medium on aligned or random collagen I patterns. Freshly isolated fascicles or TDCs served as native controls. In both systems, the majority of tendon-specific genes were similarly attenuated in serum-rich culture. Strikingly, 1-week serum-deprived culture-independent of cell morphology-converged TDC gene expression toward native levels. This study reveals a dynamic serum-responsive tendon cell phenotype. Extracting fascicles or TDCs from their native environment causes large changes in cellular phenotype, which can be limited and even reversed by serum deprivation. We conclude that serum-derived factors override matrix-integrity and cell morphology cues and that serum-deprivation stimulates a more physiological microenvironment for in vitro studies